CN114289088A - Microfluidic chip and multi-step reaction method - Google Patents
Microfluidic chip and multi-step reaction method Download PDFInfo
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- CN114289088A CN114289088A CN202210049479.2A CN202210049479A CN114289088A CN 114289088 A CN114289088 A CN 114289088A CN 202210049479 A CN202210049479 A CN 202210049479A CN 114289088 A CN114289088 A CN 114289088A
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Abstract
The application provides a micro-fluidic chip includes upper chip and lower chip, and the lower chip is provided with fluid channel, and the upper chip is provided with the micropore, and the volume of micropore is configured to be greater than fluid channel's volume. The number of the micro-holes is multiple, the micro-holes are arranged in multiple rows on the upper chip, and the number and the arrangement positions of the fluid channels on the lower chip are matched with the micro-holes. The upper chip and the lower chip are made of glass materials and are prepared by a wet etching method. After the hydrophobic treatment, the upper chip and the lower chip are assembled in the organic oil phase. The application also provides a multistep reaction method, the reaction liquid can be subjected to multi-step sample loading by adopting the microfluidic chip, and the time interval between two adjacent sample loading can be adjusted as required to match different reaction processes.
Description
Technical Field
The application relates to the field of particle preparation, in particular to a microfluidic chip and a multi-step reaction method.
Background
Regularly Clustered interspaced short palindromic sequences (CRISPRs) have been developed in recent years for a number of uses as part of the immune system of prokaryotes. In the field of nucleic acid molecule detection, the method has very high detection specificity and does not need a complex external temperature control system, thereby showing very wide application prospects. The basic principle of CRISPR-assisted nucleic acid detection systems is: by taking Cas12 as an example, by designing a crRNA sequence for specifically recognizing a target DNA molecule, when a system contains the target molecule, the crRNA can specifically recognize the target molecule, and then activate the trans-cleavage activity of Cas12, Cas12 can randomly cleave ssDNA in the reaction system, and the presence of the target molecule can be recognized by using a ssDNA probe labeled with a fluorescent molecule in the system or using other methods. However, the reaction has the disadvantages of poor reaction kinetics, high detection lower limit, and incapability of absolute quantification of target molecules. The digital nucleic acid detection method can be used for absolutely quantifying the target molecules in the reaction system. The digitalized nucleic acid detection needs to divide a reaction system into thousands or even tens of thousands of micro-droplets, the number of nucleic acid molecules in each micro-droplet is in accordance with Poisson distribution and is mostly 0 or 1, the micro-droplets containing the nucleic acid molecules can display positive signals after the reaction is finished, and the nucleic acid molecules can be absolutely quantified by counting the number of the micro-droplets with the positive signals. By utilizing the confinement effect in the micro-droplets (namely, a single molecule is confined in a micro-reaction system, and the relative concentration of the molecule is increased compared with that of a general system), the digital CRISPR detection can be realized, but the method has high requirements on the volume size of the droplets, and at least the droplet with pico-upgrade is required to complete the detection, which brings high requirements on a droplet generation part. The method using pre-amplification can solve the problem, and the reaction conditions, reaction reagents and the like of many pre-amplification systems are not compatible with the CRISPR detection system, so that a set of digital nucleic acid detection system capable of carrying out multi-step loading needs to be developed.
The core of the digital nucleic acid detection system is the generation of micro-droplets. The common methods for generating micro-droplets include three methods, namely a chip method, a micro-droplet method and a microfluidic chip method, and a plurality of commercial droplet generation products based on the methods, such as a QuantStudio 3D digital PCR system of ThermoFisher Scientific, a QX200 droplet digital PCR system of BioRad, a Crystal digital PCR system of STILLA, and the like, have been developed at present, and uniform droplets can be rapidly and stably generated by using the methods. The above-mentioned droplet generation mode can be used only for one-time droplet generation, and it is difficult to perform multi-step operations of droplets in the same chip. Many reactions require multiple steps to complete and are difficult to perform in the above systems.
The slide chip (SlipChip) is a novel micro-fluidic chip, and micro-droplets can be generated by the relative sliding of an upper chip and a lower chip. The sliding chip comprises an upper sub-chip and a lower sub-chip. The lower surface of the upper sub-chip and the upper surface of the lower sub-chip are provided with micropores. In the initial position, the upper and lower sub-chips are assembled together with the micro-wells of the upper and lower sub-chips partially overlapping to form a communicating fluid conduit. After the solution is injected into the chip, the upper sub-chip and the lower sub-chip slide relatively, and the micropores are not partially overlapped with each other, so that a large amount of liquid drops are generated. (reference: Lab Chip 20099: 2286-2292, CN104722342B)
The self-partition sliding chip is a novel displacement type micro-fluidic chip (Biosensors and Bioelectronics, Volume 155,1May, 2020; CN109046484B) capable of spontaneously generating liquid drops by surface tension, and the upper and lower sub-chips adopt a communicating pipe type design and generate liquid drops by position movement and the action of surface tension. Compared with the traditional sliding chip, the chip is simple to operate and high in operation fault tolerance.
However, the micro-droplet generating chip in the prior art has a similar technical problem that it cannot handle the reaction which needs multiple steps to complete. Or to produce microdroplets requiring multiple charges.
Therefore, those skilled in the art have motivated to develop a microfluidic chip to solve the technical problems of the prior art, and to provide a multi-step reaction method using the microfluidic chip.
Disclosure of Invention
In order to achieve the above object, the present application provides a microfluidic chip, including an upper chip and a lower chip, wherein the lower chip is provided with a fluid channel, the upper chip is provided with a micro-hole, and the volume of the micro-hole is configured to be greater than the volume of the fluid channel by more than 1.5 times.
Further, the number of the micro holes is plural, a plurality of the micro holes are arranged in plural rows on the upper chip, and the number of the fluid passages and the arrangement position on the lower chip are matched with the micro holes.
Furthermore, the upper chip and the lower chip are made of glass materials and are prepared by a wet etching method.
Further, the upper chip and the lower chip are subjected to a hydrophobization treatment.
Further, the upper chip and the lower chip are assembled in an organic oil phase.
The application also provides a multi-step reaction method, which adopts the microfluidic chip and comprises the following steps:
combining the upper chip and the lower chip to ensure that the fluid channel is not overlapped with the micropore;
injecting a first liquid into the fluid channel;
moving the upper chip and the lower chip relatively to enable the fluid channel to be overlapped with the micropores to obtain micro-droplets;
moving the upper chip and the lower chip relatively to ensure that the fluid channel is not overlapped with the micropore;
injecting a second liquid into the fluid channel;
and step six, repeating the step three to obtain the micro liquid drops generated by the multi-step reaction.
And further, repeating the steps from four to six to realize multi-step sample adding of the micro-droplets.
Furthermore, biological and chemical reactions can be carried out in the micro-droplets generated in the step six.
Further, in the second step, the first liquid is a nucleic acid amplification reaction liquid containing a loop-mediated isothermal nucleic acid amplification system.
Further, in the fifth step, the second liquid is a CRISPR reaction liquid without a template.
Compared with the prior art, the application has at least the following beneficial effects:
1. by adopting the microfluidic chip provided by the application, the reaction liquid can be subjected to multi-step sample loading, and the time interval between two adjacent sample loading can be adjusted as required to match different reaction processes.
2. The application provides a micro-fluidic chip simple structure, easy processing.
3. The microfluidic chip provided by the application can be used for simultaneously carrying out synchronous multi-step fusion operation on thousands of liquid drops.
4. The microfluidic chip provided by the application can be compatible with various liquid systems with different surface properties, the requirement on the liquid surface property of the liquid drop generation of the chip is low, various solutions containing surfactants with different concentrations can be compatible, and the compatibility to liquids with different viscosities is also high.
5. The application provides a little fluidic chip's fusion error rate is low and easily distinguish and not fuse the liquid droplet, and the chip adopts the mode of one-to-one application of sample to carry out the liquid droplet fusion, and the efficiency that the liquid droplet fuses depends on liquid droplet generation efficiency, does not receive other factors such as liquid velocity of flow to disturb, and can distinguish easily according to the liquid droplet size and not fuse the liquid droplet.
Drawings
FIG. 1 is a schematic diagram of the structure and process of use of an embodiment of the present application;
FIG. 2 is a schematic diagram of the structure and process of use of an embodiment of the present application;
FIG. 3 is a graph of the signal-to-noise ratio of the detection obtained using the reaction method of the present application;
fig. 4 is a schematic diagram of CRISPR detection at different concentrations;
FIG. 5 is a graph of concentration versus dilution factor.
Detailed Description
The technical contents of the preferred embodiments of the present application will be more clearly and easily understood by referring to the drawings attached to the specification. The present application may be embodied in many different forms of embodiments and the scope of the present application is not limited to only the embodiments set forth herein.
Example 1
As shown in fig. 1, the sliding microfluidic chip provided in this embodiment is composed of an upper chip 2 (shown in a dotted line in fig. 1A) and a lower chip 1 (shown in a solid line in fig. 1A). The upper chip 2 and the lower chip 1 are made of glass materials and are prepared by a wet etching method. The prepared upper chip 2 and lower chip 1 are subjected to hydrophobization treatment before use, so that the hydrophobic state is ensured before use. The upper chip 2 and the lower chip 1 are assembled in an organic oil phase. Wherein the lower chip 1 is provided with a fluid channel 11, the upper chip 2 is provided with a micro-hole 21, and the volume of the micro-hole 21 is set to be much larger than the volume of the fluid channel 11. Preferably, the volume of the micropores 21 is 10 times or more the volume of the fluid channel 11. In the present embodiment, it is preferable that the number of the wells 21 is plural, the plural wells 21 are arranged in one row (as shown in fig. 1) or plural rows (as shown in fig. 2) on the upper chip 2, and the number of the fluid channels 11 and the arrangement position on the lower chip 1 are matched with the wells 21.
Fig. 1 shows a method for performing a multi-step reaction by using this embodiment, which specifically includes:
step one, combining the upper chip and the lower chip to ensure that the fluid channel is not overlapped with the micropore. As shown in fig. 1A, in the initial state, the micropores 21 and the fluid channel 11 do not overlap with each other, and the liquid can flow along the fluid channel 11 and does not enter the micropores 21 due to the organic oil phase and the hydrophobic surface.
And step two, injecting the first liquid into the fluid channel 11 by using a pipette. As shown by the hatched portion in fig. 1B, the first liquid will fill the fluid channel 11 of the lower chip 1.
And step three, relatively moving the upper chip and the lower chip to enable the fluid channel 11 to be overlapped with the micropore 21. As shown in fig. 1C, since the micropores 21 provide an expansion space, the first liquid will spontaneously break off to form droplets under the action of surface tension, and enter the space of the micropores 21 from the space of the fluid channel 11.
And step four, repeating the step one, and carrying out the first reaction. As shown in FIG. 1D, the upper chip 2 and the lower chip 1 are relatively moved to the initial state again, since the volume of the micro-hole 21 is much larger than that of the fluid channel 11. When moving to the initial state, the droplets formed in step three will remain completely within the micro-pores 21, with no liquid remaining in the corresponding fluidic channels 11.
And fifthly, injecting second liquid into the fluid channel by adopting a pipette. As shown in fig. 1E, the pipette is again used to inject the second liquid into the fluid channel 11. The second liquid will fill the fluid channels 11 of the lower chip 1.
And step six, repeating the step three, and carrying out a second reaction to obtain micro droplets generated by the multi-step reaction. As shown in fig. 1F, the upper chip 2 and the lower chip 1 are relatively moved again to the initial state, and the second liquid and the droplets formed by the first liquid are mixed to form new droplets.
By using the method of this embodiment, the micro-droplet mixing of at least two reaction solutions can be accomplished. Alternatively, it is also possible to add more kinds of liquids in steps to participate in the reaction.
The preparation method of the upper chip 2 and the lower chip 1 in this embodiment:
and (3) carrying out chip structure design by using AutoCAD software, and preparing a corresponding mask. First, a mask was placed over soda lime glass, which had been coated with a chrome layer and a photoresist layer, and exposed to parallel ultraviolet light. Subsequently, the exposed glass was immersed in a 0.1mol/L sodium hydroxide solution for 1 minute to remove the photoresist portion that reacted with the ultraviolet light. The glass was then transferred to a dechroming solution for 1 minute, so that the photoresist had been removed and the bare chromium layer was removed. The dechroming solution contained 0.6mol/L perchloric acid and 0.365mol/L cerium ammonium nitrate in water. Subsequently, the treated glass was rinsed thoroughly with deionized water and air dried. Since structures with different depths, such as the fluid channels 11 and the micro-holes 21, need to be etched inside the chip, a second mask is placed over the glass and exposed to parallel uv light. Subsequently, the exposed glass was immersed in a 0.1mol/L sodium hydroxide solution for 1 minute to remove the portion of the photoresist that reacted with the UV light, leaving the chromium layer temporarily. Subsequently, the treated glass was rinsed thoroughly with deionized water and air dried. And then, placing the treated glass in a constant-temperature glass etching solution for etching, wherein the glass etching solution comprises 1mol/L hydrofluoric acid, 0.5mol/L ammonium fluoride and 0.75mol/L nitric acid solution. The desired micro-holes may be formed on the glass chip by the wet etching method. The depth of the micro-holes on the glass chip can be controlled by the time of the wet etching. And after etching to the first required depth, taking out the chip, cleaning the chip by using deionized water, putting the chip into a chromium removing solution, removing the chromium layer exposed by the second exposure, and putting the chip into a glass etching solution again for etching. The prepared chip needs to be subjected to corresponding surface treatment. The surface of the processed glass chip needs to be subjected to hydrophobic treatment, and the specific method comprises the following steps: firstly, fully cleaning the surface of glass by using deionized water, and drying the glass; secondly, the glass chip is placed in a plasma cleaning instrument for surface plasma cleaning and activation. Finally, the glass chip is placed in a container containing dichlorodimethylsilane for gas phase silanization reaction. And (3) washing the treated chip with chloroform, acetone and absolute ethyl alcohol, and drying by blowing to perform the next experiment.
In other similar embodiments, the chip material may also be quartz glass, plastic, ceramic, metal, inorganic material, fibrous material, polymer, and the like.
In other similar embodiments, the chip may be fabricated by dry etching, micromachining, 3D printing, thermoforming, pressure forming, injection molding, and the like.
In a similar embodiment, a plurality of rows of wells 21 may be provided on the upper chip 2 and a plurality of rows of fluid channels 11 (shown in FIG. 2) may be provided on the lower chip 1. The number of droplets participating in the reaction at the same time can be increased. With suitable arrangement, this embodiment can simultaneously realize multi-step reaction operation of thousands of droplets.
Example 2
This example uses the fluidic chip provided in example 1 in a digital CRISPR reaction with pre-amplification.
First, in the second step, a nucleic acid amplification reaction solution containing a LAMP system is injected into the fluid channel 11 of the lower chip 1. Preferably, SARS-CoV-2 is used as a reaction template. 25 μ L of LAMP reaction mixture contains 2.5 μ L10X isothermal amplification buffer, 6mM magnesium sulfate, 8UBst2.0 hot start DNA polymerase, 1.4mM each of dNTPs, 1mgmL-1 bovine serum albumin, 1% Tween-20, 5 μ LSARS-CoV-2cDNA, 1.6 μ M upstream inner primer, 1.6 μ M downstream inner primer, 0.8 μ M upstream circular primer, 0.8 μ M downstream circular primer, 0.2 μ M upstream outer primer, 0.2 μ M downstream outer primer.
After the LAMP reaction mixture is injected into the fluid channel 11, the microwell 21 and the fluid channel 11 are overlapped with each other to generate a droplet in the third step, and the droplet containing the LAMP reaction mixture is completely moved into the microwell 21 by sliding back to the loading state in the fourth step, leaving an empty fluid channel 11. The flow control chip is placed on a thermal cycler, and nucleic acid amplification is carried out in microdroplets containing the template, wherein the reaction temperature is 65 ℃ and the reaction time is 30 minutes.
In step five, a second liquid is injected using a pipette gun, the second liquid being a CRISPR reaction solution without template. 20 μ L CRISPR reaction contains 4 μ L10XTOLO buffer 3, 1% Tween-20, 1 μ M fluorescent probe, 500 nCRRNA and 1 μ MCas12a nuclease. After the reaction solution is filled, the micropores 21 and the fluid channels 11 are overlapped with each other again through the sixth step, at this time, the CRISPR reaction solution forms droplets and contacts with the reaction solution after the LAMP reaction, if the original sample contains a corresponding template, the microdroplets containing the template can be amplified into a large number of products from a single copy in one single droplet through the LAMP reaction, the products can be used as the template of the CRISPR reaction, the Cas12a in the reaction solution is activated, the negative cutting activity of the reaction solution is started, the fluorescent probe in the random cutting system can detect the change of the fluorescent signal by using a fluorescent microscope or other fluorescent detection equipment, the change is a positive hole, and the microdroplets without the template have no change, and are negative holes. The concentration of the original check sample can be quantitatively determined by counting the number of positive wells and negative wells.
Using this protocol, we performed quantitative detection of SARS-CoV-2 nucleic acid with the following primer sequences, crRNA sequences and fluorescent probe sequences:
the digital nucleic acid detection using this method showed good detection signal-to-noise ratio, and as shown in FIG. 3, the fluorescence intensity of the positive well after the reaction was over 8 times higher than that of the negative well. In this example, the detection capability of the reaction system was verified by using nucleic acid samples of different concentrations, and the original samples were diluted 10 respectively0.5、101、101.5、102、102.5、103Performing two-step digital CRISPR detection and concentration calculation on the chip in the reaction system without the template sample after the multiplication, wherein the fluorescence pictures after the reaction are shown in FIG. 4, wherein A-E are respectively dilution multiples of 100.5，101，101.5，102，103And the SARS-CoV-2cDNA of negative control is subjected to the fluorescence image after the digital CRISPR, and the reaction result shows good linearity. As shown in fig. 5, the calculated concentration has good agreement with the dilution factor.
The foregoing detailed description of the preferred embodiments of the present application. It should be understood that numerous modifications and variations can be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the concepts of the present application should be within the scope of protection defined by the claims.
Claims (10)
1. A microfluidic chip comprises an upper chip and a lower chip, and is characterized in that the lower chip is provided with a fluid channel, the upper chip is provided with micropores, and the volume of each micropore is configured to be more than 1.5 times larger than that of the fluid channel.
2. The microfluidic chip according to claim 1, wherein the number of the micro wells is plural, a plurality of the micro wells are arranged in a plurality of rows on the upper chip, and the number and the arrangement position of the fluid channels on the lower chip are matched with the micro wells.
3. The microfluidic chip according to claim 2, wherein the upper chip and the lower chip are made of glass materials and are prepared by a wet etching method.
4. The microfluidic chip according to claim 3, wherein the upper chip and the lower chip are subjected to a hydrophobic treatment.
5. The microfluidic chip of claim 4, wherein said upper chip and said lower chip are assembled in an organic oil phase.
6. A multi-step reaction method using the microfluidic chip according to claim 5, comprising the steps of:
combining the upper chip and the lower chip to ensure that the fluid channel is not overlapped with the micropore;
injecting a first liquid into the fluid channel;
moving the upper chip and the lower chip relatively to enable the fluid channel to be overlapped with the micropores to obtain micro-droplets;
moving the upper chip and the lower chip relatively to ensure that the fluid channel is not overlapped with the micropore;
injecting a second liquid into the fluid channel;
and step six, repeating the step three to obtain the micro liquid drops generated by the multi-step reaction.
7. The multistep reaction process of claim 6 wherein steps four to six are repeated to effect multistep loading of the microdroplets.
8. The multistep reaction process of claim 6 wherein biological and chemical reactions are carried out in the microdroplets formed in step six.
9. The multi-step reaction process according to claim 6, wherein in the second step, the first liquid is a reaction solution for nucleic acid amplification comprising a loop-mediated isothermal nucleic acid amplification system.
10. The multistep reaction process of claim 6 wherein in step five said second liquid is a template-free CRISPR reaction solution.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130281316A1 (en) * | 2012-04-24 | 2013-10-24 | SlipChip, LLC | Slip-induced compartmentalization |
| CN108837718A (en) * | 2018-06-11 | 2018-11-20 | 上海交通大学 | A kind of high throughput microlayer model gradient dilution device and method |
| US20190118177A1 (en) * | 2017-09-25 | 2019-04-25 | California Institute Of Technology | Device for additive delivery of reagents and related methods and systems |
| CN110632064A (en) * | 2019-09-20 | 2019-12-31 | 南京大学 | Multi-element paper-based sliding microfluidic chip based on cerium metal organic framework |
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- 2022-01-17 CN CN202210049479.2A patent/CN114289088A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130281316A1 (en) * | 2012-04-24 | 2013-10-24 | SlipChip, LLC | Slip-induced compartmentalization |
| US20190118177A1 (en) * | 2017-09-25 | 2019-04-25 | California Institute Of Technology | Device for additive delivery of reagents and related methods and systems |
| CN108837718A (en) * | 2018-06-11 | 2018-11-20 | 上海交通大学 | A kind of high throughput microlayer model gradient dilution device and method |
| CN110632064A (en) * | 2019-09-20 | 2019-12-31 | 南京大学 | Multi-element paper-based sliding microfluidic chip based on cerium metal organic framework |
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