WO2023023492A1 - Procédés, compositions et dispositifs de fabrication de nanoparticules lipidiques solides et de supports lipidiques nanostructurés - Google Patents

Procédés, compositions et dispositifs de fabrication de nanoparticules lipidiques solides et de supports lipidiques nanostructurés Download PDF

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Publication number
WO2023023492A1
WO2023023492A1 PCT/US2022/074985 US2022074985W WO2023023492A1 WO 2023023492 A1 WO2023023492 A1 WO 2023023492A1 US 2022074985 W US2022074985 W US 2022074985W WO 2023023492 A1 WO2023023492 A1 WO 2023023492A1
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fluid
channel
fluidic
port
microfluidic
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PCT/US2022/074985
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English (en)
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Deepak E. SOLOMON
Yadir A. GUERRERO
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Unchained Labs
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/52—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips
    • B01L9/527—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips for microfluidic devices, e.g. used for lab-on-a-chip
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • 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
    • B01L3/50273—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 characterised by the means or forces applied to move the fluids
    • 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
    • B01L3/502746—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 characterised by the means for controlling flow resistance, e.g. flow controllers, baffles
    • 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
    • B01L2200/0605—Metering of fluids
    • 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
    • B01L2200/0621—Control of the sequence of chambers filled or emptied
    • 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
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/02—Investigating particle size or size distribution
    • G01N15/0205—Investigating particle size or size distribution by optical means
    • G01N15/0227—Investigating particle size or size distribution by optical means using imaging; using holography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1484—Optical investigation techniques, e.g. flow cytometry microstructural devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1493—Particle size

Definitions

  • This disclosure is generally related to the manipulation of fluids in a microfluidics environment.
  • Lipid based nanoscale delivery vehides have obvious advantages over polymeric based delivery methods that include components that are generally regarded as safe and their ease of manufacture.
  • the most widely used lipid-based nanoparticle for gene delivery are liposomes, these bilayer particles are versatile in their ability to encapsulate hydrophilic as well as hydrophobic drugs and are relatively safe compared to other non-viral vectors.
  • ionizable Cationic Lipid Nanopartides are the most advanced form of liposomes that utilize biphasic lamellar structures to encapsulate gene therapy cargo (mRNA, siRNA or DNA) [REF), the ionizable nature of these lipids allow the vectors to maintain a near-neutral charge at neutral pH and a positive charge at addle pKa allowing uptake in the endosomal space of the cell.
  • the ability to maintain near neutral charge under physiological conditions also allows the Ionizable Cationic Lipid Nanoparticles to have a better safety profile compared to purely cationic delivery vehicles. Recent studies have also shown that the transfection efficiency of these delivery nanopartide vectors can be as high as 90% for cell lines that range from respiratory to cancer ceils’.
  • nanopartide vectors have enabled transfection of several cell types in-vitro and in-vivo there are still some hurdles that need to be overcome, some of these Include (a) The use of an alcohol phase for their manufacture which requires additional post processing steps for removal. *(b) Stability issues that prevent the nanoparticle from being stored at room temperature or limit their shelf life, (c) Increase in PDI and size after post-processing steps to remove solvent, (d) Limited concentrations that can be formulated without appreciable changes in the quality attributes of the nanopartide vectors, (e) Limited ability for controlled release.
  • This disdosure provides fluidic devices that are useful in the production of particles, such as microparticles and nanopartides, and peptides enclosed in nanoparticles.
  • an instrument comprising a piston pump, or in illustrative embodiments a series of piston pumps, fluidly connected to fluid lines that are connected to one or a series of cartridge nests that are connected to the fluid lines.
  • Each cartridge nest is adapted to and/or configured to secure a disposable microfluldic cartridge according to any aspect or embodiment provided herein, such that no manual connection of tubing to the cartridge is required for a user. Further details regarding a non-limiting illustrative embodiment of such an instrument are provided in HG. 28 and FIG. 29.
  • this disclosure provides a fluidic device (e.g., a microfluidic instrument) that comprises a first port; a first fluid transport channel In direct fluid communication With the first port, a reaction well; an overflow channel; a second fluid transport channel in direct fluid communication with the overflow channel; a fluidic constriction channel in direct fluid communication with the reaction well and the second fluid transport channel; and, a second port in direct fluid communication with the second fluid transport channel (e.g., as illustrated in FIGS. 1, 2, and 1O-14A) , or a fluidic device assembly comprising at least two of such microfluldic devices, or comprising other microfluidic devices provided herein, including in parallel or in serial.
  • methods for using such fluidic devices e g., microfluidic instrmuments
  • to produce partides are provided,
  • the nanoparticles that are produced by mixing a melted lipid and aqueous stream can be flown into straight channels for laminar cooling eg. Fig. 41.
  • the laminar cooling channel is designed such that there is minimal mixing of the streamlines in order to obtain homogenous solid lipid nanopartides.
  • the straight channels can be cooled to between -4 degrees C to 20 degrees C which are electronically controlled by heating elements around the microfluidic channels.
  • the mxing channel is typically heated to above the melting point of the lipid (35 degrees C to 120 degrees C) wherein the immediate codling of this heated lipid mixture produces solid lipid nanoparticles,
  • Some variations of the method may include moving a first fluid at a first flow rate through the microfluidic instrument in a first laminar flow stream comprising a melted lipid and moving a second fluid at a second flow rate through the microfluidic instrument in a second laminar flow stream comprising an aqueous phase.
  • the first flow rate, the second flow rate, or both the first and second flow rates may be used to control the size of the nanopartides that are produced.
  • the first flow rate, the second flow rate, or both the first and second flow rates may be set at a target flow rate to achieve a target diameter of the nanoparticles produced by the microfluidic instrument.
  • the products produced from either just mixing or mixing and cooling the reaction products can be further flown into straight channel sections where the size attributes can be monitored in a continous manner using an absorbance based measurement.
  • FIG. 1 illustrates an exemplary fluidic device that can be used to produce nanopartides.
  • This exemplary fluidic device is composed of a first port (part 1), first fluid transport channel (part 1A), reaction well (part 2), overflow channel (part 3), fluidic constriction channel (part 4), second fluid transport channel (part SA), and second port (parts).
  • FIGS. 2A-2C illustrates the introduction and removal of fluids from an exemplary fluidic device that can be used to produce nanoparticles.
  • FIG.2A Step one: introduction of first fluid into fluidic device to fill reaction well 2 (solid coloring representing the firstfluid (e.g., organic solvent solution comprising dissolved lipids or polymer solution);
  • FIG. 2B Step two: removal of first fluid (e.g., organic solvent solution comprising dissolved lipids or polymer solution);
  • FIG. 2C Step three: introduction of second fluid (e.g., aqeous buffer or water-soluble synthetic polymer solution) into fluidic device to mix with first solution thereby producing nanoparticles.
  • second fluid e.g., aqeous buffer or water-soluble synthetic polymer solution
  • FIG. 3 provides the size distribution plot of five batches of liposomes produced using an exemplary fluidic device.
  • FIG. 4 provides transmission electron microscopy of liposomes produced using an exemplary fluidic device.
  • FIG. 5 provides results obtained using fluidic devices having fluidic constriction channels of different widths (relative to each device) and washing rates.
  • FIG.6 shows the average number-weighted size of the lipid-based micelles prepared using D5PE- PEG dissolved in ethanol as the first fluid and PBS as the second fluid.
  • FIG. 7 shows the average number-weighted size of the polymeric micelles prepared using PEG* PLGA dissolved in ethanol as the first fluid and PBS as the second fluid.
  • FIG. 8 shows the average number-weighted size of the polymeric micelles prepared using PEG* PLGA dissolved In acetone as the first fluid and distilled water as the second fluid.
  • FIG. 9 shows the average number-weighted size of the polymeric micelles prepared using PEG* PLGA dissolved in ethanol as the first fluid and PBS as the second fluid.
  • FIG.10 illustrates an exemplary fluidic device including six pairs of two pillars 7 distributed within the reaction well 2 between the junction With the first fluid transport channel 1A and the fluid constriction channel 4 that can be used to produce nanoparticles.
  • FIG. 11 illustrates an exemplary fluidic device in which the shape of the reaction well 2 was changed by reducing the curvature of the well oh one side to alter flow patterns inside the reaction well 2 that can be used to produce nanopartides.
  • RG. 12A illustrates an exemplary fluidic device in which multiple fluidic device subunits are connected in series, wherein the first fluid transport channel 1A of one device in the series is continuous with the second fluid transport channel 5A of the next device in the series.
  • RG. 128 illustrates an exemplary fluidic device in which multiple fluidic device subunits are connected in series, wherein the first fluid transport channel 1A of one device in the series is continuous with the Second fluid transport channel 5A of the next device in the series, but wherein the overflow channels 3 of each subunit are on the opposite side of at least two, but optionally each, successive fluidic device subunit.
  • FIG. 13 illustrates an exemplary fluidic device comprising multiple fluidic device subunits connected to one another by a common fluid transport channel 9 which is connected to a common port
  • RG. 14A illustrates an exemplary embodiment of a fluidic device comprising multiple fluidic device subunits with two inlet channels (12, 14) having associated separate inlet ports (11 and 13, respectively) that form a Y junction in fluid communication with the first fluid transport channel 1A of the first fluidic device in a series of fluidic devices.
  • FIG. 148 provides the number-weighted size distribution for liposomes formulated using these first and second fluids in the device of RG. MA.
  • RGS . 15 and ISA illustrate an exemplary fluidic device in which 1 is a first port; 1A is a first fluid transport channel; 1A1 is a straight section of the first fluid transport channel 1A; 1A2 is a rounded section of the first fluid transport channel 1A; 2 is a reaction well; 4 is a fluidic constriction channel; 3A is passive pressure sensing channel; 3 is a second port; SA is a second fluid transport channel; 58 Is a third fluid transport channel; SC is an interface channel segment; and 6 Is a third port.
  • RGS. 16A-16C provide diagrams illustrating fluid flow while filling a device according to FIG. 15.
  • FIG. IGA shows initial introduction of a first fluid into the device through the first port 1.
  • FIG. 168 shows further filling of the device, partially filling the reaction well 2 and third fluid transport channel 58.
  • FIG. 16C shows complete filling of the reaction well 2 with first fluid and partial filling of the third fluid transport channel SB.
  • FIGS. 17A-17C provide diagrams illustrating fluid flow when fluid is withdrawn from a device according to RG. IS. RG. 17A: The first fluid fills most of the device as depicted in FIG. 16C.
  • RG. 178 A negative pressure is applied to the first port 1, causing the first fluid to begin to recede from the device towards the first port 1.
  • the geometry and dimensions of the reaction well 2, passive pressure sensing channel 3A» fluidic constriction channel (not numbered here but the part corresponding to 4 in FIG. 15) and fluid transport channels (SA, 58, SC, and 1A) prevents fluid in the reaction well 2 from leaving the device.
  • FIG. 17C Due to the design of the device, a small volume of first fluid has been captured in the reaction Well 2. At this stage using this device embodiment, essentially no other parts of the device retain any fluid (i.e. the rest of the device is empty).
  • FIGS. 18A-18D provide diagrams illustrating steps in an embodiment for using the device according to FIG. 15.
  • Top left panel FIG 18A A second fluid (e.g. human sweat) enters the device with reaction well 2 filled with test solution from the third port 6 via positive applied pressure, and enters the third fluid transport channel SB. Not illustrated as a separate figure, fluid from the second fluid reaches the reaction well 2, where it interacts with the captured first fluid.
  • Top right panel FIG 188 Fluid from the second fluid continues to flow through the device towards the first port 1 where it exits the device.
  • FIGS. 18A and Ito depict precipitate development in the device illustrated in FIG.15.
  • FIG 18C As second fluid (e.g., sweat) mixes with firstfiuid (e.g.
  • FIG Ito Precipitate PPT continues to grow the entire length of the second fluid transport channel SA. as more second fluid flows into the device and interacts with first fluid. Precipitate grows until it eventually blocks the second fluid transport channel, inhibiting incoming flow.
  • FIG. 19 shows a screenshot (still frame) from a video showing precipitate formation ("PPI”) following the interaction of the first and second fluids.
  • the device used to produce the precipitate shown in this figure included an optional thin channel 7 extending from the passive pressure sensing channel to the first fluid transport channel, not shown in FIG. 15. The width of that channel was the same width as the pressure sensing channel. Removing this optional thin channel 7 had no signficant effect on the functionality of the device.
  • FIG 20 illustrates a further exemplary embodiment of a fluidic device comprising multiple fluidic device subunits in series, with two inlet channels (12, 14) having associated separate inlet ports (11 and 13, respectively) that form a Y junction in fluid communication with the firstfiuid transport channel 201A of the first fluidic device in the series of fluidic devices.
  • the microfiuidic device shown in RG. 20, as a non-limiting example. Include 12 microfiuidic device subunits as 4 rows of 3 microfluidic device subunits each, with each microfiuidic device subunit connected in a series. As demonstrated in the Examples herein, the design in FIG.
  • the device with small dimensions functions the same as the design with large dimensions but is capable of forming smaller nanoparticles due to its reduced dimensions as provided in the Examples herein.
  • FIGS. 21A and 218 illustrate microfluidic flow (FIG. 21A) and mixing (FIG. 218) within an exemplary microfluidic device similar in design to the microfluidic device of FIG. 20.
  • An increased magnification view of one of the microfluidic devices is shown in the inset of FIG. 21A.
  • a representative image of micropartides produced using such a device is shown in the inset of FIG. 218, with the bar representing 1,000 nm.
  • FIGS.22A and 228 provides data generated using the a microfiuidic device with the design shown in FIG. 1.
  • FIG. 22A is a graph showing the effective diameter and polydispersity index (PDI) of four batches (Formulation Number 1-4) of liposomes formulated in the device and analyzed by DLS.
  • FIG.228 is a graph showingthe effective diameter/size and polydispersity index (PDI) of liposomes generated with identical input first liquid and second liquid, but different flow rates.
  • PDI effective diameter and polydispersity index
  • FIGS.23Ato23D provide data generated using the a microfluidic device with the large dimension embodiment of the design shown in FIG. 20.
  • FIG. 23A is a graph showing the effective diameter and polydispersity index (PDI) of three batches (Formulation Number 1-3) of liposomes that were made using the device and analyzed by DIS.
  • FIG. 238 is a graph showing the effective diameter and PDI of liposomes generated holding all parameters constant, but inputting a first fluid and second fluid at different flow rates.
  • FIG. 23C is a graph showing the effective diameter and PDI of liposomes generated with identical input first fluid and second fluid, but different flow rate ratios of an input stream of the first fluid to an Input stream of the second fluid
  • FIG. 23D is a graph showing the effective diameter and PDI of liposomes collected at different points during the process of flowing IL of combined first fluid and second fluid through the fluidic device.
  • FIGS.24A to 24C provide data generated using the a microfluidic device with the small dimension embodiment of the design shown in FIG. 20.
  • FIG. 24A is a graph showing the effective diameter and polydispersity index (PDI) of four batches (Formulation Number 1-4) of liposomes formulated in the device and analyzed by DLS.
  • FIG. 248 is a graph showing the effective diameter and PDI of liposomes generated with identical input first fluid and second fluid, but different flow rates.
  • RG. 24C Is a graph showing the effective diameter and PDI of liposomes generated with identical input first fluid and second fluid, but different flow rate ratios of an input stream of the first fluid to an input stream of the second fluid.
  • HG.25 provides data generated using the a microfluidic device with the large embodiment of the design shown in FIG.20 to precipitate proteins. Precipitate efficiency Is graphed for protein precipitation experiments performed at different flow rate rations of BSA to TCA.
  • FIG. 26 illustrates a further exemplary embodiment of a fluidic device similar in design to the device shown in FIG. 20 and FIG.21A that farter includes an interface tracking channel for quality control.
  • FIG.27 illustrates a scale-up fluidic system that includes 5 fluidic device assemblies99 in parallel.
  • FIG. 28 provides an illustrative instrument for making partides according to an aspect provided herein.
  • the instrument is illustrated with piston pumps and a dosed damping manifold.
  • FIG. 29 provides an illustrative instrument for making partides according to an aspect provided herein.
  • the instrument is illustrated with 5 microfluidic cartridges according to any of the embodiment provided herein, loaded into cartridge nests that connect directly to fluid lines.
  • RG.30 is a flow diagram of an exemplary process of manufacturing SLNs and NLCs using rapid heating and cooling of microfluidic assemblies.
  • RG.31 illustrates a NeoNano Server , a temperature controlled system -4-85 °C with a positive displacement pump, tor the manufacture of solid lipid nanopartides, and a workflow diagram.
  • RG32 is a graph of comparison of nanopartide properties, size control, with conventional methods of preparation using a Glycerol distearate (Precirol) as a lipid and comparison to cooling on microfluidic chip assembly.
  • Precirol Glycerol distearate
  • RG33 is a graph of comparison of nanoparticle properties with conventional methods of preparation, with cooling on microfluidic chip assaembly where size can be controlled with flow rate on cooling chip without the need for excess dilution.
  • Nanoparticles are cationic solid lipid nanopartides, and lipid used is Glycerol distearate ⁇ Octadecylamine (Cationic Lipid ⁇ .
  • FIG34 is a graph of comparison of nanoparticle properties with conventional methods of preparation, with cooling on microfluidic chip assaembly where size can be controlled depending on flow rate on cooling chip without the need for excess dilution.
  • Nanopartides are cationic solid lipid nanopartides, and iipid used is Glycerol monostearate.
  • FIG35 is a graph of comparison of nanoparticle properties with conventional methods of preparation, with cooling on microfluidic chip assaembly where size can be controlled without depending on flow rate on cooling chip without the need for excess dilution.
  • Nanopartides are cationic solid Ifpid nanopartides, and lipid used is Glycerol distearate plus Octadecylamtne (Cationic Upld).
  • HG36 is a graph of comparison of nanoparticle properties with conventional methods of preparation, with cooling on microflutdicchip assaembly where size can be controlled without depending on flow rate on coding chip without the need for excess dilution.
  • Nanopartides are cationic solid lipid nanopartides, and lipid used is Glycerol distearate + Octadecylamine (Cationic Lipid). A particle diameter profile.
  • FIG37 is a graph of comparison of nanoparticle properties with conventional methods of preparation, with cooling on microfluidic chip assaembly where size can be controlled without depending on flow rate on cooling chip without the need for excess dilution.
  • Nanopartides are cationic solid lipid nanopartides, and iipid used is Glycerol distearate ⁇ Octadecylamine (Cationic lipid). A PDI profile. Formulation 2.
  • FIG38 is a graph of comparison of nanoparticle properties with conventional methods of preparation, with cooling on microfluidic chip assaembly where size can be controlied without depending on flow rate on coding chip without the need for excess dilution, and cationic solid lipid nanoparticles.
  • Nanopartides are cationicsolid lipid nanopartides, and lipid used is Glycerol distearate + Octadecylamine (Cationic Lipid).
  • FIG.39 is a graph of comparison of nanoparticle properties with conventional methods of preparation, with cooling on microfluidic chip assaembly where size can be controlled without depending on flow rate on cooling drip without the need for excess dilution.
  • Nanopartides are cationic solid lipid nanopartides, and lipid used is Glycerol distearate * Octadecylamine (Cationic Lipid).
  • FIG.40 is a graph of stability of nanopartides manufactured using the NeoNano, a microfluidic device as exemplified in FIGS. 30 -31.
  • FK5.41 illustrates an exemplary microfluidic coding subunit device with straight channels.
  • an instrument comprising a volumetric pump(s), in non-limiting illustrative embodiments a plurality of volumetric piston pumps, fluidly connected to fluid lines that are connected to one or a series of cartridge nests that are connected to the fluid lines.
  • Each cartridge nest is adapted to and/or configured to secure a disposable microfluidic cartridge according to any aspect or embodiment provided herein, such that no manual connection of tubing to the cartridge is required for a user.
  • two piston pumps are provided per cartridge nest, such that each piston pump is adapted to and/or configured to drive fluid through one input port of a cartridge when it is loaded into a cartridge nest. Further details regarding a non-limiting illustrative embodiment of such ah instrument are provided in FIG. 28 and FIG. 29.
  • a fluidic cartridge or in illustrative embodiments a plurality (e.g. 2-12, 2-10, 2-8, 4-8, 4-6, or 5, as non-limiting examples) of microfluidic cartridges each comprising one, or in illustrative embodiments a series of microfluidic device subunits provided herein, are removably loaded and/or secured onto a cartridge nest in the following manner: On the face of each cartridge nest, there is a deformatie gasket that has through holes for the 3 ports (2 inlet and 1 outlet).
  • the cartridges When the cartridge is placed into the nest and the door is closed, the cartridges are pressed up tightly against the gasket, such that the through holes on the cartridge align with the through holes on the gasket.
  • the fluid lines connect to the undertide of the nest and line up with the 3 through holes on the gasket and the cartridge.
  • the fluid will go directly from the 2 Inlet fluid lines into the cartridge, and the formulation (fluid output) will go directly from the cartridge into the outlet fluid line.
  • a plurality of cartridges are loaded and fluidly connected to the pumps via tubing upon loading the cartridges onto the cartridge nests without any additional user intervention.
  • the heated surfaces and fluid lines throughout are heated to maintain temperature within the instrument ⁇ /* 10*C, with temperature ranging from 37- 100’C and the cooled surfaces are cooled to maintain temperature within the instrument +/- 2*C, with temperature ranging from -4 to 20*C .
  • the instrument has the following features: Flow rate- controlled piston pumps (xlO); 2 pumps per cartridge for the two input streams; Flow rate for each pump can be controlled between 0.1 and 30mL/min. Further non-limiting features are indicated on FIG. 28 and FIG. 29.
  • fluidic devices e.g., microfluidic instruments
  • the devices indude highly efficient mixing that is partially responsible for providing the devices the ability to solve numerous problems in the art
  • the fluidic devices are easy to use and provide consistent results from batch to batch and within a batch.
  • exemplary embodiments of fluidic devices provided herein can be used to produce particles, for example nanoparticles, with the ability to control particle size and can be used for straightforward scale-up from microlilters to liters, with consistent results and an optional continuous flow process.
  • exemplary embodiments of fluidic devices provided herein can be used to produce protein precipitates that allow for continuous precipitation of proteins without the need for an incubation period and that can be used to produce protein precipitates of lower concentrations than traditional batch incubation/agitation methods, thus reducing the chance for undesirable structural changes in precipitated proteins of interest.
  • a "fluidic device" of tills disclosure is a device titrough which one or more fluids can be transported and / or moved through the same. The movement of the one or more fluids can be, for instance, through passages formed within and / or upon such a device. Illustrative fluidic devices of this disclosure are illustrated in FIGS. 1, 2, 10-14A, 15-19, 20, 21, and 26.
  • the fluidic device can be a millifluidic, microflufdic, nanofluidic, or picofluidic device in which the amount of fluids within, stored within or moving within said device can be in milliliter, microliter, nanoliter, and / or picoliter amounts.
  • the reaction well is configured to hold milliliters (ml) of a fluid. In other embodiments, the reaction well is configured to hold microliters (pl) of a fluid. In other embodiments, the reaction well is configured to hold nanoliters (nl) of a fluid. In other embodiments, the reaction well is configured to hold picoliters (pl) of a fluid.
  • a fluidic device presented herein can be a millifluidic, microfluidic, nanofluidic, or picofluidic device. In illustrative embodiments, the fluidic device is a microfluidic device.
  • the fluidic devices described herein typically comprise multiple parts or regions therein through Which fluids can move and/or in which fluids can be stored and/or manipulated.
  • Channels and other parts e.g. reaction wells
  • Parts and/or regions within fluidic devices and fluidic circuits herein can include, for ecample, one or more ports, one or more air valves (eg., associated with or connected to a port), one or more channels that can form a fluidic connection, one or more high resistance air valve constriction channels, one or more reaction wells, one or more overflow channels, one or more pressure sensing channels, and one or more fluid transport channels.
  • the fluidic device is typically positioned upstream (relative to movement of air or fluid through the fluidic device) of the fluidic connection.
  • the fluidic device also includes one or more inlets and / or outlets (e.g ⁇ , ports) that may perform as an inlet, an outlet, or both.
  • the different parts and/or regions typically communicate with one another either directly or indirectly with respect to fluids moving through the same (e.g., the parts or regions are in "fluid connection,” “fluid communication” or “fluidic communication* with one another (e.g., the parts or re@ons "fluidly communicate* with one another)).
  • Direct communication between parts and/or regions means that a fluid moves directly from one part or region to another without passing through an intermediary part or region, which can be referred to herein as "direct fluidic communication”.
  • fluidic constriction channel 4 is in direct fluidic communication with reaction well 2, and fluid transport channel SA.
  • Indirect communication in contrast, means that fluid moves from one part or region to another through an intermediary part or region, referred to herein as “indirect fluidic communication,* "indirect fluid communication,” or "indirect fluid connection.”
  • X reaction well 2 is in indirect fluidic communication with fluid transport channel SA as the two parts or regions are each directly connected to fluidic constriction channel 4 but not to one another.
  • the parts of the fluidic device illustrated in RG. IS may also be arranged to be in fluidic communication with one or more other parts of such a fluidic device.
  • individual fluidic devices can also be connected to one another in a series, which sometimes can be referred to herein as a "fluidic system," a “fluidic assembly,* ora series of microfluidic device subunits. Examples of multiple fluidic devices or device subunits connected to one another in series are shown in FIGS. 12A, 128, 14A, 20, 21A, and 26. tn such embodiments, each fluidic device can be attached to one another though a fluid transport channel. For instance, RG.
  • FIG. 12A shows a first fluidic device connected to a second fluidic device through fluid transport channels SA and 1A, which collectively can be referred to as "intradevice fluid transport channel",
  • the second fluid transport channel of the first fluidic device e.g., SA in FIG. 12A
  • the fluid transport channels are typically in direct fluidic communication with one anotiter.
  • a fluidic device can include multiple fluidic devices, also referred to in such configurations as fluidic device subunits, connected in series, wherein the first fluid transport channel of a device in a series is continuous with the second fluid transport channel of the next device in the series (e.g., 1A and SA as Illustrated in FIGS. 12A and 128).
  • the reaction well of some of the or each fluid transport channels can be in fluid communication with an air control valve.
  • the fluidic devices described herein typicaily include a "fluidic constriction channel" (part 4 in figures that illustrate a microfluidic device) in direct fluidic communication with reaction well and a fluid transport channel.
  • a fluidic constriction channel 4 typically has a smaller diameter or width than a diameter or width of the reaction well and an overflow channel in the same fluidic device, or fluidic device subunit in embodiments that include a fluidic device comprising more than one fluidic device subunit.
  • a "fluidic constriction channel” has a size and shape relative to a reaction well and overflow channel of the same fluidic device, or the same fluidic device subunit for fluidic devices comprising more than one fluidic device subunits, that makes the fluidic device capable of, operable to, effective for, and adapted to retain fluid for a longer time period in the reaction well as fluid is introduced into the fluidic device, for example when the volume of fluid introduced into the fluidic device exceeds the combined capacity of its channels and wells.
  • the fluidic constriction channel has a size and shape relative to a reaction weii and overflow channel of the same fluidic device, or the same fluidic device subunit for fluidic devices comprising more than one fluidic device subunits, to retain fluid in the reaction well when liquid is removed from the fluidic device.
  • the fluidic device can retain fluid in a reaction well and the fluidic contriction channel when a negative pressure is applied through a first port 1 of a microfluidic device or microfluidic device subunit that is full of fluid.
  • die fluidic constriction channel is directly connected to reaction well 2 opposite a first fluid transport channel 1A, and has a smaller diameter or width, typically less than one-fifth and in some embodiments less than one-sixth, one-seventh, one- eighth, one-ninth, or one-tenth the diameter or width of each of the following components: the first fluid transport channel 1A, the reaction well 2, a second fluid transport channel SA, directly connected to the fluidic constriction channel 4 opposite the reaction well 2, and an overflow channel 3 that connects the first fluid transport channel 1A to the second fluid transport channel 5A as provided herein.
  • first and second inlet port also called an input port herein
  • first and second port channel ports sometimes called coflowtng fluidic devices herein
  • the size and configuration of the fluidic constriction channel relative to the first fluid transport channel 1A, the reaction well 2, the second fluid transport channel SA, and the overflow channel 3 within the same fluidic device are such that the device is capable of, operable to, or adapted to effectively, or more effectively mix a first fluid and a second fluid entering the device through different ports connected to the same reaction well through a channel.
  • the difference in widths (Which is directly a difference in hydrodynamic resistances) between different parts of fluidic devices herein, for example between the fluidic constriction channel 4 and the other parts listed above, causes a differential pressure drop at any two regions where smaller and larger channels meet, for example where the reaction well 2 and the fluidic constriction channel 4 meet.
  • This unstable flow in illustrative embodiments, is not “turbulent”, and thus makes fluidic devices herein that have such structure, designed to, operable to, capable of, and adapted to transform, or effective for transforming, an input laminar flow fluid stream into an unstable flow, but in illustrative embodiments not a turbulent flow. Furthermore, these properties thus makes fluidic devices herein that have such structure and are used to make partides (e.g.
  • microfluidic devices that are used to make microparticles or nanopartides), effective for controlling particle size and adapted to control particle size, gives them the ability to control monkeye size, and makes them operable to control particle size.
  • Such effective or more effective mixing results in relatively uniform, or more uniform particle sizes.
  • Reynolds Number ratio of inertial to viscous forces. Flows below Re of 2100 are usually accepted as laminar and above it is turbulent.
  • Illustrative embodiments of fluidic devices herein are effective for, adapted to, capable of, and operable to achieve a Re of less than 2000, less than 1500, less than 1000, or in further illustrative embodiments, less than 500.
  • fluidic constriction channel 4 can be referred to herein as "fluid constriction channel", "fluid connection channel”, fluid connection channel
  • the "reaction well* is typically a compartment or region (e.g., a depression) of the fluidic device into which in illustrative embodiments a first fluid (i.e. liquid) (e.g. an initial reagent (e.g., lipids in an organic solvent or a protein)) can be mixed with a second, third, fourth, or more fluid, or in which two or more fluids that are simultaneously input into a device herein are retained for longer periods than those traveling through an overflow channel, such that they can mix, or in which a first fluid can be stored until a second fluid is flowed into the device for example to mix in tire reaction well or to interact with a fluid in the reaction well and fluidic constriction channel.
  • a first fluid i.e. liquid
  • an initial reagent e.g., lipids in an organic solvent or a protein
  • the shape of the reaction well is configured for production of a particular particle size, or precipitate detection reaction.
  • a reaction well can have many different shapes and configurations, for example any of the foltowing shapes: angular, square, rectangular, trapezoidal, circular, triangular, and/or the like such as cylindrical Exemplary reaction wells, and shapes thereof include part 2 in figures herein that illustrate a fluidic device.
  • a device herein comprises a reaction well configured to hold, contain, or retain, operable to hold, contain, or retain, capable of retaining, adapting, or holding, or adapted to hold, contain, or retain a volume between 100 pl and 10 ml, between 1 ni and 10 ml, between 1 pl and 10 ml, between 1 nl and 10 ml, between 1 pl and 450 pi, between 5 ni and 15 nl, between 15 nl and 35 nl, between 100 nl and 1 ml, between 100 nl and 100 pl, between 1 pl and 1 ml, between 5 pl to 30 pl, between 10 pl and 1 ml, between 1 pl and 500 pl, between 10 pl and 500 pl, between 10 pl and 250 pl, between 10 pl and 200 pl, between 10 pl and 100 pl or between 10 pl and 50 pl, or about 10 pl.
  • An "overflow channel* of any of toe fluidic devices described herein provides a path through which fluid flows around a reaction well.
  • the overflow channel(s) is typically connected to, and in illustrative embodiments in direct fluidic communication with a fluid transport channel and / or reaction well as shown for example in FIGS.1 (e.g., overflow channel 3) or Fig.10 (overflow channel 3).
  • An overflow Channel typically follows a rounded shape around at least a portion of a reaction well, and thus provides a rounded path for fluid that does not enter the reaction well, for example if fluid is input into a device in excess of toe volume of toe reaction well, to flow around toe reaction well.
  • a fluid transport channel such as for example, parts 1A and 5A of any of the figures herein that illustrate a fluidic device is a channel through which fluids move in a fluidic device herein, typically between a port, an overflow channel, a reaction well, and/or a fluidic constriction channel. Accordingly, such fluid transport channels can be in direct fluidic communication with, for instance, a reaction well and/or an overflow channel. Such fluid transport channels can alternatively be in direct fluidic communication with, for instance, an overflow channel and a fluidic restriction channel. Such fluid transport channels can also be connected to one or more ports through which fluid can enter or exit the fluid transport channel.
  • An "intradevice transport channel” can be a fluid transport channel formed between devices or device subunits that are connected to one another (e.g., in fluidic communication with one another) for example in series.
  • Fluidicdevices provided herein in certain illustrative embodiments comprise an "air control valve" which is a valve through which air can enter or leave the fluidic device.
  • a valve can allow air to move into, or alternatively out of, the fluidic device when open to the surrounding atmosphere.
  • an air control valve can be used to control which reaction well(s) are filled with a fluid that is introduced into a fluidic device, in a series of microfluidic device subunitsthat include such reaction wells. This control is accomplished by independently opening or dosing an air control valve connected to a reaction well as described in the International Patent Application publication WO 2018/200896 Al).
  • the pressure sensing channel can function similarly to the passive air control valve.
  • fluids input into fluidic devices herein have various compositions and can include, but are not limited to a fluid for the production of particles, a sample, such as a protein sample or a test deodorant sample, a protein precipitant, one or more buffers, water, and/or one or more wash solutions, in some embodiments, the fluid may be air but the term fluid Is typically used herein to indicate a liquid. Air is therefore typically referred to as such.
  • a sample such as a protein sample or a test deodorant sample
  • a protein precipitant one or more buffers, water, and/or one or more wash solutions
  • the fluid may be air but the term fluid Is typically used herein to indicate a liquid. Air is therefore typically referred to as such.
  • suitable fluids can be those known for such manufacture, for example an organic solvent, typically including one or more lipids, a polymer solution, water, or one or more aqueous buffers.
  • a pocket of air can be Introduced between a fluid or fluids, producing an "air plug",
  • the fluid between air plugs can be referred to as a "fluidic slug".
  • the same or different fluids can also be introduced into the same or different ports during operation of the fluidic device, as discussed further herein.
  • this disclosure provides a fluidic device that includes a first port; a first fluid transport channel in direct fluid communication with the first port, a reaction well; an overflow channel; a second fluid transport channel in direct fluid communication with the overflow channel; a fluidic constriction channel in direct fluid communication with the reaction well and the second fluid transport channel; and, a second port in direct fluid communication with the second fluid transport channel
  • a fluidic device that includes a first port; a first fluid transport channel in direct fluid communication with the first port, a reaction well; an overflow channel; a second fluid transport channel in direct fluid communication with the overflow channel; a fluidic constriction channel in direct fluid communication with the reaction well and the second fluid transport channel; and, a second port in direct fluid communication with the second fluid transport channel
  • fluidic device subunits and include a first port (part 1), first fluid transport channel 1A, reaction well 2, overflow channel 3, fluidic constriction channel 4, second fluid transport channel SA, and second port S, and optional pillars 7 in FIG. 10.
  • a first port part 1
  • first fluid transport channel 1A reaction well 2
  • overflow channel 3 overflow channel 3
  • fluidic constriction channel 4 second fluid transport channel SA
  • second port S and optional pillars 7 in FIG. 10.
  • Exemplary size ranges for each part of such a fluidic device is provided in Table 1, as well as sizes of non-limiting exemplary devices of FIGS. 1 and 20. It is noteworthy with respect to Table 1 and the dimensions provided for FIG.
  • the third fluid transport channel refers to the channels linking two wells in a series (labeled as parts SA and 1A), which can also be referred to as intradevice transport channels
  • the second fluid transport channel refers to channel 205A which is the channel of the final microfluidic device subunit in the series that is in direct fluidic communication with the outlet (i.e. second port 5).
  • the heights (or diameters) of the various parts are the same, but tn some embodiments the heights may differ (in some embodiments, e.giller the height of the fluidic constriction channel can be from 50-500 pm while the height of the other parts can range from 100-2,000 pm).
  • the dimensions shown in Table 1 can be applied to such fluidic devices but can also be modified within the non-limiting exemplary indicated ranges to fit the user's heeds.
  • a variety of combinations of heights, depths, widths (or diameters in the case of a circular channel), and lengths may be used for each part in the device to achieve desired functionality.
  • the fluidic devices are used to make partides, such as micropartides or nanoparticles, to make protein precipitates.
  • fluidic devices provided herein can include an air control valve, but in certain embodiments do not include an air control valve.
  • FIG. 20 (large)/FIG.20 (small) $
  • First fluid Length 5900pm/5900pm/2360pm I
  • Length 1000-10000 pm, 2000-7500 p _m, or transport Width; 1200 pm/1300 pm/520 pm 2000-10000 pm channel Width; 300-2300 pm, 400-2000 pm, 300- 1500 pm, or 1000-2000 pm
  • Width 1200pm/1200pm/480pm Width: 300-2300 pm, 400-2000 pm, 300- i 1500 pm, or 1200-2000 pm
  • Second fluid Length 5460pm/4500pm/1800pm
  • Width 4000pm/4000pm/1600pm 2500-10000 pm, or 5000-12000 pm Width: 1000-7000 pm, 1500-5000 pm, or 3000-6000 pm f Design Features Measurements Measurements
  • Exemplary device of FIG. 1/ Non-limiting exemplary ranges weee ; jWmwem FImGw.m 2m0wm (leawerwgiwew)i/weFwIwGeew.m 2e0mi (Nswmwemawllw)eemmw ji
  • the fludic device for producing a reaction product such as particles or a protein precipitant
  • a reaction product such as particles or a protein precipitant
  • the fludic device for producing a reaction product can be adapted to, configured to, and operable to regulate the mixing process of a first fluid trapped in the reaction well and a second fluid that washes through the device, for example after the second fluid is delivered into the device via a syringe pump.
  • a syringe pump for example, any number of pillars can be used and positioned as desired in the reaction well 2.
  • one or more pillars may be positioned in tire reaction well 2 proximal to (i.e., nearer to) tire junction between the reaction well 2 and the fluid connection 4, or proximal to (i.e., nearer to) the junction between the reaction welt 2 and the first fluid transport channel LA.
  • the reaction well 2 comprises: a) a first opening leading to fluidic constriction channel 4 and a second opening leading to the first fluid transport channel 1A, and wherein the at least one pillar is positioned: i) distally to the first opening and proximally to the second opening; ii) distally to the second opening and proximally to the first opening; or Hi) central to the first and second openings; b) at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15 or 16 pillars; and/or, c) three pillars positioned distallytothe first opening and proximally to the second opening; three pillars positioned distally to the second opening and proximally to tire first opening; or, an even number of pillars positioned in pairs distributed between the first and second openings.
  • reaction well 2 For instance, in one illustrative embodiment, six pairs of 100 pm-diameter pillars (made of the same material as at least most of the other parts of the fluidic device) were essentially evenly distributed within the reaction well 2 (FIG. 10, pair closest to the fluidic constriction channel 4 being labeled part 7).
  • shape of the reaction well was changed slightly (FIG. 11) by reducing the curvature of the well on one side to alter flow patterns inside the well.
  • Other variations on the basic design and these modifications may also be suitable as can be determined by those of ordinary skill in the art.
  • the fluidic device for producing a reaction product such as particles and/or a protein precipitant can comprise a first port; a first fluid transportchannel in direct fluid communication With the first port, a reaction well; and, an overflow channel; a second fluid transport channel In direct fluid communication with the overflow channel; a fluidic constriction channel in direct fluid communication with the reaction well and the second fluid transport channel; and, a second port in direct fluid communication with the second fluid transport channel; wherein: the overflow channel 3 has a length of between 8,000 and 15,000 gm.
  • the fluidic constriction channel 4 has a width or diameter of 50-500 pm, in illustrative embodiments 50-250 pm, or about 100 pm;
  • the reaction weli 2 comprises one or more of one or more lipids, an organic solvent, an alcohol, acetonitrile, one or more polymers, an aqueous buffer, a mixture thereof, and/or nanoparticles in solution; and/or, optionally the reaction well 2 comprises at least one pillar, optionally having a diameter of 50-250 pm, 50-150 pm or about 100 pm, wherein each pillar is the same or different from any other pillar and optionally has a circular, triangular, or rectangular shape; the ratio of resistance between the reaction well and overflow channel is 0.067-1, 0.2 to 0.5, 0,2 to 0.3, or 0.25, -the ratio of resistance between the overflow channel and fluidic constriction channel is 0.2-12.5, for example about 1.5 to 5, or for example 1.82; and/or,
  • a fluidic device for producing partides can comprise a first port, a first fluid transport channel 1A in fluid connection with a first port 1, a reaction weli 2, an overflow channel 3, a fluidic constriction channel 4; and, a second fluid transport channel SA in fluid connection with a second port 5; wherein: the first fluid tiansport channel 1A is in direct fluidic communication with the overflow channel 3 and the reaction well 2; the overflow channel 3 is further in direct fluidic communication with the second fluid transport channel SA and the fluidic constriction channel 4; and, the fluidic constriction channel 4 is In direct fluidic communication with the reaction well 2 and the overflow channel 3; wherein: the overflow channel 3 has a length of between 8,000 and 15,000 pm, optionally about 10,900 pm; the fluidic constriction channel 4 has a width or diameter of 50-1000 pm, optionally about 100 pm; optionally the reaction well 2 comprises one or more of one or more lipids, an organic solvent, an alcohol, acet
  • a fluidic device useful for produdng nanoparticles can have a height of about 300 pm to about 500 pm, in an illustrative embodiment about 500 pm;
  • a first fluid transport channel 1A has a length of from about 2000 pm to about 10,000 pm, in the illustrative embodiment about 5900 pm, and/or a width or diameter of about 1000 pm to about 2000 pm, in the illustrative embodiment about 1200 pm;
  • an overflow channel 3 has a length of from about 8000 pm to about 15,000 pm, in the illustrative embodiment about 10,900 pm, and/or a width or diameter of about 1200 pm to about 2000 pm, in the illustrative embodiment about 1200 pm;
  • a second fluid transport channel 5A has a length of from about 2000 pm to about 10,000 pm, in the illustrative embodiment about 1500 pm, and/or a width or diameter of about 1000 pm to about 2000 pm, in the il
  • the fluidic device may comprise within at least the reaction well 2 a nanoparticle or a population of nanoparticles, optionally wherein said nanoparticle(s) is a lipid-based nanoparticie(s) or polymeric nanopartide(s).
  • Height and width dimensions provided herein are typically for rectangular channels and diameter dimensions are for circular channels.
  • channels can take on different shapes, and that if other channel shapes are implemented dimensions provided herein for rectangular or circular channels can be adapted to provide similar results with other channel shapes.
  • the different parts and sections of the microfluidic channel(s) are typically the same shape but can differ, and In one illustrative embodiment, have a rectangular shape.
  • diameter means “effective diameter”, or “hydraulic diameter”, for embodiments having channels or sections therein, that have a shape other than circular.
  • the diameter of a circular channel typically does not exceed the height of a fluidic device comprising the channel.
  • multiples of such fluidic devices i.e., fluidic device subunits
  • FIGS.12A-12B, 13, 14A, 20, and 21A can be connected in series and/or in parallel as shown in the illustrative embodiments of FIGS.12A-12B, 13, 14A, 20, and 21A. For instance, as shown in FIG. 12A and FIG.128 (and FIG.
  • each subunit is on the opposite side of at least two, but optionally each, successive fluidic device subunit (See, e.g., FIG. 128, FIG. 20, and FIG. 21A).
  • Such configuration reduces the footprint of such device.
  • such configuration is used in fluidic devices that are cassettes or cartridges, for example plastic disposable cassettes or cartridges.
  • such configuration having overflow channels on opposite sides provides better mixing of two fluids in the device, as the flow path is more disruptive because fluid cannot go only through the overflow channels.
  • each of said multiple device subunits are connected in parallel and can comprise a first fluid transport channel 1A but not a first port 1 (except for the first device in the series 10), wherein: at least two of said multiple devices are connected to one another by a first common fluid transport channel 8 connected to the first fluid transport channel 1A of each of said multiple devices to form a device subunit; and, where multiple device subunits are present in the device, at least two of said device subunits are connected to one another by a second common fluid transport channel 9 which is connected to a common port 10. While FIG.
  • 13 shows four fluidic devices linked in series to one another, in some embodiments, additional fluidic devices (e.g., five or more, such as but not limited to eight, 12, 32 fluidic devices) may be linked to one another (e.g., as may be desired by the user), and can include a single common port between all of the devices, or subsets of such fluidic device subunits can be in fluid communication with a number of common ports.
  • additional fluidic devices e.g., five or more, such as but not limited to eight, 12, 32 fluidic devices
  • additional fluidic devices e.g., five or more, such as but not limited to eight, 12, 32 fluidic devices
  • additional fluidic devices e.g., five or more, such as but not limited to eight, 12, 32 fluidic devices
  • additional fluidic devices e.g., five or more, such as but not limited to eight, 12, 32 fluidic devices
  • a fluidic device can include a first fluid transport channel 1A (part 201A in FIG. 20) in fluid communication with at least first and second port channels (12, 14) that terminate in a first and second port channel ports, respectively (11, 13).
  • Such port channel ports (11, 13) are configured to, adapted to, and operable to, permit liquids to be introduced, inserted, flowed, injected, or pushed into, or pulled or withdrawn from the fluidic device, similar to port 1 in other configurations of fluidic devices herein.
  • a fluidic device can include multiple fluidic devices (i.e., fluidic device subunits) fluidly connected In series to one another, each of said multiple fluidic devices in the series comprises a first fluid transport channel 1A in fluid communication with at least first and second port channels (12, 14) that terminate in a first and second port channel ports, respectively (11, 13); the first fluidic device (first device subUnit) in the series comprises a second fluid transport channel SA in fluid communication with the first fluid transport channel 1A of a second fluidic device in the series; the second fiulcfic device in the series, and subsequent devices in the series if present (e.g.
  • subunits 2, 3 and 4 in RG. 14A comprise a second fluid transport channel 5A in fluid communication with the first fluid transport channel 1A of tire next fluidic device in the series; and, the second fluid transport channel SA of the last fluidic device in the series (last subunit) terminates in an outlet port 5. It is contemplated that 2, 4, 6, 8, 10, 12, 20, 30, 40, 50, 75, 100, or more fluidic device subunits can be placed in series. The total fluid volume that is input into the devices when they are used in a method is determined by the detired volume of reaction product (e.g.
  • nanoparticle formulation or protein precipitate but can be, e.g., approximately one to 10,000 ml, one to 5,000 ml, one to 2,000 ml, one to 1,000 ml, one to 200 ml, such as one ml, 10ml, 100 ml, 1,000 ml,
  • first and second fluids e.g., a lipid-based or polymer-based first fluldand a second fluid being an aqueous solution or buffer, or an aqueous solution and/or buffer and/or water-soluble polymer solution, respectively
  • first and second fluids e.g., a lipid-based or polymer-based first fluldand a second fluid being an aqueous solution or buffer, or an aqueous solution and/or buffer and/or water-soluble polymer solution, respectively
  • Tubing can be connected at the outlet port 5 that can lead into a collection container.
  • fluidic devices herein that comprise fluidic device subunits can be referred to as fluidic device assemblies, some of which are coflowing fluidic device assemblies if they are also coflowing fluidic devices as discussed herein.
  • one or more passive air valves can be included in order to separately drive fluid into or out of a particular or a particular group of reaction wells or fluidic devices. The operation and configuration of passive air valves is disclosed In WO 2018/200896, incorporated herein by reference in its entirety.
  • Fluidic devices herein can be formed in cassettes or cartridges, such as disposable cassettes of cartridges, for example disposable plastic cassettes or cartridges.
  • microfluidic device assemblies with microfluidic device subunits are formed in a disposable microfluidic cartridge.
  • Such cassettes or cartridges can have different sizes and shapes, such as, but not limited to, recrtangular, square, or circular, and in some illustrative embodiments are rectangular In shape wit h widths between 10 mm and 250 mm or between 20 mm and 150 mm, or 50 mm and 150 mm, length between 10 mm and 250 mm, 50 mm and 250 mm, 100 mm and 250 mm, or 50 mm and 150 mm, and a thickness/depth of between 1 mm and 10 mm, 2 mm and 5 mm, or 1 mm and 2mm.
  • the cartridge or cassette can be 75.5 mm x 50 mm x 3 mm, 75.5 x 25 x 3 mm, or 90 mm x 50 mm x 7.5 mm.
  • Some aspect provided herein are commercial products comprising two or more disposable cassettes or cartridges each comprising a- fluidic device provided herein. Methods for making such cartridges and plastic components for such cartridges or cassettes are known in the art.
  • a fluidic device provided herein that includes a single first inlet port 1 or 10 (e.g., as illustrated in FIGS. 1, 2A-2C, and 10-13 ⁇ can be used in methods to produce a reaction product, such as nanopartides (e.g., liposomes, lipid micelles, polymer-comprising nanoparticles wherein lipids or polymers are found in the envelope, solid lipid nanopartides (SIN), or nano-structured lipid carriers (NLC)), or a protein precipitate by inputting a first fluid and a second fluid into the fluidic device.
  • a reaction product such as nanopartides (e.g., liposomes, lipid micelles, polymer-comprising nanoparticles wherein lipids or polymers are found in the envelope, solid lipid nanopartides (SIN), or nano-structured lipid carriers (NLC)
  • a protein precipitate by inputting a first fluid and a second fluid into the fluidic device.
  • the fluidic device as diown in FIGS, 1, 2A-2C, and 10-13, include a first port 1, first fluid transport channel 1A, reaction well 2, overflow channel (part 3), fluidic constriction channel 4, second fluid transport channel SA and second port (part 5).
  • a method that comporises three-steps can be used, as illustrated in FIGS. 2A-2C and further described herein, it will be understood for devices that include multiple fluid device subunits and a single input port, for example the devices of FIGS. 12 and 13, that description in the following paragraphs that refer to a single channel or reaction well, relate to each identical part of the subunits therein.
  • the first fluid e.g ., an organic solvent solution for lipid- based nanopartides, a polymer solution for polymer-based nanoparticles, or a lipid (e.g. a melted lipid), in illustrative embodiments a lipid in a liquid state that does not comprise an organic solvent, for solid lipid nanoparticles (SIN) or nano-structured lipid carriers (NIC)); indicated as a solid fill within the fluidic device
  • the first fluid is introduced into the fluidic device through the first port 1 or 10 (RG, 13) where it enters a first fluid transport channel 1A, and then enters the reaction well 2 and the overflow channel 3 concurrently.
  • the reaction well 2 and fluidic constriction channel 4 will be filled completely with the first fluid as excess fluid continues to travel through the overflow channel 3.
  • the fluid in the fluidic constriction channel 4 and the overflow channel 3 then meets at the junction between the overflow channel 3 and a second fluid transport channel 5A, and a combined stream flows through the second fluid transport channel 5A and exits the fluidic device through the second port 5.
  • all parts of the fluidic device are filled with the first fluid.
  • the first fluid is trapped in the reaction well 2 and fluidic constriction channel 4.
  • excess first fluid is removed from the other parts of the fluidic device (i.e., overflow channel 3, second fluid transport channel SA), by applying negative pressure at a port (eg., first port 1), so that the fluid retracts back through the second fluid transport channel SA and continues retracting back through overflow channel 3 toward first port 1.
  • a port eg., first port 1
  • the first fluid will travel through the overflow channel 3 only due to the stronger capillary effects in the fluidic constriction channel 4 compared to the overflow channel 3.
  • the first fluid moves through the first fluid transport channel 1A, thereby creating a fluid-air interface at the opening of the reaction well 2, and is withdrawn from the device through the first port 1 (remaining in the reaction well 2 and fluidic constriction channel 4.
  • a second fluid different than the first fluid, for example as discussed herein for the production of nanoparticles or a protein precipitate, is introduced into the fluidic device (e.g., at a flow rate of from 1 to 30 ml/minute, optionally from 5 to 20 ml/minute or 10 to 20 ml/minute) and mixed with the first fluid to produce nanoparticles.
  • second fluid is introduced through the first port in this step; or, wherein fluidic multiple devices are fluidly connected to one another in series or parallel, greater than 1000 pl aqueous buffer or water can be introduced through the first port 1 in this third step, It Is noted that as more second fluid (e.g., aqueous buffer) is washed through the device, fewer nanopartides will remain in the well, and eventually all the contents will be replaced with just the second fluid.
  • second fluid e.g., aqueous buffer
  • a tower volume of the second fluid e.g., 100 pl where 100 pl to 200 pl is typical
  • the contents of the reaction well 2 will be replaced with the mixture of nanoparticles (e.g., in ethanol and aqueous buffer), but most of the mixture will exit through the second port(s) 5.
  • This third step can employ a syringe pump prepared by connecting tubing from a syringe pump filled with the second fluid to a port (e.g., first port 1). Tubing can also be connected to the second port 5 that feeds into a collection container.
  • the syringe pump can be set to a flow rate between 1 and BOml/min, as non-Hmiting examples, and the second fluid pumped into the fluidic device through first fluid transport channel lAand into reaction well 2, replacing the first fluid that was trapped in the reaction well 2 and fluidic constriction channel 4.
  • the method for malting nanoparticles can include: a) filling the fluidic device by introducing an organic solvent solution comprising dissolved lipids or a polymer solution thru the first port 1 into the fluidic device; b) trapping the organic solvent comprising dissolved lipids or the polymer solution In a reaction well 2 and a fluidic constriction channel 4 connected therewith by applying negative pressure at the first port 1 to remove some of tire organic solvent solution or polymer solution from the fluidic device; and, c) introducing an aqueous buffer into the reaction well 2 through the first port 1 to mix with and replace the organic solvent comprising dissolved lipids or the polymer solution, wherein mixing of the organic solvent comprising dissolved lipids or the polymer solution and the aqueous buffer forms nanopartides.
  • Illustrative methods for producing nanoparticles using the illustrative device of FIG. 1 are disclosed for example in Example 1 and Example 2 herein.
  • a method for producing a reaction product using a fluidic device that includes a first fluid transport channel 1A in fluid communication with at least first and second port channels (12, 14) that terminate in first and second port channel ports, respectively (11, 13), wherein: a first fluid is introduced into a first fluid transport channel 1A of the fluidic device thru the first port channel port 11; and a second fluid that is different from thefirst fluid is introduced into the first fluid transport channel 1A thru the second port channel port 13, wherein a reaction well 2 of the device is in direct fluidic communication with the first fluid transport channel 1A, wherein a fluidic constriction channel 4 is in direct fluidic communication with the reaction well 2, and wherein some of the second fluid flows into the reaction well 2 and some (usually the remainder) of the second fluid flows around the reaction well 2 into an overflow channel 3 of the device, and wherein the first fluid mixes with some of the second fluid in the reaction well 2, thereby producing the reaction product
  • Such devices used for this aspect are typically coflowing fluidic devices and such aspect can be referred to herein as a method for producing a reaction product using a coflowing flulidic device.
  • Such coflowing fluidic devices typically have a Y junction that connects the first and second port channels (12, 14) at the first fluid transport channel of the fluidic device.
  • the method further includes collecting the reaction product through the second port 5.
  • Such embodiments can be accomplished by inputting more total fluid (i.e. first fluid and second fluid) into the device than the total volumetric capacity of the device. In such a method it is believed that fluid moves through the device as shown in FIG. 21A.
  • the device that performs this method is configured to and operable to guide (and capable of and adapted for guiding) fluid entering the device through the first fluid transport channel 1A, into the reaction well 2 and the overflow channel 3.
  • such properties of the device are believed to be due to the difference in the resistance ratios of the reaction well 2, the overflow channel 3, the reaction well 2 and foe fluidic constriction channel 4, which along With the reaction well 3, in illustrative embodiments is also filled With the first fluid and provides mixing with the second fluid as It is mixing within the reaction well 2.
  • Exemplary devices for performing such a method are provided in FIGS. 14A, 20, 21A, 21B, and 26.
  • one fluid is an organic solvent solution comprising dissolved lipids, a polymer solution comprising at least one polymer dissolved in a solvent, a protein solution, or a lipid (e.g. a melted lipid).
  • a lipid in a liquid state that does not comprise an organic solvent is an organic solvent solution.
  • the other (or another) fluid e.g.
  • first fluid is an organic serfvent solution comprising dissolved lipids and the method is a method for making partides, or a water-soluble synthetic polymer solution where the first fluid comprises at least one polymer dissolved in a solvent and the method is a method for making particles, a protein precipitant where the first fluid comprises a protein and the method is a method for precipitating proteins, or a lipid In a liquid state that does not comprise an organic solvent and the method is malting, forming or producing solid lipid nanopartides (SIM) or nano-structured lipid carriers (NIC);.
  • SIM solid lipid nanopartides
  • NIC nano-structured lipid carriers
  • the fluidic device comprises a series of fluidic device subunits each having attributes provided hereinabove for the device in this method, and in illustrative embodiments substantially identical or identical, for example with respect to reaction well 2, overflow channel 3, and fluidic constriction channel 4, as disclosed hereinabove.
  • fluid which is typically a fluid stream created by input of the first fluid and the second fluid into the device, flows through an upstream fluidic device subunit Into a second fluidic transport channel SA of the upstream fluidic device subunit it enters a first fluidic transport channel 1A of a downstream fluidic device subunit as shown in FIG. 21A herein.
  • Such methods are continuous flow methods, and fluidic devices that indude fluidic device subunits can be considered continuous flow systems.
  • Such continuous flow methods and systems can include, for example fluid reservoirs for holding a first fluid and a second fluid respectively, as well as a pumping system that is adapted to and operable to input fluid into the fluidic device through the first port channel port 11 and the second channel port 13, such as through tubing that connects the fluid reservoirs to the port channel ports.
  • Such continuous flow systems and methods providing the ability to scale up methods provided herein such that methods can be used to produce between 10 ul and 10 U between 100 ul and 101, between 250 ul and 101, between 1 ml and 5L, or between 1ml and 21, or between 1ml and 11, for example, of a reaction product solution or suspension.
  • Example 3 herein demonstrates such a method that successfully produced 11 of reaction product (nanopartides).
  • the method and system can be used to scale up based on the number of linked fluidic devices to almost unlimited scale-up potential.
  • two or more fluidic devices herein which each can be referred to as a fluidic device assembly 99 as they each include more than 1 fluidic device subunit, in this case in series, can be connected in parallel.
  • a vessel comprising a first fluid for example a first fluid reservoir 110
  • a vessel comprising a second fluid for example a second fluid reservoir 130
  • fluidic device assemblies 99 of a fluidic system through a first port channel port and a second channel port of each fluidic device assembly, such as through tubing 111, 131 that connects the fluid reservoirs 110, 130 to the port channel ports.
  • An outlet port of each assembly 99 can be connected and in fluid communication, for example with tubing 50, to a collection vessel 200.
  • Such devices can permit scale-up from an individual microfluidic device assembly comprising microfluidic device subunits in series. Two, three, four, five, ten, twenty or more microfluidic device assemblies can be connected in parallel in such a configuration to form a large-scale fluidic system that produces reaction products with similar characteristics to those produced by each fluidic device assembly.
  • User- controllable parameters such as the relative flow rate of a first fluid stream comprising the first fluid and a second fluid stream comprising the second fluid, the total flow rate of the combined stream of the first fluid stream and the second fluid stream, the dimensions of the device and the subunits of the device, the relative dimensions of the parts in the device, for example the relative width of the fluidic constriction channel compared to the reaction well and the overflow channel, can be set or optimized for a given first fluid and second fluid to consistently produce particles of a similar desired size.
  • the desired size/diameter is of a range that is set by a desired size/diameter of less than about 1 urn, 750 nm, 600 nm, 500 nm, or 200 nm, and greater than 50 nm, 75 nm, 100 nm, 150 nm, or 200 nm.
  • the first fluid and the second fluid, or the combined first fluid and the second fluid can be input into the fluidic device at a flow rate between 0.1 mi/minute and 50 ml/minute, or between 0.5 ml/minute and 25 ml/minute, or between D.5 ml/minute and 20 ml/minute, or between 1.0 ml/minute and 20 ml/minute, or between 0.5 ml/minute and 10 ml/minute, or between 0.5 ml/minute and 5 ml/minute, or between 0.5 ml/minute and 1.0 mi/minute, or exactly or about 0.5, 0.75, 0.8, 0.9, 1.0, 5, 10, 15, or 20 ml/minute.
  • a flow rate ratio can be used of between 1:20, 1:10, 1:5, 1:2, or 1:1 between the flow rates of the first fluid stream and the second fluid stream.
  • HG, 20 provides an exemplary coflowing fluidic devices of small and relative large dimensions as provided in Table 1, that include fluidic device subunits in series that can be used to produce a reaction product (e.g. nanopartides) according to the method aspect provided immediately above.
  • the first well 2O2A in the series each has a connected fluidic Constriction channel 204A with a length of 500um and a width of 300um.
  • the second well 2028 in the series has a fluidic constriction channel 2O4B with a length of SOOurn and a width of 200um. Every other well inthe series has a fluidic constriction channel with a length of SOOum and a width of lOOum (as stated in Table 1).
  • the wider fluidic constriction channel in the first two wells helps limit air-bubble formation when initially filling the device with fluid.
  • the small coflowing device e.g. small dimension version of the device of HG.
  • a first fluidic constriction channel and a second constriction channel of a first subunit and second subunit respectively are configured to, adapted to, or operable to, reduce air-bubble formation for example by having a larger width than other fluidic constriction channels in the fluidic device.
  • the first fluidic constriction channel can have a width that is 1.5 to 5 times, 2 to 4 times, and in illustrative embodiments, 3 times larger than the other fluidic constriction channels in the fluidic device
  • the second fluidic constriction channel can have a width that is 25% to 50%, 30% to 40%, and in illustrative embodiments 33% smaller than the first fluidic constriction channel, and 1.25 to 3 times, 1.5 to 2.5 times, and in illustrative embodiments, 2 times larger than the other fluidic constriction channels in the fluidic device.
  • the mixing of a first fluid and a second fluid can result in the formation of nanopartides, for example when the first fluid is lipid in liquid form, in illustrative form without an organic solvent and the second fluid is an aqueous buffer.
  • the method can be used to make, form or produce solid lipid nanopartides (SIN) or nano-structured lipid carriers (NIC).
  • instruments such as those of FIG. 28 and FIG. 29
  • devices herein can be configured such that a first fluidic device assembly comprising multiple fluidic device subunits (e.g, the microfiuidic device assembly shown in FIG. 20) can be thermally connected to a temperature regulating device or assembly that can be used to heat the channels and any liquid contained therein, to a target temperature or temperature range at between 37 and 100"C, or cool the channels and any liquid contained therein to a target temperature ranging from -4 to 20*C.
  • the heating target temperature or target temperature range used for a method depends on the melting temperature of the lipid in liquid form loaded into the microfluidic device assembly.
  • Such heating target temperature or target temperature range can be set at, or at a range centered around, a temperature that is 10-20’C above the temperature of the lipid.
  • an additional port channel port can be connected to first fluidic device assembly such that molecules to be loaded in or on the nanopartides can be loaded into the assembly.
  • some methods herein indude loading a molecule or biomolecule into a channel of the first fluidic device assembly to form nanoparticles, for example SLNs or NLCs comprising the molecule or biomoiecule within and/or on the SLNs or NLCs;
  • biomolecules can include nudeic acids (e.g. DNA or RNA), proteins, or carbohydrates.
  • the biomolecules are nudeic add vectors or other polynucleotides.
  • a second fluidic device assembly comprising multiple fluidic device subunits (e.g. the microfluidic device assembly shown in FIG. 20) can be fluidly connected in series to the first fluidic device assembly.
  • a cool liquid e.g. cool water
  • the second fluidic device assembly can be thermally connected to a temperature cooling device or assembly to cool the emulsion as it enters and after it enters, is transported or otherwise flows into the second fluidic device assembly.
  • toe temperature cooling device is used to set the temperature of toe second fluidic device assembly, or channels and wells therein, at below toe melting temperature of the lipid, or between -4 and 10*C, 0 and 10*C, 2 and 10*C, or 3 and 5*C.
  • lipid nanoparticles for example SLNs and NLCs, in illustrative embodiments less than 100 nm in diameter, are fomed within the cooled fluidic device assembly.
  • any of the fluidic devices and fluidic device assemblies disclosed herein that include a first fluid transport channel 1A in fluid communication with at least first and second port channels (12, 14) that terminate in a first and second part channel ports, respectively (11, 13) (coflowing fluidic devices), as illustrated for example in FIGS, 21A and 21B
  • mixing of the first and second fluids can occur in the reaction well 2 as wed as the overflow channel 3 as illustrated in FIG. 21B with different line patterns represening the first fluid, the second fluid, and a mixtureof the first fluid and second fluid, and mbting shown by line swirls in the reaction well 2.
  • Such mixing typically occurs when the first fluid and the second fluid are introduced into the microfluidic device, each through a different port of the first and second port channel ports (11, 13).
  • fluidic devices provided herein accomplish, are effective for providing, are capable of providing, are operable to provide, and/or are adapted to provide rapid mixing of a first fluid and a second fluid such as an organic fluid and an aqueous fluid, leading to the production of uniform reaction products (e.g. particles, such as nanoparticles or microparticles, or protein precipitates).
  • the device assembly can further comprise third, fourth, fifth, etc. fluid transport channels in fluid communication with corresponding third, fourth, fifth, etc input ports, respectively, and in fluid communication typically through one or more additional channels to one or more reaction wells.
  • additional input fluids third, fourth, fifth, etc. fluids
  • a first fluid is a protein solution and a second fluid is a protein precipitant
  • efficient mixing as a result of the design of fluidic devices of this aspect, as described for example in FIG. 21B, allows in illustrative embodiments, for continuous precipitation of protein as some fluids from the input fluid stream create by the input of the first fluid and the second fluid flow through the device via the reaction well 2 as illustrated In FIG. 21A without the need tor an Incubation period.
  • the applied flow rate ratios provided herein can result in precipitant concentrations of 2%, 1.33%, 0.67%, and 0.36%, which are lower than the typical range used in standard methods. This is beneficial, as precipitants can cause undesirable structural changes in the proteins of interest.
  • the device design described for this aspect could be incorporated into a continuous purification workflow, for example allowing for the extraction of a high yield expression product.
  • the type of partides formed, for exampie microparticles or nanopartides is dependent on the type of first and second fluids utilized.
  • the first fluid is an organic solvent solution comprising at least one organic solvent and at least one lipid and the second fluid is an aqueous buffer (optionally including additional components), in some embodiments, the first solution can comprise at least one lipid selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC); cholesterol; l,2-Dilauroyi-sn-glycero-3- phosphocholine(DLPC); l,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC); 1,2-Distearoyl-sn-glycero- 3-phosphocholine (D5PC); l,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1,2-Dimyristoyl-sn-glycero- 3-phosphoethanolamme (DMPE); l,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE); 1,2- Diole
  • DPPC dipal
  • the organic solvent can be selected from the group consisting of ethanol, methanol and chloroform, ethyl acetate, isopropanol, and hexane (preferably ethanol).
  • the dissolved lipids can comprise DPPC, cholesterol and DOTAP, and the organic solvent solution can comprise ethanol.
  • the second fluid is typically an aqueous buffer (e.g., any buffer having an effective buffering capacity at the pH range needed for nanopartide synthesis (e.g., physiological buffer) with or without a salt), and/or can be selected from foe group consisting of or based upon bidne (2-(8is(2-hydroxyethyl)amino)acetic add), carbonate, cacodylate (Dimefoylarsenic acid), Hepes (4-(2-hydroxyethy$)-l*piperazineethanesulfonic acid), isotonic sucrose, MMEESS (2-(N- morpholino)ethanesulfonic add), MOPS (3-(N-morpholino)propanesulfonic add), phosphate-buffered saline (PBS), PIPES (Piperazine-N,N'-bis(2-ethanesulfonic acid), potassium phosphate, saline solution,
  • aqueous buffer e.g
  • TAPS (
  • TCS (2-([l,3-dihydroxy-2-
  • Trls(hydroxymethyl)methyfamino]-2-hydroxypropanesuffonic acid and/or Tris (e.g., Tris(hydrpxymethyl)aminomethane or, 2-Amino-2-(hydroxymethyl)propane-l,3-dioi; e.g., TAE (Tris- acetate EDTA), TBE (Tris-borate-EDTA); TAPSO (3-(N-Tris(hYdroxymethyl)rhethylamiho]-2- hydroxypropanesulfonic acid); and in some embodiments is preferably PBS.
  • the mixture of these first and second fluids as described herein produces nanopartides comprising a lipid membrane surrounding the aqueous buffer (and any additional components).
  • polymer-based nanoparticles can be produced using these methods wherein the first fluid can be a polymer solution comprising at least one polymer and at least one solvent (e.g., acetonitrile) and the second fluid is a water-soluble polymer Solution comprising at least one water- soluble polymer and any additional components).
  • the first fluid can be a polymer solution comprising at least one polymer and at least one solvent (e.g., acetonitrile) and the second fluid is a water-soluble polymer Solution comprising at least one water- soluble polymer and any additional components).
  • the polymer can be selected from the group consisting of polyiactic acid (PLA), poly-i-lysine (PLL), polyglutamic acid (PGluA), poiyglycolic acid (PGA), polyethylene glycol (PEG), polycaprolactone (POL), polyaspartate (PAA), poly(d,l- lactide-co-glycolic) add (PLGA), cydodextrins (CD), and N-(2-hydroxypropyl)-methacrylamide copolymer (HPMA), a natural polymer, chitosan, heparin, albumin, dextran, gelatin, alginate, collagen, a mixture thereof, and/or other suitable polymers, and/or mixtures thereof.
  • PLA polyiactic acid
  • PLL poly-i-lysine
  • PGluA polyglutamic acid
  • PGA poiyglycolic acid
  • PEG polyethylene glycol
  • POL polycaprolactone
  • PAA polya
  • the solvent can be selected from the group consisting of dichloromethane and ethyl acetate, benzyl alcohol, cyclohexane, acetonitrile, and acetone, or other suitable solvent.
  • the second fluid may be an aqeuous solution (e.g,, water or an aqedus buffer such as PBS) or water- soluble synthetic polymer solution comprising, for instance, poly(vinyl alcohol) or didecyldimethylammonium bromide.
  • the mixture of these first and second fluids produces nanoparticles comprising a polymer-based membrane surrounding the water-soluble polymer solution (and any additional components).
  • the nanopartides produced by these methods can also be characterized by any suitable technique to determine, for instance, size, polydispersity index (PDI), or zeta potential, optionally as measured using a technique such as dynamic light scattering (DIS) or transmission electron microscopy (TEM).
  • PDI polydispersity index
  • DIS dynamic light scattering
  • TEM transmission electron microscopy
  • the nanoparticles have a diameter of between 5 nm and 500 nm, or less than 600 nm.
  • a microfluidic device having a single inlet port 1 can be used in a method for producing particles where a second fluid is input into the fluidic device after a reaction well 2 of the device is filled with a first fluid, and the fluidic constriction channel of the fluidic device has a width or diameter of less than 400 pm and the flow rate used to input the second fluid into the device is greater than 5 ml/minute, for example between 5 ml/minute and 20 ml/minute or between 10 ml/minute and 20 ml/minute or between 5 ml/minute and 10 ml/minute.
  • the nanoparticle is lipid-based and in some embodiments the nanopartides are polymer-based.
  • the particles are comprised of a metal. Such metal can include, but Is not limited to, silver, gold and copper.
  • the particles are metallic nanoparticles.
  • other types of nanopartides may also be produced using these fluidic devices and methods.
  • the second fluid in such embodiments may comprise additional components such as, but not limited to, one or more detectable agents, therapeutic agents, nucleic acid-base compounds (e.g., DMA, RNA, and derivatives thereof), proteins (including but not limited to therapeutic proteins), immunomodulatory nucleic acids, proteins, and/or other compounds (e.g., vaccines), and/or other suitable additional components as may be understood by those of ordinary skill in the art
  • Nanopartides, especially liposomes may also be further processed by, for instance, treating the same with polyethylene glycol (e g., PEGylation) and/or mannosyiating the same.
  • Liposomes may also be anionic, neutral, or cationic depending at least in part on the type of lipid utilized.
  • one of the fluids input into the device is a protein solution. It is contemplated that any protein can be included in such protein solution. In some embodiments, such protein is an industrial protein, a control protein for a diagnostic assay or a therapeutic protein. Concentrations of proteins in a fluid input into the device can be any concetration used for such protein for protein predpitation using conventional batch stir/incubate methods. For example, the concentration can be between 0.1 and 100 mg/ml, for example between 1 and 50 mg/ml, for example between 1 and 25 mg/ml, for example between 1 and 10 mg/ml In such embodiments, the other fluid in illustrative embodiments Is a protein precipitant.
  • the protein precipitant can be a neutral salt such as ammonium sulfate, a mineral add, such as hydrochloric add or sulfuric add, a misdble solvent such as ethanol or methanol, a non-ionic hydrophilic polymer, such as a dextran or a polyethylene glycol, a polyeiectrolyte such as Alginate, carboxymethycellulose, polyacrylic add, tannic add or polyphosphates, trichloroacetic add, phenol, ammonium acetate/methanol, methanol chloroform.
  • a neutral salt such as ammonium sulfate
  • a mineral add such as hydrochloric add or sulfuric add
  • a misdble solvent such as ethanol or methanol
  • a non-ionic hydrophilic polymer such as a dextran or a polyethylene glycol
  • a polyeiectrolyte such as Alginate, carboxymethycellulose
  • Concentrations of protein precipitants used can be the same as those used for traditional mix/incubate reactions.
  • TCA can be used at a concentration range of 4-20%.
  • the protein precipitant does not alter protein structure.
  • the protein precipitant can be a polyethylene glycol, such as PEG 6000.
  • Other embodiments of such methods are also contemplated as being suitable for use with the fluidic devices provided herein, as will be understood by those of ordinary skill in the art.
  • the fluidic devices provided herein including fluidic device(s) within a cartridge, arid fluidic circuits therein, can be fabricated using, for example, but not limited by, various soft lithographic microembossing techniques.
  • a variety of fabrication micro-forming methods that utilize, for example, but are not limited to, micro-milling, micro-stamping, and micro-molding, can be matched to substrate material properties.
  • the fluidic devices and cartridges can be injection molded using a suitable plastic.
  • a substrate can be an optically transmissive polymer, providing good optical transmission from, for example at least about 85% to 90% optical transmission over a wavelength range of about 400nm to about SOOnm.
  • polymeric materials having good optical transmission properties for the fabrication of various embodiments of a fluidic device or circuit include organosilicon polymers.
  • a fluidic device presented herein is composed of hydrophobic materials.
  • the fluidic device is composed of hydrophobic materials such as polystyrene, polycarbonate, polyfmethyl methacrylate) (PMMA), and/or pofydimethylsiloxane (PDMS), polypropylene, cydic-olefin polymers (COP), cydic-olefin copolymers (COC), polystyrene polymers, polycarbonate polymers, acrylate polymers, and the like.
  • hydrophobic materials such as polystyrene, polycarbonate, polyfmethyl methacrylate) (PMMA), and/or pofydimethylsiloxane (PDMS), polypropylene, cydic-olefin polymers (COP), cydic-olefin copolymers (COC), polystyrene polymers, polycarbonate polymers, acrylate polymers, and the like.
  • hydrophobic materials such as polystyrene, polycarbonate, polyfmethyl methacrylate) (P
  • the fluidic device has a height of between about any of 100, 125, 150, 175, 200, 225, or 300 pm on the low end of the range and about any of 200, 225, 250, 275, 300, 400 and 500 pm on the high end of tire range.
  • the fluidic device has a height of about any of 100-500 pm (e.g., about any of 100, 150, 200, 250 300, 350, 400 450, 500, or 300-500 pm).
  • the fluidic device can have a height of between about any of 100, 200, 300, 325, 350, 375, 400, and 425 pm on the low end of the range and about any of 400, 425, 450, 475, and 500 pm on the high end of the range.
  • the first fluid transport channel and the second fluid transport channel are each about 400 microns in length, or about 2,000 to 10,000 pm, or about 5,900 pm (as in the fluidic devices of FIGS. 1 and 10-14A).
  • the overflow channel has a length between about any of 400, 425, 450, 475, 500, and 525 pm on the low end of the range and about any of 500, 525, 550, 575, 600, and 625 pm on the high end of the range, or about 8,000 to 15,000 pm, or about 10,900 pm (as in the fluidic devices of FIGS. 1 and 10-14A).
  • the overflow channel has a length between about any of 400 and 625 pm. Other sizes may also be suitable as may be derived from this specification or the examples, and/or otherwise determined by these of ordinary skill in the art.
  • on-device liquid handling for performing methods using fluidic devices herein can be externally actuated in manual or automated mode using standard laboratory liquid handling equipment
  • a pressure applied at or between ports can be used as a motive force for moving liquids, for example, from part of a fluidic device to another part of that or another fluidic device.
  • a motive force for on-device liquid handling can be externally actuated by applying a decreased or negative pressure at a port or between ports or by applying an increased or a positive pressure at a po rt or between ports.
  • on-device liquid handling for various embodiments of components, devices and methods of this disclosure can be externally actuated using any manual or automated standard laboratory liquid handling equipment, such as by manual or automated pipetting systems utilizing solid or liquid displacement, that can provide a pressure from between about 720 torr to about 800 torr, which is about */- 40 torr from 1 standard atmosphere of pressure.
  • coflowing devices provided herein include a QC subassembly as illustrated in FIG. 26.
  • Such subassembly typically includes a quality control channel 261.
  • Such quality control channel 261 is typically in indirect fluidic connection with a second fluid transport channel 205* of the final microfluidic device subunit in the senes.
  • the method further comprises analyzing the reaction product using a QC subassembly.
  • reaction product formulation while most of the reaction product formulation is output for collection through a final transport channel 270 in direct fluidic communication with the second fluid transport channel 205* of the final microfluidic device subunit In the series, a fraction of the reaction product formulation is guided into the quality control channel 261 through a reaction product QC channel 265 also in direct fluidic communication with the second transport channel 205* of the final microfluidic device subunit in the series.
  • the reaction product formulation output from the fluidic device and a reference fluid with known rheological properties are added as inputs into a Y-junction formed by the reaction product QC channel 265 and a reference QC fluid channel 266.
  • the wjdth/diameter of the reaction product QC channel 265 is less titan 1/2, 1/3, or 1/4 the width/diameter of a the final transport channel 270.
  • observation channel section 269 formed in a portion of the quality control channel 261
  • an interface between a fluid stream from the fraction of the reaction product guided to the quality control channel and a fluid stream from a reference fluid are observed, monitored, and/or tracked for quality control of the reaction product
  • the observation channel section 269 is typically as an at least partially transparent section, and observation is performed using, for example a microscope or other imaging system.
  • the widths of each fluid stream provided by the reaction product passing through the quality control channel 261 can be observed, measured, and tracked, in illustrative embodiments over time, to observe the quality of the reaction product, for example by quantifying formulation consistency over time. If the size or volume fraction of the reaction product (e.g.
  • This QC application can be utilized in as in-line quality control, for example in large-volume production (e.g. production of volumes greater than 100 ml, 250 ml, 500 ml, or 11, for example IL to 10L), to ensure that particle size is staying relatively constant Over time.
  • large-volume production e.g. production of volumes greater than 100 ml, 250 ml, 500 ml, or 11, for example IL to 10L
  • a 5% or greater change in width of a fluid stream is indicative of particle inconsistency and can be used as a an acceptance cutoff for example. It is notworthy that the QC subassembly of FIG.
  • 26 is itself a separate aspect of the invention that can be in direct fluidic communication with virtually any flutdlc device, especially microfluidic device, and can be used to monitor intra-lot quality control of a reaction product made using the fluidic device, such as a particle (e.g. microparticle or nanoparticle) reaction product.
  • a QC subassembly provided herein Is operable for, effective for and/or adapted for determining the quality and consistency of a reaction product over time.
  • a detection system for performing in-line quality control when using fluidic devices herein to produce reaction products, or for detecting the formation of a precipitate, can be in optical communication with the fluidic device for example at the observation channel section 269.
  • the observation channel section 269 Is ideally transparent, for example transparent glass or transparent plastic.
  • a detection system can include an image recording and processing system.
  • the image recording and processing system can comprise at least a light source, a recording device (e.g., a camera), and an Image processor communicably coupled to the imaging device that determines a width of the fluid stream and/or other properties of the fluid based on for example two or more images using one or more algorithms.
  • the light source and recording device are typically positioned to capture two or more images of the fluid stream through the observation channel section 269.
  • Suitable, exemplary image processors e.g., imaging processing systems
  • a fluidic system herein can include a smartphone, a tablet, a personal digital device, a computer pad, a netbook, and/or a computer having imaging processor and/or digital camera integrated therein, or a camera per se.
  • the camera may be one of a Charge-Coupled Device (CCD) or Complimentary Metal-oxide Semiconductor (CMOS) camera.
  • CMOS Complimentary Metal-oxide Semiconductor
  • Suitable light sources can include, in some embodiments, at least one Light Emitting Diode (LED) or LED panel.
  • the at least one LED may be a colored LED.
  • An excitation filter may filter the at least one LED.
  • At least one such light source (e.g., LED or LED panel) may be symmetrically positioned off-axis from the camera with reference to the array.
  • the system can also indude an emission filter for filtering light entering the camera;
  • a device in illustrative embodiments a microfluidic device for detecting a reaction product.
  • a device Is effective for determiningand/or detecting and operable to determine and/or detect a reaction product or whether a first fluid and a second fluid react by forming a reaction product.
  • a related aspect provided herein is a method for using such a device to form, detect, measure, and/or analyze a reaction product (eg. a precipitate) of one or more components of a first solution and one or more components of a second solution (and possibly additional components of additional solutions).
  • a reaction product eg. a precipitate
  • Such a device for detecting a reaction product is illustrated in HG. IS herein.
  • a device for detecting a reaction product can be intended for use, for example, in chemical formulation and/or precipitation studies, with specific focus on the detection and study of fiddle compound interactions that may involve, for non-limiting example, precipitation development.
  • a device for detecting a reaction product includes three fluidic transport channels and an interface channel segment in fluidic communication with each other (l.e., first, second, and third fluid transport channels and interface channel segment), a reaction well, a fluidic constriction channel, and a passive pressure sensing channel.
  • the reaction well is in fluidic communication with the second and third transport channels at the interface channel segment via the fluidic constriction channel.
  • the exemplary device also contains three tnput/output ports for entry and exit of fluid.
  • the device is designed to provide passive, on-chip capture of a specific volume of a first fluid (i.e., first fluid droplet) and to allow input of a second fluid into the system following capture, typical ly passive capture, of the first fluid droplet. Interaction and reaction between first and second fluids occur within the device and can be monitored for a range of time periods.
  • a device for detecting a reaction product can be useful, for example, In the field of chemical formulation development.
  • the device allows detailed analysis and measurements that provide more accurate, repeatable, and high throughput studies of the interaction of components of two or more fluids, which can be members of a library of compounds.
  • a particular embodiment of this aspect of a device for analyzing a reaction product finds use in the development and study of anti-perspirant compounds by testing the interaction of potential anti-perspirant compounds or formulations with compositions representing sweat compounds under physioiogically-relevant conditions.
  • This exemplary device is optimized to closely match the dimensions of an eccrine sweat pore, allowing for dose mimicking of in vivo sweat conditions.
  • Such a device in illustrative embodiments includes a first port 1; a first fluid transport channel 1A, optionally having a relatively straight or straight section 1A1 and an optionally rounded section 1A2; a reaction well 2; a fluidic constriction channel 4; a passive pressure sensing channel 3A; a second port 3; a second fluid transport channel SA; a third fluid transport channel SA, an interface channel segment SC and, a third port 6.
  • the second fluid transport channel SA is in direct fluidic communication with the first fluid transport channel 1A at an end of the first fluid transport channel opposite the first port;
  • the fluidic constriction channel 4 is in direct fluidic communication with the reaction well 2 and an Interface channel segment SC directly connecting the second fluid transport channel 5A and the third fluid transport channel SB, wherein the width of the interface channel segment is identical to the width of the fluid transport channel to which it is directly connected;
  • the reaction well 2 is in directfluidic connection with the passive pressure sensing channel 3Aat an end of the passive pressure sensing channel opposite the second port 3;
  • the passive pressure sensing channel 3A extends from the reaction well 2 opposite the fluidic constriction channel 4 and terminates at the passive pressure sensing channel port 3; and the first fluid transport channel 1A is not in direct fluidic communication with the reaction well 2.
  • First fluid transport channel 1A Length 3000 -10,000 pm t Width 15 -1000 pm
  • Fluidic constriction channel 4 Length 10 - 500 pm Width 15 -500 pm Depth 15-300 pm
  • Third fluid transport channel SB Length 1500 -4000 pi Width 15 - 100 pm Depth 15 -100 pm [0110]
  • Various dimensions of parts of a fluidic device for detecting a reaction product can have relative tolerances, as disclosed in the following paragraphs.
  • the width of the second and third fluid transport channels (5A and SB) are different or in illustrative embodiments the same.
  • the Width of the second and third fluid transport channels (5A and SB) can be between 3/200 and the same width of the first fluid transport channel 1A, for example at a section in direct fluidic communication with (directly connected to) the second fluid transport channel, in illustrative embodiments, the width of the second fluid transport channei SA is between 3/200 and 1/1.5, or between 1/100 and X, or between 1/10 and >4, or between 1/10 and 1, or between 1/8 and %, or between 1/5 and H, or between % and 1/2 the width of the first fluid transport channel 1A, for example at a segment thereof in direct fluidic communication with (directiy connect to) the second fluid transport channel. Such a segment for example, can be the first fluid transport channel.
  • the depth of the second and third fluid transport channels (5A and SB) are different or in illustrative embodiments the same.
  • the depth of the second and third fluid transport channels (5A and 58) can be between 3/70 and the same depth of the first fluid transport channel 1A, for example at a section in direct fluidic communication with (directly connected to) the second fluid transport channel.
  • the width of the second fluid transport channel 5A is between 3/70 and 1/2 or between 1/8 and 1/2 the width of the first fluid transport channel 1A, for example at a segment thereof in direct fluidic communication with (directiy connect to) the second fluid transport channel.
  • the width and depth of an end of the interface channel segment 5C directly connected to the second fluid transport channel 5A is the same as the width and depth of the second fluid transport channel SA and the width and depth of an opposite end of the interface channei segment SC directly connected to the third fluid transport channel SB is identical to the width and depth of the third fluid transport channel SB.
  • the interface channel segment SC can have a narrowing or widening width and/or depth.
  • the width and depth of the interface channel segment, the second fluid transport channel, and the third fluid transport channel are the same.
  • the length of the interface channei segment SC is typically equal to the width of the fluidic constriction channel 4.
  • the length of the fluidic constriction channel 4 Is between .0025 to 1.25, and in illustrative embodiments between >0025 and .025 the length of the second and/or third fluid transport channels SA and SB.
  • the width of the fluidic constriction channel 4 is between .1 to 33 times, and in illustrative embodiments .25 to 4 times the width of the second and/or third fluid transport channels SA and SB.
  • the width and/or depth of the fluidic constriction channel 4 are the same as those of the second and/or third fluid transport channels 5A and SB.
  • the hydraulic diameter of the second and third fluid transport channels 5A and 58 are the same or different and between 3/105 to 1/1 the hydraulic diameter of the first fluid transport channel 1A.
  • the hydraulic diameter of the second and third fluid transport channels are the same.
  • the hydraulic diamater of the second fluid transport channel 5* is between 1/6 and 1/1 the hydraulic diameter of the third fluid transport channel 58.
  • the hydraulic diameter of the second and third fluid transport channels are the same or different and between 3/105 to 1/1 the hydraulic diameter of the first fluid transport channel 1A.
  • the hydraulic diameter of the second and third fluid transport channels are the same.
  • 5A and 5B are the same.
  • a device for analyzing a reaction product one of the aspects provided herein, such as the device depicted in FIG. 15, the device can have a direct fluidic connection between the first fluid transport channel and the reaction well (not shown in FIG. 15).
  • a device according to these embodiments can indude a direct connection between the first fluid transport channel and the reaction well (i.e. these structures can be In direct fluidic communication) as disclosed herein, for example in relation to other aspects, such as, but not limited to those shown for a device for making nanoparticles as depicted in FiG. 1.
  • the first fluid transport channel is not in direct fiddle communication with the reaction well (as depicted for ecample, in FIG. 15).
  • the second fluidic transport channel Is in direct fluidic communication with the first port at the opposite end from the end of the second fuidic transport channel that is in direct contact (direct fluidic communication) with the interface channel segment.
  • a microfluidic device for deleting a reaction product typically indudes a passive pressure sensing channel as illustrated as part 3A of FIG. 15.
  • the passive pressure sensing channel 3A can be adapted for, designed to, and/or effective for measuring the amount or flow-inhibiting strength of a reaction product (eg. precipitate) in one of the other channels of the device, especially the second fluid transport channel SA, the interface channel SC, and/or the fluidic constriction channel 4.
  • the reaction well 2 typically contains the first fluid.
  • a fluid-air interface forms in the passive pressure sensing channel 3A that exhibits an inherent capillary pressure. This capillary pressure relies directly on the surface tension of the first fluid, the contact angle of the first fluid with the device material, and the dimensions of the passive pressure sensing channel 3A.
  • the passive pressure sensing channel can be in fluid contact with the reaction well 2 at various regions of the reaction well 2.
  • the passive pressure sensing channel Is in fluid contact with the reaction Well 2 at a side of the reaction well 2 opposite a side of the reaction well in fluid communication with the fluidic constriction channel 4.
  • the passive pressure sensing channel 3A extends from the reaction well 2 opposite the fluidic constriction channel 4 and terminates at the passive pressure sensing channel port 3.
  • the passive pressure sensing channel 3A has a smaller width (eg.
  • the hydrodynamic resistance of the passive pressure sensing channel 3A is between 1.01 and 5xl0 7 , 1.5 and 4.8xl0 7 , 2 and 1x10 s , 10 and IxlO 3 , or 10 and 100 times the hydrodynamic resistance of each of the interface channel segment, the second fluid transport channel and the third fluid transport channel.
  • the passive pressure sensing channel 3* terminates at the second port 3.
  • a passive pressure sensing channel can have various geometries, segments, and angles between segments provided that it can perform the function provided herein.
  • a passive pressure sensing channel can be a straight channel, or can indude at least one, or have between 1 and 10, 1 and 5, or 1 and 2, or 1 bend, rounded orientation, and/or curve.
  • a passive pressure sensing channel includes at least two segments, wherein at least a first segment extends horizontally or at an angle from the reaction well 2, and at least one second segment extends from the first segment at other than a straight line, in illustrative embodiments, at least one second segment extends from the first segment at an angle of between 1 and 180 degrees, 30 and 160 degrees, 40 and 130 degrees, 40 and 120 degrees, or 45 and 130 degrees with respect to the first pressure sensing channel segment.
  • a passive pressure sensing channel comprises at least three segments, wherein at least a first segment extends horizontally or at an angle from the reaction well 2, at least one second segment extends from the first segment at other than a straight line and optionally at an angle of between 1 and 180 degrees, 30 and 160 degrees, 40 and 130 degrees, 40 and 120 degrees, or 45 and 130 degrees with respect to the first segment, and at least one third segment extends from the second segment at other than a straight line and optionally at an angle of between 1 and 180 degrees, 30 and 160 degrees, 40 and 130 degrees, 40 and 120 degrees, or 45 and 130 degrees with respect to the second segment.
  • the second fluid transport channel 5A extends from the third fluid transport channel SB at an angle of between 1 and 180 degrees.
  • the second fluid transport channel SA, the interface channel segment SC and the third fluid transport channel SB together form a straight or other than straight fluidic path.
  • the fluidic constriction channel 4 is at an angle relative to the second and/or third fluid transport channels (5A, SB).
  • the angle can be between 25 and 155 degrees, 30 and 145 degrees, 45 and 135 degrees, 60 and 120 degrees.
  • the angle is between 70 and 110 degrees, 80 and 100 degrees, 85 and 95 degrees, 88 and 92 degrees, about 90 degrees, or 90 degrees.
  • the hydrodynamic resistance ratios of channels within a microfluidic device for detecting a reaction product are typically effective for, operable for, adapted for, and/or provide that when the first fluid transport channel, the second fluid transport channel, the interface channel segment, the fluidic constriction channel, the reaction weli, and optionally a portion of the third fluid transport channel, are filled with a fluid, and a negative pressure is applied at the first port for a period of time or a positive pressure is applied at the third port for a period of time, the fluid is trapped in the reaction well and optionally the fluidic constriction channel, but removed from the rest of the device.
  • the hydrodynamic resistance ratios of channels within a microfluidic device for detecting a reaction product are as follows: the passive pressure sensing channel has 1.01 to 5xl0 7 ,1.01 to 4,8xl0 7 , 10 to 1x10*, 100 to 1x10*, or 100 to 1x10 s , times the resistance of the second or third fluid transport channels: the fluidic constriction channel has 4.0x10* to 2.5, 1x10* to 1, 1x10* to 0.1, or 1x10'* to IxlO* 2 times the resistance of the second or third fluid transport channels; the fluidic constriction channel has 2x10* to 700, 2x10 s to 100, 2xl0" 2 to 10, or 2x10* to 1 times, the resistance of the reaction well; the reaction well has 7xl0 7 to 0.99, 7x10* to 0,1, 7x10 s to 0.01 times the resistance of the second or third fluid transport channel; and/or the
  • the fluidic constriction channel 4 in embodiments of a device for detection a reaction product, such as that illustrated in FIG. IS, can have dimensions and a phytical makeup similar to fluidic constriction channels of other devices disclosed herein.
  • the fluidic constriction channel can be Composed of a neutral or slightly hydrophilic material.
  • the fluidic constriction channel 4 is comprised of a hydrophobic material.
  • the microfluidic device has a precipitate therein.
  • the second fluid transport channel, the third fluid transport channel, the interface channel segment, the channel, and/or the second fluid transport channel can include a precipitate therein.
  • at least the second fluid transport channel comprises a precipitate therein.
  • the reaction well 2 and optionally the fluidic constriction channel 4 of a microfluidic device for detecting a reaction product are filled with fluid, but the rest of the device is empty.
  • the volume of the reaction well has a volume of between 1 nl and 450 nl, 5 nl and 250 nl, 5 nl and 100 nl, 10 nl 50 nl or between 15 and 35 nl.
  • this disclosure provides microfluidic assemblies comprising at least two of the fluidic devices illustrated in FIGS. 15-19 (e.g., fluidic device subunits).
  • the microfluidic assembly comprises an array of between 2 and 256 of such fluidic devices, optionally between 4 and 64 of the devices.
  • the fluidic device subunits of the array are not fluidly connected, and in some embodiments these are fluidly connected. In some embodiments, less than all of the fluidic device subunits may be fluidly connected to one another. In some embodiments, multiple microfluidic assemblies are fluidly connected to one another.
  • the fluidic device subunits (or microfluidic assemblies) of the array are grouped into two or more groups, wherein devices of the same group are fluidly connected.
  • fluidic devices that are fluidly connected can comprise a first port and/or a third port that functions as the third port or first port respectively of the next device in fluid communication in the group; or wherein the first port may serve as a universal first port for all of the devices In the group or each device will have an Independent first port, and the third port may serve as the universal third port for all devices in the group or each device will have an independent third port.
  • the microfluidic assembly is a disposable cartridge. Other embodiments of microfluidic assemblies are also possible as would be understood by those of ordinary skill in the art
  • Certain asects provided herein are methods for detecting, measuring, forming, or analyzing a reaction product, in illustrative embodiments, a precipitate, or methods for detecting whether a first fluid and a second fluid react, or methods for detecting whettier components of a first fluid react with components of a second fluid, or methods for detecting an interaction of a first fluid and a second fluid, using a device referred to herein as a microfluidic device tor detecting a reaction product, for example as illustrated in FIG. 15.
  • a device referred to herein as a microfluidic device tor detecting a reaction product for example as illustrated in FIG. 15.
  • Such methods in the following paragraphs for ease of reference will be referred to as methods for detecting a reaction product. Part numbers referenced in the following paragraphs related to methods for detecting a reaction product are shown in FIG.15.
  • Such methods can involvethe following steps: a. introducing a first fluid into the device typically through the first port; b. trapping a volume of the first fluid in the reaction well 2, in illustrative embodiments by capturing a droplet of a volume, optionally a pre-defined volume, of the first fluid in the reaction well 2; c. introducing a second fluid (i.e.
  • a second solution into the device so that it can interact with the trapped volume of the first fluid, typically into third fluid transport channel SB and the interface channel segment 5C, typically through the third port 6 such the first and second fluids mix in at least part of the interface channel segment SC and/or the fluidic constriction channel 4 to form a reaction product Of one Or more components of the first fluid and one or more components the second fluid; and optionally, but typically d. detecting the reaction product
  • Step of introducing a first fluid into the device, or filling the fluidic device with the first fluid is an optional step, since it is envisioned that a device could be supplied to a user wherein the reaction well Is pre-fiiied, for example.
  • filling the fluidic device with the first fluid is accomplished by using a positive pressure to inject the first fluid through the first port.
  • a volume of a first solution Including one or more test compound(s) or compound(s) of Interest is loaded into the device, for example through the first port.
  • a first solution Including one or more test compound(s) or compound(s) of Interest (e.g., members of a library of candidate compounds, or a potential anti-perspirant solution) is loaded into the device, for example through the first port.
  • the step of introducing a second fluid (i.e. a second solution) into the device so that it can interact with the trapped volume of the first fluid typically involves delivering the second fluid into the third fluid transport channel SB and the interface channel segment SC, typically thru the third port 6.
  • the second fluid can be introduced into the third fluid transport channel at a flow rate of between G.01 nl/min and 1 ml/min, 0.05 nl/min and 100 nl/min, 0.05 nl/min and 50 pl/min, 1 nl/min and 25 pl/min, 100 nl/min and 1 pl/min, 1 pl/min and 100 pl/min, or lul/min and 10
  • the composition of the second fluid is not intended to be limited, and can include, as a nonlimiting example, members of a candidate compound library, nucleic acids, proteins, carbohydrates or lipids. Furthermore, the Composition of the first fluid and the second fluid can be switched. In other embodiments, mammalian sweat, an articial sweat, or other sweat-based compound can be the second compound.
  • a sweat-based compound can be any fluid designed to mimic sweat containing critical sweat compounds, including but not limited to artificial sweat and/or simulated body fluid including a variety of dissolved salts in distilled water along with a small amount of BSA as a model protein (e.g., between 0.01 and 1% BSA).
  • the first and second fluids mix in at least part of the interface channel segment 5C and/or the fluidic constriction channel 4 to form a reaction product of one or more components of the first fluid and one or more components the second fluid.
  • formation of the reaction product results in an increase in pressure in at least one channel within the device, and the increased pressure is detected.
  • the reaction product forms a plug, such as a precipitate plug, that blocks flow through one or more channels of the devices and in illustrative embodiments, this blockage of flow is detected and/or measured using the passive pressure sensing channel 3A.
  • this fluid can be detected and optionally measured, thus detecting the formation of the plug and optionally providing the ability to measure the strength of the blockage caused by the plug.
  • the passive pressure sensing channel 3A priorto the introduction of the second fluid into the third fluid transport channel SB, the passive pressure sensing channel 3A is filled with air and does not comprise fluid, such that a fluid-air interface is present at the point at which the reaction well 2 and the passive pressure sensing channel 3* connect.
  • the passive pressure sensing channel forms a sensitive sensor that is capable of, adapted for, and/or designed to measu re the strength of pressure build-up in the device upon formation of a reaction product that iribhibits flow in the device, for example inhibiting flow in the second transport channel, the interface channel segment, the fluidic constriction channel, the first transort channel, and/or the third transport channel.
  • Such reaction product can be a thickened fluid, a gel, a polymer, a hardened product, an aggregated product, and in illustrative embodiments, a precipitate
  • a camera is used to visualize and record the formation of a thickened fluid, a gel, a polymer, a hardened product, an aggregated product, and in illustrative embodiments, a precipitate.
  • a physical reaction product may not result from the mixing of the fluids, but the interaction between the two fluids is still important to monitor (e.g., visually or by analyzing the flow of fluids).
  • Video images can then be analyzed using known methods for analysis and measurement of such structures for example using detection systems similar to those discussed herein for microfluidic devices for producing a reaction product such as particles.
  • the reaction product forms a plug that stops flow through at least one of the channels of the device, for example the second fluid transport channel, the interface channel segment, or foe fluidic constriction channel.
  • the reaction product that forms is a fluorescent product, a colored product, or exhibits a change of color, any of which can be detected.
  • a camera in illustrative embodiments, a video camera, can be optically connected to any channel in the device, and in illustrative embodiments is optically connected to the interface channel segment, the fluidic constriction channel, the second fluid transport channel, and/or the passive pressure senring channel.
  • a camera in illustrative embodiments, a video camera, can be optically connected to any channel in the device, and in illustrative embodiments is optically connected to the interface channel segment, the fluidic constriction channel, the second fluid transport channel, and/or the passive pressure senring channel.
  • FIG. 16 An exemplary method for detecting a reaction product Is explained with reference to parts as labeled in FIG. IS.
  • the first fluid (Le. foe fluid to be captured in the device (i.e. trapped in the reaction well)) is introduced into foe device. Fluid entry for this initial loading, in this illustrative example, occurs at the first port 1.
  • a first fluid is passed through this port via positive applied pressure and into the first fluid transport channel (1A, e g.> section 1A1) (FIG. 16 step A).
  • the first fluid transport channel is not in direct fluidic communication with the reaction well.
  • Fluid continues moving through the first fluid transport channel (e.g., from 1A1 into 1A2) into foe second fluid transport channel SA, where it then readies the interface channel segment 5C followed by the third fluid transport channel SB.
  • fluid begins to fill the fluidic constriction channel 4, reaction well 2 and the third fluid transport channel 5B, based in large part by the ratio of hydrodynamic resistances between these parts (FIG. 16 step B).
  • the continuous channel fanned by the third fluidic transport channel SB, the interface channel segment 5C, and the second fluid trasnport channel SA forms a T junction.
  • excess fluid is typically removed to initiate capture of a volume (e.g. droplet capture) of the first fluid in the device.
  • Droplet capture in this device is passive in nature; due to the constructed geometry of the device, as illustrated in FIG. 15 and FIGs. 17 steps A-C, a reproducible volume of tire first fluid is captured in the reaction well.
  • excess fluid is typically removed from the device, and a negative pressure is applied at the first port 1. Fluid retreats from the first fluid transport diannel 1A in a direction opposite to its loading direction (i.e., fluid retreats towards the first port 1).
  • fluid in the reaction well 2 does not retreat towards the first port 1 and remains in the reaction well 2 (FIG. 17 step B).
  • the geometry of the device includes the dimensions of the device. The width, length, and depth of each channel play a role in the hydrodynamic resistance of each channel. The ratios of hydrodyanmic resistances between channels play a role in the movement of fluid in the device and in the capture of the first fluid in the reaction well.
  • reaction well 2 remains fall of tire first fluid, while excess fluid continues to retreat from the third fluid transport channel SB thru the interface channel segment SC to the second fluid transport channels 5A, to the first fluid transport channel (through the curved section 1A2 and straight section 1A1), and the first port 1 until only the reaction well 2 and fluidic constriction channel houses any liquid in the system (FIG. 17 step C) (i.e., a first fluid droplet is captured and housed in the reaction well 2 ) and fluid connecton channel 4).
  • a positive pressure is applied from the third port instead of a negative pessure from the first port to capture a volumen of the first fluid in the reaction well 2.
  • a second fluid is then introduced into the device to interact with the first fluid droplet captured and housed in the reaction well 2.
  • This second fluid is passed through the third port 6 via a positive applied pressure and enters the third fluid transport channel SB (FIG. 18 panel A).
  • As fluid passes through part SB it reaches the interface channel segment 5C and the opening of the reaction well 2 (at fluidic constriction channel 4), where a fluid-air Interface of the first fluid exists.
  • the incoming second fluid meets with and interacts with (i.e., mixes with) the first fluid housed in the reaction well 2 and fluidic constriction channel 4.
  • first fluid and second fucid first occurs in the interface channel segment SC, the fluidic constriction channel 4 and in the entrance to tire reaction well 2 at the junction between the fluidic constriction channel 4 and the reaction well 2.
  • Mixed first fluid and second fluid continues to flow from the interface channel segment SC towards the second fluid transport channel SA.
  • This mixed fluid continues to flow from the second fluid transport channel SA to and through the first fluid transport channel 1A and finally to the first port 1, where fluid exits the device (FIG. 18 panel B).
  • the second fluid is introduced into the device through the third port 6 for a variable period of time and in variable volume, depending on the particular use. It is noteworthy that in illustrative embodiments of the device aspect provided in FIGS. 15-18 herein, the fluidic constriction channel 4 allows for mixing of fluids but does not promote complete washing and/or quick complete fluid replacement
  • a precipitate e.g, a plug
  • this precipitation plug will grow over time as the first and second fluids continue to mix and Interact.
  • the growth profile of the precipitation plug depends on the nature of the second fluid, the nature of the first fluid, the temperature of the device, the flow rate of the incoming second fluid, and theoretically the humidity of the device.
  • the growth rate also partially depends on the sizes of the second and third fluid transport channels, the interface channel segment, the fluid connection channel, and the reaction welt If the reaction well has a larger opening as opposed to a smaller opening, there will be more mixing of the first and second fluids. It Is believed that this different mixing volume and mixing rate could have an effect on precipitate formation as well. As the precipitate plug grows over time, in some cases it blocks the flow of the second fluid through the second fluid transport channel 5A (FIG. 18 panel D) (i.e., it inhibits incomingflow of second fluid towards the first port 1). thus increasing pressure buildup within the device.
  • This pressure buildup is exerted on the fluid in the reaction well 2 and is thus also exerted on the fluid-air interface located at the entrance of the passive pressure sensing channel 3A.
  • This fluid-air interface in the passive pressure sensing channel exhibits an inherent capillary pressure.
  • This capillary pressure relies directly on the surface tension of the first fluid, the contact angle of the first fluid with the device material, and the dimensions of the passive pressure sensing channelSA.
  • the pressure build-up induced by the precipitate plug increases and exceeds the above-mentioned capillary pressure, it overcomes the capillary pressure holding the fluid interface at the beginning of passive pressure sensing channel 3A.
  • fluid begins to flow through the passive pressure sensing channel 3A and out of the device through the second port 3, where it can be detected (e.g., visually such as by eye or by using an imaging device such as a camera).
  • an imaging device such as a camera
  • the flow of fluid through it provides a built-in indicator of precipitate plug strength.
  • the capillary pressure exhibited by the fluid resting at the beginning of passive pressure sensing channel 3A can be modified by changing the depth and width of the channel (e.g., using the ranges listed below or any other suitable depth and/or width).
  • this built-in pressure sensor can be adjusted to match pressures that may be relevant in different industrial fields.
  • Non-limiting, exemplary ranges of suitable dimensions for the passive pressure sensing channel 3A are shown in Table 2.
  • a fluidic device comprising: a) a first port; b) a first fluid transport channel in fluid connection with: i. the first port; ii. a reaction well; and, iii. an overflow channel; c) a second fluid transport channel in fluid communication with the overflow channel; d) a fluidic constriction channel in fluid communication with the reaction well and the second fluid transport channel; and e) a second port in fluid communication with the second fluid transport channel.
  • a fluidic device assembly comprising at least two microfluidic devices in a disposable cartridge, wherein each fluidic device comprises: a) a first port; b
  • the at least two microfluidic devices are connected in serial, in some embodiments of the fluidic device assembly aspect immediately above, the at least two microfluidic devices are connected in parallel.
  • a fluidic device assembly comprising a series of fluidic devices, wherein each fluidic device of the series comprises: a first fluid transport channel in direct fluid communication with a reaction well and an overflow channel; a second fluid transport channel in direct fluid communication with the overflow channel; and a fluidic constriction channel in direct fluid communication with the reaction well and the second fluid transport channel, wherein: the first fluid transport channel of a first fluidic device in the series is in fluid communication with at least a first port channel and a second port channel, wherein said first port channel and said second port channel terminate in a first port channel port and typically a second port channel port, respectively; a second fluid transport channel of the first fluidic device in the series is in fluid communication With a first fluid transport channel of a second fluidic device in the series; the second fluid transport channel in the second fluidic device in the series, and subsequent devices in the series if present, are in fluid communication with the first fluid transport channel of the next fluidic device in the series; and typically, the second fluid
  • the first port channel is filled with a lipid in an organic solvent or a polymer dissolved in a solvent, and wherein the second port channel is filled with an aqueous solution.
  • the first port channel is filled with a protein and the second port channel is filled with a protein precipitant.
  • the fluidic device or a fluidic device of the fluidic device assembly further comprises a lipid in an organic solvent or a polymer dissolved In a solvent, and an aqueous solution; or wherein the fluidic device further comprises particles.
  • the fluidic device or fluidic device assembly further comprises a protein precipitate.
  • the reaction welifs comprises one or more of one or more lipids, an organic solvent, an alcohol, acetonitrile, a polymer, an aqueous buffer, a mixture thereof, and/or nanoparticles in solution.
  • the device assembly can further comprise third, fourth, fifth, etc. fluid transport channels in fluid communication With corresponding third, fourth, fifth, etc. input ports, respectively, and in fluid communication typically through one or more additional channels to one or more reaction wells.
  • additional input fluids third, fourth, fifth, etc. fluids
  • fluidic systems comprising multiple (e.g. 2, 3, 4, 5, 10, 15, 20, etc.) fluidic device assemblies, for example fluidic device assemblies (such as those immediately above) comprising a series of fluidic devices, where the fluidic device assemblies in illustrative embodiments, are fluidly connected or linked in parallel.
  • fluidic devices herein that are effective producing particles provide a system that is effective for, adapted to, and operable to produce different size partides by controlling certain parameters when such fluidic devices are used to produce particles.
  • Such parameters include, for example, a first flow rate of a stream of the first fluid as it introduced Into the fluidic device, a second flow rate of a stream of the second fluid as it introduced into the fluidic device, a ratio of the first flow rate to the second flow rate, a combined flow rate of the combined first and second streams, overal dimensions of the fluidic device used to perform a method for producing particles, wherein larger size parts of the fluidic device provide larger partides than smaller size parte, a width of the fluidic constriction channel of the fluid device used to perform a method for producing particles, or combinations thereof.
  • a fluidic device comprising: a) a first port; b) a first fluid transport channel in fluid connection with: i. the first port; ii. a reaction well; and, ill an overflow channel; c) a second fluid transport channel in fluid communication with the overflow channel; d) a fluidic constriction channel in fluid communication with the reaction well and the second fluid transport channel; and e) a second port in fluid communication with the second fluid transport channel, wherein the fluidic device comprises in the reaction well, an aqueous solution and either a lipid in an organic solvent or a polymer dissolved In a solvent; wherein the fluidic device further comprises partides; or wherein the fluidic device comprises in the reaction well, a lipid in an organic solvent or a polymer dissolved In a solvent, and an aqueous solution, and the fluidicdevice further comprises partides.
  • a fluidic device comprising: a ⁇ a first port; b) a first fluid transport channel in fluid connection with: i. the first port; ii. a reaction well; and,
  • an overflow channel Hi. an overflow channel; c) a second fluid transport channel in fluid communication with the overflow channel; d) a fluidic constriction channel In fluid communication with the reaction well and the second fluid transport channel; and e) a second port in fluid communication with the second fluid transport channel, wherein the fluidic device further comprises a protein precipitate.
  • the fluidic device or fluidic device assembly operable to produce, and effective for producing a reaction product, unless already stated therein or incompatible therewith, the fluidic device or fluidic device assembly is in a disposable cartridge.
  • the fluidic device or fluidic device assembly does not comprise a passive air control valve, or comprises a passive air control valve, for example in fluidic communication with a reaction or each reaction well.
  • the width or effective diameter of the fluidic constriction channel(s) is between 10 pm and 500 pm, 50 pm and 250 pm, 50 pm and 300 pm, 50 pm and 200 pm, or 50 pm and 150 pm, or the width or effective diameter of the fluidic constriction channei(s) is at least 50 pm and smaller than tire width or effective diameter of each of the following components: the first fluid transport channel 1A, the reaction well 2, a second fluid transport channel 5A, directly connected to the fluidic constriction channel 4 opposite the reaction well 2, and an overflow channel 3 that connects the first fluid transport channel 1A to the second fluid transport channel 5A as provided herein.
  • the fluidic constriction channel is less than one-fifth and in some embodiments less than one-sixth, one-seventh, one-eighth, one-ninth, or one-tenth the diameter or width of the above-stated components.
  • the width, diameter or effective diameter of the fluidic constriction channels is less than, approximately 0.15 to approximately 0.30 times, the width, diameter, or effective diameter of toe reaction well.
  • the reaction well(s) comprises at least one pillar, optionally having a width, diameter, or effective diameter of about 100 urn, and optionally a circular, triangular, or rectangular shape.
  • toe reaction well(s) comprises at least two, three, four, five, six, seven, eight, nine, 10, 11, 12,
  • the fluidic device or the fluidic devices within the fluidic device assembly is/are capable of, adapted to, and/or operable to transform an input laminar flow fluid stream into an unstable flow, but not a turbulent flow.
  • the reaction well is configured to hold, contain, or retain, operable to hold, contain, or retain, capable of retaining, adapting, or holding, or adapted to hold, contain, or retain a volume between 100 pl and 10 ml, between 1 nl and 10 ml, between 1 pl and 10 ml, between 1 nl and 10 ml, between 1 pl and 450 pl, between 5 nl and 15 nl, between 15 nl and 35 nl, between 100 nl and 1 ml, between 100 nl and
  • pl 100 pl, between 1 pl and 1 ml, between 5 pl to 30 pl, between 10 pl and 1 ml, between 1 pl and 500 pl, between 10 pl and 500 pl, between 10 pl and 250 pl, between 10 pl and 200 pl, between 10 pl and 100 pl or between 10 pl and 50 pl, or about 10 pl.
  • a method for producing a reaction product using a microfluidic device comprises: a) introducing a first fluid into a first fluidic channel of the microfluidic device through an inlet port; b) introducing a second fluid into the first fluidic channel of the microfluidic device, in illustrative embodiments through a second inlet port; and c) producing the reaction product by mixing the first fluid and the second fluid in a reaction well of the microfluidic device that is fluidly connected to the first fluidic channel.
  • the microfluidic device is a microfluidic device assembly comprising two or more fluidic devices, and the first fluid and second fluid are introduced in all the microfluidic devices of the device assembly and/or in illustrative embodiments the reaction product forms by mixing the first fluid and the second fluid in the reaction well of each microfluidic device of the device assembly.
  • the above method aspect is performed using any fluidic device or fluidic device assembly herein, unless incompatible therewith, as non-limiting examples any of the fluidic devices or fluidic device assemblies provided herein in this Exemplary Embodiments section, for example a fluidic device assembly comprising two or more fluidic devices fluidly connected in paralid, or in illustrative embodiments, fluidly connected in series.
  • the reaction product in some embodiments is a protein predptant.
  • the reaction product in illustrative embodiments comprises microparticles or is microparticles,
  • the reaction product is continuously harvested from the fluidic device.
  • the method further comprises monitoring consistency of the reaction product over time by measuring the width of a fluid stream of the reaction product and the width of a fluid stream of a reference fluid.
  • at least IL 2L or 51 of particles e.g.
  • micropartides and hanopartides or between IL and 10L IL and 5L IL and 2L or 2L and 5L of partides are harvested from the fluidic device and/or between 11 and 10L IL and 5L IL and 2L, or 2L and 5L of combined first, second, and optionally third, fourth etc fluid are fed into the fluidic device.
  • Further scale-up is provided herein by performing such method using multiple (e.g. 2, 3, 4, 5, 10, 15, 20, etc.) fluidic device assemblies, for example fluidic device assemblies comprising a series of fiuidic devices, where the fluidic device assemblies in illustrative embodiments, are linked in parallel.
  • Such methods in illustrative embodiments can be used to produce different size particles in a controlled manner, by setting certain parameters such as, for example, a first flow rate of a stream of the first fluid as it introduced into the fluidic device, a second flow rate of a stream of the second fluid as it introduced into the fluidic device, a ratio of the first flow rate to the second flow rate, a combined flow rate of the combined first and second streams, overai dimensions of the fluidic device used to perform the method, wherein larger size parts of the fluidic device provide larger particles than smaller size parts, a width of the fluidic constriction channel of the fluid device used to perform the method, or combinations thereof,
  • a method for producing a reaction product using any fluidic device assembly herein, unless incompatible therewith, as non-limiting examples any of the fluidic device assemblies provided herein in this Exemplary Embodiments section comprising: a. filling the fluidic device by introducing a first fluid through the first port Into the fluidic device; b. trapping the first fluid in the reaction well and the fluidic constriction channel connected therewith by applying negative pressure at the first port to remove some of the first fluid from the fluidic device; and, c. introducing a second fluid into the reaction well through the first portto mix with and replace the first fluid, wherein mixing of the first fluid and the second fluid produces a reaction product.
  • the first fluid is essentially removed from the first fluid transport channel, the overflow channel, and the second fluid transport channel before introducing the second fluid into the reaction well.
  • the input of the first fluid and the second fluid creates a laminar flow of the first fluid and the second fluid, and the method further comprises transforming the laminar flow of the first fluid and the second fluid into an unstable flow, but typically not a turbulent flow.
  • the first fluid comprises a protein
  • tire second fluid is a protein precipitant
  • the reaction product comprises a protein precipitate comprising the protein
  • a method Or process that indudes the steps illustrated in RG. 2 is provided herein, typically using a fluidic device provided herein.
  • a first fluid e.g., an organicsolvent solution comprising a lipid for lipid-based nanopartides or a polymer solution for polymer-based nanoparticles; indicated as a solid fill within the fluidic device
  • a second fluid e.g., as described herein for the production of nahoparticles
  • the first fluid comprises an organic solvent solution comprising dissolved lipids, or a polymer solution comprising at least one polymer dissolved in a solvent and the second fluid comprises water or an aqueous buffer where the first fluid Is an organic solvent solution comprising dissolved lipids, or a water-soluble synthetic polymer solution where the first fluid comprises at least one polymer dissolved in a solvent, and wherein the reaction product is a solution or suspension of particles.
  • the device is a microfluidic device and the solution or suspension of particles is a solution or suspension of nanoparticles.
  • the partides are comprised of a metal.
  • Such metal can indude, but is not limited to, silver, gold and copper.
  • the particles are metallic nanoparticles.
  • the dissolved lipids can comprise at least one lipid selected from the group consisting of dipalmitoylphosphatidyfcholine (DPPC); cholesterol; 1,2-Diiauroyl-sn- glycero-3-phosphocholine (DLPC); l,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC); 1,2-Distearoyl- sn-glycero-3-phosphocholine (DSPC); l,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1,2-Dimyristoyl- sn-glycero-3-phosphoethanolamine (DMPE); l,2-Dipatmitoyl-sn-glycero-3-phosphoethanolamine (DPPE); l,2-Dioleoyl-sn-glycero-3-phosphophospho
  • the first fluid Comprises an organic solvent solution comprising dissolved lipids
  • the dissolved lipids comprise at least two different types of lipids, optionally selected from the group consisting of DPPC, cholesterol and DOTAP.
  • the first fluid comprises an organic solvent solution comprising dissolved lipids
  • the organic solvent is selected from the group consisting of ethanol, methanol and chloroform, ethyl acetate, isopropanol, and hexane
  • the first fluid comprises an organic solvent solution comprising dissolved lipids
  • the dissolved lipids comprise DPPC, cholesterol and DOTAP
  • the organic solvent solution comprises ethanol.
  • the aqueous buffer Is a physiological buffer, optionally phosphate-buffered saline.
  • the polymer is selected from the group consisting of polylactic add (PLA), poly-l-)ysine (PH), polyglutamic add (PGluA), polyglycolic acid (PGA), polyethylene glycol (PEG), polycaprolactone (PCL), polyaspartate (PAA), poiy(d,l- lactide-co-glycolic) add (PLGA), cydodextrins (CD), and N-(2-hydroxypropyl)-methacrylamide copolymer (HPMA), a natural polymer, chitosan, heparin, albumin, dextran, gelatin, alginate, collagen, and a mixture thereof.
  • the solvent is selected from the group consisting of dichloromethane and ethyl acetate, benzyl alcohol, cyclohexane, acetonitrile, and acetone.
  • the water-soluble synthetic polymer solution comprises poly(vinyl alcohol) or didecyldimethylammonium bromide; and the aqueous solution is optionally water or phosphate-buffered saline.
  • the reaction Well or one or more reaction wells can comprise nanopartides, optionally wherein said nanopartides are lipid-based nanopartides or polymeric nanopartides.
  • the lipid-based nanopartides comprise at least one lipid selected from the group consisting pf dipalmitoylphosphatidylcholine (DPPC); cholesterol; l,2-Dilauroyl-sn-glycero-3- phosphocholine (DLPC); l,2-Dimyristoyl-sn-glycero «3*phosphocholine (DMPC); 1,2-Distearoyl-sn-glycero- 3-phosphochoiine (DSPC); l,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1,2-Dimyristoyl-sn-giycero- 3-phosphoethanolamine (DMPE); l,2-Dipalmitoyl-sn ⁇ ycero-3-phosphoethanolamine (DPPE); 1,2- Dioleoyl-sn-glycero-3-phosphoethanoiamine (DOPE); l,2-Dimyristoyl-sn-sn-
  • the solvent is selected from the group consisting of dichloromethane and ethyl acetate, benzyl alcohol, cydohexane, acetonitrile, and acetone and/or the water-soluble synthetic polymer solution comprises poly(vinyl alcohol) or didecyldimethylammonium bromide; and the aqueous solution is optionally water or pbosphate-buffered saline.
  • the aqueous solution or water-soluble synthetic polymer solution comprises a nucleic acid molecule, detection agent, or a therapeutic agent that is enveloped within the nanoparticle upon mixture of the water-soluble synthetic polymer and the polymer solution.
  • the aqueous buffer or water soluble polymer solution, respectively is introduced into the fluidic device at a flow rate of from one to 30 ml/minute, optionally from five to 20 ml/minute or 10 to 20 ml/minute.
  • the fluidic constriction channel of the fluidic device(s) has a width or diameter of less than 400 urn and the flow rate Is greater than 5 ml/minute.
  • the nanopartides have a diameter of between 5 nm and 500 nm. Furthermore, such methods can further include characterizing the properties of the nanopartides, optionally wherein said properties comprise size, polydispersity index (PDI), or zeta potential, optionally as measured using dynamic light scattering (DIS) or transmission electron microscopy (TEM).
  • PDI polydispersity index
  • DIS dynamic light scattering
  • TEM transmission electron microscopy
  • particles e.g. microparticles or nanoparticles
  • the nanoparticles including nanoparticles in a microfluidic device herein can have a diameter of less than 600 nm, for example between 5 nm and 500 nm.
  • the nanoparticles can comprise at least one detection agent and/or at least one therapeutic agent.
  • the fluidic device comprises a first input port, between 100 to 1000 pl, optionally 100 to 200 pl, of the first fluid is Introduced through the first port; or wherein multiple fluidic devices are fluidly connected to one another in series or parallel, greater 1000 pl aqueous buffer or water, or between 400 pl and 5 ml of aqueous buffer or water is introduced through the first port.
  • the first fluid transport channel comprises a diameter distal to the first port of about four times its diameter proximal to the first port; b) the diameter of the reaction well is approximately twice the diameter of the fluid transport channel proximal to the first port; c) the length of the reaction well is approximately one third the length of the first fluid transport channel; d) the diameter of the overflow channel is approximately 0.4 to 0.75 times the diameter of the first fluid transport channel distal to the first port; e) the length of the overflow channel is at least about 0.9 times the length of the first fluid transport channel; f) the second fluid transport channel comprises a diameter distal to the first port of about two times its diameter proximal to the second port; and/or g) the length of the second fluid transport channel is approximately equivalent to approximately
  • the fluidic device is comprised of PDMS wherein the diameter of the overflow channel is approximately 0.6 times the diameter of the first fluid transport channel distal to the first port; or the fluidic device is comprised of PDMS wherein the diameter of the overflow channel is approximately 0.6 times the diameter of the first fluid transport channel distal to the first port; or the fluidic device is comprised of COC wherein the diameter of the overflow channel is approximately 0.5 times the diameter of the first fluid transport channel distal to the first port.
  • the diameter of the fluidic constriction channel is approximately 0.15 to approximately 0.30 times the diameter of the reaction well; the diameter of the fluidic constriction channel is approximately 150-225 pm, optionally wherein the fluidic constriction channel is comprised of PDMS; the diameter of the fluidic constriction channel is approximately 175-200 mu; the diameter of the fluidic constriction channel is approximately 160-215 pm, optionally wherein the fluidic constriction channel is comprised of COC; the diameter of the fluidic constriction channel is approximately 0,2-0.25 times the diameter of the reaction well; the diameter of the fluidic constriction channel is approximately 0.1-0.2 times the diameter of the second fluid transport channel at the point at which the fluidic constriction channel and the second fluid transport channel contact one another; the length of the fluidic constriction channel Is approximately 0.1-0.25 times the length of the
  • the fluidic constriction channel is comprised of ROMS and has a length of approximately 0.1- 0.175 times, optionally 0.125-0.150 times, the length of the reaction well; or the fluidic constriction channel is comprised of COC and has a length of approximately 0.11-0.13 times the length of the reaction well.
  • the fluidic device has a height of about 300 pm to about 500 pm, optionally about 500 pm;
  • the first fluid transport channel has a length of from about 2000 urn to about 10,000 urn, optionally about 5900 pm, and/or a width or diameter of about 1000 um to about 2000 um, optionally about 1200 urn;
  • the overflow channel has a length of from about 8000 um to about 15,000 um, optionally about 10,900 um, and/or a width or diameter of about 1200 um to about 2000 um, optionally about 1200 um;
  • the second fluid transport channel has a length of from about 2000 um to about 10,000 um, optionally about 1500 um, and/or a width or diameter of about 1000 um to about 2000 um, optionally about 1500 um;
  • the reaction well has a length of from about 5000 um to about 12,000 um, optionally about 7460 um, and/
  • a fluidic device comprising: a first fluid transport channel comprising a straight segment, said first fluid transport channel in fluid connection with a first port and optionally comprising a section having a rounded orientation; a second fluid transport channei; a fluidic constriction channel; a reaction well; a passive pressure sensing channel in fluid connection with a second port; a third fluid transport channel in fluid connection with a third port; and an interface channel segment; wherein: the second fluid transport channel is in direct fluidic communication with the first fluid transport channel; the fluidic constriction channel is in direct fluidic communication with the reaction well and the interface channel segment connecting the second fluid transport channel and the third fluid transport channel; and the reaction well is in direct fluidic connection with the passive pressure sensing channel.
  • such fluidic device is effective for determining and/or detecting and operable to determine and/or detect a reaction product or whether a first fluid and a second fluid react by forming a reaction product
  • the width of the second and third fluid transport channels are the same or different and between 3/200 and the same width of the first fluid transport channel, optionally wherein the width of the second and third fluid transport channels are the same.
  • the depth of the second and third fluid transport channel are the same or different and between 3/70 and the same depth of the first fluid transport channel. In some embodiments of the immediately above aspect, or any fluidic device herein that is effective for detecting, and operable to detect whether a first fluid and a second fluid react by forming a reaction product, the depth of the second and third fluid transport channels are the same.
  • the width and depth of an end of the interface channel segment directly connected to the second fluid transport channel is the same as the width and depth of the second fluid transport channel and the width and depth of an opposite end of the interface channel segment directly connected to the third fluid transport channel is identical to the width and depth of the third fluid transport channel, optionally wherein the width and depth of the interface channel segment, the second fluid transport channel, and the third fluid transport channel are the same,
  • the length of the interface channel segment is equal to the width of the fluidic constriction channel.
  • the hydraulic diameter of the second and third fluid transport channels are the same or different and between 3/105 to 1/1 the hydraulic diameter of the first fluid transport channel, optionally wherein the hydraulic diameter of the second and third fluid transport channels are the same.
  • the hydraulic diamater of the second fluid vansport channel is between 1/6 and 1/1 the hydraulic diameter of the third fluid transport channel, optionally wherein the hydraulic diameter of the second and third fluid transport channels are the same.
  • the length, width and depth of the fluidic constriction channel is between 10-500 urn, 15-500 um, and 15-300 um
  • the length, width and depth of the interface channel segment is between 15*500 um, 15-100 um, and 15*100 um, respectively
  • the length of the fluidic constriction channel is between .0025 to 1.25 times the length of the second and/or third fluid transport channels
  • the width of the fluidic constriction channel is between ⁇ 1 to 33 times the width of the second and/or third fluid transport channels
  • the width and/or depth of the fluidic constriction channel are the same or different from those
  • the passive pressure sensing channel has a smaller width and/or depth compared to the interface channel segment, the second fluid transport channel and the third fluid transport channel, such that the hydrodynamic resistance of the passive pressure sensing channel is at least 1.01 times the hydrodynamic resistance of each of the interface channel segment, the second fluid transport channel and the third fluid transport channel, and optionally the hydrodynamic resistance of the passive pressure sensing channel is between 1,01 and 5x107 times the hydrodynamic resistance of each of the interface channel segment, the second fluid transport channel and the third fluid transport channel.
  • the passive pressure sensing channel terminates at the second port and: a) is a straight channel; b) comprises at least one bend, rounded orientation, and/or curve; c) comprises at least two pressure sensing channel segments, wherein at least a first pressure sensing channel segment extends horizontally or at an angle from the reaction well, and at least one second pressure sensing channel segment extends from the first segment at other titan a straight line, optionally at an angle Of between 1 and 180 degrees or 40 to 120 degrees with respect to the first pressure sensing channel segment; or d) comprises at least three pressure sensing channel segments, wherein at least a first pressure sensing channel segment extends horizontally or at an angle from the reaction well, at least one second pressure sensing channel segment extends from the first pressure sensing channel segment at other than a straight line and optionally at an
  • any fluidic device herein that is effective for detecting, and operable to detect whether a first fluid and a second fluid read by forming a readion produd: a) the second fluid transport channel is in direct fluidic communication with the first fluid transport channel at an end of the first fluid transport channel opposite the first port; b) the fluidic constridion channel is in dired fluidic communication with the readion well and an interface channel segment SC directly connecting the second fluid transport channel and the third fluid transport channel, wherein the width of the interface channel segment is identical to the width of the fluid transport channel to which it is directly connected; c) the reaction well is in direct fluidic connection with the passive pressure sensing channel at an end of the passive pressure sensing channel opposite the second port; d) the passive pressure sensing channel extends from the readion well opposite the fluidic constriction channel and terminates at the passive pressure sensing channel port; and e) the first fluid transport channel is not in direct fluidic communication with the reaction
  • the second fluid transport channel comprises a precipitate therein.
  • the readion well and optionally the fluidic constriction channel are filled with fluid, but the rest of the device is empty.
  • mkrafluidic assembly comprising at least two of the fluidic devices of the immediately above aspect or embodiments, or at least two of any fluidic devices herein that each are effective for detecting, and operable to detect whether a first fluid and a second fluid react by forming a reaction product.
  • a method for deteding a readion produded formed by a readion of a first fluid and a second fluid using a microfluidic device comprising a passive pressure sensing channel can include the following steps: a. optionally introducing the first fluid into the device typically through a first port; b. trapping a volume of the first fluid in a reaction well, in illustrative embodiments by capturing a droplet of a volume, optionally a pre-defined volume, of the first fluid in the reaction well; c. introducing the second fluid (i.e.
  • a second solution or a second liquid into the device so that it can interact with the trapped volume of the first fluid, such that the first and second fluids mix in at least part of ab interface channel segment and/or a fluidic constriction channel to form a reaction product of one or more components of the first fluid and one or more components of the second fluid; and optionally, but typically, d.
  • detecting tiie reaction product wherein in illustrative embodiments, the reaction product is a precipitate.
  • the reaction product can be detected for example, in a second fluid transport channel.
  • the microfluidic device is any of the above fluidic devices comprising a passive pressure sensing channel, or any fluidic device herein that is effective for such method and/or comprises a passive pressure sensing channel
  • a method for determining (or detecting) whether a first fluid and a second fluid react by forming a reaction product using a fluidic device of any of the above fluidic devices comprising a passive pressure sensing channel, or any fluidic device herein that is effective for such method and/or comprises a passive pressure sensing channel can include the following steps, with reference to a non-limiting example provided in FIG. 18: a. optionally introducing a first fluid into the device typically through a first port; b.
  • a volume of the first fluid in a reaction well 2 trapping a volume of the first fluid in a reaction well 2, in illustrative embodiments by capturing a droplet of a volume, optionally a pre-defined volume, of tiie first fluid In the reaction well 2; c. introducing a second fluid (i.e. a second solution or a second liquid) into the device so that it can interact with the trapped volume of the first fluid.
  • a second fluid i.e. a second solution or a second liquid
  • the second fluid is introduced into the device typically into a third fluid transport channel SB and an interface channel segment SC, typically thru a third port 6 such that the first and second fluids mix in at least part of the interface channel segment SC and/or a fluidic constriction channel 4 to form a reaction product of one or more components of the first fluid and one or more components of the second fluid; and optionally, but typically, d, detecting the reaction product, wherein in illustrative embodiments, the reaction product Is a precipitate.
  • the reaction product can be detected for example, in the second fluid transport channel SA.
  • a method for determining (or detecting) whether a first fluid and a second fluid react by forming a reaction product using a fluidic device of the above fluidic device comprising a passive pressure sensing channel, or any fluidic device herein that is effective for such method and/or comprises a passive pressure sensing channel said method comprising: a> filling the fluidic device with the first fluid through the first port by positive pressure; b. trapping a volume of the first fluid in the reaction well and the fluidic constriction channel by applying negative pressure at the first port or by applying positive pressure at the third port, to remove some of the first fluid from the fluidic device; c.
  • a method for determining (or detecting) whether a first fluid and a second fluid react by forming a reaction product using a fluidic device of the above fluidic device comprising a passive pressure sensing channel, or any fluidic device herein that is effective for such method and/or comprises a passive pressure seising channel said method comprising: a. trapping a volume of a first fluid in the reaction well; and b.
  • a method for determining (or detecting) whether a first fluid and a second fluid react by forming a reaction product using a fluidic device of the above fluidic device comprising a passive pressure sensing channel, or any fluidic device herein that is effective for such method and/or comprises a passive pressure sensing channel said method comprising: a. trapping a volume of a first fluid in the reaction well; and b.
  • reaction product formation results in a thickened fluid, a polymer, a gel, a hardened product, an aggregated product, a fluorescent product, a colored product, or a change of color.
  • reaction product forms a precipitate.
  • the passive pressure sensing channel is filled with air and does not comprise fluid, such that a fluid-air interface is present at the point at which the reaction weii and the passive pressure sensing channel connect.
  • the fluidic device is filled with a first fluid thru the first port by positive pressure.
  • fluid flowing into the passive pressure sensing channel is detected by detecting fluid exiting the passive pressure sensing channel, optionally wherein said fluid is detected visually, optionally Using a camera.
  • fluid enters the passive pressure sensing channel optionally wherein the precipitate is detected by detecting the fluid in the passive pressure sensing channel.
  • the first fluid or the second fluid is mammalian sweat, or an artificial sweat fluid.
  • the second fluid is introduced into the third fluid transport channel at a flow rate of between 0.01 nl/min and 1 ml/min, optionally between 1 nl/min and 25 u/min.
  • Ranges can be expressed herein as from about one particular value, and/or to about another particular value. When such a range Is expressed, another aspect Includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent about or approximately, ft will be understood that the particular value forms another aspect It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Ranges (e.g., 90-100%) are meant to indude the range per se as well as each independent value within the range as if each value was individually listed. All references cited within this disclosure are hereby incorporated by reference into this application in their entirety. A skilled artisan will appreciate that where the specification provides an approximate value or range, the exact value or range is within the scope of the current specification as well.
  • This example illustrates the production of nanopartides using a fluidic device illustrated in FIG.1, which indudes a first port 1, first fluid transport channel 1A, reaction well 2, overflow channe! 3, fluidic constriction channnel 4, second fluid transport channel SA, and second port 5.
  • the device used in this example had the approximate dimensions provided for the exemplary device of FIG. 1 in Table 1.
  • /DOTAP in a ratio of 67:30:3 was dissolved in ethanol at a concentration of lOmg/ml and used as the first fluid and was loaded into the fluidic device (steps one and two).
  • the second fluid used was phosphate-buffered saline (PBS) and was introduced into the device at a flow rate of 20 ml/minute (step 3).
  • PBS phosphate-buffered saline
  • FIG. 3 3) and transmission electron microscopy (TEM) (FIG. 4).
  • the 100 pm-wide fluidic constriction channel produced liposomes hairing pupe diameters of about a 100 nm diameter whether the washing speed was 5 or 10 ml/min.
  • Fluidic devices having larger fluidic constriction channels 300 and 400 pm
  • tire fluidic devices having a SCO or 400 pm fluidic constriction channel produced liposomes having pulpe diameters of about 400 nm.
  • the fluidic devices having a 300 or 400 pm fluidic constriction channel produced liposomes having diameters of about 500 nm or 600 nm, respectively.
  • liposomes having a particle diameter of 600 nm can be too large to be useful (e g., for clinical use).
  • the fluidic constriction channel of the fluidic device should have a width of less than about
  • Distilled water (the second fluid) was then washed through the device, mixing with the first fluid to form polymeric micelles.
  • the average number-weighted size of the micelles was determined to be 30.97 nm with a polydispersity index (PDI) of 0.255 (FIG. 9).
  • PDI polydispersity index
  • DPPC/Chol/DOTAP dissolved in ethanol (lOmg/mL) was introduced into the fluidic device through the first inlet port 11 at a flow rate of Sml/min, as PBS was introduced into the fluidic device through the second inlet port 13 at a flow rate of 20mL/min. Liposomes were thereby produced. Number-weighted size distribution for liposomes formulated using these first and second fluids in this device was determined; the liposomes had an average size of 226 J nm with a PDI of 0.153 (RG. 148).
  • This example farther illustrates the production of nanopartides using a single subunit fluidic device with a single input port (i.e. inlet port or first port) 1 illustrated in RG. 1.
  • the process described above and illustrated in RG. 2 was used with the device of RG. 1 with dimensions provided in Table 1 for such device, with an exemplary DPPC/Cholesterol lipid formulation.
  • a lipid formulation of DPPC/Cholesterol in a ratio of 60:40 was dissolved in ethanol at a concentration of lOmg/ml and used as the first fluid and was loaded into the fluidic device (steps 1 and 2).
  • the second fluid used was reagent grade water that was Introduced into the device at a flow rateof 10 ml/minute using a syringe pump (step 3). Mixture of the first and second fluids in the fluidic device and following these steps resulted in liposomes being present in the reaction well 2. These liposomes were washed out of the reaction well 2 by inputting water into the first port 1 and analyzed using dynamic light scattering (DLS). The effective diameter of four batdies of liposomes formulated in the device and analyzed by DLS Is shown in RG.22A; the liposomes had an average size of 145 nm with a PDI of 0.2. The DLS data demonstrates that this fluidic device and method reproducibly generated consistent formulations.
  • DLS dynamic light scattering
  • FIG. 22B The effect of total flow rate on particle size is demonstrated in FIG. 22B.
  • DPPC/Cholesterol in a ratio of 55:45 dissolved in ethanol at a concentration of lOmg/mt was used as the first fluid and was loaded into the fluidic device (steps 1 and 2).
  • the second fluid used was reagent grade water which was introduced into the device at a flow rate ranging from 1 to 20 ml/minute (step 3).
  • a faster total flow rate resulted in the formation of smaller nanoparticles.
  • microfluidic device designs similar to RG. 1 provide efficient and flexible devices for preparing partides with sizes that can be controlled by using different, controlled flow rates.
  • This example illustrates the production of nanopartides using a coflowing fluidic device, or fluidic assembly, having a series of fluidic device subunits as illustrated in FIG. 20.
  • the channel dimensions of two versions (small dimension version and larger relative dimensions version) that were prepared according to toe device design shown in FIG. 20 are listed in Table 1.
  • the small design in Figure 20 functions the same as the large design of figure 20 but is capable of forming smaller nanoparticles due to the reduced dimensions of the parts of the device.
  • the three batches of collected liposome nanoparticles were analyzed by DL5 (FIG. 23A), The effective diameter for three batches of liposomes formulated using these first and second fluids In this detrice was determined.
  • the liposomes that were produced had a Hpid concentration of 1 mg/ml, an average size (i,e. diameter) of 169.4 nm with a PDI of 0.15.
  • the DIS data demonstrates that this large design fluidic detrice of FIG. 20, and method using the same, reproducibly generated consistent formulations.
  • microfluidic device designs similar to F1G. 20 provide efficient and flexible devices for preparing particles with consistent batch to batch reproducibility for pulpe size.
  • the effective diameter for each batch of liposomes formulated using these first and second fluids in this device at these flow rates was determined using DIS.
  • the lipid concentration of the produced liposome nanopartides was 1 mg/mt.
  • the data (FIG. 23B) demonstrates that particle size decreased as total flow rate increased.
  • microfiuidic device designs similar to FIG. 20 provide an efficient and flexible device for preparing particles with different sizes by altering flow rates of streams of liquids input into the device.
  • lipid phase was introduced into a fluidic device made according to the large dimension embodiment of the design of FIG. 20 through the first inlet port 11 as about 2.73 ml of reagent grade water (i.e. second fluid) was introduced into the fluidic device through foe second inlet port 13 using a peristaltic pump.
  • the flow rates of the two streams were varied such that the total flow rate of both streams combined was held constant at 8mL/min, but the ratio between the water and lipid phases was varied from 1:1 to 10:1.
  • microfluidic device designs similar to FIG. 20 provide an efficient and flexible device for preparing particles with different sizes by holding a combined flow rate ci a first fluid stream and a second fluid stream constant, but varying the relative flow rates of the first fluid stream to the second fluid stream input into the device.
  • IL of formulation was prepared by continuously inputting the first fluid and second fluid into the device until IL of liposome nanoparticle solution was collected through port 5. Thirteen samples were collected throughout the formulation process. Each sample was measured using DLS. The lipid concentration of the produced liposome nanopartides was 1 mg/ml.
  • the data (RG. 23D) demonstrates a high degree of uniformity across large batdies of nanopartide formulations.
  • microfluidic device designs similar to FIG, 20 provide efficient and flexible devices for preparing particles In volumes that can be scaled up to liters of monkeye solutions or suspensions by inputting larger volumes of fluids into the device and collecting output microparticle solutions and suspensions as more first fluid and second fluid are being input into the device and microparticles are being formed within the device.
  • a device made according to the small dimension embodiment of the design of FIG. 20 was also used to produce liposomes.
  • Approximately 0.27 ml of DPPC/Cholesterol in a ratio of 70:30 dissolved in ethanol at a concentration of lOmg/mL (i.e. first fluid) was introduced into the small dimension fluidic device according to FIG. 20 through the first inlet port 11 at a flow rate of L4mL/min, as about 2.73 ml of reagent grade water (i.e. second fluid) was introduced into the fluidic device through the second inlet port 13 at a flow rate of 13.6ml/min using a peristaltic pump. Liposomes were thereby produced.
  • the effective diameter for three batdies of liposomes formulated using these first and second fluids in this device was determined using DLS (FIG. 24A).
  • the lipid concentration of the produced liposome nanopartides was 1 mg/ml.
  • the liposomes had an average size of 83 nm with a PDI of 0.19.
  • the 015 data demonstrates that this fluidic device and method reproducibly generated consistent formulations.
  • microfluidic device designs similar to FIG. 20 but with different dimensions, provide efficient and flexible devices for preparing partides with consistent batch to batch reproducibility for particle size.
  • microfluidic device designs similar to FIG. 20 at different dimensions provide an efficient and flexible device for preparing partides with different sizes by altering a total flow rate of combined streams of fluids input into the device.
  • lipid phase was introduced into a fluidic device made according to the small dimension embodiment of the design of FIG. 20, through the first Inlet port 11 as about 2.73 ml of reagent grade water was introduced into the fluidic device through the second inlet port 13 using a peristaltic pump.
  • the flow rates of the two streams were varied such that the total flow rate of both streams combined was held constant at 15mL/min, but the ratio between the water and lipid phases was varied from 3:1 to 10:1.
  • the lipid concentration of the produced liposome nanopartides was between 3.33mg/ml to Img/ml at the 3:1 and 10:1 ratios, respectively.
  • the effective diameter for each batch of liposomes formulated using these first and second fluids in this device at these flow rates was determined using DIS.
  • the data (FIG. 24C) demonstrates that crumble size decreased as flow rate ratio increased between the water stream and the lipid stream.
  • a fluidic device made according to the small dimension embodiment of the design of FIG, 20 was used to precipitate proteins from solutions and quantify precipitation efficiency.
  • the two input fluid streams were (1) different protein solutions and (2) applicable precipitants known to precipitate a protein of interest from a protein solution.
  • BSA Bovine Serum Albumin
  • TCA Trichloroacetic add
  • Bovine Gamma Globulin in PBS and 4% TCA were used as first Add and second fluid inputs, respectively, and input into the device.
  • Bovine Gamma Globulin was precipitated at total flow rates of 500 pl/min and 2ml/min with an efficiency of 99,1696 and 9958% when the two incoming streams were delivered at a 1:1 flow rate ratio.
  • Protein precipitant concentrations were determined to be 2%, 1,33%, 0.67% and 0.36% at the 4 different flow rate ratios above (1:1, 2:1, 5:1, 10:1) Using BSA. Thus, protein precipitant concentrations were low, but precipitate was formed with very high effedency. Low protein precipitant concentration is beneficial because precipitant can have damaging effects on the protein.
  • devices with the general design of FIG. 20 were effective for producing protein precipitations using proteins with very different molecular weights and characteristics.
  • devices provided herein, for example with the general design of FIG. 20, can be used to provide devices that are effective for, adapted for, and operable for use in methods that produce a continuous stream of a suspension of a precipitate of a target proteln(s) when streams of a protein solution and a protein preciptant solution are simultaneously input into the device.
  • the exemplary device illustrated in FIGS. 15 and ISA and similar devices disclosed herein can be used for reaction product detection, measurement, and analysis, for example in compound precipitation studies.
  • a device according toFIGS. 15 and ISA was designed to model the in vivo conditions of a sweat gland to study interaction of active compounds found in a solution including compounds useful as anti-perspirants (e.g., the first fluid, which is trapped in the reaction well 2 during use) with a solution mimicking sweat solution (e.g., the second fluid).
  • Eccrine sweat glands have a pore diameter of around 20-60 pm (Bretagne, 2017), and the second and third fluid transport channels (SB and SAJ of this exemplary device were accordingly designed to provide a similar geometry, having in this illustrative embodiment, a channel width and depth of 60 pm each for this application (but can be within a range, for example as provided in Table 2)..
  • the reaction Well 2 in this exemplary device also has a channel depth of 60 pm, but this is not an absolutely required depth and can be Within a range of, e.g., 10%.
  • the method disclosed in this Example was carried out by capturing a first fluid (i.e., the solution including potential anti-perspirant compound(s)) in a reaction well 2 and then introducing the second fluid (i.e., human sweat) via a third port 6 to interact with the first fluid, essentially as described hereinabove with reference to FIGS.15-19.
  • a first fluid i.e., the solution including potential anti-perspirant compound(s)
  • the second fluid i.e., human sweat
  • the fluidic device used in this Example had the following dimensions: a) depth of device: ⁇ 60 pm; b) third fluid transport channel SB: 60 pm width, 1675 tun length; c) second fluid transport channel 5 A: 60 pm width, 600 pm length; d) reaction well 2: 460 pm width (widest), 830 pm length; e) pressure sensing channel 3A: 40 pm width, ⁇ 2525 pm length; and, f) first fluid transport channel 1A: 460 pm width, "'12250 pm.
  • a camera was used to capture movement of fluids Into and out of the device on video, with a stiff frame from the video shown in HG. 19. The temperature of the device was maintained at 37*C with a commercially available temperature controller.
  • a first fluid containing a commercialiy-available anti-perspirafit active compound (aluminum/zirconium tetrachlorohydrex, aluminum/zirconium pentachlorohydrate, or aluminum chlorohydrate) was the first fluid loaded into the reaction well by using positive pressure to introduce approximately 5 pi through the first port 1.
  • excess first fluid was removed from the device by applying a negative pressure at the first port with a standard manual pipette, leaving approximately 20 nl of active compound captured in the reaction well.
  • Real mammalian sweat collected from a healthy individual, was added to the device through the third port 6 with an applied positive pressure at a flow rate of 1 pJ/min using a standard syringe pump.
  • a precipitate plug formed and continued to grow in the second fluid transport channel 5A, eventually completely blocking the incoming flow as observed by video analysis (FIG. 19).
  • Representative precipitate ares data measured from video images from these precipitates formed using a first fluid comprising aluminum/zirconium pentachlorohydrate and human sweat as the second fluid is shown in Table 3 below (flow rate of 1 pi/min, constant temperature of 37°C):
  • This example illustrates the production of solid lipid nanoparticles (SLNs) and Nano-structured lipid carriers (NLCs) using an exemplary fluidic device assemblies and method herein.
  • SSNs solid lipid nanoparticles
  • NLCs Nano-structured lipid carriers
  • Influence of mixing and coding parameters on size and polydispersity of SIN and NIC nanopartides may be dependent on one or more of; a) Cooling rates; b) Laminar and Turbulent mixing conditions on cooling the microemulsians; and e) rate of addition of different components for the formulation of the micro-emulsions.
  • tn order to optimize cooling on-chip we optimized the design in order to Increase cooling and decrease conductive mixing as we showed that some formulations had better polydispersity with reduced mixing
  • tn Order to eliminate mixing we first designed several iterations without mixing motifs these microfluidic channels were designed to be conducive to laminar flow (Fig. 41). We designed several channels and measure their mixing performance on chip using micron sized fluorescent beads and recorded their streamlines using a high speed camera. The vorticity was measured as a function of relative change in direction of the streamlines across a fixed unit area.
  • Mixing rate In order to study mixing rates We used a microfluidic device as previously described s , that achieves >90% mixing in a range of fiow rates.

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Abstract

L'invention concerne des dispositifs fluidiques et des ensembles de dispositifs fluidiques, comprenant des dispositifs microfluidiques et des cartouches les comprenant, qui peuvent être utilisés pour fabriquer des particules ou des précipités de protéines, ou pour surveiller la formation de précipités. Les dispositifs fluidiques peuvent comprendre des canaux qui relient un puits réactionnel à un orifice d'entrée et à un orifice de sortie, et un canal de constriction fluidique qui peut être conçu pour aider à retenir des fluides dans le puits réactionnel et/ou à favoriser le mélange à l'intérieur du puits réactionnel. Dans certaines variantes, les dispositifs fluidiques peuvent être interconnectés en ensembles fluidiques qui peuvent être utilisés dans des procédés de traitement continu.
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US11745181B2 (en) 2017-08-09 2023-09-05 Unchained Labs Devices and methods for bioassay
US11759781B2 (en) 2017-11-10 2023-09-19 Unchained Labs Integrated fluidic circuit and device for droplet manipulation and methods thereof
US11857957B2 (en) 2017-04-28 2024-01-02 Unchained Labs Fluidic devices with reaction wells and uses thereof
US11998885B2 (en) 2018-10-26 2024-06-04 Unchained Labs Fluidic devices with reaction wells and constriction channels and uses thereof

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US20190054467A1 (en) * 2007-07-13 2019-02-21 Handylab, Inc. Microfluidic cartridge
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US11857957B2 (en) 2017-04-28 2024-01-02 Unchained Labs Fluidic devices with reaction wells and uses thereof
US11745181B2 (en) 2017-08-09 2023-09-05 Unchained Labs Devices and methods for bioassay
US11759781B2 (en) 2017-11-10 2023-09-19 Unchained Labs Integrated fluidic circuit and device for droplet manipulation and methods thereof
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