WO2020081720A1 - Electronic system for capture and characterization of particles - Google Patents

Electronic system for capture and characterization of particles Download PDF

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Publication number
WO2020081720A1
WO2020081720A1 PCT/US2019/056586 US2019056586W WO2020081720A1 WO 2020081720 A1 WO2020081720 A1 WO 2020081720A1 US 2019056586 W US2019056586 W US 2019056586W WO 2020081720 A1 WO2020081720 A1 WO 2020081720A1
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WIPO (PCT)
Prior art keywords
electrodes
module
particles
sample
counter
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PCT/US2019/056586
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French (fr)
Inventor
Mark Reed
Shari YOSINSKI
David Peaper
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Yale University
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Publication of WO2020081720A1 publication Critical patent/WO2020081720A1/en

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0645—Electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0848—Specific forms of parts of containers
    • B01L2300/0851—Bottom walls
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/16—Surface properties and coatings
    • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0424—Dielectrophoretic forces

Definitions

  • the invention is generally directed to electronic devices for capturing and characterizing particles, including biological material, in a sample.
  • Detection of biological material, such as microorganisms, in a biological sample often requires the capture and identification of live microorganisms.
  • rapid identification of live microorganisms in samples remains a challenge. It requires culturing live microorganisms from samples and then assaying them for identification, such as through staining and/or microscopic evaluation. This can take days and hinder diagnosis and treatment.
  • PCR polymerase chain reaction
  • LODs range from 100 to over 1000 CFU/mL for relevant pathogens.
  • the devices typically include one or more units, each unit having a trap module containing at least two electrodes, and a primary microchannel with an inlet and an outlet.
  • the trap module itself may be used for capture and identification of particles.
  • Each unit of the devices may also include an identification module containing at least two electrodes and a secondary microchannel with an inlet and an outlet.
  • the trap module is typically fluidically connected with the identification module. Generally, the dimensions of the identification module are substantially smaller than the dimensions of the trap module.
  • the devices may include an integrated counter module.
  • the counter module is fluidically connected to the trap module or to the identification module.
  • the counter module may include a constriction, such as a microchannel, an inlet to the microchannel and an outlet.
  • the microchannel is typically arranged to provide fluid flow over two or more electrodes.
  • the counter module does not include a microchannel and is constrictionless.
  • the sample from the trap module or the identification module flows over the two or more electrodes of the counter module.
  • the particles are captured over the electrodes of the trap module or the identification module. The particles then flow over the counter electrodes and are assayed by the operability of the counter electrodes.
  • the portable particle counter devices typically include a region with two or more electrodes and a fluidic channel overlaying the electrodes. A sample is passed over the one or more electrodes for obtaining the characteristics of particles.
  • the counter device may include a constriction, such as a microchannel, an inlet to the microchannel and an outlet.
  • the microchannel if present, is typically arranged to provide fluid flow over two or more electrodes.
  • the counter device does not include a microchannel and is constrictionless.
  • the sample flows over the two or more electrodes of the counter module.
  • the sample is constricted over the electrodes by the operability of the counter electrodes.
  • the trap module and the identification module may be connected fluidically, via electrodes, or both fluidically and via electrodes.
  • the electrodes connecting the trap module with the identification module may be patterned to focus biological material onto the identification module.
  • the device has a plurality of units, such as at least two, at least three, at least four, at least five, or at least six units.
  • the at least two electrodes of the trap module and the identification module typically have a width between about 1 nm and about 10 cm, between about 1 nm and about 1 cm, between about 1 nm and about 1 mm, or between about 10 nm and about 1000 pm.
  • the electrodes may be separated from one another by a distance as wide as the width of the electrodes. In other aspects, the at least two electrodes are separated from one another by a distance that is less or more than the width of the electrodes.
  • the distance separating the at least two electrodes may be between about 0.01 times and 10 times of the width of the electrodes.
  • the primary microchannel may have a height as high as the distance between the at least two electrodes of the trap module.
  • the at least two electrodes in the trap module and the identification module may be arranged in any two-dimensional pattern, such as in linear, angled, circular, rectangle, cube, triangle, zigzag, and oval pattern.
  • the electrodes may be pitched with respect to the microfluidic channel.
  • the electrodes may be coated with an insulator.
  • the method generally includes flowing a sample with the biological material of interest through the trap module of the device at a flow rate generally between about 0.01 pL/min and 10 mL/min.
  • the method also includes supplying an alternating current (AC) to the electrodes of the trap module at a frequency specific to the biological material.
  • the method operates under the principle that applying an AC to the at least two electrodes of the trap module at a frequency sufficient to provide a dielectrophoretic (DEP) force to the biological material at which its Clausius- Mossotti (CM) factor is maximum, traps the biological material in the device.
  • DEP dielectrophoretic
  • a device with more than one unit may be used in a single method, and more than one species of biological material may be trapped by the device. Typically, the different species are trapped in the different units of the device.
  • the method may include further condensing the trapped biological material in the identification module. The condensing of the biological material from the trap module into the identification module may occur with the use of focusing electrodes connecting the trap module with the identification module. The trapped biological material may be moved onto an identification module by applying an AC signal to the focusing electrodes connecting the trap module with the identification module.
  • the condensing of the biological material from the trap module into the identification module may occur via a microfluidic shuttle system.
  • the shuttle system of the device includes closing the outlet of the primary microchannel, opening the inlet of the secondary microchannel, stopping the AC applied to the two electrodes of the trap module, and applying an AC to the two electrodes of the identification module to condense the particles from the trap module in the identification module.
  • the particles in the identification module may then be further processed.
  • the further processing include microscopic examination without staining, microscopic examination with staining, or assaying of the particles in the counter module.
  • the assaying typically includes counting the particles, characterizing particle size, shape, activation state, division state, metabolic state, live/dead determination, surface marker expression, and others.
  • the condensing of the particles from the trap module into the identification module may also occur by a combination of focusing electrodes and the microfluidic shuttle system.
  • the devices may be used with biological samples, for example, to detect contamination of biological samples.
  • the devices may also be used in the food industry, for example, to detect contamination or to monitor pathogen levels.
  • the devices may be used to monitor the environment, such as monitoring the presence of polluting particles, or the presence of a biological material of interest in waters or in industrial runoff.
  • the devices rapidly capture, identify and/or characterize particles of diverse origin and composition. Exemplary particles include live cells, microorganisms, macro molecules, nanoparticles, microparticles, and other particulates in the environment, food, or a biological sample.
  • the biological material of interest may be a eukaryotic or prokaryotic cell, microorganisms, organelles, vesicles (such as exosomes, endosomes, phagosomes, and liposomes), macromolecules, or biomarkers.
  • the microorganisms captured and identified by the device and methods include bacteria, viruses, fungi, and parasites.
  • Figure 1 is a line graph showing the frequency (f (Hz)) dependence of the Clausius-Mossotti (CM) factor, for a homogeneous particle and medium. At low frequencies the DEP force is positive (pDEP); at high frequencies the DEP force is negative (nDEP).
  • Figure 2A is a diagram showing DEP condenser electrode structure (diameter 500 pm).
  • Figures 2B, 2C, and 2D are diagrams showing different geometries of electrodes: a linear medium interdigitated finger pairs (IDE) ( Figure 2B), an angled IDE ( Figure 2C), and a linear small IDE ( Figure 2D).
  • Figure 2E is a diagram of a typical pair of interdigitated electrode in a device with an electrode-electrode gap of 25 pm, sixteen electrode fingers, and a 1 mm channel width. The microfluidic channel sidewalls are visible as parallel vertical lines on the left and right boundaries of the image.
  • Figure 3 A is a diagram showing a selective capture of E. coli (1, arranged in concentric rings) and red blood cells (2).
  • Figures 3B and 3C are graphs showing the calculated CM factor for E. coli ( Figure 3B) and red blood cells (RBC, Figure 3C) as a function of frequency (f (Hz)). The samples were tested at different conductivities (S/m).
  • Figure 4 A is a cut-away diagram showing one example of the 3 -unit, 2- stage (a trap module and an identification module) condenser device.
  • Each unit 20 includes a trap module 22, large circular first stage condensers 24 to capture the target microorganisms as they flow through the large microchannel (top to bottom).
  • Figures 4B, 4C, and 4D are diagrams showing different device design types.
  • Type A includes a selective capture step followed by microfluidic shuttling of particles to a capture region for identification ( Figure 4B).
  • Type B utilizes a selective shuttle to move particles into side channels where they are captured for identification ( Figure 4C).
  • Type C utilizes a selective shuttle to move particles to a spatially confined section of the channel where they are then captured for identification ( Figure 4D).
  • Figures 5A and 5B are bar graphs showing the particle capture locations along a channel.
  • the Number of particles captured in region versus Particle position (pm) for 46 pm-width channel is shown in Figure 5A and for 100 pm width channel is shown in Figure 5B.
  • Figure 6 is a diagram showing the circuit model for dielectrophoretic capture.
  • the function generator, VFG sources a voltage with an output impedance characterized by Rout.
  • the series resistance of the electrode leads is followed by the ionic double-layer capacitance and the solution resistance before returning along an identical path to the negative terminal of the function generator.
  • Figures 7A and 7B are graphs showing the measured impedance (Z) as a function of frequency (Freq. (Hz)) for typical DEP structures (25 pm gap), connected in series with a 2kQ resistor without ( Figure 7A) or with a 50 nm oxide deposition.
  • the legends indicate different conditions: air - top line, deionized water (DI) - second line down, O.OOlx PBS - third line down, O.Olx PBS - fourth line down, O.lx PBS - fifth line down, and lx PBS - sixth line down.
  • DI deionized water
  • Figure 8 is a graph showing the measured capture efficiency as a function of series resistance, for a 200 nm-oxide device in O.Olx PBS.
  • Figure 9 is a graph showing capture efficiency is predicted to increase monotonically up to a point as the number of IDE increases, verified by a COMSOL prediction.
  • Figures 10A and 10B are graphs showing DEP capture of
  • Figures 11 A- 11E are diagrams showing counter structure with three electrodes, and a bead flowing over the counter structure.
  • Figure 12 is a graph showing the output signal (V) over time (s) for the bead.
  • Figures 13A and 13B are graphs showing a sample output signal (V1-V2) over time (s) for a set of captured beads before and during the flow of the beads over the counter electrode structures.
  • Figure 14 is a graph showing the output signal (Vi-V2 (mV)) over time (s) for a sample with naive and activated T cells, and demonstrates that the counter discriminates between the two cell populations, such that the number of cells in the desired population may be counted.
  • Figure 15 is a line graph showing modeling the expected trajectory of particles in a fluid channel subject to DEP forces. Model predicts throughput scales with channel width without any indication of a tradeoff in performance.
  • Figures 16A-16D are graphs showing model’s prediction for
  • Figure 17A is a circuit diagram of the DEP capture and a circuit model prediction for the DEP performance with various numbers of interdigitated electrode fingers (Nf) ( Figures 17B).
  • Figure 17C is a graph showing the predictions for impedance model velocity ratio, COMSOL model velocity ratio, and experimental velocity ratio.
  • Figures 18A and 18B are graphs showing DEP performance at different flow rates.
  • Figure 18A shows the number of particles captured at 10 MHz (60seconds/flow rate) at different flow rates (m ⁇ /min).
  • Figure 18B shows the intensity of fluorescently labeled cells remaining captured (%) at different flow rates (pl/min).
  • Figure 19A is a diagram showing particles flowing through a
  • FIG. 19B is a graph showing tracking equilibrium particle velocity along the direction of fluid flow probes the DEP force magnitude.
  • Figure 19C is a graph showing changes in resistance (W) at different frequencies (kHz); the electrochemical impedance measurements extract circuit parameters characterizing the electrodes.
  • Figure 19D is a graph showing the DEP force experienced by passing particles is proportional to the squared magnitude (dashed line) of the voltage across the solution resistance element. With increasing series resistance, the ratio of the particles velocities off and on the DEP region (squares) approaches unity, indicating decreasing DEP force magnitude.
  • Figure 20 shows initially, the equilibrium velocity (squares) over the DEP electrodes decreases with an increasing number of electrode fingers until influence of the decreasing voltage outweighs the increasing number of interactions with DEP force. Changing the number of electrode fingers alters device performance.
  • Figure 21 shows the solution resistance of the channel decreases with increasing channel width and with thus the magnitude of the DEP voltage (dashed line). Increasing throughput by increasing width sacrifices DEP efficiency.
  • Figure 22 is a graph showing the impedance measurements for the coated (squares) and uncoated (circles) devices in O.lx PBS solution.
  • Figures 23A and 23B are graphs showing the expected voltage (dashed lines) differs greatly when comparing devices with ( Figure 23 A) and without ( Figure 23B) the 200 nm deposited oxide as a function of the signal frequency. This effect is observed in the equilibrium velocity ratios (Exp., solid lines) at lower signal frequencies.
  • Figure 24A is a diagram showing a top-down view of the metallization pattern for two chips 50a and 50b, each of which contains one capture module 52 and several counter modules 60.
  • a microchannel (not shown) is positioned over the capture module 52 and the counter modules 60.
  • Figure 24B is a diagram showing an enlarged perspective view of one of the counter modules 60, showing the microchannel 54 over the electrodes 62, 64, 66, and a particle 68 flown over the electrodes 62, 64, 66. Electrode length (1), gap between the electrodes (g) and microchannel width (w) are shown.
  • Figure 24C is a diagram showing a top view of a counter module/device 70 showing the electrode structure 72 with a microfluidic channel 74 aligned and bonded.
  • Figure 25A is a diagram illustration of the fluild lines emanating from the planar electrode geometry bom out by COMSOF simulation ( Figure 25B) of the electric field profile for a pair of planar sensing electrodes.
  • Figure 26A is a graph showing simulation of the impedance variation (Zc, W) at a given position (pm) for an insulating sphere passing over planar electrodes with a 40 pm inter-electrode gap as a function of vertical displacement from the electrodes.
  • Figure 26B is a graph showing changes in voltage (V diff (pV)) over time (ms) with a bead transit over planar electrodes, the data from the bead transit event demonstrating the expected behavior.
  • Figure 27A is a diagram of a top view of a“constrictionless” counter unit/device 100.
  • the counter electrodes 110 are projecting slightly into the microfluidic constriction region 120 of 1 mm.
  • Figure 27B is a graph showing the representative trace of change in voltage (V1-V2 (pV)) over time (ms) from a 4.45 pm bead in O.Olx PBS passing over the counter 100.
  • Figure 27C is a diagram showing the integration of the constrictionless counter 100 with lateral- displacement structures, such as a DEP condensing module 130, to count particles from the entirety of the sample
  • Figure 27D is a diagram of the electronic connection of the counter unit/device with the data acquisition, processing, and display devices.
  • Figure 28 A is a histogram of the peak heights (V 1/3 ) of events (Counts (dim.)) acquired during the experiment flowing bead populations of different sizes (4.45 pm (1), 6.42 pm (2), 8.87 pm (3)), as well as Gaussian fits of the histogram data to estimate the dispersion of the sensor events.
  • the dashed vertical line represents the detection threshold of the algorithm for this dataset.
  • Figure 28B is a graph showing linear regression of the particle diameter (pm) to the peak height (V 1/3 ).
  • Figures 29A and 29B are diagrams showing incoming particles trapped on the DEP electrode structure (Figure 29 A) are then subsequently released for enumeration ( Figure 29B).
  • Figures 30A and 30B are diagrams showing sample handling by a device with the DEP capture, separate, and two counter modules and permitting buffer exchange. Shown are an incoming sample and an adjacent exchange buffer stream flowing through the device without ( Figure 30A) and with ( Figure 30B) a dielectrophoresis signal applied to the separator electrodes.
  • Figures 30C-30F show histograms of Normalized counts over cell diameter (V 1/3 ). Without lateral separation (DEP off), both species of particles pass through the left coulter counter constriction region while not passing through the right counter ( Figures 30C and 30D). When a DEP force is applied (DEP ON), lateral separation drives particles into the exchange buffer stream, producing counts from the right counter structure ( Figures 30E and 30F).
  • Figures 30G and 30H are graphs showing counter data from a DEP capture, separate, and two counter device and a mixture of unactivated and activated cells flown through it.
  • the graphs show Counts of cells of various diameters (AR/R) i ⁇
  • AR/R various diameters
  • Figure 30G When the signal is OFF, only three peaks are detected: debris peak (1), unactivated cells peak (2), and minor activated cells peak (3)
  • Figure 30G When the signal is ON, four peaks are detected: debris peak (1), unactivated cells peak (2), activated cells peak (3), and activated dimers peak (4) (Figure 30H).
  • particle refers to micro- or nano-sized particles, including naturally occurring particles, synthetic particles, polymeric particles, biological material, and environmental particles.
  • biological material refers to eukaryotic or prokaryotic cells, organelles, vesicles, macromolecules, biomarkers, and microorganisms.
  • microorganism refers to microscopic organisms such as bacteria, fungi, algae, protozoa, and viruses.
  • the term“unit” refers to a segment of a device containing all the components to trap and condense one species of biological material for identification.
  • microchannel or“microfluidic channel” generally refers to a channel with at least one of a height and width dimension below two millimeters, such as below 1500 microns, for fluid flow.
  • Microchannels may be primary (in the trap module) or secondary (in the identification module), with each microchannel having an inlet and an outlet.
  • the primary and secondary microchannels may share inlets and/or outlets. For example, one inlet may serve multiple microchannels with multiple outlets.
  • the term“substantially” refers to a significant degree of similarity, difference, or effect.
  • “substantially smaller than” in a context of a comparison may refer to a measurement that is significantly smaller than the same measurement in another sample.
  • “not substantially” refers to an insignificant degree of similarity, difference, or effect.
  • substantially similar typically refers to a similarity of about 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; while substantially different typically refers to a difference above about 5%, such as about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85% or about 90%.
  • the term“subject” refers to, for example, animals.
  • the subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian.
  • a mammal e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent
  • a fish e.g., a bird or a reptile or an amphibian.
  • the term“trap” refers to a segment in the device (e.g., trap module) for capture, or to an action of capturing, of a biological material with the device. Trapping may include separating the biological material of interest from other biological materials in a sample, and capturing the biological material of interest in the trap module.
  • the term“identify” or“identification” refers to an action of verifying or identifying the captured/trapped biological material of interest. Identification typically occurs after a biological material of interest has been captured or captured and condensed in the identification module.
  • the term“condenser” or“condense” generally refers to a device component that captures the biological material of interest over a surface, or an action that reduces the surface area over which the biological material of interest is distributed.
  • the devices are typically portable devices, and include a trap module, an identification module, and, optionally, a counter module.
  • the counter module may be a stand-alone portable counter device.
  • a sample with particles is typically flowed over the trap module, where the trap module traps the particles of interest.
  • the trapped particles may then be shuttled from the trap module with a first condenser to the identification module with a second condenser to condense the particles into a smaller filed and aid with further identification.
  • the trapped or the condensed particles may further pass through a counter module of the device.
  • the trapped particles from the trap module, or condensed particles from the identification module, may then be flown through the counter module for further assaying of the particles.
  • the sample with particles may be passed through the counter device.
  • An exemplary device 10 (without a counter module) is presented in Figure 4A.
  • the device typically includes a device body 12 segmented into one or more units 20. Each unit includes a trap module 22, and an identification module 26.
  • the trap module 22 generally includes at least one condenser 24 (first condenser) containing electrodes and a primary microchannel 14.
  • the identification module 26 typically includes at least one condenser 28 (second condenser) containing electrodes, and at least one secondary microchannel 18.
  • the microchannels include an inlet and an outlet.
  • Figure 4A is a cut-away diagram (i.e., the top of the microfluidic channel is removed), and the microfluidic channels are the recesses in the material forming the device.
  • the inlet is at the top
  • the outlet is at the bottom
  • three outlets for the shuttle are on the left.
  • Embodiments of counter modules or counter devices are shown in Figures 11A-11E, 24A-25A, 27A-27D, 29A, 29B, 30A, and 30B.
  • the counter module is configured to operate independently of the trap module and the identification module, but may be integrated with the trap module or identification module into a device. If separate from the trap module or the identification module, the counter module is referred to as counter device.
  • the substrate for the modules and the microchannels form the body of the device, while the electrodes form the condensers or the counters.
  • the inlet of the secondary microchannel permits shuttling of the biological material trapped in the trap module to the second condenser of the identification module.
  • the devices may have any three-dimensional shape, such as cuboid, spherical, oval, pyramidal, hexagonal, etc.
  • Suitable device dimensions for cuboid-shaped devices include a length between 0.01 mm and 100 mm, a width between 0.01 mm and 100 mm, and a height between 0.001 mm and 10 mm.
  • Suitable dimensions for spherical or oral device include a diameter of a device between 0.01 mm and 100 mm.
  • the devices may include a plurality of units, such as at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 units.
  • microfluidic devices may be scaled up for larger fluidic devices.
  • the trap module is a region in the device containing a bottom surface, walls, and, optionally, a top surface.
  • the top surface is formed by the device lid.
  • the bottom surface of the trap module has a surface area sufficient to accommodate the electrodes of the first condenser.
  • the surface area of the bottom surface is typically greater than 0.1 mm 2 , such as between about 0.15 mm 2 and about 1 mm 2 , or about 0.2 mm 2 and about 1 mm 2 , such as about 0.2 mm 2 , about 0.25 mm 2 , about 0.3 mm 2 , about 0.35 mm 2 , about 0.4 mm 2 , about 0.45 mm 2 , about 0.5 mm 2 , about 0.55 mm 2 , or about 0.6 mm 2 .
  • the height of the walls of the trap module may be between 1 pm and 1000 pm.
  • the trap module has a surface area between about 0.1 mm 2 and about 0.4 mm 2 , such as about 0.2 mm 2 , and a height of the wall of about 100 pm.
  • microfluidic devices may be scaled up for larger fluidic devices.
  • At least two electrodes of the trap module form the first condenser.
  • the electrodes are typically formed of a conducting or a semiconducting metal, such as gold, silver, chromium, aluminum, copper, platinum, or alloys containing gold, silver, chromium, aluminum, copper, or platinum. Under an applied AC, at least one of the electrodes is negatively charged and at least one of the electrodes is positively charged.
  • the two or more electrodes of the trap module are typically metal leads with a width between about 1 nanometer (nm) and about 10 cm, between about 1 nanometer (nm) and about 10 mm, such as between about 1 nm and about 1000 micrometers (pm), or between about 1 pm and about 1000 pm.
  • Preferred ranges for electrode widths include between about 1 nm and 1000 pm, more preferably between about 10 nm and about 1000 pm, such as between about 20 nm and 500 pm.
  • Exemplary electrode widths include widths between about 1 nm and about 900 nm, between about 500 nm and about 1000 pm, between about 1 pm and about 900 pm, between about 1 pm and about 800 pm, between about 1 pm and about 700 pm, between about 1 pm and about 600 pm, between about 1 pm and about 500 pm, between about 1 pm and about 400 pm, between about 1 pm and about 300 pm, between about 1 pm and about 200 pm, between about 1 pm and about 100 pm, or between about 1 pm and about 50 pm, such as about 25 pm.
  • exemplary electrode widths include widths between about 1 pm and about 90 pm, between about 1 pm and about 80 pm, between about 1 pm and about 70 pm, between about 1 pm and about 60 pm, between about 1 pm and about 50 pm, between about 1 pm and about 40 pm, between about 1 pm and about 30 pm, between about 1 pm and about 20 pm, between about 1 pm and about 10 pm, or between about 1 pm and about 5 pm, such as about 2.5 pm.
  • the electrodes of the first condenser can have any length sufficient to form a surface area for capturing the biological material of interest in numbers sufficient for identification at a specific flow rate.
  • the first condenser may have a surface area sufficient to capture over 50%, such as about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 100% of the particles of interest in the sample.
  • the electrodes may have a length between about 1 nm and 10 cm, such as between about 10 nm and 10 cm.
  • Suitable electrode lengths include a length between about 1 mih and about 20 000 mih (2 cm), such as between about 1 mih and about 15 000 mih, between about 1 mih and about 10 000 mih, between about 200 mih and about 2 000 mih, between about 500 mih and about 2000 mih, such as about 500 mih, about 600 mih, about 700 mih, about 800 mih, about 900 mih, about 1000 mih, about 1100 mih, about 1200 mih, about 1300 mih, about 1400 mih, about 1500 mih, about 1600 mih, about 1700 mih, about 1800 mih, about 1900 mih, or about 2000 mih.
  • the device has a design such that the spacing between electrodes may be between about 1 nanometer (nm) and about 10 cm, between about 1 nanometer (nm) and about 10 mm, such as between about 1 nm and about 1000 micrometers (pm), between about 10 nm and about 1000 pm, such as between about 10 nm and about 900 pm, between about 10 nm and about 800 pm, between about 10 nm and about 700 pm, between about 10 nm and about 600 pm, between about 10 nm and about 500 pm, between about 10 nm and about 400 pm, between about 10 nm and about 300 pm, between about 10 nm and about 200 pm, between about 10 nm and about 100 pm, between about 1 pm and about 500 pm, between about 1 pm and about 400 pm, between about 1 pm and about 300 pm, between about 1 pm and about 200 pm, between about 1 pm and about 100 pm, between about 10 pm and about 500 pm, between about 10 pm and about 400 pm, between about 1 pm and about 300 pm, between about 1 pm and about 200 pm
  • the height of the channel is about the same as the electrode spacing and the length of the electrode region is typically adjusted for the efficiency in capturing the target biological material.
  • the electrodes may be coated with an insulator to increase operating voltage.
  • the electrodes may be "focused" (i.e., pitch changed).
  • the electrodes may be patterned to allow for lateral movement and separation within the channel.
  • the devices operate with flow rates between about 0.01 pL/min and about 10 mL/min, such as about 0.01 pL/min and about 5 mL/min, about 0.01 pL/min and about 1000 pL/min, about 0.01 pL/min and about 800 pL/min, about 0.01 pL/min and about 500 pL/min, about 0.01 pL/min and about 300 pL/min, about 0.01 pL/min and about 200 pL/min, about 0.01 pL/min and about 150 pL/min, about 0.01 pL/min and about 100 pL/min, about 0.01 pL/min and about 50 pL/min, about 0.01 pL/min and about 10 pL/min, or about 0.01 pL/min and about 1 pL/min.
  • the electrodes are typically arranged on the bottom surface of the trap module, but may be present on the walls, the lid of the device, or the
  • the electrodes may be positioned in any arrangement.
  • the electrodes are arranges in any two-dimensional patterns, such as in linear, angled, circular, semi-circular, rectangle, cube, triangle, zigzag, and oval patterns. Examples of electrode arrangements in devices are shown in Figures 4A-4D.
  • the two or more electrodes of the trap module may have the same width and may be separated from one another by a distance as wide as the width of the electrodes or by a distance that is less than or more than the width of the electrodes.
  • the electrodes may be separated by a distance that is between about 0.01 times and 10 times the width of the electrodes, such as about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 times the width of the electrodes.
  • electrodes may be separated by a distance between about 10 nm and 1000 pm, such as between about 10 pm and 500 pm.
  • the device includes at least one primary microfluidic channel.
  • the height of the micro fluidic channel may be between about 1 nm and about 10 cm, between about 1 nm and about 10 mm, between about 1 nm and about 1000 pm, between 10 nm and about 1000 pm.
  • Exemplary heights for the primary microfluidic channel may be between about 10 nm and about 900 pm, between about 10 nm and about 800 pm, between about 10 nm and about 700 pm, between about 10 nm and about 600 pm, between about 10 nm and about 500 pm, between about 10 nm and about 400 pm, between about 10 nm and about 300 pm, between about 10 nm and about 200 pm, between about 10 nm and about 100 pm, between about 1 pm and about 1000 pm, between about 1 pm and about 500 pm, between about 1 pm and about 400 pm, between about 1 pm and about 300 pm, between about 1 pm and about 200 pm, or between about 1 pm and about 100 pm, such as about 1 pm, about 10 pm, about 20 pm, about 30 pm, about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 100 pm, about 150 pm, or about 200 pm.
  • the width of the microfluidic channel may be between about 1 nm and about 10 cm, between about 1 nm and about 10 mm, between about 1 nm and about 1000 pm, between 10 nm and about 1000 pm, between about 1 pm and about 10 000 pm, such as between about 10 pm and about 10 000 pm, between about 10 pm and about 9 000 pm, between about 10 pm and about 8 000 pm, between about 10 pm and about 7 000 pm, between about 10 pm and about 6 000 pm, between about 10 pm and about 5 000 pm, between about 10 pm and about 4 000 pm, between about 10 pm and about 3 000 pm, between about 10 pm and about 2 000 pm, between about 10 pm and about 1 000 pm, or between about 10 pm and about 500 pm.
  • Figure 4A is a cut-away diagram showing an exemplary device with its structure.
  • the top fluidic cover is removed to enable visualization.
  • the top of the microfluidic channel is removed, and the microfluidic channels are the recesses in the material forming the device.
  • the inlet is at the top, the outlet is at the bottom, and three outlets for the shuttle are on the left.
  • An example of a primary microfluidic channel 14 is shown in Figure 4A.
  • the trap module may be used for capture and identification of biological material, such that the trap module may also be the identification module.
  • each unit of the devices may include an identification module containing at least two electrodes and a secondary microchannel with an inlet and an outlet.
  • the trap module is typically connected with the identification module through focusing electrodes and/or a microfluidic shuttle system.
  • the dimensions of the identification module are substantially smaller than the dimensions of the trap module.
  • the identification module includes the same components and geometry as that of the trap module, but with smaller dimensions.
  • the identification module is a region in the device containing a bottom surface, walls, and, optionally, a top surface.
  • the top surface is formed by the device lid.
  • the bottom surface of the identification module has a surface area sufficient to accommodate the electrodes of the second condenser.
  • the surface area of the bottom surface is typically greater than 0.01 mm 2 , such as between about 0.01 mm 2 and about 1 mm 2 , or about 0.015 mm 2 , about 0.02 mm 2 , about 0.025 mm 2 , about 0.03 mm 2 , about 0.035 mm 2 , about 0.04 mm 2 , about 0.045 mm 2 , or about 0.05 mm 2 .
  • the height of the walls of the identification module may be between about 1 pm and about 1000 pm, between about 1 pm and about 900 pm, between about 1 pm and about 1000 pm, between about 1 pm and about 900 mih, between about 1 mih and about 800 mih, between about 1 mih and about 700 mih, between about 1 mih and about 600 mih, between about 1 mih and about 500 mih, between about 1 mih and about 400 mih, between about 1 mih and about 300 mih, between about 1 mih and about 200 mih, between about 1 mpi and about 100 mih, between about 1 mih and about 50 mih, such as about 1 mih, about 10 mpi, about 20 mih, about 30 mpi, about 40 mih, about 50 mih, about 60 mpi, about 70 mih, about 80 mpi, or about 100 mih.
  • the identification module has a surface area between about 0.015 mm 2 and about 0.05 mm 2 , such as about 0.025 mm 2 , and a height of the wall of about 0.1 mm.
  • At least two electrodes form the second condenser.
  • the electrodes are typically formed of a conducting or semiconducting metal, or a combination of conducting or semiconducting metals.
  • Exemplary metals include gold, silver, chromium, aluminum, copper, platinum, or alloys containing gold, silver, chromium, aluminum, copper, or platinum.
  • At least one of the electrodes is negatively charged and at least one of the electrodes is positively charged.
  • the two or more electrodes of the second condenser typically have a width between about 1 nm and about 500 pm.
  • Exemplary electrode widths include widths between about 1 nm and about 100 pm, between about 1 nm and about 50 pm, between about 1 nm and about 40 pm, between about 1 nm and about 30 pm, between about 1 nm and about 20 pm, between about 1 nm and about 15 pm, or between about 1 nm and about 10 pm, such as between about 1 nm and about 5 pm.
  • identification module include between about 1 pm and about 100 pm, between about 1 pm and about 50 pm, between about 1 pm and about 40 pm, between about 1 mih and about 30 mih, between about 1 mih and about 20 mih, between about 1 mih and about 15 mih, or between about 1 mih and about 10 mih, such as between about 1 mih and about 5 mih.
  • the electrodes of the second condenser can have any length sufficient to form a surface area for capturing a particle of interest in numbers sufficient for identification of the biological material at a specific flow rate.
  • the second condenser may have a surface area sufficient to capture over 50%, such as about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 100% of the biological material of interest in the sample.
  • the electrodes of the identification module may have a length between about 1 nm and 10 cm, such as between about 10 nm and 10 cm. Suitable electrode lengths include a length between about 1 pm and about 20 000 pm (2 cm), such as between about 1 pm and about 15 000 pm, between about 1 pm and about 10 000 pm, between about 200 pm and about 2 000 pm, between about 500 pm and about 2000 pm, such as about 500 pm, about 600 pm, about 700 pm, about 800 pm, about 900 pm, about 1000 pm, about 1100 pm, about 1200 pm, about 1300 pm, about 1400 pm, about 1500 pm, about 1600 pm, about 1700 pm, about 1800 pm, about 1900 pm, or about 2000 pm.
  • Suitable electrode lengths include a length between about 1 pm and about 5000 pm, between about 100 pm and about 1 000 pm, between about 200 pm and about 1000 pm, such as about 200 pm, about 300 pm, about 350 pm, about 400 pm, about 450 pm, about 500 pm, about 550 pm, about 600 pm, about 650 pm, about 700 pm, about 750 pm, about 800 pm, about 850 pm, about 900 pm, about 950 pm, or about 1000 pm.
  • the electrodes are typically arranged on the bottom surface of the identification module, but may be present on the walls, the lid of the device, or the combinations thereof.
  • the electrodes may be positioned in any arrangement.
  • the electrodes are arranged in a two-dimensional pattern, such as in linear, angled, circular, semi-circular, rectangle, cube, triangle, zigzag, and oval patterns.
  • the two or more electrodes of the identification module may have the same width and may be separated from one another by a distance as wide as the width of the electrodes or by a distance that is less than or more than the width of the electrodes.
  • the electrodes may be separated by a distance that is between about 0.01 times and 10 times the width of the electrodes, such as about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 times the width of the electrodes.
  • the secondary microfluidic channels stem off the primary channel.
  • Each secondary microfluidic channel includes an outlet.
  • An example of a secondary microfluidic channel 18 is shown in Figure 4A.
  • the primary and/or secondary microfluidic channels may be disposable.
  • the microfluidic channels may be formed of the same material as the device body, attached to the device body, and be readily removable.
  • the disposable microfluidic channels may help with microscopy following condensing of the biological material in the identification module.
  • the capture and identification device may include a counter module.
  • the counter module may be a stand-alone counter device.
  • the counter device typically provides a rapid assaying of the particles by counting the particles, characterizing particle size, shape, activation state, division state, metabolic state, live/dead determination, and/or surface marker expression.
  • Alternative names for the counter device used herein include counter device, counter, coulter-type counter, coulter counter particle detection systems, or counter structure.
  • the counter device typically includes an electrode arrangement.
  • the electrode arrangement may be overplayed with a microfluidic channel, forming a constricted counter device.
  • the counter device does not include a microfluidic channel over the electrode arrangement, forming constrictionless counter device.
  • the counter module or the counter device includes a three- electrode arrangement.
  • the three electrodes are parallel to one another, separated by a gap.
  • the counter electrodes are planar electrodes with a sample flown over the planar electrodes in a microchannel.
  • the three electrode arrangement includes a left sensing electrode, a right sensing electrode, and a middle reference electrode.
  • An example is shown in Figures 11A-11E, 24B, and 27D.
  • Figures 11A-11E show a passing particle nears the sensing region (Figure 11 A) and then enters the fluidic channel (Figures 11B-11D) before finally exiting the sensing region (Figure 11E).
  • the electrodes typically have a width between about 1 pm and about 1000 pm, such as between about 10 pm and about 900 pm, between about 10 pm and about 800 pm, between about 10 pm and about 700 pm, between about 10 pm and about 600 pm, between about 10 pm and about 500 pm, between about 10 pm and about 400 pm, between about 10 pm and about 300 pm, between about 10 pm and about 200 pm, or between about 10 pm and about 100 pm.
  • the electrodes may be formed of gold, silver, chromium, aluminum, copper, platinum, and their alloys.
  • the electrodes may be coated or uncoated. Typical coatings include passivating materials such as Si0 2 or Si 3 N 4 ; or thin film plastics, such as parylene or photoresist or thermoplastics.
  • the counter module or counter device typically includes a microfluidic channel overlaying at least a portion of the electrodes.
  • the microfluidic channel may include an inlet, an outlet, and a constriction region.
  • the microfluidic channel may not have a constriction region, forming a constrictionless counter module or a constrictionless counter device.
  • the constriction of the microfluidic channel may have a width between about 1 pm and about 100 pm, such as about 1 pm and about 10 pm, about 1 pm and about 20 pm, about 1 pm and about 30 pm, about 1 pm and about 40 pm, about 1 pm and about 50 pm, about 1 pm and about 60 pm, about 1 pm and about 70 pm, about 1 pm and about 80 pm, or about 1 pm and about 90 pm at a region overlaying at least a portion of the electrodes.
  • the microfluidic channel without a constriction may have a width between about 100 pm and about 10000 pm at a region overlaying at least a portion of the electrodes.
  • the microfluidic channel of the counter module is typically in a fluidic connection with the trap module or the identification module.
  • the counter module’s microfluidic channel may be connected to the primary microfluidic channel or the secondary microfluidic channel. This connection establishes a fluidic connection between the modules of the device.
  • fluid flows through the fluidic connection at via a flow rate between about 0.01 pL/min and about 10 mL/min. The fluid typically flows at different flow rates in different modules of the device.
  • Embodiments of counter modules or counter devices are shown in Figures 11A-11E, 24A-25A, 27A-27D, 29A, 29B, 30A, and 30B.
  • the counter module is configured to operate independently of the trap module and the identification module, but may be integrated with the trap module or identification module in a DEP device. If separate from the trap module or the identification module, the counter module is referred to as counter device.
  • Exemplary DEP devices with counter modules are shown in Figures 24A, 24B, 29A, 29B, 30A, and 30B (with the counter’s microfluidic channel constriction) and Figure 27C (without the counter’s microfluidic channel constriction).
  • Exemplary counter devices are shown in Figures 24C (with the counter’s microfluidic channel constriction) and 27C (with the counter’s microfluidic channel constriction), referred to as“constrictionless” counters.
  • Figures 30A and 30B show a schematic of a device with the DEP capture module and two counter modules.
  • the device captures, separates, and counts particles of interest from a mixture of particles in a sample. These devices provide characterization of the total particle population in a sample, as well as characterization of the particles of interest, simultaneously.
  • the counter module or the counter device operates with an alternating current (AC) having a frequency between 10 kHz and 10 MHz and a peak-to-peak voltage amplitude between 0.5 V and 10 V.
  • AC alternating current
  • the counter module or the counter device may be connected to one or more of a function generator, instrumentation amplified, lock-in amplifier, oscilloscope, a data processor, and a display monitor.
  • FIG. 27D An exemplary counter module or a counter device connected to signal acquis ion, amplification, and display means is presented in Figure 27D.
  • Figure 27D shows the circuit diagram 200 of the complete three-electrode structure of the sensing region, driven by the sine wave output 210 of the function generator 220. The resulting voltage at the left and right sensing electrodes is measured by the PCB -mounted instrumentation amplifier 230 before the signal is fed to the lock-in amplifier 240 whose output signal is measured by the oscilloscope 250, controlled during acquisition by a MATLAB routine in a processor 260.
  • the device is battery powered, for portable use, in settings such as in hospitals, laboratories, homes, and in the field.
  • the power supply through a function generator, provides an AC voltage for DEP capture modules and/or counter modules of the device or to the counter devices.
  • the devices may include a lid or an insert covering the trap module, the identification module, or both.
  • the devices are designed to be portable and include an inlet adapted for connecting with a sample delivery line.
  • the devices may also include an outlet adapted for sample exit from the device.
  • the device inlet and the device outlet may be the primary microchannel inlet and the primary microchannel outlet.
  • the device may be detached from the sample delivery line and used in biological material identification steps.
  • the identification module may be detachable. The user may detach the identification module and use it in biological material identification steps.
  • the device is designed as a disposable device for single use. In other aspects, the device is designed as a reusable device.
  • the device is typically formed of a body, electrodes, and optionally, a lid and microfluidics.
  • Exemplary materials for forming the device body include polymers such as polycarbonate, thermoplastics, polydimethyl sulfone (PDMS), polyurethane (PU), polyethylene oxide (PEO), polybutylene terephthalate (PBT), polyether sulfone (PES), polyimide (PI) polymers, TEFLON®/PTFE, polystyrene, cyclic olefin copolymer (COC), and block copolymers and asymmetric blends thereof.
  • polymers such as polycarbonate, thermoplastics, polydimethyl sulfone (PDMS), polyurethane (PU), polyethylene oxide (PEO), polybutylene terephthalate (PBT), polyether sulfone (PES), polyimide (PI) polymers, TEFLON®/PTFE, polystyrene, cyclic olefin copolymer (COC), and block copolymers and asymmetric blends thereof.
  • Exemplary materials for forming the device body also include silicon, metal, ceramic glass, or glass variations, such as low-expansion glass.
  • Exemplary materials for forming the electrodes include gold, silver, chromium, aluminum, copper, platinum, and their alloys.
  • the devices utilize low cost, mass production materials as alternatives to PDMS which is not preferred for medical devices, such as polycarbonate, thermoplastics, etc.
  • thermoplastic materials for injection molding include Cyclic Olefin Co-polymer (COC). It is also optically transparent, which allows for easy imaging for inspection.
  • PMMA polymethylmethacrylates
  • Acrylite® Evonik Rohm GmbH LLC, Darmstadt, Germany
  • Lucite® Lucite International, Inc, Cordova, TN
  • R-Cast R-Cast
  • Plexiglas® and Altuglas® (Arkema France Corp., Colombes, France), Optix, Perspex®, Oroglas, Cyrolite ® (Evonik Cyro LLC, Parsippany, NJ), and
  • the electrodes may be passivated from the fluid with an insulator, allowing for higher operating voltages and thus more efficient capture.
  • the passivation may be with chemical vapor depositions (CVD) deposited insulators, such as Si0 2 or Si 3 N 4 ; or thin film plastics, such as parylene or photoresist or thermoplastics.
  • CVD chemical vapor depositions
  • Suitable methods for making the devices include lower cost and higher throughput fabrication approaches, such as roll-to-roll, embossing, etc.
  • Suitable materials for forming the devices include polymers for the device body and conductors for the electrodes.
  • Methods of making the device body include three-dimensional printing, micromolding, deep ion etching, nano imprint lithography, micromachining, laser etching, three dimensional printing, or stereolithography, patterning, photolithography, etching, CNC micromachining, thermal bonding, and chemical bonding.
  • Three-dimensional (3D) printing is a process of making 3D objects from a digital model.
  • 3D printing is an additive process, where successive layers of material are laid down in different shapes. After each layer is added the“ink” is polymerized, typically by photopolymerization, and the process repeated until a 3D object is created.
  • the 3D printing workflow can be described in three sequential steps: 1) the powder supply system platform is lifted and the fabrication platform is lowered one layer; 2) a roller spreads the polymer powder into a thin layer; 3) a print-head prints a liquid binder that bonds the adjacent powder particles together.
  • Two kinds of 3D printing techniques may be used: inkjet printing with the typical printers.
  • Nanoimprint lithography is a fast and cost-efficient technique for fabricating nanostructures.
  • the procedure of NIL is to stack multiple layers of such structures on top of each other; that is, a finished double -layer of structures is covered with a spacer-layer which is planarized using the chemical- mechanical polishing so that a second layer can be processed on top.
  • Methods of making the electrodes include liftoff metallization, etching, shadow evaporation, or direct printing.
  • the devices are typically machine-assembled for accuracy and reproducibility.
  • the turnaround cycle for modularized computer-aided design (CAD) and machining is relatively quick. It is easy and rapidly customizable according to user’s individual needs.
  • Computer numerical controlled (CNC) machines such as lathes and mills, may be used to manufacture the components of the device body and electrodes.
  • the devices used for dielectrophoresis may be fabricated in a cleanroom facility.
  • the electrode structures are lithographically patterned in photoresist atop a Borooat-33 glass wafer.
  • Metallization with 200 nm of aluminum follows. Devices are either allowed to form a native oxide upon exposure to atmosphere or are subsequently coated in layer of 200 nm of plasma-enhanced chemical vapor deposition (PECVD) silicon dioxide as an insulating coating. The wafer is then diced and cleaned. At this point, the chips are available for use.
  • PECVD plasma-enhanced chemical vapor deposition
  • Figure 2E shows a diagram of a typical pair of interdigitated electrode. This particular device has an electrode-electrode gap of 25 pm, sixteen electrode fingers, and a 1 mm channel width.
  • An imprint mold may be used to fabricate the microfluidic channels.
  • an SET-8 photoresist may be photolithographically defined to create a nominally 20 pm feature height for the microfluidic channels.
  • Polydimethylsiloxane (PDMS, Dow Corning Sylgard 184) may be mixed in a 10:1 ratio and poured over the mold. The mixture and mold may be de-bubbled for thirty minutes in a vacuum chamber before being cured for one hour at 70°
  • the PDMS“wafer” may be subsequently peeled from the mold. Individual microfluidic channels may be cut from the mold, cleaned, and hole-punched to form inlet and outlet ports. The microfluidic channels may then be bonded to the individual chips after UV-ozone treatment by heating the aligned PDMS-chip combination in an oven for fifteen minutes at 70° C, after which devices are ready for use.
  • An interdigitated electrode structure is shown in Figure 2E. The microfluidic channel sidewalls are visible as parallel vertical lines on the left and right boundaries of the image.
  • An exemplary method of making the counter module or the counter device includes photoresist coating, pattern definition, metal deposition, and a lift-off process. This simplicity compared to alternative electrode geometries significantly reduces per-device fabrication cost.
  • a microfluidic channel of a desired width and height may be formed by photolithography and bonded to a counter device such that the three counter electrodes projected slightly into the width of the channel (constrictionless counter module/device), as seen in Figure 27A,or overlay the electrodes, as seen in Figure 24C.
  • Sterilization techniques include gas treatment (e.g., ethylene oxide), ionizing radiation, sonication, surface treatment (e.g., surfactant), rinsing in sterile water, and autoclaving.
  • the nanosensor-based electrical devices for rapid biological material screening described herein overcome these obstacles.
  • the technique minimizes false positives and negatives and does not require complex sample processing.
  • the device provides a rapid, miniaturized and portable biosensing platform suitable for use in diverse settings, such as in hospitals, food and drug testing laboratories, or in the field.
  • DEP dielectrophoresis
  • EO electroosmosis
  • DEP dielectrophoretic
  • FDEP dielectrophoretic force
  • grad E the gradient of the spatially varying electric field E(r,o )
  • K(co) is called the Clausius-Mossotti (CM) factor, which depends on the permeabilities and conductivities of the cell and the surrounding media.
  • CM Clausius-Mossotti
  • the organism viability can be maintained through the DEP flow cell allowing further culturing or assaying of the organism for infection control or epidemiologic purposes.
  • a sample of interest is applied to the device via the
  • microfluidic channel inlet An electric field is applied to the electrodes form the power source at a frequency suitable to trap a biological material of interest.
  • the biological material may be used for identification in the trap module.
  • the biological material may be further condensed on the identification module, if present.
  • the condensing of the biological material on the identification module may be accomplished through microfluidic focusing or with focusing electrodes.
  • microfluidic focusing the microchannel outlet may be closed, the secondary channel inlet may be opened, the field in the trap module may be turned off, and the filed in the identification module may be turned on.
  • This staged“shuttle” transfer technique allows the trapped biological material from the trap module to be transferred to the identification module with a coincident reduction in FOV to allow further inspection and identification of the biological material. Focusing the biological material with the focusing electrodes may be achieved through various device and electrode designs. Examples of device and electrode designs are presented in Figures 4B, 4C, and 4D, and in Table 1 below.
  • Design types A, B, and C correspond to the diagrams shown in Figures 4B, 4C, and 4D, respectively.
  • Inspection and identification may be via microscopy, staining, molecular techniques (such as flow cytometry; immuno staining; staining; cell
  • DEP permits manipulating the position of particles within the device for the capture and concentration of rare targets from within the sample or to physically separate out the target from the sample background.
  • Dielectrophoresis is the forced exerted by an electric field acting on the dipole moment of a charge-neutral particle.
  • the particle's polarizability governs its response to the external field and depends on both the mobility of charge within the particle (conductivity) as well as the particle's ability to accumulate charge (permittivity).
  • positive and negative charge carriers within the particle re-arrange. This spatial arrangement of opposing charge distributions constitutes a dipole.
  • Dielectrophoresis boasts enormous appeal for point-of-care diagnostics.
  • Cells, viruses, and other biomarkers are permealizable and therefore experience the dielectrophoretic force.
  • the actuating mechanism is the interaction of an applied electric field with a particle in solution.
  • Microelectrode structures are readily fabricated to manipulate the target within the sample. Different cell species have differing frequency responses, allowing some selectivity of the target analyte through the choice of operating frequency. Dielectrophoretic manipulation does not rely upon the presence of chemical binding elements to selectively interact with the desired analyte, and in this manner is said to be label-free. The ease of fabrication and lack of a need for additional chemical treatments greatly simplifies some aspects of implementation for point-of-care diagnostics.
  • Detection of biological agents at very low concentrations is limited by diffusion of the target to the sensing element.
  • the electric field gradient generated for dielectrophoresis reaches microns into solution, actively driving analyte motion to overcome diffusion limitations on the measurement time- scale. These limitations are exacerbated by sample dilution, which is often required to manipulate the sample conductivity into a suitable regime for other detection mechanisms. Dilution reduces the concentration of the target analyte, demanding a compensatory increase in sensitivity.
  • Dielectrophoresis may be used to capture and concentrate the target from solution either before or after dilution to bolster the local concentration of analyte, reducing demands on sample volume throughput and thereby decreasing the time-to-results.
  • CM factors for various components of a whole blood sample show different crossover frequencies for each component, as well as different frequencies for various components in a sample. This may be used with a device where the biological material of interest is separated, or differentially trapped, from other blood components. Additionally, a trapping structure can serve as a concentrator, which would pull the bacteria from the blood sample. Different biological materials in general have different CM factors, providing for selection of different biological materials by setting the appropriate frequency. To be selective for a given species in a sample containing a mix of species, the CM factor is typically maximum for the species of interest, and is negative for the others.
  • the CM factors can be determined for living organisms, while molecular methods detect nucleic acids from both viable and dead pathogens.
  • the CM factor for a given species of bacteria may be computed with the knowledge of the physical structure of the bacteria (e.g. number of cell walls, membranes, shape, size, and internal conductivity of the bacteria).
  • CM factor for a given organism may be determined experimentally by monitoring the capturing efficiency under conditions of constant solution conductivity, flow rate, and applied voltage while manipulating the applied frequency. CM factors may be determined individually for each cell type by seeing what frequency effectively captures the cells, at a given solution conductivity, as detailed in Figures 10A and 10B.
  • electroosmosis may be used capture the biological material.
  • EO is generally not frequency selective.
  • the selectivity may be provided by alternating between EO and DEP modes.
  • the devices permit rapid capture of particles, such as a biological material, from a sample.
  • the sample may be a biological sample, food sample, or an environmental sample.
  • the capture, shuttling and condensing occurs within 30 seconds, 1 minute, 1.5 minutes, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes following sample application.
  • the captured and condensed biological material is then ready for microscopic observation and pathogen identification.
  • Exemplary biological samples include diluted or undiluted blood, plasma, spinal fluid, urine, stool, tear, saliva, sputum, mucus, or exudate.
  • Exemplary food samples may be obtained from the food industry and may be liquids from food preparation, storage, or cooking, runoff from meat processing, and runoff from washing of fruits and vegetables.
  • Exemplary environmental samples may be obtained from monitoring the environment and may include waters from streams, rivers, lakes, seas, pools, swimming pool samples, drinking water, industrial water, industrial runoff, rainwater runoff, or stream water.
  • the devices may capture, identify, and characterize particles, such as plastic micro- and nano-particles, and biological material.
  • the sample is a blood sample with a central line associated bloodstream infections (CLABSI).
  • CLABSI central line associated bloodstream infections
  • the samples may be diluted with water or buffer with a sufficient ionic strength and conductivity to permit biological material trapping.
  • the biological material may be diluted in a buffer with the ionic strength substantially the same as the ionic strength of phosphate buffered saline (PBS, lx PBS), or 0.001 x PBS, 0.002 x PBS, 0.003 x PBS, 0.004 x PBS, 0.005 x PBS, 0.006 x PBS, 0.007 x PBS, 0.008 x PBS, 0.009 x PBS, 0.01 x PBS, 0.02 x PBS, 0.03 x PBS, 0.04 x PBS, 0.05 x PBS, 0.06 x PBS, 0.07 x PBS, 0.08 x PBS, 0.09 x PBS, 0.1 x PBS, 0.2 x PBS, 0.3 x PBS, 0.4 x PBS, 0.5 x PBS, 0.6 x PBS
  • the biological material of interest may be a eukaryotic or prokaryotic cell, microorganisms, organelles, vesicles (such as exosomes, endosomes, phagosomes, and liposomes), macromolecules, or biomarkers.
  • the microorganisms captured and identified by the device and methods include bacteria, viruses, fungi, and parasites.
  • Exemplary genera of microorganisms include Staphylococcus, Enterococcus, and Klebsiella, Enterobacter,
  • the device may be used to detect weaponized bacterial CBW agents such as Bacillus anthracis and Yersinia pestis.
  • the devices typically capture the biological material in the trap module.
  • the biological material may then shuttled into the identification module, where the biological material is condensed to a surface area suitable for microscopic observation.
  • Observation and identification of condensed particles may be via microscopy, staining, molecular techniques (such as flow cytometry;
  • the particles may be identified using microscopy.
  • Methods of particle, such as biological material, identification using microscopy are known in the art. These include direct pathogen observation under a microscope. Other methods include staining the captured and condensed biological material with suitable stains and observing them under a light, a fluorescence, or a confocal microscope.
  • the microscopy utilizes high power compound microscopes permitting detection of biological material.
  • suitable microscopes include phase-contrast microscopes, travelling microscopes, epifluorescence microscopes, confocal microscopes, two-photon microscopes, and
  • the microscopes will provide a maximum field of view (FOV) between about 10 mm and about 0.185 mm (185 pm), such as about 0.185 mm, about 0.18 mm, about 0.2 mm, about 0.22 mm, about 0.25 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.625 mm, about 0.9 mm, about 1 mm, about 1.25 mm, about 1.8 mm, about 2 mm, about 2.5 mm, about 4.5 mm, about 5 mm, about 6.25 mm, about 9 mm, or about 10 mm.
  • the staining methods will vary with the nature of the biological material, and/or the mode of detection.
  • the staining of the biological material occurs on the
  • Suitable staining methods include Gram staining by Gram method, acid- fast staining, staining for flagella, endospore staining, Ziehl-Neelsen stain, haematoxylin and eosin (H&E) staining, Papanicolaou staining, Periodic Acid Shiff (PAS) staining, and Romanowsky stains.
  • Suitable dyes for staining the pathogens include Basic Fuchsin, Rose Bengal Sodium Salt, Safranin T, Malachite Green oxalate salt, Resazurin sodium salt, 3,3prime-Diethyloxacarbocyanine iodide, 2-Naphthyl caprylate, 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI), and Malachite green.
  • the counter module or the counter device provides for rapid assaying of particles from samples.
  • the assaying may include qualitative and quantitative characterization of particles of interest.
  • the qualitative and quantitative characterization includes assaying the number of particles of interest, characterizing particle size, shape, activation state, division state, metabolic state, live/dead determination, surface marker expression, and others.
  • the devices are suitable for detecting and identifying particles, such as pathogens, from various samples, including biological samples. Detection and identification may help with diagnosis, and/or inform of suitable therapies.
  • the devices are particularly suitable for capture and identification of pathogenic bacteria, fungi, viruses, and parasites.
  • bacteria detectable by the device examples include commensal bacteria, oral and skin bacteria, or pathogenic bacteria.
  • Commensal bacteria typically include healthy gut microbiota, such as Lactobacillus and Bifidobacteria, Enterococcus faecium, or Lactobacillus pentosus WE7.
  • a non-limiting list of exemplary probiotic microorganisms includes Lactic acid bacteria (LAB), such as Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Lactococcus spp., Bifidobacterium spp. and
  • Pathogenic bacteria include bacteria from the genera Bacillus,
  • Bartonella Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia and Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio, and Yersinia.
  • viruses detectable by the devices include viruses of virus families Adenoviridae, Papillomaviridae, Parvoviridae, Herpesviridae,
  • Poxviridae Hepadnaviridae, Polyomaviridae, Anelloviridae, Reoviridae, Picomaviridae, Caliciviridae, Togaviridae, Arenaviridae, Flaviviridae,
  • Orthomyxoviridae Paramyxoviridae, Bunyaviridae, Rhabdoviridae, Filoviridae, Coronaviridae, Astroviridae, Bomaviridae, Arteriviridae, and Hepeviridae.
  • Examples of parasites detectable by the devices include protozoa, such as plasmodium, amoeba, babesia, Balatidium coli, blastocystis, coccida,
  • Entamoeba histolytica giardia, Cystoisospora belli, Leishmania, Naegleria fowleri, Rhinosporidium seeberi, Toxoplasma gondii, Trichomonas vaginalis, and Trypanosoma.
  • the parasites may include those which cause Acanthamoebiasis, Babesiosis, Balantidiasis, Blastocystosis, Coccidiosis, Amoebiasis, Giardiasis, Isosporiasis or cystosporiasis, Leishmaniasis, Primary amoebic
  • PAM meningoencephalitis
  • Acanthamoebiasis is typically caused by tiny ameba can affect the eye, the skin, and the brain. It exists all over the world in water and soil. Individuals can become infected if they clean contact lenses with tap water. Babesiosis comes from parasites babesia that are spread by ticks. It affects the red blood cells. The risk is highest in summer in the Northeast and upper Midwest of the United States.
  • Balantidiasis is passed on by Balatidium coli, a single-cell parasite that usually infects pigs but can, in rare cases, cause intestinal infection in humans. It can be spread through direct contact with pigs or by drinking contaminated water, usually in tropical regions.
  • Blastocystosis is caused by Blastocystis and affects the intestines.
  • the blastocystis enters humans through the fecal-oral route. A person can get it by eating food or drink contaminated with human or animal feces where the parasite is present.
  • Coccidiosis is caused by coccida and affects the intestines. Coccidia is passed on through the fecal-oral route. It is found around the world. It can also affect dogs and cats, but these are different kinds. Dogs, cats, and humans cannot normally infect each other.
  • Amoebiasis is caused by the parasite Entamoeba histolytica. It affects the intestines. It is more likely in tropical regions and in areas with high population density and poor sanitation. It is transmitted through the fecal-oral route.
  • Giardiasis is caused by giardia, or "beaver fever" affects the lumen of the small intestine. If humans ingest food or water contaminated with feces, dormant cysts may infect the body.
  • Isosporiasis or cystosporiasis is caused by the Cystoisospora belli, previously known as Isospora belli. It affects the epithelial cells of the small intestine. It exists worldwide and is both treatable and preventable. It is passed on through the fecal-oral route.
  • Leishmaniasis is a disease that is passed on by parasites of the
  • Leishmania family It can affect the skin, the viscera, or the mucous membranes of the nose, mouth, and throat. It can be fatal.
  • the parasite is transmitted by types of sandflies.
  • Primary amoebic meningoencephalitis (PAM) is passed on through a free-living ameba known as Naegleria fowleri. It affects the brain and the nervous system, and it is nearly always fatal within 1 to 18 days. It is transmitted through breathing in contaminated soil, swimming pools, and contaminated water, but not from drinking water.
  • Malaria is caused by different types of plasmodium, which affect the red blood cells. It exists in tropical regions and is transmitted by the Anopheles mosquito.
  • Rhinosporidiosis is caused by Rhinosporidium seeberi. It mainly affects the mucous of the nose, conjunctiva, and urethra. Polyps result in nasal masses that need to be removed through surgery. Bathing in common ponds can expose the nasal mucous to the parasite.
  • Toxoplasmosis is a parasitic pneumonia caused by the parasite
  • Toxoplasma gondii It affects the liver, heart, eyes and brain. It occurs worldwide. People can become infected after ingesting raw or undercooked pork, lamb, goat, or milk, or though contact with food or soil that is
  • Trichomoniasis also known as “trich” is a sexually transmitted infection (STI) caused by the parasite Trichomonas vaginalis. It affects the female urogenital tract. It can exist in males, but usually without symptoms.
  • STI sexually transmitted infection
  • Trypanomiasis (Sleeping sickness) is passed on when the tetse fly transmits a parasite of the Trypanosoma family. It affects the central nervous system, blood, and lymph. It leads to changes in sleep behavior, among other symptoms, and it is considered fatal without treatment. It can cross the placenta and infect a fetus during pregnancy.
  • Chagas disease is caused by the parasite Trypanosoma cruzi and affects the blood, muscle, nerves, heart, esophagus and colon. It is transmitted through an insect bite. Over 300,000 people in the U.S. have the parasite that can lead to this disease. 2. Detecting Bloodstream Infections
  • the devices may be used with intravenous lines, central lines, or for testing the fluids or therapeutics provided to patients, for presence or absence of contaminating microorganisms.
  • the contaminating microorganisms may be bacteria, viruses, fungi, or parasites.
  • CLABSIs Central line-associated bloodstream infections
  • a central line (also known as a central venous catheter) is a catheter (tube) that doctors often place in a large vein in the neck, chest, or groin to give medication or fluids or to collect blood for medical tests.
  • Intravenous catheters also known as IVs
  • Central lines are different from IVs because central lines access a major vein that is close to the heart and can remain in place for weeks or months and be much more likely to cause serious infection.
  • Central lines are commonly used in intensive care units.
  • the bloodstream infections may be caused by staphylococci (both Staphylococcus aureus (as well as methicillin-resistant S. aureus ) and the coagulase-negative staphylococci), enterococci, aerobic Gram-negative bacilli and yeast.
  • staphylococci both Staphylococcus aureus (as well as methicillin-resistant S. aureus ) and the coagulase-negative staphylococci
  • enterococci enterococci
  • aerobic Gram-negative bacilli When aerobic Gram-negative bacilli are assessed as a group, their frequency follows that of the staphylococci.
  • Certain pathogens are associated with specific host, treatment, and catheter characteristics. S aureus infections are disproportionately represented in infections of hemodialysis catheters.
  • Gram negative bacilli have been associated with infections of patients with cancer, and they are typically the pathogens recovered in instances of infusate
  • Gram-negative bacilli and yeast have been affiliated with catheters placed in femoral veins, while Candida have been associated with infections of lines used for administration of parenteral nutrition.
  • the devices are particularly suited for detecting and identifying microorganisms from various genera, such as from Staphylococcus,
  • Exemplary microorganisms detected by the devices include S. aureus , S. epidermidis, E. faecium, C. albicans , and E. coli.
  • the devices may be used for detecting, identifying, and characterizing biological material, including eukaryotic cells of interest, in a sample.
  • the devices may be operated at a frequency to capture the biological material of interest, which then may be condensed in the identification module, and/or flown to the counter module for characterization.
  • the biological material in the identification module may then be further processed.
  • the further processing include microscopic examination without staining, microscopic examination with staining, or assaying of the biological material in the counter module.
  • the assaying typically includes counting the particles, characterizing particle size, shape, activation state, division state, metabolic state, live/dead determination, surface marker expression, and others.
  • the devices may be used for routine, diagnostic, therapeutic, or as needed screenings to detect the presence or absence of contaminating microorganisms.
  • the screenings may be of food, water sources, beverages, and liquid therapeutics for contamination with microorganisms.
  • the screening may be of environmental samples, such as swimming pool samples, drinking water, industrial water, rainwater runoff, or stream water. V. Kits
  • Kits containing one or more devices, sterile lids, staining reagents, and instructions for use are provided.
  • the devices in kits may be pre-assembled for immediate use, or include instructions for assembly at the point of use by the end user.
  • the kits may include a chart with CM factors for different particles, such as eukaryotic cells or microorganisms, and the corresponding settings on the device at which the device may be used for capture and identify a biological material of interest.
  • kits may provide disposable, single-use devices, or devices for repeat use.
  • kits provide sterile, pathogen-free devices, and instructions for the device use, cleaning, and sterilization for repeat use.
  • Example 1 A biological material identification device.
  • the devices were made by a photolithography step with liftoff metallization.
  • the method includes electrode pattern fabrication using a bi-layer lift-off process.
  • the steps of the process include step 1: on a silicon wafer with 3 pm Si0 2 spin LOR10A and S1808; step 2: expose and develop features with ME312:DI 1:1; step 3: evaporate Ti 5 nm and Au 40 nm; step 4: lift off gold using 1165 Microposit remover; step 5: PDMS bonding to silicon substrate with oxygen plasma at 100 W for 30 seconds at 350 mTorr.
  • Electrodes for material capture There are two important parameters in the design of the electrode.
  • the spacing of the electrode was about 25 microns, as it gave very efficient capture.
  • the height of the microfluidic channel was approximately the same height as the spacing.
  • the electrodes used for the described tests were passivated.
  • the length of the trapping region was adjusted such that at the target flow rate, typically between 1 pL/min and 100 pL/min, all of the bacteria from the test sample were trapped. Subsequent in-line traps at a lower flow rate condensed the bacteria into a smaller spatial region.
  • the overall dimensions of a device may vary, the devices may have a width between 8 mm and 25 mm, length between 20 mm and 75 mm, and thickness between 0.8 mm and 5 mm.
  • Condensing the biological material to an identification unit Condensing the biological material to an identification unit.
  • the approach was to conventionally stain and identify them.
  • the larger about 2 cm condenser is incompatible with a microscope field-of-view (FOV) of about 180 microns.
  • FOV microscope field-of-view
  • a second condensation step onto a second-stage, 180 micron condenser was performed. This was done by first closing the outlet of the primary channel, opening secondary channels that create flow over the second stage condensers, releasing the field of the 1st stage, and then turning on the 2nd stage field(s).
  • This staged“shuttle” transfer technique allowed all targets from the first stage to then be transferred to the second stage, with a coincident reduction in FOV to allow microscope inspection.
  • the target bacteria were then fixed to the second stage by drying (with a hot plate, or integrated heater on bottom of slide), at which point the field can be removed.
  • the bacteria could also be fixed by high electric field.
  • Typical electrode structures are shown in Figures 2A-2D, and typical device structures are shown in Figures 4A-4D. Their operability is summarized in Table 1.
  • Figure 2A shows a circular electrode structure; but these can be of different shapes, such as linear, angled, or tapered structures.
  • the length of the trapping region was adjusted such that at the target flow rate (typically, between 0.01 pL/min and 200 pL/min), all of the bacteria from the test sample were trapped.
  • Figure 4A is a cut-away diagram showing the device structure, with the top fluidic cover removed to enable visualization.
  • the top of the microfluidic channel is removed, and the microfluidic channels are the recesses in the material forming the device.
  • the inlet is at the top, the outlet is at the bottom, and three outlets for the shuttle are on the left.
  • the microchannel can be made from PDMS, which would allow easy removal after condensing to allow microscope immersion inspection.
  • Figure 4A shows the larger 1st stage / smaller 2nd stage configuration, and the secondary fluid channel that enabled flow over the 2nd stage.
  • the three sets of condensers were not for redundancy, but to capture three distinct bacterial targets, selected by frequency.
  • Example 2 CM factor dependence on the AC frequency and voltage.
  • the unique CM factor frequency dependence for a specific biological material of interest can be measured by quantifying a particle’s acceleration in the presence of an applied AC signal, for a range of frequencies.
  • Figure 1 illustrates a typical CM factor frequency dependence, showing the crossover from pDEP to nDEP.
  • the frequency at which this crossover occurs is generally different for different cell types.
  • the devices utilize the frequency dependence of the sign of the CM factor between different biological materials to separate the biological material of interest from a test sample.
  • Example 3. E. coli capture with an exemplary device.
  • the device was as described in Example 1.
  • the device having an electrode structure shown in Figure 2A, was used with green fluorescent E. coli, which were trapped at the electrode edges where the region of highest electric field gradient and, therefore, maximum pDEP trapping force occurs.
  • Example 4 E. coli capture and separation from RBC with an exemplary device.
  • Separating different species of organisms based on their CM factor may also be done by first determining the CM factor individually for each species. This may be done by seeing what frequency effectively captures the cells, at a given solution conductivity, as shown in Figures 10A and 10B.
  • Example 5 Modifying electrode microchannel dimensions does not change the device performance.
  • Figures 5 A and 5B show that widening the electrodes from 46 mhi to 100 mhi results in particle capture occurring farther down the length of the channel, and scales linearly with the electrode width.
  • the channel length necessary to achieve various capture rates is shown in Table 2. The overall length for 100% capture did not exceed 1 cm.
  • the data in Table 2 show that the number of electrodes needed to achieve capture remain relatively constant and so the channel length necessary scales linearly with the electrode width.
  • Example 6 Methods to Improve circuits for AC dielectrophoretic devices.
  • dielectrophoretic (DEP) approaches require high throughput, typically at flowrates on the order of 100 pL/min, which often imply large electrode areas.
  • DEP dielectrophoretic
  • decreased capture efficiency may be observed as structures expand in cross- section area, as well encountering problems related to Joule heating. Decline in capture efficiency and the Joule heating both originate from the same underlying phenomenon: as the cross-sectional area of the capture electrodes exposed to solution increases, the magni tude of the solution resistance element term decreases. This causes the proportion of the applied voltage dropping across the solution resistance to decrease accordingly.
  • This example provides a method to modify the coupled microchip circuit design for highly efficient DEP systems, for both positive and negative DEP approaches.
  • FIG 6 shows a simplified circuit model for a DEP system.
  • a function generator is used to supply an AC voltage, which generates the electric field gradients necessary for dielectrophoretic capture
  • This voltage source has an internal output impedance, and the combination is enclosed in the dashed lines shown in Figure one.
  • This voltage source is applied across an interdigitated electrode structure, such as in Figure 2 A.
  • the electrical lead-ins of the structure have some resistance, here represented by eiec .
  • the voltage across the solution resistance element in Figure 6 is the sole determinant of capture force. However, if the solution resistance becomes comparable to the other resistances and impedances (including possible parasitic capacitances), the electrode and double layer impedances will divide the voltage (attenuate the voltage across the solution resistance element), resulting in decreased DEP efficiency .
  • the transfer function that expresses ratio of voltage across solution as a function of the voltage applied by the function generator (VFG) is lim
  • Figures 7 A and 7B show a typical measurement for a DEP structure as a function of frequency, with a series 2 kit resistor.
  • Figure 8 shows a capture efficiency from a DEP liner interdigitized electrode array.
  • the device had an oxide coating, and the ionic strength was adjusted so that the solution resistance would dominate. Under the condition of low series resistance, DEP capture efficiency can be unity.
  • capture efficiency optimization is a complex interplay between the solution resistance determined by the application, an optimized electrode design to minimize lead in resistance, and a limit that the source resistance must be much less than the solution resistance.
  • DEP capture is performed at the lowest applied voltage that still results in maximal capture, to minimize extraneous Joule heating.
  • the power per unit area is roughly constant/independent of width. Simply placing multiple IDE structures in parallel, or even if they were entirely adjacent with separate buffered drive circuits would not suffice. The power dissipation at the critical capture voltage would remain constant for the whole width.
  • the substrate should be of high thermal conductivity to minimize Joule heating of the sample.
  • Electronic cell counters such as those provided by Beckman Coulter® (Beckman Coulter, Inc., Brea, CA), may be adapted for biological material quantification.
  • the current COMSOL simulations may be extended to different specifications in terms of cell types, ionic strengths, channel widths/heights, flow-rates and linear velocities with a focus on obtaining the minimum number of electrodes (and as a consequence chamber length) required to achieve 100% capture. It should also be refined to take-account of the‘bouncing’ of bacteria observed between electrodes at higher flow-rates.
  • the simulations may be conducted with the following experimental parameters: fluid conductivities: 0 - 10 S/m; channel widths: 10-10,000 pm (1 cm); channel heights: 1 - 1000 pm; flow rates: 0.01 - 1000 pL/min; and linear velocities: 0.2 - 2xl0 / pm/sec.
  • an on-chip (in-line with DEP capture regions) Coulter counter that can count: (i) individual latex beads; (ii) E. coli ; and (iii) Mycobacterium smegmatis, may be developed.
  • the device will utilize narrow microfluidic channels, then determine the maximum width at which these will work, typically about or less than 1 mm.
  • the device may be used to investigate the detection efficiency as a function of flow rate, electrode spacing/width, and solution conductivity .
  • the device may be able to distinguish between bacteria, bacterial clumps, and sputum.
  • Coulter counter sensing techniques extract information about particle size from voltage spikes generated as the particle passes between two or more sensing electrodes.
  • Laminar flow in microfluidic channels is used to confine the sample flow so that it passes through a narrow sensing region.
  • a problem inherent to laminar flow in microfluidic channels is the creation of a parabolic velocity profile in the directions transverse to fluid flow'. This creates a dispersion in particle velocities, creating additional inhomogeneity in the voltage spikes detected for a single species (atop any natural inhomogeneities due to variance in particle size).
  • DEP dielectrophoresis
  • the proposed DEP-focused particle counter system presents the following improvements on conventional coulter counter particle detection systems:
  • Expanded species selectivity ability - DEP force magnitude and direction depends on the electrochemical properties of the particle and thus would allow for preferential counting of a particular species within a mixed population based not just on size differences, but also on electrochemical differences between species of the same size.
  • a sinusoidal voltage signal of 5 VPP amplitude at 4 MHz frequency was applied to interdigitated gold electrodes (25 pm width, 25 pm gap, electrode gap lengths 5-6 times the diameter of the target analyte may be useful for capture performance).
  • Solution was flown over the electrodes at 0.5 pL/min, while a number of beads were captured on the el ectrode.
  • the signal was then removed, and the captured beads were released into the solution flow.
  • the captured packet of beads begun to move downstream in the channel.
  • the downstream counter structure (with three electrodes arrangement, Figures 11A-11E) was running.
  • a sinusoidal excitation signal of 1 V rms amplitude (although the values may range between 1 mVrms and 10 Vrms) at 70 kHz frequency (although the values for frequency may range from 10 kHz to 10 MHz, dependent on the system and the desired application) was applied to the middle electrode.
  • the top and bottom electrodes in the three- electrode structure were the sensing electrodes.
  • Appropriately-configured measurement circuitry transduced the electrochemical impedance between the left (right) sensing electrode and center excitation electrode into a voltage V i (V2).
  • Example 8 Modeling for Increasing throughput through the DEP device.
  • the model predicts the throughput scales with the channel width without any indication of a tradeoff in performance (Figure 15).
  • the model predicts the throughput scales with an increase channel height or in flow speed ( Figures 16A-16D).
  • Example 9 Experimental testing of modeling shows a trade-off in scaling and no linear increase throughput through the DEP device
  • Described are a seri es of investigations to demonstrate ho w device performance is impacted by design variations from the perspective of this voltage transmission framework.
  • the competition between the Stokes force and the DEP force to shift the equilibrium velocity of incident particles flowing over the DEP electrodes was used.
  • the magnitude of this shift was determined by the competition between the DEP and Stokes force acting on the particle in that region.
  • FIG. 19A and 19B depicting the position as a function of time as a particle passes over the interdigitated electrode (IDE) array, located at xl.
  • IDE interdigitated electrode
  • FIG. 19B depicting the position as a function of time as a particle passes over the interdigitated electrode (IDE) array, located at xl.
  • sequential image analysis was performed to track and trace the position of particles frame-by-frame from recorded videos.
  • the beads were fluorescently-tagged, and therefore fluorescence was employed for imaging with a laser excitation source and optical filter to maximize the particle -background contrast.
  • the change in equilibrium velocities occurring between xl and x2 as the particle as it passes over the array is proportional to the magnitude of the DEP force.
  • the fractional change in velocity that particles experience when subjected to DEP forces over the device are extracted as
  • Fluorescent beads were passed over the interdigitated electrodes for particle tracking video analysis.
  • the polystyrene beads (Polysciences, Inc. 17867-5) were 1.77 mhi in diameter and fluoresced green under excitation.
  • the beads were diluted 4,000-fold in O.lx PBS and passed at a flow rate of 0.4 pL/min The low flow rate was chosen to ensure a sufficient number of frames were recorded per particle transit.
  • the dilution was chosen to ensure a high number of beads passing during recordings while not being so high as to overwhelm the tracking algorithm computationally.
  • the O.lx PBS buffer was chosen to reduce the solution resistance and thereby emphasize the significance of design variations on device performance in contrast to lower-conducti vity solutions. As can be seen from inspection of Eqn. 7, the largest influence of electrode design is expected to be seen when the solution resistance is comparable to the electrode resistances.
  • dieiectrophoretic force is in opposition to this drag force.
  • the flow rates were chosen such that the magnitude of the two forces would be comparable to improve detection.
  • Tektronix A FG3252 function generator was used to provide the AC voltage signal necessary to produce a DEP force. Both output channels were used, sourcing sine waves between 0.1 -20 MHz configured to be 180° of phase with respect to each other, a mode of operation known as bipolar DEP. Each output channel was configured to expect a 50 W load impedance and fed directly into a dual-channel, high-frequency power amplifier (Tabor Electronics 9250). Typical voltage amplitudes were 1.2 Vpp for the Tektronix function generator with a subsequent ten-fold increase in amplitude provided by the Tabor amplifier. These amplitudes were chosen such that the incoming beads experienced significant slowing over the DEP electrodes without becoming captured to render the measurements sensitive to shifts in the DEP force.
  • the dielecrophoretic force varies not only as the particles pass over the electrodes but also depends on the particles’ height within the channel.
  • the laminar flow profile of a microfluidic channel is fastest in the center, thereby introducing variance in the drag force arising from vertical height as well as the lateral position within the channel.
  • Increasing the number of electrode structures within the fluidic region is another strategy for improving device performance, particularly for capture. Particles not captured by the first pair of electrode structures have additional chances to be captured during subsequent interactions with the DEP force as they pass over the repeating electrode sub-units. Accordingly, COMSOL simulations predict asymptotically-increasing capture probability as the number of repeating sub-units is increased .
  • Increasing fluidic channel width is a common tactic to increase volumetric throughput for DEP-actuated devices. Increasing the width produces a commensurate decrease in the solution resistance of the fluidic region.
  • a microfluidic channel of varying widths (0.5, 1.0, and 2.0 mm) was placed over identically- fabricated electrode structures, figures not shown. The volumetric flowrate (0.2, 0.4, and 0.8 pL/min.) was correspondingly adjusted to maintain a constant linear velocity - keeping the Stokes’ force constant across all three channel widths.
  • Increasing channel height is another means of increasing volumetric throughput at constant linear flowrate.
  • the fringing electric fields between planar metal electrodes driving the DEP capture decay in strength with increasing vertical distance above the electrode surface.
  • the fraction of cells passing far above the electrode surface scarcely experience the DEP force.
  • Insulating layers are preferable to inhibit electrolysis at the electrode solution interface, reduce the likelihood of cell adhesion, and reduce the probability ⁇ of electrode corrosion by the sample.
  • These protective coatings introduce an additional series impedance in-line with the solution resistance and therefore impact the magnitude of the DEP force between the electrodes.
  • the voltage transmission model also directly" informs physical design limits on the effective capacitance permissible when coating the electrodes with a protective, insulating layer.
  • Capacitive coupling ( Csub ) through the substrate arises between the DEP electrodes.
  • Csub is an extensive quantity, depending upon the electrode density (the inter-electrode gap length) and the total area of the electrode structure.
  • the dielectric properties of the substrate also impact this term, which forms in parallel with the solution impedance and interfacial capacitance.
  • Csnb sets an upper bound on the operational frequency for DEP capture. For the typical structures fabricated on glass, the capacitance is negligible.
  • a large pseudo-capacitance forms at the electrode-solution interface in conductive solutions. Ion concentration (solution conductivity) and device area govern the magnitude of the pseudo-capacitance The effects of variations in Qo were considered, the series combination of this pseudo-capacitance with the capacitance of a protective coating deposited over the device region The impedance of the smaller capacitor dominates series capacitor combinations.
  • the deposited coating is the determining factor. As the thickness of the coating increases, the effective capacitance decreases shifting the curves rightward. This is in line with the results from Figures 23A and 23B.
  • the maximal permissible coating capacitance is determined by the solution resistance of the device and the desired operating frequency -- ⁇
  • Example 10 Design considerations for a portable counter module or counter device.
  • the terms particle and cell are used interchangeably.
  • the small capacitance of cell membranes gives the appearance of an insulating particle in the measurement signal for sufficiently low operating frequencies, typically below 1 MHz.
  • the impedance-based flow cytometer (that may be used as a counter module or a counter device) adopts a three-electrode design, modeled after the cytometer presented by N.N. Watkins, et ah, among others (Watkins et al., Lab Chip, 11(8): 1437-47 (2011); Gawad et ah, Lab Chip, l(l):76-82 (2001)).
  • the circuit operates as an impedance bridge.
  • a sinusoidal excitation signal (VAC) at the middle electrode drives current flow through solution to the left and right sensing electrodes.
  • Each of the sensing electrodes is connected to circuit ground by a resistor, henceforth referred to as the bridge resistor ( R br ).
  • each sensing electrode Vi , V2
  • R so/n the ratio of the bridge resistor to the solution impedance between the excitation and sensing electrodes.
  • the solution impedances and bridge resistors are perfectly symmetric and thus both sensing electrodes are at identical potentials.
  • the solution impedance is temporarily increased, reducing the voltage measured at the sensing electrode.
  • the process repeats as the particle subsequently passes between the excitation electrode and the other sensing electrode. In this manner, a passing particle generates a characteristic voltage signal encoding information about both its velocity and its size.
  • Figures 11A-12 depict the process by which a typical counter signal is generated in a three-electrode geometry.
  • the left- and right-most electrodes serve as sensing elements, monitoring the impedance between them and the middle electrode at which an external voltage is applied.
  • the solution resistance is increased due to the volume displaced by the particle.
  • the solution resistance returns to its normal operating state.
  • the process repeats as the particle flows between (d) the middle and right-most electrodes before finally exiting (e) the sensing region.
  • the output of this configuration (f) is a voltage signal proportional to the difference in resistance between the left and right sensing regions (R L -R R ).
  • the cell membrane capacitance renders cells electrically indistinguishable from insulating particles.
  • researchers have also begun to use elevated frequencies in the MHz regime as part of their excitation signal.
  • the impedance of the membrane capacitance is significantly reduced, allowing researchers to probe the inner conductivity of the cell cytoplasm. In this manner, cell populations of comparable size but differing in physiology may be discriminated from one another, enhancing the counter's capabilities.
  • planar microelectrodes for impedance-based sensing confers multiple advantages over other more-complicated geometries.
  • the electrode definition requires only a few steps: photoresist coating, pattern definition, metal deposition, and a lift-off process. This simplicity compared to alternative electrode geometries significantly reduces per-device fabrication cost.
  • the ease of fabrication simplifies combining the impedance sensor with additional sensing modalities (e.g., target capture, target recognition) into a single microfluidic sensing platform (Valera et al., Lab Chip, 18(10): 1461- 1470, (2016)).
  • the additional resistive sensing element formed by the third electrode transforms the characteristic output signal from a single voltage peak to an antisymmetric peak structure.
  • the elapsed time between the local maxima and minima of the antisymmetric structure reduces uncertainty in transit time measurements during flow conditions, compared to extracting particle velocity information from the full-width at half-maximum (FWHM) of a two-electrode configuration.
  • Design of the fluidic constriction is an integral aspect of the
  • the Coulter principle depends upon the displaced volume of conductive solution by a passing particle. Therefore, the ratio of the volume of the target analyte to that of the sensing region, colloquially called the filling factor, strongly determines sensor
  • a blocked channel effectively halts the device's ability to count particles until the blockage is removed.
  • large hydraulic pressures build up after clog formation. The resultant pressures can cause catastrophic mechanical failure of the fluidic channel, posing a significant biohazard to the end user when dealing with biological samples.
  • Particle densities between 0.1-1 million/mL function best for acquiring a significant number of events within a reasonable measurement time-frame without presenting excessive clogging risk (for 20 pm x 20 pm channel cross- sectional areas).
  • planar electrode geometry adapted in the sensing set-up greatly simplifies the device fabrication process.
  • a single mask and a single metallization layer is all that is required for the counter sensing electrodes, reducing fabrication complexity and cost per sensing device.
  • the planar electrode geometry limits the size resolution performance of the counter structure.
  • an electric field forms when an electric potential is applied across the two electrodes.
  • the electric field that forms is non-homogenous, as shown in Figures 25A and 25B.
  • the solution conductivity remains uniform over the entire sensing volume, different regions of the solution have nonidentical contributions to the impedance between the two electrodes.
  • the magnitude of the impedance-based signal acquires a marked vertical dependence, as can be seen in Figures 26 A and 26B. This dependence produces a 20% dispersion in signal magnitude at fixed particle size, corresponding to a 7% uncertainty in diameters.
  • Solutions to the vertical dependence require either manipulation of the incoming particle stream or overhauling the electrode design.
  • researchers have implemented solutions using acoustic waves to focus the particles into the middle of the channel, sheath flows, and negative dielectrophoresis.
  • structuring the electrodes in three dimensions can greatly simplify the electric field profile at the cost of complicating device fabrication.
  • microfabricated planar electrodes offers an alternative solution: the fringing electric field of the planar electrode geometry.
  • the density of electric field lines arcing from electrode to electrode falls off rapidly with distance from the electrode surface. This has directly observable consequences for the sensor signal as a function of particle height within the channel.
  • planar electrodes which just barely project into the side of a wide ( ⁇ l mm) microfluidic channel would only enumerate particles passing through the narrow width of solution flowing over the electrodes. Because of the self-limiting nature of the fringing electric fields, the sensing region does not feel the effects of the entire width of the channel.
  • the impedance-based cell counter aims to compete with fluorescence- based cytometers. Incorporating simultaneous optical imaging within the measurement system enables direct comparison to fluorescence-based approaches and simultaneous real-time verification of fluidic performance during the development process. To this end, all of the sensing experiments were conducted on the viewing stage of an Olympus BX51 microscope equipped with 5x, lOx, and 40x objectives as well as multiple filter lenses for fluorescence imaging. An Olympus DP70 camera system allows for image and video capture for later analysis. An Xcite Series 120Q laser source provides an intense source for fluorescence imaging.
  • PCB printed circuit board
  • a metal sample mount was then designed to mate with the PCB.
  • a groove milled out of the sample mount allows devices to easily be loaded underneath the spring-loaded connector from the side.
  • a slot (shaded blue) recessed in the center of the milled-out groove has been machined with lateral tolerances much tighter than the contact pads' pitch to mechanically ensure in plane alignment.
  • Vertical alignment with the springloaded pins is likewise mechanically determine by the vertical displacement between the bottom of the slot and the height of the PCB.
  • the combination of the PCB and sample mount thus provides a secure and robust connection between the device and the coaxial connections on the PCB.
  • Sample alignment in all three dimensions is achieved by the physical structure, removing a significant barrier to reliability and ease-of-use.
  • the metal sample mount and the ground plane of the PCB form a Faraday cage around the device to shield the device from external
  • sample mount was also machined with a second, larger contact area, to permit interfacing device geometries which do not conform to the milled socket while still handling alignment in the vertical plane.
  • a sinusoidal voltage source is required for the three-electrode bridge circuit.
  • An Agilent 33120A function generator was used for the counter measurements as opposed to the built-in function generator of the lock-in amplifier.
  • the Agilent 33120A demonstrated lower noise floors and higher spectral purity than the sine wave generator of the SR830 lock-in amplifier, as measured on a network analyzer.
  • the excitation signal a 70 kHz sinusoid with 1 V rms amplitude, was rarely varied during the course of development.
  • the output signal from the bridge circuit was monitored during experiments with a Stanford Research Systems SR830 lock-in amplifier.
  • Lock- in amplifiers exploit the orthogonality of sine and cosine functions to extract the amplitude of a specific frequency component of the input signal with high fidelity. This enables detection of the small changes in the bridge resistance during particle transit events expected at low filling factors.
  • the transit time of the particles over the counter structure dictates the necessary sampling rate for measuring the bridge voltage. From the perspective of the Nyquist criterion, the minimum sampling frequency is 2/ St, where St is the transit time of a particle passage.
  • St is the transit time of a particle passage.
  • researchers typically aim for a minimum of 20 datapoints per event, requiring sampling rates of 10-1000 kHz depending upon desired flowrate and constriction geometry. To satisfy this condition, a
  • Tektronix DPO4104 was employed to record the analog voltage signals from the rear panel of the lock-in amplifier. Furthermore, extracting particle size information from the shape of the voltage signal requires a sufficient number of datapoints per particle trace, with minimums in the literature between 10 and 20 points. Transit times of 0.1 ms correspond to sampling rates between 100 - 200 kHz which is hardly a stringent requirement in a laboratory setting, however there exists economic incentive to minimize the necessary sample rate when producing portable systems. The sampling rate also must be increased with increasing expected event frequency to resolve abnormal signatures arising from contemporaneous transits.
  • the printed circuit board comes equipped with the ability to interface with up to six counter structures. Each has a single Texas Instruments OPA- 2227 operational amplifier configured as a dual-channel unity-gain voltage follower. A gain -bandwidth product of 8 MHz more than exceeds the necessary operating frequency of the Coulter counters. For a balanced bridge being driven by the typical 1 V rms amplitude, the equivalent peak-to-peak voltage occurring at either node is 2.83 Vpp. Given the specified slew-rate of 2.3 V/ps, operation up to 0.8 MHz is possible. A dual-channel op-amp is chosen to avoid variance among individual integrated circuits which would contribute to a differential signal between the two terminals.
  • instrumentation amplifier can be configured to provide additional gain of the differential signal, elevating the signal of interest further over the suppressed background signal between the two amplifiers.
  • a New Era syringe pump (NE-1000) was used. Typical sample flow rates range from 0.1 - 5.0 pL/min.
  • microfluidic constriction for the counter region presents a hydraulic resistance, generating large back-pressures in the fluidic channel as flow velocity increases.
  • the backpressure splits open the tubing inlet or breaks the adhesion between the channel and the substrate, causing leaks.
  • increasing the flowrate reduces the mean time to clog formation within the channel, a problem exacerbated by the rigidity of polystyrene beads used for calibration experiments.
  • Curve fitting on the histogram bin counts extracts the standard deviation of the background noise which determines the threshold levels for peak detection of the individual trace.
  • a coincidence detection for particle recognition was employed: within a finite time window (the expected transit time based on flow rate and constriction geometry), the voltage signal must cross the positive threshold level with a rising and then falling edge before crossing the negative threshold with a falling and then rising edge. If all four of these crossings occur within the finite time window (the expected transit time based on flow rate and constriction geometry), the voltage signal must cross the positive threshold level with a rising and then falling edge before crossing the negative threshold with a falling and then rising edge. If all four of these crossings occur within the
  • Curve-fitting of the antisymmetric peak structure within the event extracts the voltage amplitude (proportional to measured particle size) and elapsed time (measured velocity) between the maxima and minima of the signal.
  • a two-dimensional histogram is then constructed of the velocity and size information to look at the dispersion in recorded events along both dimensions, reflecting both physical effects arising from the channel geometry (parabolic flow velocity profile, height dependence of the signal amplitude) as well as uncertainties arising from variances in the fitting algorithm.
  • the spread in measured values for the peak height which determines the ability to use sizing alone to distinguish amongst incident particles or species.
  • Tween-20 was added by volume as a surfactant to inhibit aggregate formation within the sample.
  • the sample was flown over the counter device at 1.0 pL/min. while recording 1600 one-second data samples.
  • the algorithm analyzed the acquired data and extracted particle transit time and peak height for each detected event. Three separate populations are clearly visible.
  • the cube root of the peak height is plotted, as it should be directly proportional to particle diameter. Constructing a histogram of the peak heights reveals a trimodal distribution, as can be seen in Figure 28A.
  • Gaussian peak fitting extracts the mean signal amplitude and uncertainty for the three populations, plotted as a function of nominal bead diameter in Figure 28B.
  • the output response time of the SR830 is dictated by the steepness of its bandpass filter as well as the integration constant chosen. For maximal signal-to-noise ratio during measurements at the targeted volumetric flow rate, a 30 ps time constant and 24 dB ./decade roll-off were chosen. Per the SR830 datasheet, this generates a 99% response time of 300 ps. No significant attenuation was observed for flow velocities up to 8.0 pL/min., or 0.5 mL/hr. A consistent function over the range of flow-rates was observed germane to the desired clinical applications of the sensor.
  • Example 12 Counter as an assay module or device.
  • the counter device allows for not just the separation and isolation of activated T-cells, but also to be able to provide a quantitative on-chip measurement of the activation. To do this, the wide variety of functions accomplished by planar metal electrodes in a fluidic environment was used.
  • the counter device separates T cells, in high cond, in relatively high viscosity, and with on-chip quantification rather than optical detection.
  • the current methods for assaying cells require different techniques that are time consuming, and/or the signal from cells is transient. These drawbacks are summarized in Table 3.
  • the counter module/device allows for on-chip quantification and assaying of the cell state without the need for stains or markers.
  • the assay is rapid, label-free, and the device is free of optical components for detecting the signals from the cells.
  • Table 3 Drawbacks of current techniques for cell assays. The main drawback is that there is no rapid, label-free method of assaying cells.
  • the impedance-based flow cytometers provide information on the number and size distribution of incident particles (Cheung et ah, Cytometry A, 65(2): 124-32 (2005)). The two subpopulations are readily resolved by the clear size differentiation (4-5 pm v. 8-12 pm) between activated and unactivated T-cells (O’Connell et al., Comparative medicine , 65(2):96-l 13 (2015)).
  • Operating a counter structure near the outlet of each stream one can count the total population of activated and unactivated cells in the laminar flow of the original sample as well as in the exchange buffer. Thereby one can quantify the efficiency and purity of the dielectrophoresis separation as well as the ratio of unactivated to activated T-cells within the sample to assess immunological status.
  • the lymphocyte sample is the lymphocyte sample.
  • the T-cells are primary cells, prepared directly from murine splenocytes, distinct from cell lines generated for modeling cell behavior under tissue culture conditions.
  • the T-cells are in an unactivated state when initially prepared.
  • unactivated T cells are exposed to activation-inducing agonist antibodies, anti-CD3 and anti-CD28, for 72 hours. Both populations are suspended in l.Ox phosphate-buffered saline with 0.1% by volume of Pluronic F-127, a surfactant from Sigma Aldrich, to minimize cell adhesion to the device.
  • samples containing a mixture of both naive and unactivated T-cells were introduced, in 1:1 and 2:1 ratios.
  • the combined signal contains a sum of both the naive and activated signatures from Figure 14.
  • Size-based discrimination as a diagnostic criterion requires the ability to differentiate between naive and activated T-cells after a prolonged period of growth. The ability to distinguish between the two was demonstrated. It remains to be seen how the growth process occurs over time for populations of T-cells after antigen exposure. The growth kinetics and size resolution of the sensor could both set a lower bound on time after antigen exposure for a detectable immune response.
  • This example demonstrates the implementation of an impedance-based sensor for particle sizing and enumeration using planar metal electrodes.
  • the sensor embodiment is suitable for lab-on-a-chip sensing applications.
  • Example 13 Integrating DEP capture and counting modules - capture and count device.
  • Dielectrophoretic electrode structures have been widely used for cell capture (Lee et ah, Biomicro fluidics, 12(5):054104, (2016); Rosenthal et al., Lab on a Chip, 6(4):508-515 (2006)) and sample concentration (Park et al, Lab on a Chip, l l(l7):2893-2900 (2011); Jubery et al, Electrophoresis, 35(5):69l-7l3 (2014); Wang et al., Dielectrophoretic separation of microalgae cells in ballast water in a microfluidic chip
  • Example 14 Devices for lateral separation of particles, optionally for buffer exchange, and/or particle counting.
  • a device with electrode arrays was designed to manipulate the lateral displacement of cells within the sample to enhance the functionality of the impedance-based assay. Dielectrophoresis and the Stokes' force have different dependencies on cell diameter. Under carefully chosen conditions, one can separate activated and unactivated T-cells. This approach has been previously demonstrated in the literature (Han and Frazier, Lab on a Chip, 8(7): 1079- 1086 (2008)).
  • Physiological samples are inherently messy. Separating the enumeration and analysis target into a parallel fluid stream isolates it from the environment containing debris and up to billions of cells per mL which include the fluidic background signal. This confers two distinct benefits. Only the purified side stream needs pass through a constriction region for enumeration, greatly reducing the clogging probability during operation.
  • Isolating the target from a high number of background count relaxes the rejection thresholds for false positives and false negatives at the same error rate in terms of events per volume. This feature is particular desirable for
  • the described devices integrate both the DEP separation and counter enumeration onto a single microfluidic chip, having established both operational capabilities separately.
  • the devices permit quantifying the separation efficiency of the assay and the purity of the sample within the exchange buffer stream as illustrated in Figures 30A-30H.
  • Two separate inlets, one connected to the sample and the other to the buffer solution, flow in side-by-side in the wider microfluidic channel before passing over the separator structure.
  • Figure 30A the parallel laminar flows continue through the device, separating at the junction before passing over a counter structure en route to two outlets in the bottom of Figures 30A and 30B.
  • An applied DEP signal drives lateral separation of the activated T- cells (large spheres) as well as a few unactivated T-cells (small spheres) into the buffer stream. These pass through the right outlet channel where they are enumerated by the right counter in Figure 30B.
  • the counter in the left channel detects both populations in the outlet stream whereas the right channel sees few, if any, events. Histograms of the detected events for both counters are shown in Figures 30C and 30D.
  • the DEP signal drives lateral separation of the activated T-cells, the bulk of the activated population in the left channel (the sample stream) is depleted and instead detected in the outlet of the buffer channel by the right channel counter. A fraction of the unactivated T-cells are also separated by the DEP signal.
  • the DEP signal therefore drives changes in the detected cell distributions as measured by both counters, shown Figures 30E and 30F. Experimental results from these assays are shown in Figures 30G and 30H.
  • Enumeration was performed using counter structures fabricated on a glass substrate to mitigate the influence of parasitic capacitances from the bonding pads, whereas the initial separation structures were fabricated on silicon wafers.
  • the combined devices have been fabricated on glass and are
  • the device with dielectrophoretic manipulation and impedance-based cell counting can be further integrated in biosensing applications by extending the result from sample in physiological saline to separation in whole blood.
  • a two-step buffer exchange process would eliminate the need for sample centrifugation prior to analysis (diagrams not shown).
  • the DEP device used in particles separation (lateral separation of beads) and buffer exchange were conducted on the same device using flow of two different buffers over the device.

Abstract

Modular portable devices for capture, identification and characterization of particles in a sample of fluid using a dielectrophoretic (DEP) and/or electroosmotic (EO) trap include a trap module, an identification module, and/or a counter module. The devices may include a shuttle system for shuttling particles trapped in the trap module to the identification module or the counter module. The devices may be used for identifying central line associated bloodstream infections (CLABSI). The devices are able to separate from a blood sample and condense the most important bacteria involved in CLABSI within minutes, in a format that allows immediate staining and identification or counting the captured cells. In addition to condensation, the technique has the ability to separate specifically-selected pathogens, simplifying and automating their rapid identification.

Description

ELECTRONIC SYSTEM FOR CAPTURE
AND CHARACTERIZATION OF PARTICLES
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of and priority to U.S.S.N.
62/746,327 filed October 16, 2018, which is hereby incorporated by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
This invention was made with Government support under Grant No. HDTRA 1-12- 1-0042 awarded by United States Department of Defense. The Government has certain rights in the invention.
FIELD OF THE INVENTION
The invention is generally directed to electronic devices for capturing and characterizing particles, including biological material, in a sample.
BACKGROUND OF THE INVENTION
Detection of biological material, such as microorganisms, in a biological sample often requires the capture and identification of live microorganisms. However, rapid identification of live microorganisms in samples remains a challenge. It requires culturing live microorganisms from samples and then assaying them for identification, such as through staining and/or microscopic evaluation. This can take days and hinder diagnosis and treatment.
Theoretically superior molecular techniques (e.g. polymerase chain reaction (PCR)) have limitations in several important respects. In the case of biological samples infected with pathogens, dead pathogens will give falsely positive results with PCR, leading to unnecessary and potentially detrimental clinical interventions. PCR for the full spectrum of potential pathogens (i.e. gram positive and negative bacteria and yeast) requires harsh cell lysis conditions including detergents, bead beating, and/or strong alkali solutions.
These conditions substantially damage nucleic acids leading to inappropriately high limits of detection (LODs). Indeed, among FDA-cleared PCR-based tests for direct detection of pathogens from primary specimens (e.g. SIMPLEXA® (Diasorin Molecular LLC, Cypress, CA), Xpert Cabra-R® (Cepheid
Corporation, Sunnyvale, CA), and FILMARRAY® (Biofire Diagnostics, LLC, Salt Lake City, UT)), published LODs range from 100 to over 1000 CFU/mL for relevant pathogens.
There remains a need for devices and methods for rapid capture and identification of biological material of interest, which remain live after capture and identification.
Therefore, it is the object of the present invention to provide devices for capturing and identifying live biological material of interest present in samples.
It is another object of the present invention to provide methods of making the devices.
It is yet another object of the present invention to provide methods of using the devices.
SUMMARY OF THE INVENTION
Devices for rapid capture of particles, including particles from
environmental and biological samples have been developed that are portable and rapidly capture, identify and/or characterize the particles, including live cells, microorganisms, macromolecules, nanoparticles, microparticles, and other particulates in the environment, food, or a biological sample. The devices typically include one or more units, each unit having a trap module containing at least two electrodes, and a primary microchannel with an inlet and an outlet. The trap module itself may be used for capture and identification of particles.
Each unit of the devices may also include an identification module containing at least two electrodes and a secondary microchannel with an inlet and an outlet. The trap module is typically fluidically connected with the identification module. Generally, the dimensions of the identification module are substantially smaller than the dimensions of the trap module. The devices may include an integrated counter module. In this embodiment, the counter module is fluidically connected to the trap module or to the identification module. The counter module may include a constriction, such as a microchannel, an inlet to the microchannel and an outlet. The microchannel is typically arranged to provide fluid flow over two or more electrodes. In other aspects, the counter module does not include a microchannel and is constrictionless. In this aspect, the sample from the trap module or the identification module flows over the two or more electrodes of the counter module. In this embodiment, the particles are captured over the electrodes of the trap module or the identification module. The particles then flow over the counter electrodes and are assayed by the operability of the counter electrodes.
Also described are portable particle counter devices. The portable particle counter devices typically include a region with two or more electrodes and a fluidic channel overlaying the electrodes. A sample is passed over the one or more electrodes for obtaining the characteristics of particles. The counter device may include a constriction, such as a microchannel, an inlet to the microchannel and an outlet. The microchannel, if present, is typically arranged to provide fluid flow over two or more electrodes. In other aspects, the counter device does not include a microchannel and is constrictionless. In this aspect, the sample flows over the two or more electrodes of the counter module. In this embodiment, the sample is constricted over the electrodes by the operability of the counter electrodes.
In devices with identification module(s), the trap module and the identification module may be connected fluidically, via electrodes, or both fluidically and via electrodes. The electrodes connecting the trap module with the identification module may be patterned to focus biological material onto the identification module.
Typically, the device has a plurality of units, such as at least two, at least three, at least four, at least five, or at least six units. The at least two electrodes of the trap module and the identification module typically have a width between about 1 nm and about 10 cm, between about 1 nm and about 1 cm, between about 1 nm and about 1 mm, or between about 10 nm and about 1000 pm. The electrodes may be separated from one another by a distance as wide as the width of the electrodes. In other aspects, the at least two electrodes are separated from one another by a distance that is less or more than the width of the electrodes. The distance separating the at least two electrodes may be between about 0.01 times and 10 times of the width of the electrodes. The primary microchannel may have a height as high as the distance between the at least two electrodes of the trap module.
The at least two electrodes in the trap module and the identification module may be arranged in any two-dimensional pattern, such as in linear, angled, circular, rectangle, cube, triangle, zigzag, and oval pattern. The electrodes may be pitched with respect to the microfluidic channel. The electrodes may be coated with an insulator.
Methods for capturing a biological material from a sample and identifying the biological material are also provided. The method generally includes flowing a sample with the biological material of interest through the trap module of the device at a flow rate generally between about 0.01 pL/min and 10 mL/min. The method also includes supplying an alternating current (AC) to the electrodes of the trap module at a frequency specific to the biological material. The method operates under the principle that applying an AC to the at least two electrodes of the trap module at a frequency sufficient to provide a dielectrophoretic (DEP) force to the biological material at which its Clausius- Mossotti (CM) factor is maximum, traps the biological material in the device. A device with more than one unit may be used in a single method, and more than one species of biological material may be trapped by the device. Typically, the different species are trapped in the different units of the device. When the device contains units with both a trap module and an identification module, the method may include further condensing the trapped biological material in the identification module. The condensing of the biological material from the trap module into the identification module may occur with the use of focusing electrodes connecting the trap module with the identification module. The trapped biological material may be moved onto an identification module by applying an AC signal to the focusing electrodes connecting the trap module with the identification module.
The condensing of the biological material from the trap module into the identification module may occur via a microfluidic shuttle system. The shuttle system of the device includes closing the outlet of the primary microchannel, opening the inlet of the secondary microchannel, stopping the AC applied to the two electrodes of the trap module, and applying an AC to the two electrodes of the identification module to condense the particles from the trap module in the identification module. The particles in the identification module may then be further processed. The further processing include microscopic examination without staining, microscopic examination with staining, or assaying of the particles in the counter module. The assaying typically includes counting the particles, characterizing particle size, shape, activation state, division state, metabolic state, live/dead determination, surface marker expression, and others.
The condensing of the particles from the trap module into the identification module may also occur by a combination of focusing electrodes and the microfluidic shuttle system.
The devices may be used with biological samples, for example, to detect contamination of biological samples. The devices may also be used in the food industry, for example, to detect contamination or to monitor pathogen levels. The devices may be used to monitor the environment, such as monitoring the presence of polluting particles, or the presence of a biological material of interest in waters or in industrial runoff. The devices rapidly capture, identify and/or characterize particles of diverse origin and composition. Exemplary particles include live cells, microorganisms, macro molecules, nanoparticles, microparticles, and other particulates in the environment, food, or a biological sample.
Generally, the biological material of interest may be a eukaryotic or prokaryotic cell, microorganisms, organelles, vesicles (such as exosomes, endosomes, phagosomes, and liposomes), macromolecules, or biomarkers. The microorganisms captured and identified by the device and methods include bacteria, viruses, fungi, and parasites.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a line graph showing the frequency (f (Hz)) dependence of the Clausius-Mossotti (CM) factor, for a homogeneous particle and medium. At low frequencies the DEP force is positive (pDEP); at high frequencies the DEP force is negative (nDEP).
Figure 2A is a diagram showing DEP condenser electrode structure (diameter 500 pm). Figures 2B, 2C, and 2D are diagrams showing different geometries of electrodes: a linear medium interdigitated finger pairs (IDE) (Figure 2B), an angled IDE (Figure 2C), and a linear small IDE (Figure 2D). Figure 2E is a diagram of a typical pair of interdigitated electrode in a device with an electrode-electrode gap of 25 pm, sixteen electrode fingers, and a 1 mm channel width. The microfluidic channel sidewalls are visible as parallel vertical lines on the left and right boundaries of the image.
Figure 3 A is a diagram showing a selective capture of E. coli (1, arranged in concentric rings) and red blood cells (2). Figures 3B and 3C are graphs showing the calculated CM factor for E. coli (Figure 3B) and red blood cells (RBC, Figure 3C) as a function of frequency (f (Hz)). The samples were tested at different conductivities (S/m). Figure 4 A is a cut-away diagram showing one example of the 3 -unit, 2- stage (a trap module and an identification module) condenser device. Each unit 20 includes a trap module 22, large circular first stage condensers 24 to capture the target microorganisms as they flow through the large microchannel (top to bottom). The captured targets are then transferred to the identification module 26 with smaller second stage condensers 28 for drying, staining, and microscope inspection and identification. Figures 4B, 4C, and 4D are diagrams showing different device design types. Type A includes a selective capture step followed by microfluidic shuttling of particles to a capture region for identification (Figure 4B). Type B utilizes a selective shuttle to move particles into side channels where they are captured for identification (Figure 4C). Type C utilizes a selective shuttle to move particles to a spatially confined section of the channel where they are then captured for identification (Figure 4D).
Figures 5A and 5B are bar graphs showing the particle capture locations along a channel. The Number of particles captured in region versus Particle position (pm) for 46 pm-width channel is shown in Figure 5A and for 100 pm width channel is shown in Figure 5B.
Figure 6 is a diagram showing the circuit model for dielectrophoretic capture. The function generator, VFG, sources a voltage with an output impedance characterized by Rout. The series resistance of the electrode leads is followed by the ionic double-layer capacitance and the solution resistance before returning along an identical path to the negative terminal of the function generator.
Figures 7A and 7B are graphs showing the measured impedance (Z) as a function of frequency (Freq. (Hz)) for typical DEP structures (25 pm gap), connected in series with a 2kQ resistor without (Figure 7A) or with a 50 nm oxide deposition. The legends indicate different conditions: air - top line, deionized water (DI) - second line down, O.OOlx PBS - third line down, O.Olx PBS - fourth line down, O.lx PBS - fifth line down, and lx PBS - sixth line down.
Figure 8 is a graph showing the measured capture efficiency as a function of series resistance, for a 200 nm-oxide device in O.Olx PBS.
Figure 9 is a graph showing capture efficiency is predicted to increase monotonically up to a point as the number of IDE increases, verified by a COMSOL prediction.
Figures 10A and 10B are graphs showing DEP capture of
microorganisms based on solution conductivity and applied frequency. The capture counts for E. coli in O.OOlx PBS, 5V (Figure 10A) and M. smegmatis in 0.01 xPBS solution, 10 V (Figure 10B) at different frequencies (Hz) are shown.
Figures 11 A- 11E are diagrams showing counter structure with three electrodes, and a bead flowing over the counter structure.
Figure 12 is a graph showing the output signal (V) over time (s) for the bead.
Figures 13A and 13B are graphs showing a sample output signal (V1-V2) over time (s) for a set of captured beads before and during the flow of the beads over the counter electrode structures.
Figure 14 is a graph showing the output signal (Vi-V2 (mV)) over time (s) for a sample with naive and activated T cells, and demonstrates that the counter discriminates between the two cell populations, such that the number of cells in the desired population may be counted.
Figure 15 is a line graph showing modeling the expected trajectory of particles in a fluid channel subject to DEP forces. Model predicts throughput scales with channel width without any indication of a tradeoff in performance.
Figures 16A-16D are graphs showing model’s prediction for
performance of DEP capture with: changes in channel height (pm) at various flow speeds (pm /s) (Figure 16A), changes in flow speed (pm /s) at various channel heights (pm ) (Figure 16B), changes in volumetric throughput (mL/hr) at various channel heights (mih ) (Figure 16C), and changes in volumetric throughput (mL/hr) at various flow speeds (pm /s) (Figure 16D).
Figure 17A is a circuit diagram of the DEP capture and a circuit model prediction for the DEP performance with various numbers of interdigitated electrode fingers (Nf) (Figures 17B). Figure 17C is a graph showing the predictions for impedance model velocity ratio, COMSOL model velocity ratio, and experimental velocity ratio.
Figures 18A and 18B are graphs showing DEP performance at different flow rates. Figure 18A shows the number of particles captured at 10 MHz (60seconds/flow rate) at different flow rates (mΐ/min). Figure 18B shows the intensity of fluorescently labeled cells remaining captured (%) at different flow rates (pl/min).
Figure 19A is a diagram showing particles flowing through a
microfluidic channel move at an equilibrium velocity, v0ff , determined by the Stokes force. Over the IDE region, the Stokes' force competes with the DEP force, reducing the equilibrium velocity von. Figure 19B is a graph showing tracking equilibrium particle velocity along the direction of fluid flow probes the DEP force magnitude. Figure 19C is a graph showing changes in resistance (W) at different frequencies (kHz); the electrochemical impedance measurements extract circuit parameters characterizing the electrodes. Figure 19D is a graph showing the DEP force experienced by passing particles is proportional to the squared magnitude (dashed line) of the voltage across the solution resistance element. With increasing series resistance, the ratio of the particles velocities off and on the DEP region (squares) approaches unity, indicating decreasing DEP force magnitude.
Figure 20 shows initially, the equilibrium velocity (squares) over the DEP electrodes decreases with an increasing number of electrode fingers until influence of the decreasing voltage outweighs the increasing number of interactions with DEP force. Changing the number of electrode fingers alters device performance.
Figure 21 shows the solution resistance of the channel decreases with increasing channel width and with thus the magnitude of the DEP voltage (dashed line). Increasing throughput by increasing width sacrifices DEP efficiency.
Figure 22 is a graph showing the impedance measurements for the coated (squares) and uncoated (circles) devices in O.lx PBS solution.
Figures 23A and 23B are graphs showing the expected voltage (dashed lines) differs greatly when comparing devices with (Figure 23 A) and without (Figure 23B) the 200 nm deposited oxide as a function of the signal frequency. This effect is observed in the equilibrium velocity ratios (Exp., solid lines) at lower signal frequencies.
Figure 24A is a diagram showing a top-down view of the metallization pattern for two chips 50a and 50b, each of which contains one capture module 52 and several counter modules 60. A microchannel (not shown) is positioned over the capture module 52 and the counter modules 60. Figure 24B is a diagram showing an enlarged perspective view of one of the counter modules 60, showing the microchannel 54 over the electrodes 62, 64, 66, and a particle 68 flown over the electrodes 62, 64, 66. Electrode length (1), gap between the electrodes (g) and microchannel width (w) are shown. Figure 24C is a diagram showing a top view of a counter module/device 70 showing the electrode structure 72 with a microfluidic channel 74 aligned and bonded.
Figure 25A is a diagram illustration of the fluild lines emanating from the planar electrode geometry bom out by COMSOF simulation (Figure 25B) of the electric field profile for a pair of planar sensing electrodes.
Figure 26A is a graph showing simulation of the impedance variation (Zc, W) at a given position (pm) for an insulating sphere passing over planar electrodes with a 40 pm inter-electrode gap as a function of vertical displacement from the electrodes. Figure 26B is a graph showing changes in voltage (Vdiff (pV)) over time (ms) with a bead transit over planar electrodes, the data from the bead transit event demonstrating the expected behavior.
Figure 27A is a diagram of a top view of a“constrictionless” counter unit/device 100. The counter electrodes 110 are projecting slightly into the microfluidic constriction region 120 of 1 mm. Figure 27B is a graph showing the representative trace of change in voltage (V1-V2 (pV)) over time (ms) from a 4.45 pm bead in O.Olx PBS passing over the counter 100. Figure 27C is a diagram showing the integration of the constrictionless counter 100 with lateral- displacement structures, such as a DEP condensing module 130, to count particles from the entirety of the sample Figure 27D is a diagram of the electronic connection of the counter unit/device with the data acquisition, processing, and display devices.
Figure 28 A is a histogram of the peak heights (V1/3) of events (Counts (dim.)) acquired during the experiment flowing bead populations of different sizes (4.45 pm (1), 6.42 pm (2), 8.87 pm (3)), as well as Gaussian fits of the histogram data to estimate the dispersion of the sensor events. The dashed vertical line represents the detection threshold of the algorithm for this dataset. Figure 28B is a graph showing linear regression of the particle diameter (pm) to the peak height (V1/3).
Figures 29A and 29B are diagrams showing incoming particles trapped on the DEP electrode structure (Figure 29 A) are then subsequently released for enumeration (Figure 29B).
Figures 30A and 30B are diagrams showing sample handling by a device with the DEP capture, separate, and two counter modules and permitting buffer exchange. Shown are an incoming sample and an adjacent exchange buffer stream flowing through the device without (Figure 30A) and with (Figure 30B) a dielectrophoresis signal applied to the separator electrodes. Figures 30C-30F show histograms of Normalized counts over cell diameter (V1/3). Without lateral separation (DEP off), both species of particles pass through the left coulter counter constriction region while not passing through the right counter (Figures 30C and 30D). When a DEP force is applied (DEP ON), lateral separation drives particles into the exchange buffer stream, producing counts from the right counter structure (Figures 30E and 30F). Figures 30G and 30H are graphs showing counter data from a DEP capture, separate, and two counter device and a mixture of unactivated and activated cells flown through it. The graphs show Counts of cells of various diameters (AR/R)i \ When the signal is OFF, only three peaks are detected: debris peak (1), unactivated cells peak (2), and minor activated cells peak (3) (Figure 30G). When the signal is ON, four peaks are detected: debris peak (1), unactivated cells peak (2), activated cells peak (3), and activated dimers peak (4) (Figure 30H). The harvest efficiency was 93.5%, the purity = 59.4%.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
As used herein, the term“particle” refers to micro- or nano-sized particles, including naturally occurring particles, synthetic particles, polymeric particles, biological material, and environmental particles.
As used herein, the term“biological material” refers to eukaryotic or prokaryotic cells, organelles, vesicles, macromolecules, biomarkers, and microorganisms.
As used herein, the term“microorganism” refers to microscopic organisms such as bacteria, fungi, algae, protozoa, and viruses.
As used herein, the term“unit” refers to a segment of a device containing all the components to trap and condense one species of biological material for identification.
As used herein, the term“microchannel” or“microfluidic channel” generally refers to a channel with at least one of a height and width dimension below two millimeters, such as below 1500 microns, for fluid flow. Microchannels may be primary (in the trap module) or secondary (in the identification module), with each microchannel having an inlet and an outlet. In some aspects, the primary and secondary microchannels may share inlets and/or outlets. For example, one inlet may serve multiple microchannels with multiple outlets.
As used herein, the term“substantially” refers to a significant degree of similarity, difference, or effect. For example,“substantially smaller than” in a context of a comparison may refer to a measurement that is significantly smaller than the same measurement in another sample. Conversely,“not substantially” refers to an insignificant degree of similarity, difference, or effect. Therefore, substantially similar typically refers to a similarity of about 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; while substantially different typically refers to a difference above about 5%, such as about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85% or about 90%.
As used herein, the term“subject” refers to, for example, animals. The subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian.
As used herein, the term“trap” refers to a segment in the device (e.g., trap module) for capture, or to an action of capturing, of a biological material with the device. Trapping may include separating the biological material of interest from other biological materials in a sample, and capturing the biological material of interest in the trap module.
As used herein, the term“identify” or“identification” refers to an action of verifying or identifying the captured/trapped biological material of interest. Identification typically occurs after a biological material of interest has been captured or captured and condensed in the identification module. As used herein, the term“condenser” or“condense” generally refers to a device component that captures the biological material of interest over a surface, or an action that reduces the surface area over which the biological material of interest is distributed.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.
Use of the term "about" is intended to describe values either above or below the stated value in a range of approx. +/- 10%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/- 5%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/- 2%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/- 1%.
II. Particle Capture and Identification Devices
The devices are typically portable devices, and include a trap module, an identification module, and, optionally, a counter module. The counter module may be a stand-alone portable counter device. A sample with particles is typically flowed over the trap module, where the trap module traps the particles of interest. The trapped particles may then be shuttled from the trap module with a first condenser to the identification module with a second condenser to condense the particles into a smaller filed and aid with further identification.
The trapped or the condensed particles may further pass through a counter module of the device. The trapped particles from the trap module, or condensed particles from the identification module, may then be flown through the counter module for further assaying of the particles.
Alternatively, the sample with particles may be passed through the counter device. An exemplary device 10 (without a counter module) is presented in Figure 4A. The device typically includes a device body 12 segmented into one or more units 20. Each unit includes a trap module 22, and an identification module 26. The trap module 22 generally includes at least one condenser 24 (first condenser) containing electrodes and a primary microchannel 14. The identification module 26 typically includes at least one condenser 28 (second condenser) containing electrodes, and at least one secondary microchannel 18. The microchannels include an inlet and an outlet. Figure 4A is a cut-away diagram (i.e., the top of the microfluidic channel is removed), and the microfluidic channels are the recesses in the material forming the device. The inlet is at the top, the outlet is at the bottom, and three outlets for the shuttle are on the left.
Embodiments of counter modules or counter devices are shown in Figures 11A-11E, 24A-25A, 27A-27D, 29A, 29B, 30A, and 30B. Typically, the counter module is configured to operate independently of the trap module and the identification module, but may be integrated with the trap module or identification module into a device. If separate from the trap module or the identification module, the counter module is referred to as counter device.
Generally, the substrate for the modules and the microchannels form the body of the device, while the electrodes form the condensers or the counters.
Typically the inlet of the secondary microchannel permits shuttling of the biological material trapped in the trap module to the second condenser of the identification module.
The devices may have any three-dimensional shape, such as cuboid, spherical, oval, pyramidal, hexagonal, etc. Suitable device dimensions for cuboid-shaped devices include a length between 0.01 mm and 100 mm, a width between 0.01 mm and 100 mm, and a height between 0.001 mm and 10 mm. Suitable dimensions for spherical or oral device include a diameter of a device between 0.01 mm and 100 mm. The devices may include a plurality of units, such as at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 units.
The dimensions of the microfluidic devices may be scaled up for larger fluidic devices.
A. Trap Module with First Condenser
The trap module is a region in the device containing a bottom surface, walls, and, optionally, a top surface. In some aspects, the top surface is formed by the device lid.
Generally, the bottom surface of the trap module has a surface area sufficient to accommodate the electrodes of the first condenser. The surface area of the bottom surface is typically greater than 0.1 mm2, such as between about 0.15 mm2 and about 1 mm2, or about 0.2 mm2 and about 1 mm2, such as about 0.2 mm2, about 0.25 mm2, about 0.3 mm2, about 0.35 mm2, about 0.4 mm2, about 0.45 mm2, about 0.5 mm2, about 0.55 mm2, or about 0.6 mm2.
The height of the walls of the trap module may be between 1 pm and 1000 pm.
In one example, the trap module has a surface area between about 0.1 mm2 and about 0.4 mm2, such as about 0.2 mm2, and a height of the wall of about 100 pm.
The dimensions of the microfluidic devices may be scaled up for larger fluidic devices.
1. Electrodes of the First Condenser
At least two electrodes of the trap module form the first condenser. The electrodes are typically formed of a conducting or a semiconducting metal, such as gold, silver, chromium, aluminum, copper, platinum, or alloys containing gold, silver, chromium, aluminum, copper, or platinum. Under an applied AC, at least one of the electrodes is negatively charged and at least one of the electrodes is positively charged.
a. Dimensions
The two or more electrodes of the trap module are typically metal leads with a width between about 1 nanometer (nm) and about 10 cm, between about 1 nanometer (nm) and about 10 mm, such as between about 1 nm and about 1000 micrometers (pm), or between about 1 pm and about 1000 pm. Preferred ranges for electrode widths include between about 1 nm and 1000 pm, more preferably between about 10 nm and about 1000 pm, such as between about 20 nm and 500 pm. Exemplary electrode widths include widths between about 1 nm and about 900 nm, between about 500 nm and about 1000 pm, between about 1 pm and about 900 pm, between about 1 pm and about 800 pm, between about 1 pm and about 700 pm, between about 1 pm and about 600 pm, between about 1 pm and about 500 pm, between about 1 pm and about 400 pm, between about 1 pm and about 300 pm, between about 1 pm and about 200 pm, between about 1 pm and about 100 pm, or between about 1 pm and about 50 pm, such as about 25 pm.
Other exemplary electrode widths include widths between about 1 pm and about 90 pm, between about 1 pm and about 80 pm, between about 1 pm and about 70 pm, between about 1 pm and about 60 pm, between about 1 pm and about 50 pm, between about 1 pm and about 40 pm, between about 1 pm and about 30 pm, between about 1 pm and about 20 pm, between about 1 pm and about 10 pm, or between about 1 pm and about 5 pm, such as about 2.5 pm.
Generally, the electrodes of the first condenser can have any length sufficient to form a surface area for capturing the biological material of interest in numbers sufficient for identification at a specific flow rate. The first condenser may have a surface area sufficient to capture over 50%, such as about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 100% of the particles of interest in the sample. The electrodes may have a length between about 1 nm and 10 cm, such as between about 10 nm and 10 cm. Suitable electrode lengths include a length between about 1 mih and about 20 000 mih (2 cm), such as between about 1 mih and about 15 000 mih, between about 1 mih and about 10 000 mih, between about 200 mih and about 2 000 mih, between about 500 mih and about 2000 mih, such as about 500 mih, about 600 mih, about 700 mih, about 800 mih, about 900 mih, about 1000 mih, about 1100 mih, about 1200 mih, about 1300 mih, about 1400 mih, about 1500 mih, about 1600 mih, about 1700 mih, about 1800 mih, about 1900 mih, or about 2000 mih.
In some aspects, the device has a design such that the spacing between electrodes may be between about 1 nanometer (nm) and about 10 cm, between about 1 nanometer (nm) and about 10 mm, such as between about 1 nm and about 1000 micrometers (pm), between about 10 nm and about 1000 pm, such as between about 10 nm and about 900 pm, between about 10 nm and about 800 pm, between about 10 nm and about 700 pm, between about 10 nm and about 600 pm, between about 10 nm and about 500 pm, between about 10 nm and about 400 pm, between about 10 nm and about 300 pm, between about 10 nm and about 200 pm, between about 10 nm and about 100 pm, between about 1 pm and about 500 pm, between about 1 pm and about 400 pm, between about 1 pm and about 300 pm, between about 1 pm and about 200 pm, between about 1 pm and about 100 pm, between about 10 pm and about 500 pm, between about 10 pm and about 400 pm, between about 10 pm and about 300 pm, between about 10 pm and about 200 pm, between about 10 pm and about 100 pm, such as about 40 pm; or about 25 pm, and not less than the size of the target biological material.
The height of the channel is about the same as the electrode spacing and the length of the electrode region is typically adjusted for the efficiency in capturing the target biological material. The electrodes may be coated with an insulator to increase operating voltage. The electrodes may be "focused" (i.e., pitch changed). The electrodes may be patterned to allow for lateral movement and separation within the channel.
The devices operate with flow rates between about 0.01 pL/min and about 10 mL/min, such as about 0.01 pL/min and about 5 mL/min, about 0.01 pL/min and about 1000 pL/min, about 0.01 pL/min and about 800 pL/min, about 0.01 pL/min and about 500 pL/min, about 0.01 pL/min and about 300 pL/min, about 0.01 pL/min and about 200 pL/min, about 0.01 pL/min and about 150 pL/min, about 0.01 pL/min and about 100 pL/min, about 0.01 pL/min and about 50 pL/min, about 0.01 pL/min and about 10 pL/min, or about 0.01 pL/min and about 1 pL/min.
b. Electrode Arrangement
The electrodes are typically arranged on the bottom surface of the trap module, but may be present on the walls, the lid of the device, or the
combinations thereof.
The electrodes may be positioned in any arrangement. Typically, the electrodes are arranges in any two-dimensional patterns, such as in linear, angled, circular, semi-circular, rectangle, cube, triangle, zigzag, and oval patterns. Examples of electrode arrangements in devices are shown in Figures 4A-4D.
The two or more electrodes of the trap module may have the same width and may be separated from one another by a distance as wide as the width of the electrodes or by a distance that is less than or more than the width of the electrodes. The electrodes may be separated by a distance that is between about 0.01 times and 10 times the width of the electrodes, such as about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 times the width of the electrodes. For example, electrodes may be separated by a distance between about 10 nm and 1000 pm, such as between about 10 pm and 500 pm.
2. Primary Microfluidic Channel
Typically, the device includes at least one primary microfluidic channel. The height of the micro fluidic channel may be between about 1 nm and about 10 cm, between about 1 nm and about 10 mm, between about 1 nm and about 1000 pm, between 10 nm and about 1000 pm. Exemplary heights for the primary microfluidic channel may be between about 10 nm and about 900 pm, between about 10 nm and about 800 pm, between about 10 nm and about 700 pm, between about 10 nm and about 600 pm, between about 10 nm and about 500 pm, between about 10 nm and about 400 pm, between about 10 nm and about 300 pm, between about 10 nm and about 200 pm, between about 10 nm and about 100 pm, between about 1 pm and about 1000 pm, between about 1 pm and about 500 pm, between about 1 pm and about 400 pm, between about 1 pm and about 300 pm, between about 1 pm and about 200 pm, or between about 1 pm and about 100 pm, such as about 1 pm, about 10 pm, about 20 pm, about 30 pm, about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 100 pm, about 150 pm, or about 200 pm.
The width of the microfluidic channel may be between about 1 nm and about 10 cm, between about 1 nm and about 10 mm, between about 1 nm and about 1000 pm, between 10 nm and about 1000 pm, between about 1 pm and about 10 000 pm, such as between about 10 pm and about 10 000 pm, between about 10 pm and about 9 000 pm, between about 10 pm and about 8 000 pm, between about 10 pm and about 7 000 pm, between about 10 pm and about 6 000 pm, between about 10 pm and about 5 000 pm, between about 10 pm and about 4 000 pm, between about 10 pm and about 3 000 pm, between about 10 pm and about 2 000 pm, between about 10 pm and about 1 000 pm, or between about 10 pm and about 500 pm. Figure 4A is a cut-away diagram showing an exemplary device with its structure. The top fluidic cover is removed to enable visualization. The top of the microfluidic channel is removed, and the microfluidic channels are the recesses in the material forming the device. The inlet is at the top, the outlet is at the bottom, and three outlets for the shuttle are on the left. An example of a primary microfluidic channel 14 is shown in Figure 4A.
B. Identification Module with Second Condenser
The trap module may be used for capture and identification of biological material, such that the trap module may also be the identification module.
In other aspects, each unit of the devices may include an identification module containing at least two electrodes and a secondary microchannel with an inlet and an outlet. The trap module is typically connected with the identification module through focusing electrodes and/or a microfluidic shuttle system.
Generally, the dimensions of the identification module are substantially smaller than the dimensions of the trap module.
Typically, the identification module includes the same components and geometry as that of the trap module, but with smaller dimensions. For example, the identification module is a region in the device containing a bottom surface, walls, and, optionally, a top surface. In some aspects, the top surface is formed by the device lid.
Generally, the bottom surface of the identification module has a surface area sufficient to accommodate the electrodes of the second condenser. The surface area of the bottom surface is typically greater than 0.01 mm2, such as between about 0.01 mm2 and about 1 mm2, or about 0.015 mm2, about 0.02 mm2, about 0.025 mm2, about 0.03 mm2, about 0.035 mm2, about 0.04 mm2, about 0.045 mm2, or about 0.05 mm2.
The height of the walls of the identification module may be between about 1 pm and about 1000 pm, between about 1 pm and about 900 pm, between about 1 pm and about 1000 pm, between about 1 pm and about 900 mih, between about 1 mih and about 800 mih, between about 1 mih and about 700 mih, between about 1 mih and about 600 mih, between about 1 mih and about 500 mih, between about 1 mih and about 400 mih, between about 1 mih and about 300 mih, between about 1 mih and about 200 mih, between about 1 mpi and about 100 mih, between about 1 mih and about 50 mih, such as about 1 mih, about 10 mpi, about 20 mih, about 30 mpi, about 40 mih, about 50 mih, about 60 mpi, about 70 mih, about 80 mpi, or about 100 mih.
In one example, the identification module has a surface area between about 0.015 mm2 and about 0.05 mm2, such as about 0.025 mm2, and a height of the wall of about 0.1 mm.
1. Electrodes of the Second Condenser
At least two electrodes form the second condenser. The electrodes are typically formed of a conducting or semiconducting metal, or a combination of conducting or semiconducting metals. Exemplary metals include gold, silver, chromium, aluminum, copper, platinum, or alloys containing gold, silver, chromium, aluminum, copper, or platinum.
Under an applied AC, at least one of the electrodes is negatively charged and at least one of the electrodes is positively charged.
a. Dimensions
The two or more electrodes of the second condenser typically have a width between about 1 nm and about 500 pm. Exemplary electrode widths include widths between about 1 nm and about 100 pm, between about 1 nm and about 50 pm, between about 1 nm and about 40 pm, between about 1 nm and about 30 pm, between about 1 nm and about 20 pm, between about 1 nm and about 15 pm, or between about 1 nm and about 10 pm, such as between about 1 nm and about 5 pm. Other suitable ranges for electrode width in the
identification module include between about 1 pm and about 100 pm, between about 1 pm and about 50 pm, between about 1 pm and about 40 pm, between about 1 mih and about 30 mih, between about 1 mih and about 20 mih, between about 1 mih and about 15 mih, or between about 1 mih and about 10 mih, such as between about 1 mih and about 5 mih.
Generally, the electrodes of the second condenser can have any length sufficient to form a surface area for capturing a particle of interest in numbers sufficient for identification of the biological material at a specific flow rate. The second condenser may have a surface area sufficient to capture over 50%, such as about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 100% of the biological material of interest in the sample.
The electrodes of the identification module may have a length between about 1 nm and 10 cm, such as between about 10 nm and 10 cm. Suitable electrode lengths include a length between about 1 pm and about 20 000 pm (2 cm), such as between about 1 pm and about 15 000 pm, between about 1 pm and about 10 000 pm, between about 200 pm and about 2 000 pm, between about 500 pm and about 2000 pm, such as about 500 pm, about 600 pm, about 700 pm, about 800 pm, about 900 pm, about 1000 pm, about 1100 pm, about 1200 pm, about 1300 pm, about 1400 pm, about 1500 pm, about 1600 pm, about 1700 pm, about 1800 pm, about 1900 pm, or about 2000 pm.
Suitable electrode lengths include a length between about 1 pm and about 5000 pm, between about 100 pm and about 1 000 pm, between about 200 pm and about 1000 pm, such as about 200 pm, about 300 pm, about 350 pm, about 400 pm, about 450 pm, about 500 pm, about 550 pm, about 600 pm, about 650 pm, about 700 pm, about 750 pm, about 800 pm, about 850 pm, about 900 pm, about 950 pm, or about 1000 pm.
b. Electrode Arrangement
The electrodes are typically arranged on the bottom surface of the identification module, but may be present on the walls, the lid of the device, or the combinations thereof. The electrodes may be positioned in any arrangement. Typically, the electrodes are arranged in a two-dimensional pattern, such as in linear, angled, circular, semi-circular, rectangle, cube, triangle, zigzag, and oval patterns.
The two or more electrodes of the identification module may have the same width and may be separated from one another by a distance as wide as the width of the electrodes or by a distance that is less than or more than the width of the electrodes. The electrodes may be separated by a distance that is between about 0.01 times and 10 times the width of the electrodes, such as about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 times the width of the electrodes.
2. Secondary Microfluidic Channels
The secondary microfluidic channels stem off the primary channel. Each secondary microfluidic channel includes an outlet. An example of a secondary microfluidic channel 18 is shown in Figure 4A.
3. Disposable Microchannels
The primary and/or secondary microfluidic channels may be disposable. The microfluidic channels may be formed of the same material as the device body, attached to the device body, and be readily removable.
The disposable microfluidic channels may help with microscopy following condensing of the biological material in the identification module.
C. Counter Module or Counter Device
The capture and identification device may include a counter module. Alternatively, the counter module may be a stand-alone counter device. The term counter device as used herein to refer to both stand-alone counter device and the counter module of the capture and identification device. This is because slight manufacturing adjustments can easily integrate the counter device with the capture and identification device to make the counter device one of its modules. The counter device typically provides a rapid assaying of the particles by counting the particles, characterizing particle size, shape, activation state, division state, metabolic state, live/dead determination, and/or surface marker expression. Alternative names for the counter device used herein include counter device, counter, coulter-type counter, coulter counter particle detection systems, or counter structure.
The counter device typically includes an electrode arrangement. The electrode arrangement may be overplayed with a microfluidic channel, forming a constricted counter device. In other aspects, the counter device does not include a microfluidic channel over the electrode arrangement, forming constrictionless counter device.
1. Counter Electrodes
Typically, the counter module or the counter device includes a three- electrode arrangement. The three electrodes are parallel to one another, separated by a gap. The counter electrodes are planar electrodes with a sample flown over the planar electrodes in a microchannel.
The three electrode arrangement includes a left sensing electrode, a right sensing electrode, and a middle reference electrode. An example is shown in Figures 11A-11E, 24B, and 27D. Figures 11A-11E show a passing particle nears the sensing region (Figure 11 A) and then enters the fluidic channel (Figures 11B-11D) before finally exiting the sensing region (Figure 11E).
The electrodes typically have a width between about 1 pm and about 1000 pm, such as between about 10 pm and about 900 pm, between about 10 pm and about 800 pm, between about 10 pm and about 700 pm, between about 10 pm and about 600 pm, between about 10 pm and about 500 pm, between about 10 pm and about 400 pm, between about 10 pm and about 300 pm, between about 10 pm and about 200 pm, or between about 10 pm and about 100 pm. The electrodes may be formed of gold, silver, chromium, aluminum, copper, platinum, and their alloys. The electrodes may be coated or uncoated. Typical coatings include passivating materials such as Si02 or Si3N4; or thin film plastics, such as parylene or photoresist or thermoplastics.
2. Microfluidic Channel
The counter module or counter device typically includes a microfluidic channel overlaying at least a portion of the electrodes. The microfluidic channel may include an inlet, an outlet, and a constriction region. The microfluidic channel may not have a constriction region, forming a constrictionless counter module or a constrictionless counter device.
The constriction of the microfluidic channel may have a width between about 1 pm and about 100 pm, such as about 1 pm and about 10 pm, about 1 pm and about 20 pm, about 1 pm and about 30 pm, about 1 pm and about 40 pm, about 1 pm and about 50 pm, about 1 pm and about 60 pm, about 1 pm and about 70 pm, about 1 pm and about 80 pm, or about 1 pm and about 90 pm at a region overlaying at least a portion of the electrodes. The microfluidic channel without a constriction may have a width between about 100 pm and about 10000 pm at a region overlaying at least a portion of the electrodes.
The microfluidic channel of the counter module is typically in a fluidic connection with the trap module or the identification module. The counter module’s microfluidic channel may be connected to the primary microfluidic channel or the secondary microfluidic channel. This connection establishes a fluidic connection between the modules of the device. Typically, fluid flows through the fluidic connection at via a flow rate between about 0.01 pL/min and about 10 mL/min. The fluid typically flows at different flow rates in different modules of the device.
3. Integration with DEP device
Embodiments of counter modules or counter devices are shown in Figures 11A-11E, 24A-25A, 27A-27D, 29A, 29B, 30A, and 30B. Typically, the counter module is configured to operate independently of the trap module and the identification module, but may be integrated with the trap module or identification module in a DEP device. If separate from the trap module or the identification module, the counter module is referred to as counter device.
Exemplary DEP devices with counter modules are shown in Figures 24A, 24B, 29A, 29B, 30A, and 30B (with the counter’s microfluidic channel constriction) and Figure 27C (without the counter’s microfluidic channel constriction). Exemplary counter devices are shown in Figures 24C (with the counter’s microfluidic channel constriction) and 27C (with the counter’s microfluidic channel constriction), referred to as“constrictionless” counters.
Figures 30A and 30B show a schematic of a device with the DEP capture module and two counter modules. In this embodiment, the device captures, separates, and counts particles of interest from a mixture of particles in a sample. These devices provide characterization of the total particle population in a sample, as well as characterization of the particles of interest, simultaneously.
4. Signal Capture, Amplification, and Display
Typically, the counter module or the counter device operates with an alternating current (AC) having a frequency between 10 kHz and 10 MHz and a peak-to-peak voltage amplitude between 0.5 V and 10 V.
The counter module or the counter device may be connected to one or more of a function generator, instrumentation amplified, lock-in amplifier, oscilloscope, a data processor, and a display monitor.
An exemplary counter module or a counter device connected to signal acquis ion, amplification, and display means is presented in Figure 27D. Figure 27D shows the circuit diagram 200 of the complete three-electrode structure of the sensing region, driven by the sine wave output 210 of the function generator 220. The resulting voltage at the left and right sensing electrodes is measured by the PCB -mounted instrumentation amplifier 230 before the signal is fed to the lock-in amplifier 240 whose output signal is measured by the oscilloscope 250, controlled during acquisition by a MATLAB routine in a processor 260.
D. Power Source
Typically, the device is battery powered, for portable use, in settings such as in hospitals, laboratories, homes, and in the field. The power supply, through a function generator, provides an AC voltage for DEP capture modules and/or counter modules of the device or to the counter devices.
E. Lid
The devices may include a lid or an insert covering the trap module, the identification module, or both.
F. Portable Design and Use
The devices are designed to be portable and include an inlet adapted for connecting with a sample delivery line. The devices may also include an outlet adapted for sample exit from the device. The device inlet and the device outlet may be the primary microchannel inlet and the primary microchannel outlet.
After the particles of interest are condensed on the second condenser, the device may be detached from the sample delivery line and used in biological material identification steps. In some aspects, the identification module may be detachable. The user may detach the identification module and use it in biological material identification steps.
In some aspects, the device is designed as a disposable device for single use. In other aspects, the device is designed as a reusable device.
III. Methods of Making the Device
A. Materials
The device is typically formed of a body, electrodes, and optionally, a lid and microfluidics.
Exemplary materials for forming the device body include polymers such as polycarbonate, thermoplastics, polydimethyl sulfone (PDMS), polyurethane (PU), polyethylene oxide (PEO), polybutylene terephthalate (PBT), polyether sulfone (PES), polyimide (PI) polymers, TEFLON®/PTFE, polystyrene, cyclic olefin copolymer (COC), and block copolymers and asymmetric blends thereof.
Exemplary materials for forming the device body also include silicon, metal, ceramic glass, or glass variations, such as low-expansion glass.
Exemplary materials for forming the electrodes include gold, silver, chromium, aluminum, copper, platinum, and their alloys.
Generally, the devices utilize low cost, mass production materials as alternatives to PDMS which is not preferred for medical devices, such as polycarbonate, thermoplastics, etc. Commonly used thermoplastic materials for injection molding include Cyclic Olefin Co-polymer (COC). It is also optically transparent, which allows for easy imaging for inspection.
Other polymers include polymethylmethacrylates (PMMA), with notable trade names including Acrylite® (Evonik Rohm GmbH LLC, Darmstadt, Germany), Lucite® (Lucite International, Inc, Cordova, TN), R-Cast,
Plexiglas® and Altuglas® (Arkema France Corp., Colombes, France), Optix, Perspex®, Oroglas, Cyrolite ® (Evonik Cyro LLC, Parsippany, NJ), and
Sumipex ® (Sumitomo Chemical Co., Ltd., Tokyo, Japan). Other polymers include polymers and copolymers containing polyacrylonitrile.
The electrodes may be passivated from the fluid with an insulator, allowing for higher operating voltages and thus more efficient capture. The passivation may be with chemical vapor depositions (CVD) deposited insulators, such as Si02 or Si3N4; or thin film plastics, such as parylene or photoresist or thermoplastics.
B. Methods of Making
Suitable methods for making the devices include lower cost and higher throughput fabrication approaches, such as roll-to-roll, embossing, etc. Suitable materials for forming the devices include polymers for the device body and conductors for the electrodes. Methods of making the device body include three-dimensional printing, micromolding, deep ion etching, nano imprint lithography, micromachining, laser etching, three dimensional printing, or stereolithography, patterning, photolithography, etching, CNC micromachining, thermal bonding, and chemical bonding.
Three-dimensional (3D) printing is a process of making 3D objects from a digital model. 3D printing is an additive process, where successive layers of material are laid down in different shapes. After each layer is added the“ink” is polymerized, typically by photopolymerization, and the process repeated until a 3D object is created.
The 3D printing workflow can be described in three sequential steps: 1) the powder supply system platform is lifted and the fabrication platform is lowered one layer; 2) a roller spreads the polymer powder into a thin layer; 3) a print-head prints a liquid binder that bonds the adjacent powder particles together. Billiet et al., Biomaterials , 33:6020-6041 (2012). Two kinds of 3D printing techniques may be used: inkjet printing with the typical printers.
(Marizza et al, Microelectrionic Engin. 111:391-395 (2013)) or nanoimprint lithography.
Nanoimprint lithography (NIL) is a fast and cost-efficient technique for fabricating nanostructures. The procedure of NIL is to stack multiple layers of such structures on top of each other; that is, a finished double -layer of structures is covered with a spacer-layer which is planarized using the chemical- mechanical polishing so that a second layer can be processed on top. Liu et al.,
J. Nanomat. Aug. (2013).
Methods of making the electrodes include liftoff metallization, etching, shadow evaporation, or direct printing.
The devices are typically machine-assembled for accuracy and reproducibility. The turnaround cycle for modularized computer-aided design (CAD) and machining is relatively quick. It is easy and rapidly customizable according to user’s individual needs. Computer numerical controlled (CNC) machines, such as lathes and mills, may be used to manufacture the components of the device body and electrodes.
1. Exemplary method of making a DEP device
The devices used for dielectrophoresis may be fabricated in a cleanroom facility. The electrode structures are lithographically patterned in photoresist atop a Borooat-33 glass wafer. Metallization with 200 nm of aluminum follows. Devices are either allowed to form a native oxide upon exposure to atmosphere or are subsequently coated in layer of 200 nm of plasma-enhanced chemical vapor deposition (PECVD) silicon dioxide as an insulating coating. The wafer is then diced and cleaned. At this point, the chips are available for use.
Figure 2E shows a diagram of a typical pair of interdigitated electrode. This particular device has an electrode-electrode gap of 25 pm, sixteen electrode fingers, and a 1 mm channel width.
An imprint mold may be used to fabricate the microfluidic channels. For example, an SET-8 photoresist may be photolithographically defined to create a nominally 20 pm feature height for the microfluidic channels.
Polydimethylsiloxane (PDMS, Dow Corning Sylgard 184) may be mixed in a 10:1 ratio and poured over the mold. The mixture and mold may be de-bubbled for thirty minutes in a vacuum chamber before being cured for one hour at 70°
C. The PDMS“wafer" may be subsequently peeled from the mold. Individual microfluidic channels may be cut from the mold, cleaned, and hole-punched to form inlet and outlet ports. The microfluidic channels may then be bonded to the individual chips after UV-ozone treatment by heating the aligned PDMS-chip combination in an oven for fifteen minutes at 70° C, after which devices are ready for use. An interdigitated electrode structure is shown in Figure 2E. The microfluidic channel sidewalls are visible as parallel vertical lines on the left and right boundaries of the image.
2. Exemplary Method of Making a Counter Module or a Counter Device
An exemplary method of making the counter module or the counter device includes photoresist coating, pattern definition, metal deposition, and a lift-off process. This simplicity compared to alternative electrode geometries significantly reduces per-device fabrication cost.
A microfluidic channel of a desired width and height may be formed by photolithography and bonded to a counter device such that the three counter electrodes projected slightly into the width of the channel (constrictionless counter module/device), as seen in Figure 27A,or overlay the electrodes, as seen in Figure 24C.
C. Sterilization
One or more sterilization procedures may be performed to sterilize the device. Sterilization techniques include gas treatment (e.g., ethylene oxide), ionizing radiation, sonication, surface treatment (e.g., surfactant), rinsing in sterile water, and autoclaving.
IV. Methods of Using the Device
Conventional biological material detection methods have proven inadequate, because they require long incubation periods and have a high cost. The nanosensor-based electrical devices for rapid biological material screening described herein overcome these obstacles. The technique minimizes false positives and negatives and does not require complex sample processing. The device provides a rapid, miniaturized and portable biosensing platform suitable for use in diverse settings, such as in hospitals, food and drug testing laboratories, or in the field.
The key technology behind the device’s approach is dielectrophoresis (DEP) and electroosmosis (EO). If an AC voltage is applied to an electrode arrangement, the gradient of the electric field will impart a net force called the dielectrophoretic (DEP) force, due to the net polarizability of the object with respect to the surrounding medium. This force is given by FDEP~Re[K(co)]VEA2, where FDEP is the dielectrophoretic force, grad E is the gradient of the spatially varying electric field E(r,o ), and K(co) is called the Clausius-Mossotti (CM) factor, which depends on the permeabilities and conductivities of the cell and the surrounding media. The sign of the (real part of the) CM factor determines whether the force is attractive (positive dielectrophoresis, pDEP) or repulsive (negative dielectrophoresis, nDEP).
The organism viability can be maintained through the DEP flow cell allowing further culturing or assaying of the organism for infection control or epidemiologic purposes.
A. Operating the Device
Generally, a sample of interest is applied to the device via the
microfluidic channel inlet. An electric field is applied to the electrodes form the power source at a frequency suitable to trap a biological material of interest.
Once the biological material is trapped, which may take a few seconds to a few minutes, the biological material may be used for identification in the trap module. Alternatively, the biological material may be further condensed on the identification module, if present. The condensing of the biological material on the identification module may be accomplished through microfluidic focusing or with focusing electrodes.
For example, in microfluidic focusing the microchannel outlet may be closed, the secondary channel inlet may be opened, the field in the trap module may be turned off, and the filed in the identification module may be turned on. This staged“shuttle” transfer technique allows the trapped biological material from the trap module to be transferred to the identification module with a coincident reduction in FOV to allow further inspection and identification of the biological material. Focusing the biological material with the focusing electrodes may be achieved through various device and electrode designs. Examples of device and electrode designs are presented in Figures 4B, 4C, and 4D, and in Table 1 below.
Table 1. Different device designs and their respective focusing of the biological material on the identification module. Design types A, B, and C correspond to the diagrams shown in Figures 4B, 4C, and 4D, respectively.
Figure imgf000036_0001
Inspection and identification may be via microscopy, staining, molecular techniques (such as flow cytometry; immuno staining; staining; cell
fractionation; DNA, RNA, and/or protein extraction; polymerase chain reaction (PCR); immunoPCR; enzyme-linked immunosorbent assay (ELISA); Western blotting; and HPLC) and/or culturing of the biological material. 1. Principles of DEP
DEP permits manipulating the position of particles within the device for the capture and concentration of rare targets from within the sample or to physically separate out the target from the sample background.
Dielectrophoresis is the forced exerted by an electric field acting on the dipole moment of a charge-neutral particle. The particle's polarizability governs its response to the external field and depends on both the mobility of charge within the particle (conductivity) as well as the particle's ability to accumulate charge (permittivity). Under the influence of an external electric field, positive and negative charge carriers within the particle re-arrange. This spatial arrangement of opposing charge distributions constitutes a dipole.
Suspending the neutral particle within a fluid medium complicates the response. An external electric field applied across the fluid will drive the re arrangement of charge in both the neutral particle as well as the fluid. Charge within the fluid will move to respond to the external electric field and counter balance the dipole of the neutral particle. Depending on the polariability of the particle and the fluid medium, the particle dipole will be either partially-, completely-, or over-balanced. The counter-balancing dictates the effective dipole moment observed by the particle in the presence of an external electric field.
In the case of a uniform electric field, no net force is exerted on the dipole. The interaction between the field and the spatial charge distribution of the dipole exerts a torque which rotates the particle into alignment with the external field. In the presence of an electric field gradient, however, the particle experiences a force acting along the gradient lines. This force, dielectrophoresis, induces motion towards either the maxima or minima of the gradient depending upon the orientation of the induced dipole.
Dielectrophoresis boasts incredible appeal for point-of-care diagnostics. Cells, viruses, and other biomarkers are permealizable and therefore experience the dielectrophoretic force. The actuating mechanism is the interaction of an applied electric field with a particle in solution. Microelectrode structures are readily fabricated to manipulate the target within the sample. Different cell species have differing frequency responses, allowing some selectivity of the target analyte through the choice of operating frequency. Dielectrophoretic manipulation does not rely upon the presence of chemical binding elements to selectively interact with the desired analyte, and in this manner is said to be label-free. The ease of fabrication and lack of a need for additional chemical treatments greatly simplifies some aspects of implementation for point-of-care diagnostics.
Detection of biological agents at very low concentrations is limited by diffusion of the target to the sensing element. The electric field gradient generated for dielectrophoresis reaches microns into solution, actively driving analyte motion to overcome diffusion limitations on the measurement time- scale. These limitations are exacerbated by sample dilution, which is often required to manipulate the sample conductivity into a suitable regime for other detection mechanisms. Dilution reduces the concentration of the target analyte, demanding a compensatory increase in sensitivity. Dielectrophoresis may be used to capture and concentrate the target from solution either before or after dilution to bolster the local concentration of analyte, reducing demands on sample volume throughput and thereby decreasing the time-to-results.
a. CM Factor
Under a nonuniform AC electric field, a particle that is more easily polarized than the surrounding medium will be attracted toward the region of highest field. However, if the particle is less polarized than the medium, the particle will be repelled from the high field region. This attraction toward the high field region is termed positive dielectrophoresis (pDEP), while the repulsion from the high field region is termed negative dielectrophoresis (nDEP). The time averaged DEP force DEP— 2ns/;!r3Re[/cM] V| £Ί2 is dependent on the permittivity of the medium Em, the radius of the particle r, the Clausius-Mossotti (CM) factor /CM, and the magnitude of the electric field gradient V\E\2. If the value of Re [/CM] is positive, the particle exhibits positive DEP properties and if the value of the Re [/CM] is negative, the particle exhibits negative DEP properties. However, if /CM = 0, the corresponding frequency defines the crossover frequency, where the particle exhibits neither + nor - DEP properties. The CM factor is defined by CM— (//, - E*m) / (e/, +2 £*m), where e/, and E*m are the complex permittivities of the particle and the medium, respectively, and can be defined as e*= e— zs/w, where s is the conductivity of the medium and <X> is the frequency of the applied field. It is clear that the permittivity effects dominate at high frequencies and the conductivity effects dominate at relatively low frequencies (Cheng et al, Biomicrofluidics, 1:021503-1 - 021503-15 (2007)).
Calculated CM factors for various components of a whole blood sample show different crossover frequencies for each component, as well as different frequencies for various components in a sample. This may be used with a device where the biological material of interest is separated, or differentially trapped, from other blood components. Additionally, a trapping structure can serve as a concentrator, which would pull the bacteria from the blood sample. Different biological materials in general have different CM factors, providing for selection of different biological materials by setting the appropriate frequency. To be selective for a given species in a sample containing a mix of species, the CM factor is typically maximum for the species of interest, and is negative for the others.
The CM factors can be determined for living organisms, while molecular methods detect nucleic acids from both viable and dead pathogens. The CM factor for a given species of bacteria may be computed with the knowledge of the physical structure of the bacteria (e.g. number of cell walls, membranes, shape, size, and internal conductivity of the bacteria).
The CM factor for a given organism may be determined experimentally by monitoring the capturing efficiency under conditions of constant solution conductivity, flow rate, and applied voltage while manipulating the applied frequency. CM factors may be determined individually for each cell type by seeing what frequency effectively captures the cells, at a given solution conductivity, as detailed in Figures 10A and 10B.
In another aspects, electroosmosis (EO) may be used capture the biological material. EO is generally not frequency selective. The selectivity may be provided by alternating between EO and DEP modes.
2. Rapid Capture
The devices permit rapid capture of particles, such as a biological material, from a sample. The sample may be a biological sample, food sample, or an environmental sample.
Generally, the capture, shuttling and condensing occurs within 30 seconds, 1 minute, 1.5 minutes, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes following sample application. The captured and condensed biological material is then ready for microscopic observation and pathogen identification.
a. Biological, Food, and Environmental Samples
Exemplary biological samples include diluted or undiluted blood, plasma, spinal fluid, urine, stool, tear, saliva, sputum, mucus, or exudate.
Exemplary food samples may be obtained from the food industry and may be liquids from food preparation, storage, or cooking, runoff from meat processing, and runoff from washing of fruits and vegetables. Exemplary environmental samples may be obtained from monitoring the environment and may include waters from streams, rivers, lakes, seas, pools, swimming pool samples, drinking water, industrial water, industrial runoff, rainwater runoff, or stream water. The devices may capture, identify, and characterize particles, such as plastic micro- and nano-particles, and biological material.
In some aspects, the sample is a blood sample with a central line associated bloodstream infections (CLABSI).
The samples may be diluted with water or buffer with a sufficient ionic strength and conductivity to permit biological material trapping. The biological material may be diluted in a buffer with the ionic strength substantially the same as the ionic strength of phosphate buffered saline (PBS, lx PBS), or 0.001 x PBS, 0.002 x PBS, 0.003 x PBS, 0.004 x PBS, 0.005 x PBS, 0.006 x PBS, 0.007 x PBS, 0.008 x PBS, 0.009 x PBS, 0.01 x PBS, 0.02 x PBS, 0.03 x PBS, 0.04 x PBS, 0.05 x PBS, 0.06 x PBS, 0.07 x PBS, 0.08 x PBS, 0.09 x PBS, 0.1 x PBS, 0.2 x PBS, 0.3 x PBS, 0.4 x PBS, 0.5 x PBS, 0.6 x PBS, 0.7 x PBS, 0.8 x PBS, 0.9 x PBS, of 1 x PBS.
b. Biological Material
Generally, the biological material of interest may be a eukaryotic or prokaryotic cell, microorganisms, organelles, vesicles (such as exosomes, endosomes, phagosomes, and liposomes), macromolecules, or biomarkers. The microorganisms captured and identified by the device and methods include bacteria, viruses, fungi, and parasites. Exemplary genera of microorganisms include Staphylococcus, Enterococcus, and Klebsiella, Enterobacter,
Pseudomonas, Escherichia, Acinetobacter, Listeria, and Candida. The device may be used to detect weaponized bacterial CBW agents such as Bacillus anthracis and Yersinia pestis.
The devices typically capture the biological material in the trap module. The biological material may then shuttled into the identification module, where the biological material is condensed to a surface area suitable for microscopic observation. 3. Particle Identification
Observation and identification of condensed particles may be via microscopy, staining, molecular techniques (such as flow cytometry;
immuno staining; staining; cell fractionation; DNA, RNA, and/or protein extraction; PCR; immunoPCR; ELISA; Western blotting; and HPLC) and/or culturing of the biological material in suitable medium, broth, or nutrient-rich agar.
The particles may be identified using microscopy. Methods of particle, such as biological material, identification using microscopy are known in the art. These include direct pathogen observation under a microscope. Other methods include staining the captured and condensed biological material with suitable stains and observing them under a light, a fluorescence, or a confocal microscope.
Generally, the microscopy utilizes high power compound microscopes permitting detection of biological material. Examples of suitable microscopes include phase-contrast microscopes, travelling microscopes, epifluorescence microscopes, confocal microscopes, two-photon microscopes, and
ultramicroscopes. Typically, the microscopes will provide a maximum field of view (FOV) between about 10 mm and about 0.185 mm (185 pm), such as about 0.185 mm, about 0.18 mm, about 0.2 mm, about 0.22 mm, about 0.25 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.625 mm, about 0.9 mm, about 1 mm, about 1.25 mm, about 1.8 mm, about 2 mm, about 2.5 mm, about 4.5 mm, about 5 mm, about 6.25 mm, about 9 mm, or about 10 mm. The staining methods will vary with the nature of the biological material, and/or the mode of detection.
Typically, the staining of the biological material occurs on the
identification module of the device.
Suitable staining methods include Gram staining by Gram method, acid- fast staining, staining for flagella, endospore staining, Ziehl-Neelsen stain, haematoxylin and eosin (H&E) staining, Papanicolaou staining, Periodic Acid Shiff (PAS) staining, and Romanowsky stains.
Suitable dyes for staining the pathogens include Basic Fuchsin, Rose Bengal Sodium Salt, Safranin T, Malachite Green oxalate salt, Resazurin sodium salt, 3,3prime-Diethyloxacarbocyanine iodide, 2-Naphthyl caprylate, 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI), and Malachite green.
4. Rapid Assay of Particles
The counter module or the counter device provides for rapid assaying of particles from samples. The assaying may include qualitative and quantitative characterization of particles of interest. The qualitative and quantitative characterization includes assaying the number of particles of interest, characterizing particle size, shape, activation state, division state, metabolic state, live/dead determination, surface marker expression, and others.
B. Particle Detection for Diagnostic Use
The devices are suitable for detecting and identifying particles, such as pathogens, from various samples, including biological samples. Detection and identification may help with diagnosis, and/or inform of suitable therapies.
1. Pathogens
The devices are particularly suitable for capture and identification of pathogenic bacteria, fungi, viruses, and parasites.
a. Bacteria
Examples of bacteria detectable by the device include commensal bacteria, oral and skin bacteria, or pathogenic bacteria.
Commensal bacteria typically include healthy gut microbiota, such as Lactobacillus and Bifidobacteria, Enterococcus faecium, or Lactobacillus pentosus WE7. A non-limiting list of exemplary probiotic microorganisms includes Lactic acid bacteria (LAB), such as Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Lactococcus spp., Bifidobacterium spp. and
Streptococcus spp., as well as certain yeasts and bacilli. Pathogenic bacteria include bacteria from the genera Bacillus,
Bartonella, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia and Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio, and Yersinia.
b. Viruses and Parasites
Examples of viruses detectable by the devices include viruses of virus families Adenoviridae, Papillomaviridae, Parvoviridae, Herpesviridae,
Poxviridae, Hepadnaviridae, Polyomaviridae, Anelloviridae, Reoviridae, Picomaviridae, Caliciviridae, Togaviridae, Arenaviridae, Flaviviridae,
Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Rhabdoviridae, Filoviridae, Coronaviridae, Astroviridae, Bomaviridae, Arteriviridae, and Hepeviridae.
Examples of parasites detectable by the devices include protozoa, such as plasmodium, amoeba, babesia, Balatidium coli, blastocystis, coccida,
Entamoeba histolytica, giardia, Cystoisospora belli, Leishmania, Naegleria fowleri, Rhinosporidium seeberi, Toxoplasma gondii, Trichomonas vaginalis, and Trypanosoma.
The parasites may include those which cause Acanthamoebiasis, Babesiosis, Balantidiasis, Blastocystosis, Coccidiosis, Amoebiasis, Giardiasis, Isosporiasis or cystosporiasis, Leishmaniasis, Primary amoebic
meningoencephalitis (PAM), Malaria, Rhinosporidiosis, Toxoplasmosis, Trypanomiasis (Sleeping sickness), and Chagas disease.
Acanthamoebiasis is typically caused by tiny ameba can affect the eye, the skin, and the brain. It exists all over the world in water and soil. Individuals can become infected if they clean contact lenses with tap water. Babesiosis comes from parasites babesia that are spread by ticks. It affects the red blood cells. The risk is highest in summer in the Northeast and upper Midwest of the United States.
Balantidiasis is passed on by Balatidium coli, a single-cell parasite that usually infects pigs but can, in rare cases, cause intestinal infection in humans. It can be spread through direct contact with pigs or by drinking contaminated water, usually in tropical regions.
Blastocystosis is caused by Blastocystis and affects the intestines. The blastocystis enters humans through the fecal-oral route. A person can get it by eating food or drink contaminated with human or animal feces where the parasite is present.
Coccidiosis is caused by coccida and affects the intestines. Coccidia is passed on through the fecal-oral route. It is found around the world. It can also affect dogs and cats, but these are different kinds. Dogs, cats, and humans cannot normally infect each other.
Amoebiasis is caused by the parasite Entamoeba histolytica. It affects the intestines. It is more likely in tropical regions and in areas with high population density and poor sanitation. It is transmitted through the fecal-oral route.
Giardiasis is caused by giardia, or "beaver fever" affects the lumen of the small intestine. If humans ingest food or water contaminated with feces, dormant cysts may infect the body.
Isosporiasis or cystosporiasis is caused by the Cystoisospora belli, previously known as Isospora belli. It affects the epithelial cells of the small intestine. It exists worldwide and is both treatable and preventable. It is passed on through the fecal-oral route.
Leishmaniasis is a disease that is passed on by parasites of the
Leishmania family. It can affect the skin, the viscera, or the mucous membranes of the nose, mouth, and throat. It can be fatal. The parasite is transmitted by types of sandflies. Primary amoebic meningoencephalitis (PAM) is passed on through a free-living ameba known as Naegleria fowleri. It affects the brain and the nervous system, and it is nearly always fatal within 1 to 18 days. It is transmitted through breathing in contaminated soil, swimming pools, and contaminated water, but not from drinking water.
Malaria is caused by different types of plasmodium, which affect the red blood cells. It exists in tropical regions and is transmitted by the Anopheles mosquito.
Rhinosporidiosis is caused by Rhinosporidium seeberi. It mainly affects the mucous of the nose, conjunctiva, and urethra. Polyps result in nasal masses that need to be removed through surgery. Bathing in common ponds can expose the nasal mucous to the parasite.
Toxoplasmosis is a parasitic pneumonia caused by the parasite
Toxoplasma gondii. It affects the liver, heart, eyes and brain. It occurs worldwide. People can become infected after ingesting raw or undercooked pork, lamb, goat, or milk, or though contact with food or soil that is
contaminated with cat feces.
Trichomoniasis, also known as "trich", is a sexually transmitted infection (STI) caused by the parasite Trichomonas vaginalis. It affects the female urogenital tract. It can exist in males, but usually without symptoms.
Trypanomiasis (Sleeping sickness) is passed on when the tetse fly transmits a parasite of the Trypanosoma family. It affects the central nervous system, blood, and lymph. It leads to changes in sleep behavior, among other symptoms, and it is considered fatal without treatment. It can cross the placenta and infect a fetus during pregnancy.
Chagas disease is caused by the parasite Trypanosoma cruzi and affects the blood, muscle, nerves, heart, esophagus and colon. It is transmitted through an insect bite. Over 300,000 people in the U.S. have the parasite that can lead to this disease. 2. Detecting Bloodstream Infections
The devices may be used with intravenous lines, central lines, or for testing the fluids or therapeutics provided to patients, for presence or absence of contaminating microorganisms. The contaminating microorganisms may be bacteria, viruses, fungi, or parasites.
Central line-associated bloodstream infections (CLABSIs) result in thousands of deaths each year and billions of dollars in added costs to the U.S. healthcare system, yet these infections are preventable.
A central line (also known as a central venous catheter) is a catheter (tube) that doctors often place in a large vein in the neck, chest, or groin to give medication or fluids or to collect blood for medical tests. Intravenous catheters (also known as IVs) are used frequently to give medicine or fluids into a vein near the skin’s surface (usually on the arm or hand), for short periods of time. Central lines are different from IVs because central lines access a major vein that is close to the heart and can remain in place for weeks or months and be much more likely to cause serious infection. Central lines are commonly used in intensive care units.
The bloodstream infections may be caused by staphylococci (both Staphylococcus aureus (as well as methicillin-resistant S. aureus ) and the coagulase-negative staphylococci), enterococci, aerobic Gram-negative bacilli and yeast. When aerobic Gram-negative bacilli are assessed as a group, their frequency follows that of the staphylococci. Certain pathogens are associated with specific host, treatment, and catheter characteristics. S aureus infections are disproportionately represented in infections of hemodialysis catheters. Gram negative bacilli have been associated with infections of patients with cancer, and they are typically the pathogens recovered in instances of infusate
contaminations. Gram-negative bacilli and yeast have been affiliated with catheters placed in femoral veins, while Candida have been associated with infections of lines used for administration of parenteral nutrition. The devices are particularly suited for detecting and identifying microorganisms from various genera, such as from Staphylococcus,
Enterococcus, Klebsiella, Enterobacter, Pseudomonas, Escherichia,
Acinetobacter, and Candida. Exemplary microorganisms detected by the devices include S. aureus , S. epidermidis, E. faecium, C. albicans , and E. coli.
3. Detection and Identification of Biological Material The devices may be used for detecting, identifying, and characterizing biological material, including eukaryotic cells of interest, in a sample. The devices may be operated at a frequency to capture the biological material of interest, which then may be condensed in the identification module, and/or flown to the counter module for characterization.
The biological material in the identification module may then be further processed. The further processing include microscopic examination without staining, microscopic examination with staining, or assaying of the biological material in the counter module. The assaying typically includes counting the particles, characterizing particle size, shape, activation state, division state, metabolic state, live/dead determination, surface marker expression, and others.
The Examples below show capture and characterization of eukaryotic red blood cells and T cells with the exemplary devices (Figures 3C and 14). An exemplary device with one capture module and two counter modules may be used to characterize cells in a mixed population (Figures 30C-30H).
4. Screening Food and Environmental Samples
The devices may be used for routine, diagnostic, therapeutic, or as needed screenings to detect the presence or absence of contaminating microorganisms. The screenings may be of food, water sources, beverages, and liquid therapeutics for contamination with microorganisms.
The screening may be of environmental samples, such as swimming pool samples, drinking water, industrial water, rainwater runoff, or stream water. V. Kits
Kits containing one or more devices, sterile lids, staining reagents, and instructions for use are provided. The devices in kits may be pre-assembled for immediate use, or include instructions for assembly at the point of use by the end user. The kits may include a chart with CM factors for different particles, such as eukaryotic cells or microorganisms, and the corresponding settings on the device at which the device may be used for capture and identify a biological material of interest.
The kits may provide disposable, single-use devices, or devices for repeat use.
Generally, the kits provide sterile, pathogen-free devices, and instructions for the device use, cleaning, and sterilization for repeat use.
The present invention will be further understood by reference to the following non-limiting examples.
Examples
Example 1. A biological material identification device.
Materials and methods
The devices were made by a photolithography step with liftoff metallization. Generally, the method includes electrode pattern fabrication using a bi-layer lift-off process. The steps of the process include step 1: on a silicon wafer with 3 pm Si02 spin LOR10A and S1808; step 2: expose and develop features with ME312:DI 1:1; step 3: evaporate Ti 5 nm and Au 40 nm; step 4: lift off gold using 1165 Microposit remover; step 5: PDMS bonding to silicon substrate with oxygen plasma at 100 W for 30 seconds at 350 mTorr.
Electrodes for material capture. There are two important parameters in the design of the electrode. The spacing of the electrode was about 25 microns, as it gave very efficient capture. The height of the microfluidic channel was approximately the same height as the spacing.
The electrodes used for the described tests were passivated. The length of the trapping region was adjusted such that at the target flow rate, typically between 1 pL/min and 100 pL/min, all of the bacteria from the test sample were trapped. Subsequent in-line traps at a lower flow rate condensed the bacteria into a smaller spatial region.
The overall dimensions of a device may vary, the devices may have a width between 8 mm and 25 mm, length between 20 mm and 75 mm, and thickness between 0.8 mm and 5 mm.
Condensing the biological material to an identification unit. Once the target biological material was captured in the first, larger condenser, the approach was to conventionally stain and identify them. However, the larger about 2 cm condenser is incompatible with a microscope field-of-view (FOV) of about 180 microns. To transfer all the captured target from this (first) stage, a second condensation step onto a second-stage, 180 micron condenser, was performed. This was done by first closing the outlet of the primary channel, opening secondary channels that create flow over the second stage condensers, releasing the field of the 1st stage, and then turning on the 2nd stage field(s).
This staged“shuttle” transfer technique allowed all targets from the first stage to then be transferred to the second stage, with a coincident reduction in FOV to allow microscope inspection. The target bacteria were then fixed to the second stage by drying (with a hot plate, or integrated heater on bottom of slide), at which point the field can be removed. The bacteria could also be fixed by high electric field.
Results
Typical electrode structures are shown in Figures 2A-2D, and typical device structures are shown in Figures 4A-4D. Their operability is summarized in Table 1.
Figure 2A shows a circular electrode structure; but these can be of different shapes, such as linear, angled, or tapered structures. The length of the trapping region was adjusted such that at the target flow rate (typically, between 0.01 pL/min and 200 pL/min), all of the bacteria from the test sample were trapped.
Figure 4A is a cut-away diagram showing the device structure, with the top fluidic cover removed to enable visualization. The top of the microfluidic channel is removed, and the microfluidic channels are the recesses in the material forming the device. The inlet is at the top, the outlet is at the bottom, and three outlets for the shuttle are on the left. The microchannel can be made from PDMS, which would allow easy removal after condensing to allow microscope immersion inspection.
Figure 4A shows the larger 1st stage / smaller 2nd stage configuration, and the secondary fluid channel that enabled flow over the 2nd stage. The three sets of condensers were not for redundancy, but to capture three distinct bacterial targets, selected by frequency.
Example 2. CM factor dependence on the AC frequency and voltage.
Materials and methods
The unique CM factor frequency dependence for a specific biological material of interest can be measured by quantifying a particle’s acceleration in the presence of an applied AC signal, for a range of frequencies.
Results
Figure 1 illustrates a typical CM factor frequency dependence, showing the crossover from pDEP to nDEP. The frequency at which this crossover occurs is generally different for different cell types. The devices utilize the frequency dependence of the sign of the CM factor between different biological materials to separate the biological material of interest from a test sample. Example 3. E. coli capture with an exemplary device.
Materials and methods
The device was as described in Example 1.
The device, having an electrode structure shown in Figure 2A, was used with green fluorescent E. coli, which were trapped at the electrode edges where the region of highest electric field gradient and, therefore, maximum pDEP trapping force occurs.
Results
A correction to the simple model of DEP was found to be necessary for cells, as the sign of the CM factor was found to not simply go from positive at low frequency to negative at high frequency (as would be seen for a
homogeneous particle), but was peaked positive at a certain frequency, and negative elsewhere. This is extremely advantageous, as it enables multiplexed individual pathogen selection. Figures 3B and 3C show this dependence (and also as a function of solution conductivity). When the solution conductivity is too high, a clear pDEP peak is lost, indicating that slightly reduced conductivity is necessary for efficient trapping.
Example 4. E. coli capture and separation from RBC with an exemplary device.
Materials and methods
For a pDEP trapping and separation demonstration, a model system of the separation of E. coli from human red blood cells (RBCs) was used. This was possible because different cells have distinct CM factors - indeed, the frequency for pDEP for the E. coli, and nDEP for the RBCs was adjusted. This allows to set the electrode frequency to attract E. coli and repel RBCs. A solution of E. coli and RBCs was passed over the same device with condenser structure as shown in Figure 2A. The RBCs were fluorescently tagged as orange, and the E. coli were green as before. Results
The result is shown in Figure 3A. At the working frequency of 10 MHz, the dielectrophoretic force repelled the RBC’s while attracting and concentrating the E. coli.
Separating different species of organisms based on their CM factor may also be done by first determining the CM factor individually for each species. This may be done by seeing what frequency effectively captures the cells, at a given solution conductivity, as shown in Figures 10A and 10B.
Example 5. Modifying electrode microchannel dimensions does not change the device performance.
Materials and methods
To support the electrode design, COMSOL simulations were performed for studying the efficiency of DEP capture of bacteria using a 2D microfluidic channel geometry with interdigitated electrodes in a cross section on the floor of the channel. First, the electric field caused by the application of AC signal to the electrodes at a given frequency and voltage was modeled. From this modeling plots were extracted of the electric field gradient, which was the relevant variable in the determination of DEP force on a particle. Secondly, the fluid flow profile was simulated for a given flow speed. The resultant flow profile was parabolic in laminar flow microchannels. Finally, 100 cells were introduced into the channel at the inlet (uniformly distributed along the height of the channel) and monitored for movement as they flowed along the channel and felt both DEP and drag forces. The electrode width and spacing dependence for 46 pm by 46 pm and 100 pm by 100 pm was compared.
The following experimental conditions were used: 40 V, 1E6 MHz, 30 pm high channel, 30 pl/min volumetric flowrate, and 0.0199 S/m fluid conductivity. Results
Figures 5 A and 5B show that widening the electrodes from 46 mhi to 100 mhi results in particle capture occurring farther down the length of the channel, and scales linearly with the electrode width. The channel length necessary to achieve various capture rates is shown in Table 2. The overall length for 100% capture did not exceed 1 cm.
Table 2. Channel length and electrode width necessary to achieve various capture rates for capturing 100 microorganisms present in the test sample,
Figure imgf000054_0001
The data in Table 2 show that the number of electrodes needed to achieve capture remain relatively constant and so the channel length necessary scales linearly with the electrode width. The data show that the length increases linearly with electrode width (width=spacing), the length increases linearly with flow velocity, the slip conditions increase the length by about 10-20%, and the capture length may be reduced by decreasing the gap spacing for a given electrode width (for 100 pm width, 25 pm gap, by about hall).
Example 6, Methods to Improve circuits for AC dielectrophoretic devices.
To outperform ELISA or PCR in time-to-diagnosis, dielectrophoretic (DEP) approaches require high throughput, typically at flowrates on the order of 100 pL/min, which often imply large electrode areas. In positive DEP mode, decreased capture efficiency may be observed as structures expand in cross- section area, as well encountering problems related to Joule heating. Decline in capture efficiency and the Joule heating both originate from the same underlying phenomenon: as the cross-sectional area of the capture electrodes exposed to solution increases, the magni tude of the solution resistance element term decreases. This causes the proportion of the applied voltage dropping across the solution resistance to decrease accordingly.
This example provides a method to modify the coupled microchip circuit design for highly efficient DEP systems, for both positive and negative DEP approaches.
Results
Figure 6 shows a simplified circuit model for a DEP system. A function generator is used to supply an AC voltage, which generates the electric field gradients necessary for dielectrophoretic capture This voltage source has an internal output impedance, and the combination is enclosed in the dashed lines shown in Figure one. This voltage source is applied across an interdigitated electrode structure, such as in Figure 2 A. The electrical lead-ins of the structure have some resistance, here represented by eiec. A double-layer capacitance,
CDL, naturally forms at the ionic solution-electrode interface. The resistance due to the conductivity of the ionic solution is given by Rsoin-
The voltage across the solution resistance element in Figure 6 is the sole determinant of capture force. However, if the solution resistance becomes comparable to the other resistances and impedances (including possible parasitic capacitances), the electrode and double layer impedances will divide the voltage (attenuate the voltage across the solution resistance element), resulting in decreased DEP efficiency .
The transfer function that expresses ratio of voltage across solution as a function of the voltage applied by the function generator (VFG) is lim
Figure imgf000055_0001
Equation (1)
To maximize efficiency of supplied power, Rsoiution must dominate. Figures 7 A and 7B show a typical measurement for a DEP structure as a function of frequency, with a series 2 kit resistor.
Figure 8 shows a capture efficiency from a DEP liner interdigitized electrode array. The device had an oxide coating, and the ionic strength was adjusted so that the solution resistance would dominate. Under the condition of low series resistance, DEP capture efficiency can be unity.
Thus, capture efficiency optimization is a complex interplay between the solution resistance determined by the application, an optimized electrode design to minimize lead in resistance, and a limit that the source resistance must be much less than the solution resistance. Under these conditions, DEP capture is performed at the lowest applied voltage that still results in maximal capture, to minimize extraneous Joule heating.
One might think that increasing the interdigitated electrode length will simply increase capture efficiency, and increasing this to very long length will ensure capture. Likewise, having parallel microfluidic paths over the same electrodes would increase throughput, and increasing this to very many parallel paths can infinitely increase throughput. However, these both are not true as this decreases solution resistance, and at some point the efficiency will drop.
Increasing the number of fingers should decrease the voltage across solution, all else held equal. This should result in a“turning over” of capture efficiency at some critical number of fingers determined by solution
conductivity and circuit parameters.
Joule heating is potentially a problem, as this would heat the solution and damage or kill the target. The power dissipated across the solution resistance is given by:
Figure imgf000056_0001
To minimize Joule heating, preferably operate at the frequency such that FDEP is maximal for a given input voltage. Consider the power dissipated per unit area of the electrode; RSOin is inversely proportional to area, therefore:
Figure imgf000057_0002
and thus: Equation (4)
The power per unit area is roughly constant/independent of width. Simply placing multiple IDE structures in parallel, or even if they were entirely adjacent with separate buffered drive circuits would not suffice. The power dissipation at the critical capture voltage would remain constant for the whole width.
This has an implication on microchip design. The substrate should be of high thermal conductivity to minimize Joule heating of the sample.
Figure imgf000057_0001
Optical methods of cell counting following DEP capture are not ideal for a low-cost, miniaturized real-world platform. Electronic cell counters, such as those provided by Beckman Coulter® (Beckman Coulter, Inc., Brea, CA), may be adapted for biological material quantification.
The current COMSOL simulations may be extended to different specifications in terms of cell types, ionic strengths, channel widths/heights, flow-rates and linear velocities with a focus on obtaining the minimum number of electrodes (and as a consequence chamber length) required to achieve 100% capture. It should also be refined to take-account of the‘bouncing’ of bacteria observed between electrodes at higher flow-rates.
The simulations may be conducted with the following experimental parameters: fluid conductivities: 0 - 10 S/m; channel widths: 10-10,000 pm (1 cm); channel heights: 1 - 1000 pm; flow rates: 0.01 - 1000 pL/min; and linear velocities: 0.2 - 2xl0/ pm/sec. For coulter-type detection, an on-chip (in-line with DEP capture regions) Coulter counter that can count: (i) individual latex beads; (ii) E. coli ; and (iii) Mycobacterium smegmatis, may be developed. The device will utilize narrow microfluidic channels, then determine the maximum width at which these will work, typically about or less than 1 mm. The device may be used to investigate the detection efficiency as a function of flow rate, electrode spacing/width, and solution conductivity . The device may be able to distinguish between bacteria, bacterial clumps, and sputum.
Coulter counter sensing techniques extract information about particle size from voltage spikes generated as the particle passes between two or more sensing electrodes. Laminar flow in microfluidic channels is used to confine the sample flow so that it passes through a narrow sensing region. A problem inherent to laminar flow in microfluidic channels is the creation of a parabolic velocity profile in the directions transverse to fluid flow'. This creates a dispersion in particle velocities, creating additional inhomogeneity in the voltage spikes detected for a single species (atop any natural inhomogeneities due to variance in particle size).
Furthermore, the theory of resistive pulse sensing underpinning the Coulter counter method assumes the particle is displacing a fixed volume of conductive solution in a uniform electric field. The classic, easily-fabricated planar electrode geometry used widely in the literature strongly violates this assumption. The electric field lines arc from planar electrode to planar electrode, creating a strong dependence in signal magnitude on vertical displacement from the sensing electrodes. Because signal magnitude is directly correlated to particle volume, this effect significantly impedes discrimination between particles of only somewhat different sizes.
Efforts to solve this problem have focused on either complex engineering of electrode geometries or solutions which greatly complicate the fluidics or device design. Such approaches are not desirable from a commercialization standpoint.
Proposed are dielectrophoresis (DEP) devices as actuators for an impedance-based flow cytometry device (colloquially, a counter). Rather than seeking to remedy the inherent limitations of planar electrode geometries, the devices exploit their spatial sensitivity to improve device performance in terms of both signal to noise and species selecti vity.
The proposed DEP-focused particle counter system presents the following improvements on conventional coulter counter particle detection systems:
1. Increased signal to noise ratio - Vertical focusing brings particles down near the sensing electrode surface, maximizing detection signal to noise.
2. Expanded species selectivity ability - DEP force magnitude and direction depends on the electrochemical properties of the particle and thus would allow for preferential counting of a particular species within a mixed population based not just on size differences, but also on electrochemical differences between species of the same size.
3. Constrained spread of signal amplitude - Vertical focusing brining particles near the sensing electrode surface will result in more uniform signal amplitude than vertically dispersed particles.
4. Constrained spread of parti cle transit time detection - Vertical focusing bringing particles near the sensing electrode surface will result in more uniform particle velocity than vertically dispersed particles.
5. Increased sample throughput - By eliminating the need for narrow vertical geometric constraints to yield sufficient signal strength, more fluid can be passed over the sensing region without sacrificing detection signal amplitude
6. Decreased particle clogging - By allowing larger vertical channel geometries over the sensing region, particles will be less likely to clog than in traditionally vertically constrained channels.
5 Materials and Methods
To demonstrate the concept of combining dielectrophoretic capture with a microfluidic cell counters for enumeration, the following experiment was performed on a chip. A stock solution containing 4.5 pm diameter polystyrene beads (Spherotech PP-40-10, SPHERO™, Spherotech, Inc., Lake Forest, IL) were diluted 2000-fold in O.Olx PBS, and 0.5% volume TWEEN®-20 was added to prevent aggregation and adhesion of the beads.
A sinusoidal voltage signal of 5 VPP amplitude at 4 MHz frequency was applied to interdigitated gold electrodes (25 pm width, 25 pm gap, electrode gap lengths 5-6 times the diameter of the target analyte may be useful for capture performance). Solution was flown over the electrodes at 0.5 pL/min, while a number of beads were captured on the el ectrode.
The signal was then removed, and the captured beads were released into the solution flow. The captured packet of beads begun to move downstream in the channel.
During this process, the downstream counter structure (with three electrodes arrangement, Figures 11A-11E) was running. A sinusoidal excitation signal of 1 Vrms amplitude (although the values may range between 1 mVrms and 10 Vrms) at 70 kHz frequency (although the values for frequency may range from 10 kHz to 10 MHz, dependent on the system and the desired application) was applied to the middle electrode. The top and bottom electrodes in the three- electrode structure were the sensing electrodes. Appropriately-configured measurement circuitry transduced the electrochemical impedance between the left (right) sensing electrode and center excitation electrode into a voltage V i (V2).
A sample with naive and activated T cells in l.Ox PBS, flowing at 0.5 pL/min, was used to demonstrate the ability of the counter to discriminate between the tw?o cell populations. Results
Passage of a polystyrene bead or cell over the electrode structures resulted in an output voltage signal, V1-V2, as shown in Figure 12.
While the solution was flowing and beads were being aggregated at the capture electrodes only a few such voltage pulses were detected, arising from beads which the upstream capture structure failed to capture. Shortly after the capture signal was turned off, however, the packet of beads that was released flowed over the counter structure. During this brief window, very many such voltage pulses (as shown in Figure 13B) were detected, allowing an enumeration of the number of beads which had been previously captured (Figures 13 A and 13B) Subsequent data analysis of the voltage trace permitted counting the number of captured heads during the experiment.
Flowing a sample with naive and activated T cells over the counter stnicture permitted counting the number of activated T cells. Since activated T cells are larger, a stronger signal was observed for the activated T cells than for the naive T cells (Figure 14).
Example 8, Modeling for Increasing throughput through the DEP device.
Increasing throughput through the DEP device is desirable for diverse clinical applications of the device.
It was tested whether the device area can be scaled up to increase throughput. The conditions tested were: an increase in channel width, wider electrode array, number of electrode fingers, increase in fluid flow, and flow speed. A modeling was performed for the expected trajectory of particles in a fluid channel subject to DEP forces.
The model predicts the throughput scales with the channel width without any indication of a tradeoff in performance (Figure 15). The model predicts the throughput scales with an increase channel height or in flow speed (Figures 16A-16D).
The circuit model predicts tradeoffs (Figures 17A-17C). Experimentally tested results show there is a trade-off (Figures 18A and
18B)
Example 9, Experimental testing of modeling shows a trade-off in scaling and no linear increase throughput through the DEP device,
Described are a seri es of investigations to demonstrate ho w device performance is impacted by design variations from the perspective of this voltage transmission framework. Operating at higher linear flow velocities, the competition between the Stokes force and the DEP force to shift the equilibrium velocity of incident particles flowing over the DEP electrodes was used. The magnitude of this shift was determined by the competition between the DEP and Stokes force acting on the particle in that region.
Figure imgf000062_0001
Particles owing in a microfluidic system quickly reach an equilibrium velocity due to the Stokes’ force exerted by the fluid medium. When passing over the interdigitated electrode arrays, the particles experiencing pDEP experience an additional force opposing their direction of motion, reducing their equilibrium velocity. For full pDEP capture, the equilibrium velocity is reduced to zero. Multiple examples in literature have attempted to map the real component of the CM factor by" analyzing cell velocities under laminar flow from microscope video recordings.
This process is illustrated in Figures 19A and 19B, depicting the position as a function of time as a particle passes over the interdigitated electrode (IDE) array, located at xl. In generating the position-time traces for the hundreds of particles passing over the IDE region, sequential image analysis was performed to track and trace the position of particles frame-by-frame from recorded videos. The beads were fluorescently-tagged, and therefore fluorescence was employed for imaging with a laser excitation source and optical filter to maximize the particle -background contrast. The change in equilibrium velocities occurring between xl and x2 as the particle as it passes over the array is proportional to the magnitude of the DEP force. The fractional change in velocity that particles experience when subjected to DEP forces over the device are extracted as
Figure imgf000063_0001
This fractional slowing as a measure of the time-averaged strength of the DEP force was monitored and compared with expected trends predicted by Eqn 7
Figure imgf000063_0002
Eq. 7
Multiple difficulties arise in extracting the precise force dieletrophoresis exerts on the passing particles. Force, proportional to acceleration, is related to the second derivative of position . Optical approaches measure the position as a functi on of time, and therefore extracting the acceleration requires
differentiating twice with respect to time. Evaluating multiple orders of numerical derivatives inherently amplifies measurement noise, here generated both by uncertainty in the position as well as uncertainties in frame-to-frame timing interval. The di electrophoretic force also acts on the particles in three dimensions and thus the top-down microscopy averages over the ensemble distribution of vertical positions within the channel. These findings were used to make best-practices recommendations for the desi gn of DEP electrode structures optimized for function in high-throughput and high-conductivity scenarios.
The Sample
Fluorescent beads were passed over the interdigitated electrodes for particle tracking video analysis. The polystyrene beads (Polysciences, Inc. 17867-5) were 1.77 mhi in diameter and fluoresced green under excitation. The beads were diluted 4,000-fold in O.lx PBS and passed at a flow rate of 0.4 pL/min The low flow rate was chosen to ensure a sufficient number of frames were recorded per particle transit. The dilution was chosen to ensure a high number of beads passing during recordings while not being so high as to overwhelm the tracking algorithm computationally. The O.lx PBS buffer was chosen to reduce the solution resistance and thereby emphasize the significance of design variations on device performance in contrast to lower-conducti vity solutions. As can be seen from inspection of Eqn. 7, the largest influence of electrode design is expected to be seen when the solution resistance is comparable to the electrode resistances.
Operating Conditions
Solution was flown through the micro fluidic channels at rates between 0.2-1.0 pL/min., depending on the width of the microfluidic channel under investigation. The linear flow speed, and thus the viscous drag force, varies inversely with channel width at a given fl ow rate. The effect of the
dieiectrophoretic force is in opposition to this drag force. The flow rates were chosen such that the magnitude of the two forces would be comparable to improve detection.
The Electronics
Tektronix A FG3252 function generator was used to provide the AC voltage signal necessary to produce a DEP force. Both output channels were used, sourcing sine waves between 0.1 -20 MHz configured to be 180° of phase with respect to each other, a mode of operation known as bipolar DEP. Each output channel was configured to expect a 50 W load impedance and fed directly into a dual-channel, high-frequency power amplifier (Tabor Electronics 9250). Typical voltage amplitudes were 1.2 Vpp for the Tektronix function generator with a subsequent ten-fold increase in amplitude provided by the Tabor amplifier. These amplitudes were chosen such that the incoming beads experienced significant slowing over the DEP electrodes without becoming captured to render the measurements sensitive to shifts in the DEP force.
Furthermore, the instantaneous forces experienced by the particles are rapidly changing. The dielecrophoretic force varies not only as the particles pass over the electrodes but also depends on the particles’ height within the channel. The laminar flow profile of a microfluidic channel is fastest in the center, thereby introducing variance in the drag force arising from vertical height as well as the lateral position within the channel. These factors combine to render evaluation of the dielectrophoretic force magnitude challenging to put in their appropriate context. The desired end functionality of dielectrophoretic capture is a change from the initial equilibrium velocity to nil in the electrode region. Equilibrium velocity shifts therefore are a suitable proxy measure of the DEP force and an experimentally-relevant metric for performance evaluation.
An expectation may be to monitoring the fraction of captured particles to evaluate performance. However, capture is an unbounded threshold condition; a bead cannot be more captured by DEP forces exceeding those necessary to reduce the equilibrium velocity. For a given input voltage, there will be a range of electrode geometries for which the voltage across the solution resistance is sufficient for high capture and a range of geometries for which the voltage is insufficient for any capture. The only nuance in the measurement lies in the interpolant regime in which some, but not all, incident particles are captured. This regime is not a priori guaranteed to span a wide range of geometries, nor include any of the extant devices for a given set of operating conditions.
Measuring changes in the equilibrium velocity, however, avoids the pitfalls of capture-efficiency based performance evaluation. Sensitivity lost due to excessive capture force is avoided entirely by eschewing capture altogether, operating the experiment below that threshold. Evaluating differing equilibrium velocities allows for making comparisons between a range of electrode geometries, all of which achieve no capture for the initial conditions chosen. Results
Series Resistance
The presence of an external resistance in series with the solution resistance element will impact the transmission of the voltage signal driving DEP. Typical origins include the output impedance of the voltage sources driving capture and the electrode leads transmitting the signal to the microfluidic region To illustrate this phenomenon, a series resistance was introduced in line with the device. For each value of the series resistance used, the particle tracking software identified the location of the fluorescent beads from frame to frame, computing the velocity in the region of the video with and without the DEP force. The measured impedance of the electrodes (Figures 19C and 19D) was used to compute the expected voltage across the solution resistance and thereby the relative strength of the DEP force the particles experienced. As expected, the equilibrium velocity over the interdigitated electrodes increases as the series resistance is increased, indicating a decrease in the strength of the DEP force on the particles.
Number of fingers
Increasing the number of electrode structures within the fluidic region is another strategy for improving device performance, particularly for capture. Particles not captured by the first pair of electrode structures have additional chances to be captured during subsequent interactions with the DEP force as they pass over the repeating electrode sub-units. Accordingly, COMSOL simulations predict asymptotically-increasing capture probability as the number of repeating sub-units is increased .
As a consequence, then, it was posited that the only upper bound on capture electrode area was the maximal permissible footprint of the device. Akin to expanding channel width, increasing the number of electrode sub-units increases the total area exposed to solution and thereby decreases the solution resistance and thus the DEP force exerted. Competition between this phenomenon and the increasing capture probability predicts that the global maxima for capture probability is achieved at a finite number of electrode sub units.
This is demonstrated by measuring the change in equilibrium velocity while doubling the number of interdigitated electrode fingers from device to device. At first, as the number of fingers - and thus repeating units --- increases, the equilibrium velocity of the particles over the DEP region decreases, as can be seen in Figure 20. Further increases in the number of electrode lingers, however, has the opposite effect, as the decreasing solution resistance reduces the magnitude of the voltage driving the DEP force. A fit in the expected form of a
Figure imgf000067_0001
F; ) interpolates the predicted voltage from Eqn. 7 plotted in Figure 20. Losses in magnitude outweigh the increasing capture probability of additional subunits, constraining the number of fingers to a geometry- and conductivity-specific optimum.
Figure imgf000067_0002
Increasing fluidic channel width is a common tactic to increase volumetric throughput for DEP-actuated devices. Increasing the width produces a commensurate decrease in the solution resistance of the fluidic region. A microfluidic channel of varying widths (0.5, 1.0, and 2.0 mm) was placed over identically- fabricated electrode structures, figures not shown. The volumetric flowrate (0.2, 0.4, and 0.8 pL/min.) was correspondingly adjusted to maintain a constant linear velocity - keeping the Stokes’ force constant across all three channel widths. Each doubling of the channel width correspondingly halves the solution resistance of the channel, consequentially decreasing the effective voltage seen across the solution (Figure 21) which is again interpolated with the fitting function a I
Figure imgf000067_0003
* : ·' l The voltage predictions of the device impedance model are contrasted with conventional approaches which do not modify the Dirichlet boundary conditions as the number of fingers are varied, here populated with data from the NF = 16 case from the previous experiment which are nominally identical to the 1 mm channel width condition.
Increasing channel height is another means of increasing volumetric throughput at constant linear flowrate. The fringing electric fields between planar metal electrodes driving the DEP capture decay in strength with increasing vertical distance above the electrode surface. The fraction of cells passing far above the electrode surface scarcely experience the DEP force.
Increasing channel heights thereby increases fractional waste of the inlet sample!.
Protective coatings
Insulating layers are preferable to inhibit electrolysis at the electrode solution interface, reduce the likelihood of cell adhesion, and reduce the probability^ of electrode corrosion by the sample. These protective coatings introduce an additional series impedance in-line with the solution resistance and therefore impact the magnitude of the DEP force between the electrodes. The voltage transmission model also directly" informs physical design limits on the effective capacitance permissible when coating the electrodes with a protective, insulating layer.
Two electrode structures differing only" in the presence of a 200 nm of PECYD silicon dioxide coating were compared to illustrate the coatings influence on device performance (Figure 22). The equilibrium velocity" for passing particles was measured while the signal frequency ranged from 100 kHz to 20 MHz. The impedance of the oxide coating varies accordingly. Figure 23A illustrates the change in equilibrium velocity as the particles pass over the DEP region for the device without the PEC VD coating. The slowing effect of the DEP force is in line with expectations from the voltage transmission perspective, neglecting variations in the CM factor of the polystyrene beads in the range of frequencies investigated when comparing to the voltage predictions but the comparison between devices at a fixed frequency remains valid. Contrast this with the performance of the device with a 200 nm PECVD coating, as seen in Figure 23B. DEP slowing rapidly vanishes at lower frequencies in a sharp transition between 7 MHz and 1 MHz where the impedance of the oxide attenuates the signal.
Therefore, at elevated physiologically-relevant conductivities, simulations to enhance performance must incorporate loading of the voltage source into the Dirichlet boundary conditions. In high conductivity it is desirable to maximize the performance to reduce operational demands (such as power and heat dissipation) while still achieving the desired functionality. This lowers the barrier to implementation for portable lab-on-a-chip applications.
As the electrode area exposed to solution or solution conductivity- increases, the consequences of the low-impedance load manifest in weakened capture and Joule heating challenges. Joule heating constraints are a particular concern for operation in physiological salinities. The optimal/maximal array size for DEP capture is demonstrated. Competition exists between the number of momentum impulses, (aNF ), and their magnitude from the applied external voltage.
Figure imgf000069_0001
where Rsoin has been redefined as p/A to make explicit the dependence of the solution resistance on the area of the channel exposed to solution. Likewise, Csub and 0o have had their area dependencies (A) separated out. Eqn. 8 and Eqn. 9 p __ T7Ϊ/ (,p\ can be combined by inserting the definition ' ! The spatial profile of the potential is dictated by the electrode geometry. If assuming a fixed geometry, therefore, one may separate the potential ^ (h) into a spatial profile ^ (^) which governs the gradient between the DEP electrodes and a circuit parameter-dependent function (Eqn. 9) which dictates the amplitude of potential multiplying the spatial function. Thus, the expression for the DEP force becomes:
Figure imgf000070_0001
The effect of these parameters was explored (data di scussed and not shown). Starting with rounded values approximating the NF = 16 device from Figure 20, the parameters were then adjusted one-by-one to illustrate how variations in each would impact device performance per Eqn 9. The value of parameters held constant were as follows: changes in V soir/V2AC due to variations in the self-capacitance of the interdigitated electrode structures at fixed device area (0.8 mm2), the interfacial capacitance due to the presence of an oxide coating, the concentration of the saline buffer solution for different device areas exposed to solution, and the area of the device exposed within the fluidic channel. Some secondary parameters were also varied within each plot to give a richer understanding of the interplay of the several variables, these values were reported directly adjacent the line to which they correspond.
Capacitive coupling ( Csub ) through the substrate arises between the DEP electrodes. Csub is an extensive quantity, depending upon the electrode density (the inter-electrode gap length) and the total area of the electrode structure. The dielectric properties of the substrate also impact this term, which forms in parallel with the solution impedance and interfacial capacitance. Csnb sets an upper bound on the operational frequency for DEP capture. For the typical structures fabricated on glass, the capacitance is negligible. Some attenuation in the DEP force magnitude is predicted at higher
frequen cies for larger values of the substrate capacitance term, constraining fabrication options.
A large pseudo-capacitance forms at the electrode-solution interface in conductive solutions. Ion concentration (solution conductivity) and device area govern the magnitude of the pseudo-capacitance The effects of variations in Qo were considered, the series combination of this pseudo-capacitance with the capacitance of a protective coating deposited over the device region The impedance of the smaller capacitor dominates series capacitor combinations.
Due to the atomic ally-thin nature of the ionic double-layer, the deposited coating is the determining factor. As the thickness of the coating increases, the effective capacitance decreases shifting the curves rightward. This is in line with the results from Figures 23A and 23B. Within the framework, the maximal permissible coating capacitance is determined by the solution resistance of the device and the desired operating frequency
Figure imgf000071_0001
--§
Figure imgf000071_0002
This simple guideline allows for protective coatings with no marked detriment to device performance. Fabrication of dielectrophoresis electrodes normally involves noble metals such as gold or platinum to minimize reactions at the electrode surface Protective electrodes enable use of cheaper metals in device design for significant cost savings.
The Clausius-Mossatti factor and physiological needs of the biological target constrain the choice of solution conductivity for DEP devices. The solution resistance and interfacial capacitance scale inversely and linearly with conductivity, respectively Changes in the solution conductivity for a fixed device design will alter the voltage driving DEP capture per Eqn 9. This effect is plotted for two order-of-magnitude variations in device area exposed to solution (data not shown). As the solution conductivity decreases, device performance becomes less and less sensitive to design variations. Conversely, performance varies as one alters the area of device (A) exposed to solution for a fixed solution conductivity(data not shown). In low conductivity regimes, the device area may be scaled aggressively before performance limitations take hold. At higher conductivities, performance is highly sensitive to device footprint for a fixed Rial.
This assumption is necessary for Eqn. 10 to hold. The spatial pattern and circuit parameters are in fact coupled through the geometry design process, which must be taken into consideration when optimizing device design.
The scaling parameters varied depend heavily on the value of the total external series resistance, Rtot. Minimizing losses from series resistances requires reducing the output impedance of the voltage source and metal leads. Increasing the width and thickness of the electrode leads while reducing length. Integrated circuit solutions for voltage sources can reduce the output impedance below the 50 W convention for benchtop electronics, mitigating some attenuation. Researchers should be aware that there are diminishing returns to these increases for the electrode leads as the series resistance contribution approaches a few W at most. Finger resistance should be primarily address through minimizing the excess finger length. Further study into the interplay of the electrode width/gap on the DEP force, but such design changes also alter the gradient profile driving the DEP capture and therefore require a more nuance and target- specific view but remains an active area of investigating for performance engineering.
Maximizing the solution resistance will improve performance, all else held constant. Possibilities include decreasing the area exposed to solution, widening the inter-electrode gap, reducing solution conductivity. This is most readily done by adjusting the conductivity of the sample solution used and helps to explain the prevalence of DEP in the literature conducted at lower
conductivity: with low conductivity/large resistance, other design considerations are unlikely to have a significant deleterious impact on performance.
Example 10. Design considerations for a portable counter module or counter device.
Materials and Methods
The terms particle and cell are used interchangeably. The small capacitance of cell membranes gives the appearance of an insulating particle in the measurement signal for sufficiently low operating frequencies, typically below 1 MHz.
The impedance-based flow cytometer (that may be used as a counter module or a counter device) adopts a three-electrode design, modeled after the cytometer presented by N.N. Watkins, et ah, among others (Watkins et al., Lab Chip, 11(8): 1437-47 (2011); Gawad et ah, Lab Chip, l(l):76-82 (2001)). The circuit, operates as an impedance bridge. A sinusoidal excitation signal (VAC) at the middle electrode drives current flow through solution to the left and right sensing electrodes. Each of the sensing electrodes is connected to circuit ground by a resistor, henceforth referred to as the bridge resistor ( Rbr ). The potential that forms at each sensing electrode ( Vi , V2) is governed by the ratio of the bridge resistor to the solution impedance ( Rso/n ) between the excitation and sensing electrodes. Under ideal operating conditions, the solution impedances and bridge resistors are perfectly symmetric and thus both sensing electrodes are at identical potentials. When a non-conductive particle passes between the excitation and sensing electrodes, the solution impedance is temporarily increased, reducing the voltage measured at the sensing electrode. The process repeats as the particle subsequently passes between the excitation electrode and the other sensing electrode. In this manner, a passing particle generates a characteristic voltage signal encoding information about both its velocity and its size. Figures 11A-12 depict the process by which a typical counter signal is generated in a three-electrode geometry. The left- and right-most electrodes serve as sensing elements, monitoring the impedance between them and the middle electrode at which an external voltage is applied. As the particle approaches (a) and enters (b) the sensing region formed between the left-most and middle electrodes, the solution resistance is increased due to the volume displaced by the particle. As the particle passes back over the middle electrode (c), the solution resistance returns to its normal operating state. The process repeats as the particle flows between (d) the middle and right-most electrodes before finally exiting (e) the sensing region. The output of this configuration (f) is a voltage signal proportional to the difference in resistance between the left and right sensing regions (RL-RR).
The AC approach
Employing a time varying voltage signal simplifies the measurement logistics compared to direct current (DC) approaches. Reference (or pseudo reference) electrodes are necessary to establish stable DC potentials in solution, but are difficult to microfabricate and cumbersome to integrate. Therefore, reference electrodes present a trade-off between simplicity of design and measurement capabilities. Without the use of a reference electrode, a drifting DC potential complicates measurement attempts with a constantly-moving baseline.
Circuit model of the cell
At sufficiently low frequencies the cell membrane capacitance renders cells electrically indistinguishable from insulating particles. However, researchers have also begun to use elevated frequencies in the MHz regime as part of their excitation signal. At elevated frequencies, the impedance of the membrane capacitance is significantly reduced, allowing researchers to probe the inner conductivity of the cell cytoplasm. In this manner, cell populations of comparable size but differing in physiology may be discriminated from one another, enhancing the counter's capabilities.
Microelectrode design
The implementation of planar microelectrodes for impedance-based sensing confers multiple advantages over other more-complicated geometries. The electrode definition requires only a few steps: photoresist coating, pattern definition, metal deposition, and a lift-off process. This simplicity compared to alternative electrode geometries significantly reduces per-device fabrication cost. The ease of fabrication simplifies combining the impedance sensor with additional sensing modalities (e.g., target capture, target recognition) into a single microfluidic sensing platform (Valera et al., Lab Chip, 18(10): 1461- 1470, (2018)).
The extended emphasis on design simplicity suggests eliminating the third electrode in favor of a two-electrode approach. Such implementation is observed in much of the early Coulter counter work (DeBlois and Bean, Review of Scientific Instruments , 4l(7):909-9l6 (1970); Wu et al., Lab Chip ,
8(11): 1943-9 (2008); and Suehiro et al., Journal of Physics D: Applied Physics , 32(2l):2814-2820 (1999)). The additional resistive sensing element formed by the third electrode transforms the characteristic output signal from a single voltage peak to an antisymmetric peak structure. The elapsed time between the local maxima and minima of the antisymmetric structure reduces uncertainty in transit time measurements during flow conditions, compared to extracting particle velocity information from the full-width at half-maximum (FWHM) of a two-electrode configuration.
Differential measurement configurations are the critical competitive advantage of three electrode approaches. The two solution impedance elements formed between the middle and the left and right electrodes, respectively, are nominally identical under all flow conditions and therefore no voltage froms across the bridge. Monitoring changes in the difference between these two impedances greatly enhances sensitivity by reducing the background signal upon which the transitory resistive pulse of a passing bead is imposed.
The fluidic constriction
Constriction diameter and signal magnitude
Design of the fluidic constriction is an integral aspect of the
microfabricated counter performance. The Coulter principle depends upon the displaced volume of conductive solution by a passing particle. Therefore, the ratio of the volume of the target analyte to that of the sensing region, colloquially called the filling factor, strongly determines sensor
performance. Consider an insulating spherical particle passing through a cylindrical volume of conductive solution. The effective change in solution resistance of the cylinder, measured between the circular faces, is given by (DeBlois and Bean, Review of Scientific Instruments , 4l(7):909-9l6 (1970); Jiang et al., Journal of Micromechanics and Microengineering , l7(2):304 (2007)):
Figure imgf000076_0001
where p is the solution conductivity, L the length and D the diameter of the cylinder, and d the diameter of the particle. Here there is an assumption L » D. Given the cubic dependence on analyte diameter, the ideal constriction is roughly equal to the diameter of the largest analyte body in the envisioned end- user sample. Matching the diameter of the constriction to the largest target analyte maximizes sensitivity for a given heterogenous sample. It assumes that no debris larger than the largest analyte exists in the solution, or else the debris must be filtered out upstream of the constriction region to prevent it from blocking the channel. Constriction diameter and clogging probability
A blocked channel effectively halts the device's ability to count particles until the blockage is removed. During actively-driven fluid flow, large hydraulic pressures build up after clog formation. The resultant pressures can cause catastrophic mechanical failure of the fluidic channel, posing a significant biohazard to the end user when dealing with biological samples.
Large debris within the sample is not the sole vector of clog formation. During normal operation, there is a finite probability that an incident particle will adhere to the side-wall of the channel. As the fluidic channel narrows down to the constriction diameter, wall-particle interactions become increasingly likely. A common failure mode observed in the fluidic channels is one such particle failing to become unstuck before a subsequent particle enters and adheres to the first. An aggregate quickly forms in the constriction region, driving jam formation and rapid onset of clogging.
Researchers have investigated the factors influencing the mean-time-to- failure (MTF) for clog formation in fluidic constrictions. Particle number density, flow rate, constriction cross-section, and constriction length all influence this failure mode, as do particle rigidity and the geometry of the narrowing region approaching the constriction (Wyss et ah, Physical Review E, 74(6):06l402 (2006); Rajan et ah, Biosens Bioelectron , 77:1062-9 (2016)). In the process of sensor development, all of these parameters to minimize clogging probability during measurement may be manipulated. Ultimately, the particle number density, rigidity, and flow rate are dictated by the end-user application. Engineering of the constriction geometry becomes the main approach to extend the MTF.
Particle concentration
Particle densities between 0.1-1 million/mL function best for acquiring a significant number of events within a reasonable measurement time-frame without presenting excessive clogging risk (for 20 pm x 20 pm channel cross- sectional areas).
Results
Planar electrode geometry
The planar electrode geometry adapted in the sensing set-up (Figures 24A-24C) greatly simplifies the device fabrication process. A single mask and a single metallization layer is all that is required for the counter sensing electrodes, reducing fabrication complexity and cost per sensing device.
The planar electrode geometry limits the size resolution performance of the counter structure. During device operation, an electric field forms when an electric potential is applied across the two electrodes. The electric field that forms is non-homogenous, as shown in Figures 25A and 25B. While the solution conductivity remains uniform over the entire sensing volume, different regions of the solution have nonidentical contributions to the impedance between the two electrodes. The magnitude of the impedance-based signal acquires a marked vertical dependence, as can be seen in Figures 26 A and 26B. This dependence produces a 20% dispersion in signal magnitude at fixed particle size, corresponding to a 7% uncertainty in diameters.
Solutions to the vertical dependence require either manipulation of the incoming particle stream or overhauling the electrode design. Researchers have implemented solutions using acoustic waves to focus the particles into the middle of the channel, sheath flows, and negative dielectrophoresis.
Alternatively, structuring the electrodes in three dimensions can greatly simplify the electric field profile at the cost of complicating device fabrication.
Example 11. Constrictionless counter
Commercial Coulter® counter devices depend upon their constriction region to maximize the filling fraction of the incoming analyte and thus the signal magnitude. The detection threshold and size resolution of the counter is dictated by this fraction. Potential drawbacks to reducing the constriction volume have already been discussed.
The implementation of microfabricated planar electrodes offers an alternative solution: the fringing electric field of the planar electrode geometry. The density of electric field lines arcing from electrode to electrode falls off rapidly with distance from the electrode surface. This has directly observable consequences for the sensor signal as a function of particle height within the channel.
For planar electrodes projecting into a wide solution channel, fringing occurs not only in the z-direction but also in the x-y plane where the electrodes terminate. Defining the x-axis along the direction of fluid flow, the density of field lines decays rapidly along the y-direction from the end of the electrodes. This spatial decay limits the volume of solution probed laterally past the end of the planar metal electrodes. In the manner, the effective volume sensed is confined electrically, rather than mechanically.
As a consequence, planar electrodes which just barely project into the side of a wide (~l mm) microfluidic channel would only enumerate particles passing through the narrow width of solution flowing over the electrodes. Because of the self-limiting nature of the fringing electric fields, the sensing region does not feel the effects of the entire width of the channel.
Thus good filling fractions may still be obtained despite the significant increase in overall channel width, eliminating the need for a constriction region.
To test this, a 1 mm-wide microfluidic channel was bonded to a counter device such that the three counter electrodes projected slightly into the width of the channel, as can be seen in Figure 27A. Polystyrene beads having a 4.45 pm diameter and 10,000x-fold diluted in O.Olx PBS were flown through the device at 12.5 pL/min. The absence of a constriction makes possible a fifty-fold increase in volumetric flow-rate compared to previous experiments, a key advantage of this technique. Sheer probability dictates that some of the beads within the sample would be expected to flow over the counter device to be detected. Indeed such an event can be observed in Figure 27B. The significance of this demonstration cannot be understated. This helps with increasing volumetric throughput 50- fold. Eliminating the need for a mechanical constriction also sidesteps a significant engineering challenge for product reliability. This demonstration only counted a small fraction of the sample within the population. Combined with lateral displacement via dielectrophoresis, as shown in Figure 27C, the entire sample volume may be interrogated by focusing all the particles to the side of the channel occupied by the counter structure.
Materials and Methods
Microscope and stage mount
Microscope
The impedance-based cell counter aims to compete with fluorescence- based cytometers. Incorporating simultaneous optical imaging within the measurement system enables direct comparison to fluorescence-based approaches and simultaneous real-time verification of fluidic performance during the development process. To this end, all of the sensing experiments were conducted on the viewing stage of an Olympus BX51 microscope equipped with 5x, lOx, and 40x objectives as well as multiple filter lenses for fluorescence imaging. An Olympus DP70 camera system allows for image and video capture for later analysis. An Xcite Series 120Q laser source provides an intense source for fluorescence imaging.
Test fixture
Previous solutions for making electrical contact to devices on the optical stage involved lengthy cabling and a multitude of solder joints. This approach posed challenges for the mechanical positioning and connection stability when forming electrical contact. The extant cabling also had significant coupling to external noise sources as well as cross-talk between wires. A mechanically- robust and properly- shielded test fixture was needed.
As part of the test fixture, a printed circuit board (PCB) was designed, which permitted electrical contact to individual pins on the device through coaxial connectors mounted on the board, while leaving sufficient clearance for the microscope objective lenses. The PCB made contact to the device via spring- loaded connectors projecting from the underside of the board.
A metal sample mount was then designed to mate with the PCB. A groove milled out of the sample mount, allows devices to easily be loaded underneath the spring-loaded connector from the side. A slot (shaded blue) recessed in the center of the milled-out groove has been machined with lateral tolerances much tighter than the contact pads' pitch to mechanically ensure in plane alignment. Vertical alignment with the springloaded pins is likewise mechanically determine by the vertical displacement between the bottom of the slot and the height of the PCB.
The combination of the PCB and sample mount thus provides a secure and robust connection between the device and the coaxial connections on the PCB. Sample alignment in all three dimensions is achieved by the physical structure, removing a significant barrier to reliability and ease-of-use.
Furthermore, the metal sample mount and the ground plane of the PCB form a Faraday cage around the device to shield the device from external
electromagnetic interference.
The sample mount was also machined with a second, larger contact area, to permit interfacing device geometries which do not conform to the milled socket while still handling alignment in the vertical plane.
The electronics
The circuit diagram of the complete three-electrode structure, driven by the sine wave output of the function generator is shown in Figure 27D. Function generators
A sinusoidal voltage source is required for the three-electrode bridge circuit. An Agilent 33120A function generator was used for the counter measurements as opposed to the built-in function generator of the lock-in amplifier. The Agilent 33120A demonstrated lower noise floors and higher spectral purity than the sine wave generator of the SR830 lock-in amplifier, as measured on a network analyzer. The excitation signal, a 70 kHz sinusoid with 1 Vrms amplitude, was rarely varied during the course of development.
Lock-in amplifier
The output signal from the bridge circuit was monitored during experiments with a Stanford Research Systems SR830 lock-in amplifier. Lock- in amplifiers exploit the orthogonality of sine and cosine functions to extract the amplitude of a specific frequency component of the input signal with high fidelity. This enables detection of the small changes in the bridge resistance during particle transit events expected at low filling factors.
An additional lock-in amplifier, the Stanford Research Systems 844, was used to characterize the impedance of the devices in the 25 kHz - 1 MHz regime. The additional order of magnitude in frequency range provided additional information about circuit electrical characteristics.
Oscilloscope
The transit time of the particles over the counter structure dictates the necessary sampling rate for measuring the bridge voltage. From the perspective of the Nyquist criterion, the minimum sampling frequency is 2/ St, where St is the transit time of a particle passage. Researchers typically aim for a minimum of 20 datapoints per event, requiring sampling rates of 10-1000 kHz depending upon desired flowrate and constriction geometry. To satisfy this condition, a
Tektronix DPO4104 was employed to record the analog voltage signals from the rear panel of the lock-in amplifier. Furthermore, extracting particle size information from the shape of the voltage signal requires a sufficient number of datapoints per particle trace, with minimums in the literature between 10 and 20 points. Transit times of 0.1 ms correspond to sampling rates between 100 - 200 kHz which is hardly a stringent requirement in a laboratory setting, however there exists economic incentive to minimize the necessary sample rate when producing portable systems. The sampling rate also must be increased with increasing expected event frequency to resolve abnormal signatures arising from contemporaneous transits.
The measurement circuit
The printed circuit board comes equipped with the ability to interface with up to six counter structures. Each has a single Texas Instruments OPA- 2227 operational amplifier configured as a dual-channel unity-gain voltage follower. A gain -bandwidth product of 8 MHz more than exceeds the necessary operating frequency of the Coulter counters. For a balanced bridge being driven by the typical 1 Vrms amplitude, the equivalent peak-to-peak voltage occurring at either node is 2.83 Vpp. Given the specified slew-rate of 2.3 V/ps, operation up to 0.8 MHz is possible. A dual-channel op-amp is chosen to avoid variance among individual integrated circuits which would contribute to a differential signal between the two terminals.
In addition to the dual-channel voltage follower, a precision
instrumentation amplifier for each counter measurement channel was introduced. An instrumentation amplifier produces a signal proportional to the difference between the two input terminals. Signals common to both terminals are subtracted out. The efficacy to which signals common to both inputs are suppressed is referred to as the common-mode rejection ratio. The
instrumentation amplifier can be configured to provide additional gain of the differential signal, elevating the signal of interest further over the suppressed background signal between the two amplifiers. The syringe pump
To flow sample through the device, a New Era syringe pump (NE-1000) was used. Typical sample flow rates range from 0.1 - 5.0 pL/min.,
corresponding to linear velocities of 5,000 - 250,000 pm/s. for particles within the constriction region and transit times across the entire length of the three electrode structure between 0.4 - 20 ms for the 20 pm x 20 pm constriction cross-sections.
Reducing processing time at a fixed sample volume requires increasing sample through-put and therefore flowrate. A single droplet of blood, roughly 30 pL in volume, requires half an hour to process at flowrates of 1.0 pL/min.
Mechanical and instrumentation limitations prevent arbitrarily increasing sample throughput. The microfluidic constriction for the counter region presents a hydraulic resistance, generating large back-pressures in the fluidic channel as flow velocity increases. As flowrates approach lOs of pL/min., the backpressure splits open the tubing inlet or breaks the adhesion between the channel and the substrate, causing leaks. Furthermore, increasing the flowrate reduces the mean time to clog formation within the channel, a problem exacerbated by the rigidity of polystyrene beads used for calibration experiments.
Results
Counter performance evaluation
Prior to performing detection of heterogeneous biological populations, performance baselines need to be established for the counter performance. The limit of detection for particle diameter as well as the resolution of diameter- based classification is of particular importance as means of discriminating between biological specimens of varying size.
To avoid the complications of handling biological material as well as eliminate population heterogeneity as a variable for evaluating the sensor response, polystyrene beads with tight size dispersions were used. These experiments allow to assess the sensitivity of the counter device over a range of solution conductivities and particle densities.
Population analysis
To quantify the resolution capabilities, repeated recording of the response of the sensor to a known input was performed. The tight homogeneity of purchased polystyrene beads was used to approximate an identical input. Many hundreds of beads were passed through the counter structure while recording the output signal. Extracting bead size and transit
time information from every recorded passage, the range of output responses was recorded, which corresponded to the given input - a particle of known size in a known solution conductivity.
The process underlying the peak detection algorithm used to generate the ensemble measurement statistics was developed (data not shown). A
representative data trace containing two bead passage events (change in voltage (V i-V2 (pV)) over time (ms)), were condensed into a histogram of counts generate a threshold parameter (horizontal lines in traces) based upon the standard deviation of the background noise. Threshold detection identifies the passage events which are then fit with to extract particle size and velocity parameters, which are mapped for thousands of such events acquired during the measurement. Each recorded data trace lasts for a second and can contain some number of bead passage events. The program constructs a histogram of the time- domain voltage signal. The output noise of the lock-in amplifier has a Gaussian profile, and allows constructing a histogram of the acquired signal. Curve fitting on the histogram bin counts extracts the standard deviation of the background noise which determines the threshold levels for peak detection of the individual trace. A coincidence detection for particle recognition was employed: within a finite time window (the expected transit time based on flow rate and constriction geometry), the voltage signal must cross the positive threshold level with a rising and then falling edge before crossing the negative threshold with a falling and then rising edge. If all four of these crossings occur within the
predefined time window, that section of the data-trace is flagged as containing an event. Curve-fitting of the antisymmetric peak structure within the event extracts the voltage amplitude (proportional to measured particle size) and elapsed time (measured velocity) between the maxima and minima of the signal.
A two-dimensional histogram is then constructed of the velocity and size information to look at the dispersion in recorded events along both dimensions, reflecting both physical effects arising from the channel geometry (parabolic flow velocity profile, height dependence of the signal amplitude) as well as uncertainties arising from variances in the fitting algorithm. Of particular interest is the spread in measured values for the peak height, which determines the ability to use sizing alone to distinguish amongst incident particles or species.
Physiological conductivity
To assess the performance of the counter, a sample containing 4.45 pm (Spherotech PP-40-10), 6.42 pm (Spherotech FP-6056-02), and 8.87 pm
(Spherotech PP-100-10) diameter beads was prepared. The l.Ox PBS buffer was filtered twice to remove particulate matter from the stock solution, and subsequently this mixture was used to dilute and rinse the beads. The nominal weight fraction of the three bead samples was used to calculate the nominal bead density per mL. The nominal densities determined the dilution ratios. The final sample contained all three bead populations with a nominal 670,000 beads/mL.
A 1.0% Tween-20 was added by volume as a surfactant to inhibit aggregate formation within the sample.
The sample was flown over the counter device at 1.0 pL/min. while recording 1600 one-second data samples. The algorithm analyzed the acquired data and extracted particle transit time and peak height for each detected event. Three separate populations are clearly visible. The cube root of the peak height is plotted, as it should be directly proportional to particle diameter. Constructing a histogram of the peak heights reveals a trimodal distribution, as can be seen in Figure 28A. Gaussian peak fitting extracts the mean signal amplitude and uncertainty for the three populations, plotted as a function of nominal bead diameter in Figure 28B.
Linear regression (dashed line) of the peak locations gives the expected signal amplitude as a function of particle size for the given constriction. The intercept of the regression with the amplitude of the threshold for peak detection gives the limit of detection for the measured sensor, here -2.8 pm. Inspecting Figure 28A, particle diameter differences can be resolved on the order of 0.5 - 1.0 pm.
Flowrate and transit time
While the signal arising due to a particle of a given volume passing through the sensing region should be independent of flow velocity, the lock-in amplifier itself places limits on the measurement bandwidth of the system. The output response time of the SR830 is dictated by the steepness of its bandpass filter as well as the integration constant chosen. For maximal signal-to-noise ratio during measurements at the targeted volumetric flow rate, a 30 ps time constant and 24 dB ./decade roll-off were chosen. Per the SR830 datasheet, this generates a 99% response time of 300 ps. No significant attenuation was observed for flow velocities up to 8.0 pL/min., or 0.5 mL/hr. A consistent function over the range of flow-rates was observed germane to the desired clinical applications of the sensor.
Example 12. Counter as an assay module or device.
Impedance cytometry as an assay technique
Consider a cylindrical volume of conductive solution with metal electrodes on opposite faces. Provided the length of the cylinder is large compared to the diameter (to ignore fringing effects), the resistance between the faces may be computed. Now if an insulating sphere is inside the conductive cylinder, the resistance increases due to the portion of the solution volume which has been displaced. The magnitude of the resistance change is
proportional to the fraction of the particle volume to cylinder volume, as shown. This is the Coulter principle, which has been used in benchtop cell counters. The membrane capacitance of cells causes them to appear insulating at DC and thus the analysis readily maps. Typical benchtop variants have a three-electrode sensing approach, two reference electrodes sourcing a high and low voltage to establish a potential gradient in the solution. A narrow constriction between one of the reference electrodes and a sensing electrode in the middle acts as the tiny cylinder of“sensing” volume, whose resistance rapidly changes as a cell flows through it. The magnitude of these resistance changes (and the resultant voltage spikes at the sense electrode) are proportional to the particle volume, or the diameter cubed.
The counter device allows for not just the separation and isolation of activated T-cells, but also to be able to provide a quantitative on-chip measurement of the activation. To do this, the wide variety of functions accomplished by planar metal electrodes in a fluidic environment was used.
The counter device separates T cells, in high cond, in relatively high viscosity, and with on-chip quantification rather than optical detection.
The current methods for assaying cells require different techniques that are time consuming, and/or the signal from cells is transient. These drawbacks are summarized in Table 3. The counter module/device allows for on-chip quantification and assaying of the cell state without the need for stains or markers. The assay is rapid, label-free, and the device is free of optical components for detecting the signals from the cells. Table 3. Drawbacks of current techniques for cell assays. The main drawback is that there is no rapid, label-free method of assaying cells.
Figure imgf000089_0001
The impedance-based flow cytometers provide information on the number and size distribution of incident particles (Cheung et ah, Cytometry A, 65(2): 124-32 (2005)). The two subpopulations are readily resolved by the clear size differentiation (4-5 pm v. 8-12 pm) between activated and unactivated T-cells (O’Connell et al., Comparative medicine , 65(2):96-l 13 (2015)). Operating a counter structure near the outlet of each stream, one can count the total population of activated and unactivated cells in the laminar flow of the original sample as well as in the exchange buffer. Thereby one can quantify the efficiency and purity of the dielectrophoresis separation as well as the ratio of unactivated to activated T-cells within the sample to assess immunological status.
The lymphocyte sample
The T-cells are primary cells, prepared directly from murine splenocytes, distinct from cell lines generated for modeling cell behavior under tissue culture conditions. The T-cells are in an unactivated state when initially prepared. To obtain activated T-cells, unactivated T cells are exposed to activation-inducing agonist antibodies, anti-CD3 and anti-CD28, for 72 hours. Both populations are suspended in l.Ox phosphate-buffered saline with 0.1% by volume of Pluronic F-127, a surfactant from Sigma Aldrich, to minimize cell adhesion to the device.
Impedance-based discrimination
After establishing the ability to separate the unactivated and activated cells, the counter device was used to count and size them. Populations of activated and unactivated T-cells are readily differentiated from one another in mixed solutions, as can be seen in Figure 14. Samples of naive and activated T- cells were prepared and flown through counter constriction regions at 0.4 pL/min. Sixteen hundred one-second data traces were acquired and analyzed to produce the results shown in Figure 14, which shows a clear distinction between the two populations.
To verify this detection further, samples containing a mixture of both naive and unactivated T-cells were introduced, in 1:1 and 2:1 ratios. The combined signal contains a sum of both the naive and activated signatures from Figure 14.
Three sequential peaks are visible in the population histogram. The broad, rightmost feature captures heterogeneous size distribution of the T-cells after activation. The concentration of activated T-cells mixed into dilution was unchanged. Accordingly, no pronounced change in the peak magnitude between the two conditions was seen. In contrast, the height of the naive cell population peak halves when the mixing concentration of naive cells in solution is reduced two-fold. Histograms of this data is not shown.
Impedance-based measurements of activation kinetics
Size-based discrimination as a diagnostic criterion requires the ability to differentiate between naive and activated T-cells after a prolonged period of growth. The ability to distinguish between the two was demonstrated. It remains to be seen how the growth process occurs over time for populations of T-cells after antigen exposure. The growth kinetics and size resolution of the sensor could both set a lower bound on time after antigen exposure for a detectable immune response.
Four distinct samples were prepared to investigate the T-cell growth kinetics. Naive cells and cells that were exposed to antigen 72 hours prior were prepared as previously described. Additional samples which had been exposed to antigen 24 and 48 hours prior were also prepared using this same protocol.
Changes in the size distributions over time are clearly visible in the four samples (data not shown). The naive distribution is the same as previously observed in Figure 14. Twenty-four hours after antigen exposure, the naive cells begin to undergo activation. The peak at 0.1 AR/R broadens, acquiring a rightward shoulder as cells within the population grow at varying rates. This process continues in the sample taken forty-eight hours after antigen exposure.
A fraction of the population has reached the fully activated state while a fraction still has yet to undergo activation and appreciably change in size. A full seventy- two hours after activation, the naive cell peak is fully suppressed only the broad activated population and debris signatures remain.
This example demonstrates the implementation of an impedance-based sensor for particle sizing and enumeration using planar metal electrodes. The sensor embodiment is suitable for lab-on-a-chip sensing applications.
Monitoring impedance changes induced by insulating particles, one can detect, count, and discriminate based on size for a wide range of particle sizes and in a range of solution conductivities.
Example 13. Integrating DEP capture and counting modules - capture and count device.
Researchers are continuously investigating additional applications of dielectrophoresis for lab-on-a-chip biosensing applications, combining it with other on-chip technologies to tackle specific design challenges and demonstrate utility in additional contexts. Dielectrophoretic electrode structures have been widely used for cell capture (Lee et ah, Biomicro fluidics, 12(5):054104, (2018); Rosenthal et al., Lab on a Chip, 6(4):508-515 (2006)) and sample concentration (Park et al, Lab on a Chip, l l(l7):2893-2900 (2011); Jubery et al, Electrophoresis, 35(5):69l-7l3 (2014); Wang et al., Dielectrophoretic separation of microalgae cells in ballast water in a microfluidic chip
Electrophoresis, (2018); Gadish and Voldman , Analytical Chemistry,
78(22):7870-7876 (2006)). Here, it was investigated how sample concentration could be used to enhance the performance of the counter subsystem. In low particle density regimes, the volume of solution per particle greatly exceeds the volume of the constriction region. DEP capture enhances the local concentration of particles within the volume of fluid above the electrodes. This presents no computational for the counter software provided that the particle density does not produce a high incidence rate for simultaneous passage of multiple particles.
At high capture efficiencies, very few particles will pass over the counter structure while the DEP signal is on. When the DEP signal is turned off, a packet of concentrated particles will leave the electrode structure and travel downstream. The process restarts when the capture signal is applied once more, in a process illustrated by Figures 29A and 29B.
To show this detection scheme in action, 4.45 pm diameter polystyrene beads were 2,000x-fold diluted in O.Olx PBS and flown through the device at 0.5 pL/min. A capture signal of 4.0 MHz and 5.0 Vpp concentrated the incoming beads at the electrode structure. The counter region was simultaneously monitored optically and electrically. While the capture signal was applied, a few single bead passage events were observed as the DEP electrodes failed to capture some beads. Shortly after the capture signal was turned off, a large number of beads was observed passing through.
Example 14. Devices for lateral separation of particles, optionally for buffer exchange, and/or particle counting.
A device with electrode arrays was designed to manipulate the lateral displacement of cells within the sample to enhance the functionality of the impedance-based assay. Dielectrophoresis and the Stokes' force have different dependencies on cell diameter. Under carefully chosen conditions, one can separate activated and unactivated T-cells. This approach has been previously demonstrated in the literature (Han and Frazier, Lab on a Chip, 8(7): 1079- 1086 (2008)).
Physiological samples are inherently messy. Separating the enumeration and analysis target into a parallel fluid stream isolates it from the environment containing debris and up to billions of cells per mL which include the fluidic background signal. This confers two distinct benefits. Only the purified side stream needs pass through a constriction region for enumeration, greatly reducing the clogging probability during operation.
Furthermore, physically filtering the incoming fluid stream in this manner greatly simplifies the computational complexity of enumeration.
Isolating the target from a high number of background count relaxes the rejection thresholds for false positives and false negatives at the same error rate in terms of events per volume. This feature is particular desirable for
background signals which are comparable in size to the intended target which necessitates additional discrimination mechanisms to distinguish between the two populations.
The described devices integrate both the DEP separation and counter enumeration onto a single microfluidic chip, having established both operational capabilities separately. The devices permit quantifying the separation efficiency of the assay and the purity of the sample within the exchange buffer stream as illustrated in Figures 30A-30H. Two separate inlets, one connected to the sample and the other to the buffer solution, flow in side-by-side in the wider microfluidic channel before passing over the separator structure. In the absence of an applied DEP signal to the separator, Figure 30A, the parallel laminar flows continue through the device, separating at the junction before passing over a counter structure en route to two outlets in the bottom of Figures 30A and 30B. An applied DEP signal, Figure 30B, drives lateral separation of the activated T- cells (large spheres) as well as a few unactivated T-cells (small spheres) into the buffer stream. These pass through the right outlet channel where they are enumerated by the right counter in Figure 30B.
In the absence of an applied DEP signal, the counter in the left channel detects both populations in the outlet stream whereas the right channel sees few, if any, events. Histograms of the detected events for both counters are shown in Figures 30C and 30D. When the DEP signal drives lateral separation of the activated T-cells, the bulk of the activated population in the left channel (the sample stream) is depleted and instead detected in the outlet of the buffer channel by the right channel counter. A fraction of the unactivated T-cells are also separated by the DEP signal. The DEP signal therefore drives changes in the detected cell distributions as measured by both counters, shown Figures 30E and 30F. Experimental results from these assays are shown in Figures 30G and 30H.
Enumeration was performed using counter structures fabricated on a glass substrate to mitigate the influence of parasitic capacitances from the bonding pads, whereas the initial separation structures were fabricated on silicon wafers. The combined devices have been fabricated on glass and are
experiencing issues with electrode integrity while applying the DEP drive signal. Also developed is a diagnostic assay combining lateral displacement with enumeration and sizing which could deliver valuable information about patient health status. The efforts centered on enumerating the ratio of activated to unactivated T-cells in physiological saline as an indicator of patient
immunological function. The device with dielectrophoretic manipulation and impedance-based cell counting can be further integrated in biosensing applications by extending the result from sample in physiological saline to separation in whole blood. A two-step buffer exchange process would eliminate the need for sample centrifugation prior to analysis (diagrams not shown). The DEP device used in particles separation (lateral separation of beads) and buffer exchange were conducted on the same device using flow of two different buffers over the device. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific
embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:
1. A device for capture and identification of micron-sized or nano-sized particles, the device comprising one or more units, each unit comprising
a trap module comprising at least two interdigitated electrodes (IDE), and a primary microfluidic channel with an inlet and an outlet, and
a second module,
wherein the second module comprises an identification module comprising at least two IDE electrodes and a secondary microfluidic channel with an inlet and an outlet, and/or a counter module.
2. The device of claim 1, wherein the identification module is in fluidic connection with the trap module and has a surface area substantially smaller than the surface area of the trap module.
3. The device of claim 1 or 2, comprising a plurality of units, such as at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 units.
4. The device of any one of claims 1-3, wherein the at least two electrodes of the trap module have a width between about 1 pm and about 1000 pm.
5. The device of any one of claims 1-4, wherein the at least two electrodes of the trap module have about the same width and are separated from one another by a distance as wide as the width of the electrodes or by a distance that is less or more than the width of the electrodes.
6. The device of claim 5, wherein the distance separating the at least two electrodes is between about 0.01 times and 10 times of the width of the electrodes.
7. The device of any one of claims 1-6, wherein the at least two electrodes of the identification module have a width between about 1 pm and 50 pm.
8. The device of any one of claims 1-7, wherein the distance separating the electrodes of the trap module is between about 10 nm and about 1000 pm.
9. The device of any one of claims 1-8, wherein the at least two electrodes of the trap module and the at least two electrodes of the identification module are arranges in any two-dimensional pattern, such as in linear, angled, circular, rectangle, cube, triangle, zigzag, and oval pattern.
10. The device of any one of claims 1-9, wherein the at least two electrodes of the trap module and the at least two electrodes of the identification module have a length between about 1 pm and 20 000 pm (2 cm) and is sufficient to form a surface area for capturing a sufficient number of particles of interest in a sample at a flow rate for identification.
11. The device of any one of claims 1-10, wherein the at least two electrodes of the trap module, and/or identification module are pitched.
12. The device of any one of claims 1-11, wherein the at least two electrodes of the trap module, and/or identification module are coated with an insulator.
13. The device of any one of claims 1-12, wherein the counter module is in fluidic connection with the trap module and/or the identification module.
14. The device of claim 1, wherein the counter module comprises at least three planar electrodes and a microfluidic channel overlaying at least a portion of the three electrodes.
15. The device of claim 13 or 14, wherein the fluidic connection is via a microfluidic channel having an inlet, an outlet, and a constriction region having a width between about 1 pm and 100 pm at a region overlaying the electrodes.
16. The device of claim 13 or 14, wherein the fluidic connection is via a channel having a width between about 100 pm and 10000 pm at a region overlaying at least a portion of the electrodes.
17. The device of claim 16 forming a sensing region electrically confined to a region around the portion of the electrodes overlaid by the fluidic channel.
18. The device of any one of claims 13-17, wherein the electrodes have a width between about 1 pm and about 1000 pm.
19. The device of any one of claims 13-18 comprising two or more counter modules, all in fluidic connection with the trap module or the identification module.
20. The device of any one of claims 13-19 operated with an alternating current (AC) having a frequency between 10 kHz and 10 MHz and a peak-to- peak voltage amplitude between 0.5 V and 10 V.
21. The device of any one of claims 13-20 operably connected to one or more of a function generator, instrumentation amplified, lock-in amplifier, oscilloscope, a data processor, and a display monitor.
22. A device for assaying particles in a sample, the device comprising at least three planar electrodes and a fluidic channel overlaying at least a portion of the three electrodes.
23. The device of claim 22, wherein the fluidic channel comprises an inlet, an outlet, and a constriction region having a width between about 1 pm and about 100 pm at a region overlaying the electrodes.
24. The device of claim 22, wherein the fluidic channel has a width between about 100 pm and about 10000 pm at a region overlaying at least a portion of the electrodes.
25. The device of claim 24 forming a sensing region electrically confined to a region around the portion of the electrodes overlaid by the fluidic channel.
26. The device of any one of claims 22-25, wherein the electrodes have a width between about 1 pm and about 1000 pm.
27. The device of any one of claims 22-26 operated with an alternating current (AC).
28. The device of claim 27, wherein the AC is between 10 kHz and 10 MHz and a peak-to-peak voltage amplitude is between 0.5 V and 10 V.
29. The device of any one of claims 22-28 operably connected to one or more of a function generator, instrumentation amplified, lock-in amplifier, oscilloscope, a data processor, and a display monitor.
30. A method for capturing and characterizing a particle from a sample comprising applying the sample to a device of any one of claims 1-21.
31. The method of claim 30, comprising applying an alternating current (AC) to the at least two electrodes of the trap module in a single unit at a frequency at which the CM factor for the particle is maximum, thereby trapping the particle in the unit.
32. The method of claim 30 or 31, wherein the particle is a biological material.
33. The method of claim 32, wherein the device comprises more than one unit, each unit adapted for capturing different species of biological material from the same sample.
34. The method of claim 33, comprising applying different AC to the at least two electrodes of the trap module in different units, each different AC at a frequency at which the CM factor for each of the different species of biological material is maximum, thereby trapping each of the different species of biological material in the different units.
35. The method of any one of claims 32-34, further comprising condensing the trapped biological material in the identification module.
36. The method of claim 35 comprising closing the outlet of the primary microfluidic channel, opening the inlet of the secondary microfluidic channel, stopping the AC applied to the at least two electrodes of the trap module, and applying an AC to the at least two electrodes of the identification module to condense the biological material from the trap module in the identification module.
37. The method of claim 35 or 36, wherein the biological material in the identification module is processed for microscopic examination.
38. The method of any one of claims 32-37 comprising flowing a sample with biological material through the device at varying flow rates in the trap module, identification module, and/or counter module, the flow rate between about 0.2 pm/sec and about 2xl07 pm/sec.
39. The method of claim 38, wherein the sample is selected from the group consisting of undiluted or diluted blood, plasma, spinal fluid, urine, stool, tears, saliva, sputum, mucus, and exudate.
40. The method of claim 38 or 39, wherein the sample is an undiluted or diluted blood sample with a central line associated bloodstream infections (CLABSI).
41. The method of claim 40, wherein the undiluted or diluted blood sample comprises a biological material selected from the group consisting of bacteria, viruses, fungi, and parasites.
42. The method of claim 40 or 41, wherein the blood sample comprises one or more microorganisms from a genus selected from the group of consisting of Staphylococcus, Enterococcus, Klebsiella, Enterobacter, Pseudomonas, Escherichia, Acinetobacter, Listeria, and Candida.
43. The method of any one of claims 32-42, wherein the biological material is a microorganism selected from the group consisting of S. aureus , S.
epidermidis, E. faecium, C. albicans , Salmonella, Listeria monocytogenes, and E. coli.
44. A method of assaying particles in a sample, the method comprising flowing the sample through the device of any one of claims 1-29.
45. The method of claim 44, wherein assaying comprises obtaining particle characteristics selected from the group consisting of number of particles, size of particles, shape of particles, activation state of particles, division state of particles, metabolic state of particles, live/dead determination, surface marker expression of particles, and combinations thereof.
46. The method of claim 44 or 45, wherein the sample is selected from the group consisting of undiluted or diluted blood, plasma, spinal fluid, urine, stool, tears, saliva, sputum, mucus, exudate, liquids from food preparation, liquids from food storage, liquids from cooking, runoff from meat processing, runoff from fruit and vegetable processing, waters from streams, rivers, waters from lakes, waters from seas, swimming pool samples, drinking water, industrial water, industrial runoff, rainwater runoff, and stream water.
47. The method of any one of claims 44-46, comprising flowing the sample through the device at varying flow rates in the trap module, identification module, and/or counter module, the flow rate ranging between about 0.2 pm/sec and about 2xl07 pm/sec.
48. The method of any one of claims 44-47, wherein the particle
characteristics are displayed on a display monitor as quantitative and/or qualitative data.
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