CN113522378B - Microfluidic chip based on electrohydrodynamics, micro sample application device and method - Google Patents
Microfluidic chip based on electrohydrodynamics, micro sample application device and method Download PDFInfo
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- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/02—Burettes; Pipettes
- B01L3/0241—Drop counters; Drop formers
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- 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
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Abstract
The invention provides a microfluidic chip based on electrohydrodynamics, a micro sample application device and a method, wherein the chip comprises: at least one capillary tube having an inlet end and an outlet end; the chip body comprises at least one sample inlet, a micro channel connected with the sample inlet, and a capillary embedding channel connected with the micro channel, wherein the capillary embedding channel is provided with an outlet end, the capillary is arranged in the capillary embedding channel and connected with the micro channel, and the length of the capillary is greater than that of the capillary embedding channel; the upper electrode is positioned on the chip body and close to the position of the capillary; a lower electrode having an insulating support, the lower electrode being located below the capillary outlet. The chip embeds the capillary into the microfluidic chip, has accurate fixing effect, simplifies the structure and can realize full-automatic sample spotting of samples.
Description
Technical Field
The invention belongs to the field of microfluidic chips, and particularly relates to a microfluidic chip based on electrohydrodynamics, a micro sample application device and a micro sample application method.
Background
Droplet generation technology is an important branch of microfluidic chip technology. Droplet-based microfluidic chip technology is compatible with a wide range of chemical and biological reagents and "electronic controls" and has good programmability and constructability. The microfluidic chip droplet generation platform can accurately control and rapidly mix samples in droplets, thereby reducing reaction time. The microfluidic chip droplet technology can accurately control generation and control of droplets, and uniform monodisperse droplets are generated, so that the microfluidic chip droplet technology becomes a high-throughput platform for biomedical and chemical research. The produced liquid drops have nL and pL grades in size and can be used as a reactor for directly synthesizing particles or carrying reagents for application in the biomedical field.
The microfluidic technology based on liquid drops has great potential in the aspects of drug transportation, biosensing and the like, and is more and more widely applied in recent years, and the advantages are as follows: the reagent consumption is low, each micro-droplet can be used as an independent micro-reaction container for various biochemical reactions, the independent control can be realized, and the huge specific surface area of the droplet has catalytic action on a plurality of reactions.
Electrohydrodynamic jet printing was by j.u.park 1 The basic principle of the Electrohydrodynamic (EHD) -based micro-droplet spray-forming deposition technique proposed and developed by the same is to apply a high voltage of several thousands to several tens of thousands of volts between a conductive nozzle (a first electrode) and a conductive substrate (a second electrode), and form a strong electric field between the conductive nozzle (the first electrode) and the conductive substrate, under the action of the electric field, the droplets at the tip of the nozzle are polarized and form positive charges on the surface of the droplets to be accumulated; due to the existence of coulomb repulsion, the charged liquid drop is gradually elongated to form Taylor cone; when the electric field force borne by the liquid drop at the tip of the nozzle exceeds the surface tension of the liquid, the tiny liquid drop with positive charge is ejected from the top of the Taylor cone to form a superfine cone jet flow with the diameter being 1 to 2 orders of magnitude smaller than the nozzle size; in combination with the control of the motion stage, precise deposition of droplets on the substrate is achieved.
The electrohydrodynamic jet printing technology is originally applied to the field of materials, and through development, the current electrohydrodynamic jet printing technology is also applied to the field of biology and has partial coupling with a microfluidic chip, but the current technology generally needs precise and complicated supporting and aligning equipment, printing nozzles with precise sizes and complex conductive electrodes, so that the technology needs complicated and precise equipment to support, and most laboratories do not have the basis for the research.
Disclosure of Invention
In order to solve the problems, the invention provides a microfluidic chip based on Electrohydrodynamics (EHD) and a micro sample application device, wherein a capillary tube is embedded in the microfluidic chip, so that the microfluidic chip has an accurate fixing effect, the structure is simplified, and the full-automatic sample application of a sample can be realized.
To achieve the above object, in one aspect, the present invention provides a microfluidic chip based on electrohydrodynamics, the chip comprising:
at least one capillary tube having an inlet end and an outlet end;
the chip body comprises at least one sample inlet, a micro channel connected with the sample inlet, and a capillary embedding channel connected with the micro channel, wherein the capillary embedding channel is provided with an outlet end, the capillary is arranged in the capillary embedding channel and connected with the micro channel, and the length of the capillary is greater than that of the capillary embedding channel;
the upper electrode is positioned on the chip body and close to the position of the capillary;
a lower electrode having an insulating support, the lower electrode being located below the capillary outlet.
In another preferred embodiment, the outlet end of the capillary tube insertion channel is glued to the outer wall of the capillary tube.
In another preferred embodiment, the material of the chip body is glass or high molecular polymer.
In another preferred example, the material of the chip body is silicate glass, quartz glass, calcium fluoride glass, PDMS (polydimethylsiloxane) or PMMA (polymethyl methacrylate), and is preferably PDMS.
In another preferred embodiment, the material of the insulating support is glass or a high molecular polymer.
In another preferred embodiment, the material of the insulating support is silicate glass, quartz glass, calcium fluoride glass, PDMS (polydimethylsiloxane) or PMMA (polymethyl methacrylate), and is preferably PDMS.
In another preferred embodiment, the chip body and the insulating support of the lower electrode are made of the same material.
In another preferred embodiment, the chip body and the insulating support of the lower electrode are in an integrally molded structure.
In another preferred embodiment, the chip body and the insulating support of the lower electrode are made of different materials.
In another preferred embodiment, the chip body is separable from the insulating substrate.
In another preferred embodiment, the diameter of the capillary insertion channel is larger than the diameter of the microchannel.
In another preferred embodiment, the capillary has an inner diameter greater than or equal to the diameter of the microchannel.
In another preferred embodiment, the inner diameter of the capillary tube is 50-700um, preferably 100um.
In another preferred embodiment, the length of the capillary is 5-10mm, preferably 6mm.
In another preferred embodiment, the capillary length is 1-5mm, preferably 2mm longer than the capillary-embedded channel.
In another preferred example, the height of the microfluidic chip channel is 10-5000um, preferably 120um.
In another preferred embodiment, the material of the upper and lower electrodes is liquid metal, salt water or solder, preferably liquid metal.
In another preferred example, the electrode is in an axisymmetric shape, and the symmetry axis is a straight line on which the capillary is located.
In another preferred embodiment, the upper and lower electrodes are spaced apart by a distance of 4-10mm, preferably 5mm.
In another preferred embodiment, the two parts of the lower electrode are spaced apart by a distance of 3-10mm, preferably 6mm.
In another preferred embodiment, the chip comprises at least one aqueous phase sample inlet and at least one oil phase sample inlet.
The chip further comprises a fiber channel for embedding an optical fiber, preferably, the fiber channel is located below the upper electrode.
In another preferred example, the electrode is externally connected with a high-voltage power supply.
In another preferred example, the chip can be used for the spotting of biological materials, material samples.
The biological sample comprises: flowers, leaves, stems, roots, seeds, etc. of plants, body fluids of animals (including humans) (e.g., urine, blood, saliva, bile, gastric juice, lymph, and other secretions of the organism, etc.), hair, muscle, and some tissue organs (e.g., thymus, pancreas, liver, lung, brain, stomach, kidney, etc.), and various microorganisms.
The material samples included: metal materials, inorganic non-metal materials, high polymer materials and composite materials.
In a further aspect the present invention provides an electrohydrodynamic based microspotting device comprising:
the micro-fluidic chip is provided with at least two sample inlets and two opposite optical fiber channels;
a laser transmitter for transmitting laser in visible light band;
the laser detector is used for detecting optical signals and converting the optical signals into electric signals;
the two optical fibers are embedded into the optical fiber channel, one of the optical fibers is connected with the laser emitter, and the other optical fiber is connected with the laser detector;
a relay for controlling the switching of the circuit;
the two poles of the high-voltage direct current power supply are respectively connected with the upper electrode and the lower electrode of the micro-fluidic chip;
the data acquisition card is used for acquiring and transmitting signals detected by the laser detector, acquiring and transmitting the signals to the computer and controlling the output voltage or current of the high-voltage direct-current power supply;
the mobile platform is used for placing the receiving container;
and the computer is connected with the data acquisition card and the mobile platform, and is used for reading the laser signals and controlling the movement of the mobile platform and the switching of the high-voltage direct-current power supply circuit through programs.
In another preferred example, the laser detector is a photodiode or a photomultiplier tube.
In another aspect, the present invention provides a method for spotting trace amounts based on electrohydrodynamics, comprising:
the micro-fluidic chip is provided with two sample inlets, wherein one sample inlet is filled with mixed liquid of oil and microorganism liquid drops, the other sample inlet is filled with pure oil phase, and the interval between the microorganism liquid drops and the microorganism liquid drops is enlarged under the action of the oil phase of the mixed liquid, so that the microorganism liquid drops enter the capillary from the micro-channel; laser emitted by the laser emitter penetrates through the capillary tube through optical fiber irradiation, is transmitted to the laser detector through the optical fiber, and is transmitted to the computer through the data acquisition card; when the liquid drops pass through the capillary, the optical signal is changed, so that the signal data displayed by a computer program can judge that one liquid drop passes through, the program controls the data acquisition card to output a set voltage, the relay is utilized to conduct the circuit of the high-voltage direct-current power supply, and the high-voltage direct-current power supply applies voltage to the upper electrode and the lower electrode of the microfluidic chip to enable the microbial liquid drops to drop out. By adjusting the magnitude of the applied voltage, the size of the dripped liquid drop and the response time of the dripped liquid drop can be controlled, and single dripping of a single liquid drop is realized; and controlling the moving platform to accurately drop the liquid drops into the receiving container.
In another preferred example, the microorganism liquid drop is a microorganism sample solution collected in the environment, a cultured microorganism solution or a mixed solution of a plurality of microorganisms.
In another preferred example, the separation distance between the microorganism liquid drops is adjusted by adjusting the flow rate of the two injection ports.
In another preferred example, the flow rate is controlled by a constant flow syringe pump or a constant pressure syringe device.
In another preferred example, the oil phase is one or more of fluorocarbon oil, mineral oil, silicone oil, vegetable oil and petroleum ether.
The invention has the beneficial effects that:
the invention discloses a microfluidic chip based on Electrohydrodynamic (EHD), and the capillary is embedded in the microfluidic chip, so that the microfluidic chip has an accurate fixing effect and a simplified structure; the method is characterized in that electro-hydrodynamic drive is adopted, high pressure is applied to a capillary, and controllable force is applied to emulsion liquid drops to form double emulsion liquid (water/oil/air liquid drops); the micro-fluidic chip can be repeatedly used, and can realize large-scale production, thereby reducing the operation cost; the use and the operation are simple, convenient and safe, and non-professional personnel can also operate skillfully.
The micro-sample application device formed by the micro-fluidic chip and the coupling external simple device can realize full-automatic sample application of the microorganism liquid drop, the device is simple, convenient, safe and easy to operate, and the technical threshold of microorganism sample application is greatly reduced.
Drawings
In the accompanying drawings, like parts and features have like reference numerals. Many of the figures are schematic and may not be to scale.
FIG. 1 is a schematic diagram of a microfluidic chip with two sample inlets;
FIG. 2 is a schematic diagram of a microfluidic chip with one injection port;
FIG. 3 is a schematic diagram of another alternative of a microfluidic chip structure;
FIG. 4 is a schematic view showing the structure of the microorganism spotting device.
The specific reference numbers are as follows:
1: a microfluidic chip; 1-1: an upper electrode; 1-2: a sample inlet; 1-2-1: an oil phase sample inlet; 1-2-2: a water phase sample inlet; 1-3: fiber channels 1-4: a capillary tube; 1-5: a lower electrode; 1-6: a chip body; 1-7: a micro flow channel; 2: a laser transmitter; 3: a laser detector; 4: a data acquisition card; 5: a relay; 6: a high voltage direct current power supply; 7: a computer; 8: and (4) moving the platform.
Detailed Description
For the purpose of facilitating understanding of the embodiments of the present invention, the following description will be made in terms of several specific embodiments with reference to the accompanying drawings, and the embodiments are not to be construed as limiting the embodiments of the present invention. Furthermore, the drawings are schematic and, thus, the apparatus and devices of the present invention are not limited by the size or scale of the schematic.
It is to be noted that, in the claims and the specification of the present patent, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, the use of the verb "comprise a" to define an element does not exclude the presence of another, same element in a process, method, article, or apparatus that comprises the element.
Example 1
Preparing a micro-fluidic chip:
the structure of the prepared microfluidic chip is shown in figure 1, and the chip is an integrated structure.
(1) The channel structure of the microfluidic chip shown in figure 1 is designed by CAD, and comprises a micro channel 1-7, a capillary embedded channel at the outlet of the micro channel, an optical fiber channel 1-3 and an electrode channel structure, and film mask printing is carried out. And dripping SU-8 photoresist on the cleaned silicon wafer for spin coating, wherein the thickness of the silicon wafer is about 120um. And then covering the mask with the mask, exposing the mask under an exposure machine, and cleaning the uncured part with a developing solution to obtain the silicon wafer template.
(2) And uniformly mixing the PDMS monomer and the curing agent according to a certain proportion to obtain the PDMS high polymer. And pouring the PDMS high polymer on a silicon wafer template, wherein the thickness of the PDMS high polymer is about 1-10mm, and drying to obtain the PDMS chip with the channel structure.
(3) And punching an oil phase sample inlet 1-2-1, a water phase sample inlet 1-2-2 and a liquid electrode sample inlet on a PDMS chip with a channel structure by using a puncher. The lower layer bonded chip is a PDMS smooth substrate with the thickness of 1-10mm and no etching pattern.
(4) And plasma bonding the upper and lower layers of chips, and standing at 70 ℃ overnight to recover the hydrophobicity of the chips. Cutting is carried out according to the designed outline of the chip bodies 1-6, and the final structure is shown in figure 1.
(5) Insert capillary 1-4 with capillary embedding passageway department, capillary length is 6mm, and the external diameter is about 360um, and the internal diameter is 100um. The chip is placed on a glue drying plate at 90 ℃ and heated, and PDMS is used for sealing the capillary and the outlet at the tail end of the microfluidic channel, so that all liquid can only flow out from the tail end of the capillary.
(6) And liquid metal is poured from the sample inlets of the upper electrode channel and the lower electrode channel to form an upper electrode 1-1 and a lower electrode 1-5.
In this embodiment, the body of the microfluidic chip and the insulating support of the lower electrode are integrated into a single structure, and in other embodiments, the chip body and the insulating support of the lower electrode may be made into separate structures. In addition, the number, arrangement structure and electrode structure of the sample inlets in the chip can be adjusted according to the requirement, and fig. 2 and 3 show other variable structures of the chip, and the protection scope of the invention is not limited to the above structures.
Example 2
An electrohydrodynamic based spotting device, as shown in fig. 4, comprising: the micro-fluidic chip 1 is provided with at least two sample inlets and two opposite optical fiber channels;
the laser emitter 2 emits laser in a visible light wave band;
a laser detector 3 for detecting an optical signal and converting the optical signal into an electrical signal; the laser detector includes, but is not limited to, a photodiode or photomultiplier tube;
the two optical fibers are embedded into the optical fiber channel, one of the optical fibers is connected with the laser emitter, and the other optical fiber is connected with the laser detector;
a relay 5 for controlling the switching of the circuit;
the two poles of the high-voltage direct current power supply 6 are respectively connected with the upper electrode and the lower electrode of the micro-fluidic chip;
the data acquisition card 4 is used for acquiring and transmitting signals detected by the laser detector, acquiring and transmitting the signals to a computer and controlling the output voltage or current of the high-voltage direct-current power supply;
a mobile platform 8 for placing a receiving container;
and the computer 7 is connected with the data acquisition card and the mobile platform, and is used for reading the laser signals, controlling the movement of the mobile platform and switching on and off the high-voltage direct-current power supply circuit through programs.
Example 3
Taking a coupling interface of the microfluidic chip applied to flow sorting and mass spectrometry after droplet culture of a microbiome as an example, the chip structure takes the chip structure prepared in example 1 (fig. 1) as an example:
1. liquid culture and amplification of microorganisms in droplets
The selective culture medium containing the microorganisms generates droplets with the common microfluidic cross-shaped droplet generation chip, the droplet size is 10-100 um, preferably 80um, the droplets are collected into an EP tube and placed in an incubator for culturing for a period of time, and the target microorganisms are propagated in large quantities and filled with the droplets.
2. Label-free growth phenotype droplet sorting based on scattered light
Cultured and cultured droplets can be sorted using a scattered light-based label-free growth phenotype droplet sorting system, and droplets that overgrow the target microorganism can be sorted and collected into a one-milliliter centrifuge tube.
3. Separation of individual droplets
The separation of the collected liquid drops is carried out by the micro-fluidic chip based on the electro-hydrodynamic liquid drop jet printing, and the specific operations are as follows:
early preparation:
(1) firstly, preparing a high-voltage direct-current power supply, a mobile platform, a sample injection pump, a computer (LABVIEW software control system running), a photodiode (or a photomultiplier), a laser transmitter, two optical fibers, a data acquisition card and a related control circuit;
(2) respectively connecting the sample injection ends of the oil phase and the oil phase containing the cultured cell liquid drop into 1-2-1 and 1-2-2 sample injection ports;
(3) respectively inserting optical fibers of a signal receiving end and a laser signal output end into two sides of the 1-3 optical fiber channels;
(4) connecting two electrodes of a high-voltage direct-current power supply 6 with an upper electrode 1-1 and a lower electrode 1-5 of the chip respectively;
(5) the microfluidic chip is fixed above the moving platform by a simple fixing device such as a clamp.
The experimental process comprises the following steps:
(1) adjusting the flow rate of the oil phase and the injection pump of the oil phase containing the cultured cell droplets to be proper and allowing the cultured cell droplets to flow out stably at intervals;
(2) the laser transmitter is turned on and a program programmed in the LABVIEW software in the computer is run.
(3) The high-voltage direct-current power supply output end is turned on after being adjusted to zero volt, then the voltage is adjusted according to the size of liquid drops to be output, and the size of the liquid drops to be output is adjusted according to the principle that: only one cultured liquid drop is in the dropped liquid drops;
(4) placing a MALDI (matrix-assisted laser desorption ionization) plate on a moving platform to collect droplets;
(5) the laser emitter 2 emits laser light through the capillary tube by fiber illumination and is transmitted to the laser detector 3 through the fiber, and then the signal is transmitted to the computer 7 through the data acquisition card 4. When the liquid drops pass through the capillary, the optical signals are changed, so that the signal data displayed by a computer program can judge that one liquid drop passes through, the program controls the data acquisition card 4 to output a set voltage, the relay 5 is utilized to conduct the circuit of the high-voltage direct-current power supply 6, and the high-voltage direct-current power supply 6 applies voltages to the upper electrode 1-1 and the lower electrode 1-5 of the microfluidic chip to enable the liquid drop to drip. By adjusting the magnitude of the applied voltage, the size of the dripped liquid drop and the response time of the dripped liquid drop can be controlled, and single dripping of a single liquid drop is realized by adjustment.
(6) Meanwhile, when the signal data displayed by the computer program can judge that one droplet passes through, the moving platform 8 is controlled by the program to move, so that the droplet is accurately dripped into a set position on the MALDI plate.
(7) And the coupling control of liquid drop signal identification, high-voltage direct-current power circuit connection and mobile platform movement is realized through an LABVIEW program.
(8) And (3) preprocessing a MALDI plate sample with the liquid drop, and performing MALDI-TOF mass spectrometry.
And (4) finishing the experiment:
if the chip can not be reused in a short time, the chip is taken down, the liquid channel is washed clean by absolute ethyl alcohol, and the chip is placed into an oven for drying and then can be recycled.
Example 4
Plate streaking of droplets:
the structure of the microfluidic chip used in this example is shown in fig. 2: the chip is of an integrated structure, only has one sample inlet 1-2, and does not need an optical fiber channel.
(1) Connecting a liquid sample inlet 1-2 with an injector containing bacterial liquid, pumping the bacterial liquid from the sample inlet 1-2 by using a pump to fill the channel, and setting the sample injection speed;
(2) respectively connecting the upper electrode 1-1 and the lower electrode 1-5 with two poles of a high-voltage direct-current power supply 6;
(3) the chip is fixed by a simple fixing device, a two-dimensional moving platform 8 is arranged below the chip, a solid culture medium or a 96-hole culture plate with a liquid culture medium is arranged on the two-dimensional moving platform 8, and a lighted alcohol lamp is arranged beside the two-dimensional moving platform to manufacture a sterile environment;
(4) and (3) opening the pump to enable the liquid to enter the capillary channel 1-3, opening the high-voltage power supply 6, and adjusting the voltage, wherein the higher the voltage is in a certain voltage range, the smaller the liquid drop generated by dripping the bacterial liquid out of the capillary is. The voltage controls the generation speed and the size of the formed liquid drop;
(5) the moving speed of the moving platform 8 is adjusted to be matched with the generation speed of the liquid drops, so that each liquid drop uniformly drops on a solid culture medium or a 96-hole plate containing a liquid culture medium;
(6) after the experiment is finished, the culture medium is placed in an incubator for culture, the chip is washed by absolute ethyl alcohol in a liquid channel, and then dried for recycling.
⑦
Example 5
As a coupling interface for flow sorting and cell sequencing after droplet culture of a microbiome, the chip structure adopted is shown in fig. 1:
1. liquid culture and amplification of microorganisms in droplets
The selective culture medium containing the microorganisms generates droplets with the common microfluidic cross-shaped droplet generation chip, the droplet size is 10-100 um, preferably 80um, the droplets are collected into an EP tube and placed in an incubator for culturing for a period of time, and the target microorganisms are propagated in large quantities and filled with the droplets.
2. Label-free growth phenotype droplet sorting based on scattered light
Cultured droplets may be sorted using a scattered light-based label-free growth phenotype droplet sorting system, and droplets that overgrow the target microorganism may be sorted and collected.
3. Separation of individual droplets
Separating the collected liquid drops by the micro-fluidic chip based on electro-hydrodynamic liquid drop jet printing specifically comprises the following operations:
early preparation:
(1) firstly, preparing a high-voltage direct-current power supply, a mobile platform, a sample injection pump, a computer (LABVIEW software control system running), a photodiode (or a photomultiplier), a laser transmitter, two optical fibers, a data acquisition card and a related control circuit;
(2) respectively connecting the sample injection ends of the oil phase and the oil phase containing the cultured cell liquid drop into 1-2-1 and 1-2-2 sample injection ports;
(3) respectively inserting optical fibers of a signal receiving end and a laser signal output end into two sides of the 1-3 optical fiber channels;
(4) connecting two electrodes of a high-voltage direct-current power supply with an upper electrode 1-1 and a lower electrode 1-5 of a chip respectively;
(5) the microfluidic chip is fixed above the two-dimensional moving platform by using a simple fixture or other fixing devices, and an ignited alcohol lamp is placed beside the two-dimensional moving platform to manufacture a sterile environment.
In the experiment:
(6) adjusting the injection pumps of the oil phase and the oil phase of the separated liquid drops containing the target microorganism to proper flow rates so that the cultured liquid drops can have larger intervals and can stably flow out;
(7) the laser emitter 2 is turned on and a program programmed in the LABVIEW software of the computer is run.
(8) The output end of the high-voltage direct-current power supply 6 is turned on after being adjusted to zero volt, then the voltage is adjusted according to the size of liquid drops to be output, and the size of the liquid drops to be output is adjusted according to the following principle: only one cultured liquid drop is in the dropped liquid drops;
(9) placing the 96-well plate on a moving platform 8 to collect liquid drops;
laser light emitted by laser emitter 2 at the wavelength (R) passes through the capillary via fiber optics and is transmitted to laser detector 3 via fiber optics, which then transmits the signal to computer 7 via data acquisition card 4. When the liquid drops pass through the capillary, the optical signal is changed, so that the signal data displayed by a computer program can judge that one liquid drop passes through, the program controls the data acquisition card to output a set voltage, the relay 5 is utilized to conduct the circuit of the high-voltage direct-current power supply 6, and the high-voltage direct-current power supply 6 applies voltage on the upper electrode 1-1 and the lower electrode 1-5 of the microfluidic chip to enable the liquid drops to drip. By adjusting the magnitude of the applied voltage, the size of the dripped liquid drop and the response time of the dripped liquid drop can be controlled, and single dripping of a single liquid drop is realized by adjustment.
4. Cell sequencing
The bacterial solution in the EP tube is sequenced, and various information of the target microorganism can be obtained through sequencing.
Example 6
The structure of the microfluidic chip used as a coupling interface for Raman flow sorting and single cell sequencing is shown in FIG. 1.
1. Raman flow sorting
The Raman flow type sorting system can perform Raman spectrum measurement on the single cells, perform liquid drop single wrapping on the single cells, judge whether the single cells are target cells or not through the Raman spectrum, and sort out liquid drops containing the target cells through dielectric medium if the single cells are the target cells.
2. Separation of droplets
The separation of the collected liquid drops is carried out by the micro-fluidic chip based on the electro-hydrodynamic liquid drop jet printing, and the specific operations are as follows:
early preparation:
(1) firstly, preparing a high-voltage direct-current power supply, a mobile platform, a sample injection pump, a computer (LABVIEW software control system running), a photodiode (or a photomultiplier), a laser transmitter, two optical fibers, a data acquisition card and a related control circuit;
(2) respectively connecting the sample injection ends of the oil phase and the oil phase containing the cultured cell liquid drop into 1-2-1 and 1-2-2 sample injection ports;
(3) respectively inserting optical fibers of a signal receiving end and a laser signal output end into two sides of the 1-3 optical fiber channels;
(4) connecting two electrodes of a high-voltage direct-current power supply with an upper electrode 1-1 and a lower electrode 1-5 of a chip respectively;
(5) the microfluidic chip is fixed above the two-dimensional moving platform by fixing devices such as simple clamps, and an ignited alcohol lamp is placed beside the two-dimensional moving platform to manufacture a sterile environment.
In the experiment:
(6) adjusting the injection pumps of the oil phase and the separated oil phase containing the droplets of the target single cells to proper flow rates, so that each droplet can have a larger interval and can stably flow out;
(7) placing a 96-hole plate containing pure water on a moving platform to collect the dripped liquid drops;
(8) the laser emitter 2 emits laser light through the capillary tube by fiber illumination and is transmitted to the laser detector 3 through the fiber, and then the signal is transmitted to the computer 7 through the data acquisition card 4. When the liquid drops pass through the capillary, the optical signal is changed, so that the signal data displayed by a computer program can judge that one liquid drop passes through, the program controls the data acquisition card to output a set voltage, the relay 5 is utilized to conduct the circuit of the high-voltage direct-current power supply 6, and the high-voltage direct-current power supply 6 applies voltage on the upper electrode 1-1 and the lower electrode 1-5 of the microfluidic chip 1 to enable the liquid drops to drip. By adjusting the magnitude of the applied voltage, the size of the dripped liquid drop and the response time of the dripped liquid drop can be controlled, and single dripping of a single liquid drop is realized by adjustment.
(9) Meanwhile, when signal data displayed by a computer program can judge that one liquid drop passes through, the moving platform 8 is controlled to move through the program, so that the liquid drop is accurately dripped into a set position hole on the 96-hole plate.
And (3) coupling control of liquid drop signal identification, high-voltage direct-current power supply connection and moving of the mobile platform is realized on the capacitor (R) through an LABVIEW program, so that each dropped liquid drop is accurately dropped into a hole of a 96-hole plate.
3. Cell sequencing
And performing single cell sequencing operation on the single cell droplets in the 96-well plate, and obtaining the gene information of the target single cell through sequencing.
Claims (10)
1. A microspotting method based on electrohydrodynamics is characterized by comprising the following steps:
respectively introducing mixed liquid of oil and liquid drops and pure oil phase into two sample inlets of the microfluidic chip to enable the liquid drops to generate intervals and enter a capillary tube through a micro-channel;
the laser emitter emits laser, the laser penetrates through the capillary tube through the irradiation of the optical fiber and is transmitted to the laser detector through the optical fiber, and the liquid drop information is judged through the optical signal;
when the liquid drop is judged to be one liquid drop, the computer controls the acquisition card to output a set voltage, the relay is utilized to conduct a circuit of the high-voltage direct-current power supply, the high-voltage direct-current power supply applies voltages to the upper electrode and the lower electrode of the microfluidic chip, and the size of the dropped liquid drop and the response time of the dropped liquid drop can be controlled and the liquid drop is dropped by adjusting the magnitude of the applied voltage;
and controlling the moving platform to accurately drop the liquid drops into the receiving container.
2. The method of claim 1, wherein the liquid in the droplet is a solution of a microorganism sample collected from the environment, a cultured solution of a single microorganism, or a mixed solution of multiple microorganisms.
3. An electrohydrodynamic-based microfluidic chip for use in the method of any one of claims 1-2, wherein the microfluidic chip comprises:
at least one capillary tube having an inlet end and an outlet end;
the chip body comprises at least one sample inlet, a micro channel connected with the sample inlet, and a capillary embedding channel connected with the micro channel, wherein the capillary embedding channel is provided with an outlet end, the capillary is arranged in the capillary embedding channel and connected with the micro channel, and the length of the capillary is greater than that of the capillary embedding channel;
the upper electrode is positioned on the chip body and close to the capillary;
a lower electrode having an insulating support, the lower electrode being located below the capillary outlet.
4. The microfluidic chip according to claim 3, wherein the chip body is made of glass or high molecular polymer; the insulating support body is made of glass or high molecular polymer.
5. The microfluidic chip according to claim 4, wherein the chip body and the insulating support of the bottom electrode are made of the same material, and the chip body and the insulating support of the bottom electrode are integrally formed.
6. The microfluidic chip according to claim 3, wherein the diameter of the capillary embedded channel is larger than that of the microchannel.
7. The microfluidic chip according to claim 3, wherein the inner diameter of the capillary is greater than or equal to the diameter of the microchannel.
8. The microfluidic chip according to claim 3, wherein the upper and lower electrodes are made of liquid metal, saline solution or solder.
9. Electrohydrodynamic-based microspotting device for use in a method according to any of claims 1-2, comprising:
the micro-fluidic chip is provided with at least two sample inlets and two opposite optical fiber channels;
a laser transmitter for transmitting laser in visible light band;
the laser detector is used for detecting optical signals and converting the optical signals into electric signals;
the two optical fibers are embedded into the optical fiber channel, one of the optical fibers is connected with the laser emitter, and the other optical fiber is connected with the laser detector;
a relay for controlling the switching of the circuit;
the two poles of the high-voltage direct current power supply are respectively connected with the upper electrode and the lower electrode of the micro-fluidic chip;
the data acquisition card is used for acquiring and transmitting signals detected by the laser detector, acquiring and transmitting the signals to the computer and controlling the output voltage or current of the high-voltage direct-current power supply;
the mobile platform is used for placing the receiving container;
and the computer is connected with the data acquisition card and the mobile platform, and is used for reading the laser signals and controlling the movement of the mobile platform and the switching of the high-voltage direct-current power supply circuit through programs.
10. The apparatus of claim 9, wherein the laser detector is a photodiode or photomultiplier tube.
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| CN202010287622.2A CN113522378B (en) | 2020-04-13 | 2020-04-13 | Microfluidic chip based on electrohydrodynamics, micro sample application device and method |
| PCT/CN2021/086739 WO2021208872A1 (en) | 2020-04-13 | 2021-04-12 | Electrohydrodynamics-based microfluidic chip, micro sample application device and method |
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| CN114149893A (en) * | 2021-11-23 | 2022-03-08 | 中国科学院青岛生物能源与过程研究所 | Particle self-clamping flow type micro-fluidic chip, manufacturing method thereof and particle self-dispersion method |
| CN114130440B (en) * | 2021-11-26 | 2022-11-04 | 哈尔滨工业大学 | Automatically controlled initiative lane pumping device based on gallium-based liquid metal liquid drop |
| CN114260035B (en) * | 2021-12-23 | 2023-04-07 | 广东工业大学 | Multilayer wrapped micro-fluidic chip and cell particle generator |
| CN117990665A (en) * | 2022-10-27 | 2024-05-07 | 中国石油天然气股份有限公司 | In-situ emulsification process description and characterization device, method and system |
| CN116169005B (en) * | 2023-02-28 | 2023-07-28 | 厦门金诺花科学仪器有限公司 | Disposable hydrophobic nano-liter sample application needle head and sample application test method |
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