Microdroplets and applications thereof, microfluidic chips, and crisper molecular detection methods

Abstract

A microdroplet and application thereof, a microfluidic chip and a CRISPR molecular detection method, the microdroplet comprises, from inside to outside, a first layer droplet, a second layer droplet and a third layer droplet, wherein the first layer droplet and the third layer droplet are both aqueous phase droplets, and the second layer droplet is an oil phase droplet; the first layer droplet and the third layer droplet are configured to be separated into double aqueous phases by the second layer droplet to form the microdroplet at a first temperature, and to be fused together to convert the microdroplet into a single aqueous phase droplet at a second temperature.The CRISPR molecular detection method of the embodiment of the present disclosure can perform the amplification stage and the detection stage in the same droplet and the closed microfluidic chip, improve the automation degree of detection, thereby improving the detection efficiency and accuracy, and can reduce the volume of the microfluidic chip, and improve the application potential of the CRISPR-Cas platform in molecular diagnosis POCT.

Description

TECHNICAL FIELD

[0001] The embodiments of the present disclosure relate to, but are not limited to, the field of biotechnology, and in particular to a microdroplet and application thereof, a microfluidic chip and a CRISPR molecular detection method. BACKGROUND

[0002] Since the first time that people realized that deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are genetic materials, the research on DNA and RNA has never stopped. In the process of the continuous development of medicine and detection technology, people also gradually realized that the detection of DNA and RNA is an essential tool and a key step to realize precision medicine. Through the detection and analysis of nucleic acids, researchers can understand the pathogenic principle of many genetic diseases, can be used to distinguish the specific type of cancer, and can be used for the diagnosis of infectious diseases.

[0003] In 2012, people discovered the potential of CRISPR technology applied to gene editing. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a repeated sequence in the genome of prokaryotes, which is an immune weapon produced in the history of life evolution, that is, viruses can integrate their own genes into bacteria and use the cell tools of bacteria to replicate their own genes. In order to remove the foreign invading genes of viruses from their own genome, bacteria have evolved CRISPR system, which can remove the virus genes from their own genome without making any noise. This is a unique immune system of bacteria, which is an acquired immune system for bacteria to resist the invasion of foreign genetic materials such as viruses. Later, CRISPR technology was confirmed to be used in the field of nucleic acid detection, and more specific detection tools for different types of nucleic acids were found. SUMMARY

[0004] The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of protection of the present disclosure.

[0005] The embodiments of the present disclosure provide a microdroplet, which comprises, from inside to outside, a first layer droplet, a second layer droplet and a third layer droplet, wherein the first layer droplet and the third layer droplet are both aqueous phase droplets, and the second layer droplet is an oil phase droplet; the first layer droplet and the third layer droplet are configured to be separated into a double aqueous phase by the second layer droplet to form the microdroplet at a first temperature, and to be fused together to convert the microdroplet into a single aqueous phase droplet at a second temperature.

[0006] In the exemplary embodiments of the present disclosure, the diameter of the first layer droplet can be 50 μm to 100 μm, the diameter of the third layer droplet can be 60.5 μm to 150 μm, and the diameter of the third layer droplet is not more than 2 to 3 times the diameter of the first layer droplet.

[0007] In the exemplary embodiments of the present disclosure, the first temperature can be 37℃ to 44℃, and the second temperature can be 55℃ to 65℃.

[0008] In the exemplary embodiments of the present disclosure, the material of the first layer droplet includes polyethylene glycol, and the material of the third layer droplet includes dextran.

[0009] In the exemplary embodiments of the present disclosure, the average molar mass of the polyethylene glycol can be 6400 g / mol to 12800 g / mol, and the average molar mass of the dextran can be 470000 g / mol to 500000 g / mol.

[0010] In the exemplary embodiments of the present disclosure, in the first layer droplet, the mass percentage of polyethylene glycol is w1, and w1 can be 3.5% to 3.85%; in the third layer droplet, the mass percentage of dextran is w2, and w2 can be 2% to 7%.

[0011] In the exemplary embodiments of the present disclosure, w1 can be 3.5% to 3.85%, and w2 can be 4% to 5.5%.

[0012] In the exemplary embodiments of the present disclosure, w1 and w2 can satisfy:

[0013] w1 = -0.34 x w2 + 5.38, wherein 2 < w2 < 7.

[0014] In the exemplary embodiments of the present disclosure, the first layer droplet can contain reaction components and samples to be tested required for RAA amplification, RPA amplification, RCA amplification, RCT amplification, NASBA amplification, HAD amplification, ERA isothermal amplification, or flow cytometry analysis, and the third layer droplet can contain reaction components required for CRISPR molecular detection.

[0015] The present disclosure also provides the use of the microdroplet as described above in RAA amplification, RPA amplification, RCA amplification, RCT amplification, NASBA amplification, HAD amplification, ERA isothermal amplification, flow cytometry analysis, or CRISPR molecular detection.

[0016] The present disclosure also provides a microfluidic chip comprising a flow channel, wherein the flow channel comprises a main flow channel and a narrowed portion, and the diameter of the main flow channel, the diameter of the narrowed portion, and the diameter of the third layer droplet of the microdroplet provided by the present disclosure satisfy:

[0017] R 2min <R3≤R 1min ;

[0018] wherein, R 1min is the minimum value of the diameter of the main flow channel; R 2min is the minimum value of the diameter of the narrowing; and R3 is the diameter of the third layer of droplets.

[0019] In the exemplary embodiments of the present disclosure, the narrowing can be arranged at a corner of the flow channel, or at a non-corner of the flow channel.

[0020] In the exemplary embodiments of the present disclosure, the narrowing can be formed by an expansion unit arranged in the flow channel, the expansion unit being configured to expand when the temperature of the microfluidic chip reaches a second temperature, where the second temperature is the same as the definition of the second temperature in the microdroplet provided in the embodiments of the present disclosure.

[0021] In the exemplary embodiments of the present disclosure, the expansion unit can be arranged at a corner of the flow channel.

[0022] In the exemplary embodiments of the present disclosure, the expansion unit can include any one or more of a polydimethylsiloxane-coated hydrogel and an aluminum-coated silicon dioxide.

[0023] In the exemplary embodiments of the present disclosure, the microfluidic chip can further include an indium tin oxide heating layer.

[0024] The embodiments of the present disclosure also provide a CRISPR molecular detection method, which includes continuously performing an amplification stage and a detection stage in the same microfluidic chip using the microdroplet as described above.

[0025] In the exemplary embodiments of the present disclosure, the CRISPR molecular detection method can include:

[0026] S10: performing a constant temperature amplification reaction in the microfluidic chip using the microdroplet at a constant first temperature, to form an amplification product in the first layer of droplets;

[0027] S20: adjusting the temperature of the microfluidic chip to a second temperature, and applying pressure to the microdroplet, so that the first layer of droplets and the third layer of droplets fuse together, thereby converting the microdroplet into a single aqueous phase droplet;

[0028] S30: adjusting and maintaining the temperature of the microfluidic chip at a constant first temperature, and performing a detection stage in the microfluidic chip using the single aqueous phase droplet.

[0029] The microdroplet of the embodiment of the present disclosure can exhibit a multi-droplet with a three-layer structure and containing a double aqueous phase at a first temperature, and can be converted into a single aqueous phase droplet at a second temperature, thereby providing the possibility of performing the amplification stage and the detection stage of the CRISPR molecular detection in the same droplet (here, the three-layer structure microdroplet and the single aqueous phase droplet converted therefrom are regarded as the same droplet system).

[0030] The CRISPR molecular detection method of the embodiment of the present disclosure can integrate the CRISPR molecular detection and the multi-droplet features into one system, so that the amplification stage and the detection stage are performed in the same droplet (here, the three-layer structure microdroplet and the single aqueous phase droplet converted therefrom are regarded as the same droplet system), thereby reducing the pipetting steps and manual operations, facilitating the simplification and automation of the process; at the same time, the entire process is performed in the droplet of the closed microfluidic chip, thereby eliminating the possibility of aerosol pollution and further improving the accuracy of the detection; the multi-droplet to single aqueous phase droplet conversion is rapidly realized through temperature change and flow channel structure change, thereby facilitating the further reduction of manual operations and the reduction of the chip volume, the improvement of the product portability, and the improvement of the application potential of the CRISPR-Cas platform in the molecular diagnosis point-of-care testing (POCT).

[0031] Other features and advantages of the present disclosure will be set forth in the following description, and in part will become more apparent from the description, or will be learned by practice of the present disclosure. Other advantages of the present disclosure will be realized and attained by those of ordinary skill in the art, including studying the following description and accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0032] The accompanying drawings are included to provide an understanding of the technical solutions of the present disclosure, and constitute a part of the specification, and are used to explain the technical solutions of the present disclosure together with the embodiments of the present disclosure, and do not constitute a limitation on the technical solutions of the present disclosure.

[0033] Figure 1 A structure schematic diagram of a microdroplet of an exemplary embodiment of the present disclosure;

[0034] Figure 2 A structure schematic diagram of a flow channel of a microfluidic chip of an exemplary embodiment of the present disclosure;

[0035] Figure 3 A structure schematic diagram of a flow channel of a microfluidic chip of another exemplary embodiment of the present disclosure;

[0036] Figure 4 A structure schematic diagram of a flow channel of a microfluidic chip of still another exemplary embodiment of the present disclosure;

[0037] Figure 5A structural schematic diagram of a flow channel of a microfluidic chip according to another exemplary embodiment of the present disclosure;

[0038] Figure 6 A flowchart of a CRISPR molecular detection method according to an exemplary embodiment of the present disclosure.

[0039] The meanings of the reference symbols in the drawings are as follows:

[0040] 1 - first layer of droplets; 2 - second layer of droplets; 3 - third layer of droplets; 10 - flow channel; 11 - main flow channel; 12 - narrowing; 20 - expansion unit. DETAILED DESCRIPTION

[0041] The implementations herein can be implemented in any of numerous ways. One skilled in the art will readily recognize that the implementations and techniques described herein can be practiced with various and alternative means. The implementations and techniques are not limited to the examples described herein. In their most general form, various aspects of the implementations can be found in the drawings and their accompanying descriptions in the detailed description. The present disclosure should not be construed as limited to only those implementations described and shown in the Figures. The embodiments and features of the present disclosure can be combined with each other as long as they do not conflict with each other.

[0042] In the drawings, the size, the thickness of the layers, or the region of constituent elements, etc. can be exaggerated for the sake of clarity. Thus, the present disclosure should not be construed as being limited to the size, the shape of the components illustrated in the drawings, and the numerical values shown in the drawings do not reflect the actual scale. In addition, the drawings schematically show ideal examples, and the present disclosure is not limited to the shapes or numerical values shown in the drawings.

[0043] At present, there are still many deficiencies in the application of CRISPR technology in the detection field, for example: (1) The CRISPR molecular detection is divided into two stages: the amplification stage and the detection stage. The reaction environments of the two stages are different, and generally need to be divided into two-step reactions, so that pipetting is required, which makes it difficult to integrate and affects the application potential of CRISPR technology in rapid detection; (2) In addition to increasing the manual operation steps, opening the cover after the amplification is completed is also easy to cause aerosol pollution, which affects the detection results and increases the risk of false positives.

[0044] Microdroplets are often used as microreactors to realize biochemical reactions, rapid mixing of reagents, and synthesis of microparticles, etc. For example, gene chips, protein chips, single-cell analysis, droplet digital PCR (ddPCR), CRISPR molecular detection, and other technologies all need to use microdroplets.

[0045] The microdroplet provided by the embodiments of the present disclosure comprises, from inside to outside, a first layer droplet, a second layer droplet and a third layer droplet, wherein the first layer droplet and the third layer droplet are both aqueous phase droplets, and the second layer droplet is an oil phase droplet; the first layer droplet and the third layer droplet are configured to be separated into aqueous two-phase by the second layer droplet to form the microdroplet at a first temperature, and to be fused together to convert the microdroplet into a single aqueous phase droplet at a second temperature.

[0046] The microdroplet of the embodiments of the present disclosure can exhibit a multi-droplet with a three-layer structure and containing aqueous two-phase at a first temperature, and can be converted into a single aqueous phase droplet at a second temperature, which provides the possibility for amplification and detection stages of CRISPR molecular detection in the same droplet (here, the three-layer structure microdroplet and the single aqueous phase droplet converted therefrom are regarded as the same droplet system), and is beneficial to integrate the CRISPR molecular detection into one system, reduce the pipetting steps and manual operation in the CRISPR molecular detection process, facilitate the simplification and automation of the process, improve the accuracy of detection, and improve the application potential of the CRISPR-Cas platform in molecular diagnosis point-of-care testing (POCT).

[0047] Figure 1 A structural schematic diagram of the microdroplet of the exemplary embodiments of the present disclosure. As shown in Figure 1 the microdroplet comprises, from inside to outside, a first layer droplet 1, a second layer droplet 2 and a third layer droplet 3, wherein the first layer droplet 1 and the third layer droplet 3 are both aqueous phase droplets, and the second layer droplet 2 is an oil phase droplet; the first layer droplet 1 and the third layer droplet 3 are configured to be separated into aqueous two-phase by the second layer droplet 2 to form the microdroplet at a first temperature, and to be fused together to convert the microdroplet into a single aqueous phase droplet at a second temperature.

[0048] In the exemplary embodiments of the present disclosure, the diameter of the first layer of droplets can be 50-100 μm, for example, the diameter of the first layer of droplets can be 50-60 μm, 60-70 μm, 70-80 μm, 80-90 μm, 90-100 μm; the diameter of the third layer of droplets can be 60.5-150 μm, for example, the diameter of the third layer of droplets can be 60.5-70 μm, 70-80 μm, 80-90 μm, 90-100 μm, 100-110 μm, 110-120 μm, 120-130 μm, 130-140 μm, 140-150 μm; and the diameter of the third layer of droplets is not more than 2-3 times the diameter of the first layer of droplets, for example, the diameter of the third layer of droplets is not more than 2 times, 2.5 times or 3 times the diameter of the first layer of droplets. When the diameter of the first layer of droplets is 50-100 μm, the diameter of the third layer of droplets is 60.5-150 μm, and the diameter of the third layer of droplets is not more than 2-3 times the diameter of the first layer of droplets, it is beneficial to keep the first layer of droplets in a relatively stable state at the first temperature, and when the temperature is adjusted to the second temperature, it is beneficial to the first layer of droplets and the third layer of droplets to tend to and be able to fuse together.

[0049] In the exemplary embodiments of the present disclosure, the first temperature can be 37-44℃, for example, can be 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃; the second temperature can be 55-65℃, for example, can be 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃. The amplification reaction of CRISPR molecular detection and the CRISPR detection reaction temperature are usually 37-44℃, so when the first temperature is 37-44℃, it is beneficial to the amplification reaction of CRISPR molecular detection and the CRISPR detection reaction, and the first layer of droplets and the third layer of droplets in the microdroplets tend to be layered, which is beneficial to being separated into a double aqueous phase, so that a relatively stable microdroplet with a three-layer structure can be formed; when the second temperature is 55-65℃, the structural stability of the microdroplet is reduced, and the first layer of droplets and the third layer of droplets in the microdroplet tend to and can be fused into a single aqueous phase within a certain time.

[0050] In the exemplary embodiments of the present disclosure, the material of the first layer of droplets includes polyethylene glycol, and the material of the third layer of droplets includes dextran.

[0051] In the exemplary embodiments of the present disclosure, the average molar mass of the polyethylene glycol can be 6400 g / mol to 12800 g / mol, for example, can be 8000 g / mol, and the average molar mass of the dextran can be 470000 g / mol to 500000 g / mol, for example, can be 482000 g / mol. When the average molar mass of the polyethylene glycol is 6400 g / mol to 12800 g / mol and the average molar mass of the dextran is 470000 g / mol to 500000 g / mol, it is beneficial to keep the first layer droplets in a relatively stable state at the first temperature, and when the temperature is adjusted to the second temperature, it is beneficial to the first layer droplets and the third layer droplets to tend to and be able to fuse together.

[0052] In the exemplary embodiments of the present disclosure, in the first layer droplets, the mass percentage of polyethylene glycol is w1, w1 can be 3.5% to 3.85%, for example, w1 can be 3.5%, 3.65%, 3.75%, or 3.85%; in the third layer droplets, the mass percentage of dextran is w2, w2 can be 2% to 7%, for example, w2 can be 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75%, 6%, 6.25%, 6.5%, 6.75%, or 7%. When w1 is 3.5% to 3.85% and w2 is 2% to 7%, it is beneficial to keep the first layer droplets in a relatively stable state at the first temperature, and when the temperature is adjusted to the second temperature, it is beneficial to the first layer droplets and the third layer droplets to tend to and be able to fuse together.

[0053] In the exemplary embodiments of the present disclosure, w1 can be 3.5% to 3.85%, and w2 can be 4% to 5.5%.

[0054] In the exemplary embodiments of the present disclosure, w1 and w2 can satisfy:

[0055] w1 = -0.34 x w2 + 5.38, wherein 2 < w2 < 7.

[0056] When w1 and w2 satisfy w1 = -0.34 x w2 + 5.38, wherein 2 < w2 < 7, it is beneficial to keep the first layer droplets in a relatively stable state at the first temperature, and when the temperature is adjusted to the second temperature, it is beneficial to the first layer droplets and the third layer droplets to tend to and be able to fuse together.

[0057] In the exemplary embodiments of the present disclosure, w1 can be 3.5% to 3.85%, w2 can be 2% to 7%, and w1 and w2 can satisfy:

[0058] w1 = -0.34 x w2 + 5.38.

[0059] In the exemplary embodiments of the present disclosure, the first layer of droplets can contain reaction components and samples to be tested required for Recombinase Aided Amplification (RAA), Recombinase Polymerase Amplification (RPA), Rolling Circle Amplification (RCA), Rolling Circle Transcription (RCT), Nuclear Acid Sequence-Based Amplification (NASBA), Helicase-Dependent Amplification (HAD), Enzymatic Recombinase Amplification (ERA), or flow cytometry analysis, and the third layer of droplets can contain reaction components required for CRISPR molecular detection. For example, the third layer of droplets can contain Cas12a enzyme, Cas13 enzyme, or Cas14 enzyme, and gRNA, probe, buffer, and other components, without containing samples to be tested.

[0060] In the exemplary embodiments of the present disclosure, the microdroplets can be formed by T-shaped channel method or capillary flow confocal method. For example, the microdroplets can be prepared by referring to the methods disclosed in Controlled formulation of monodisperse double emulsions in a multiple-phase microfluidic system, Soft Matter, 2005, 1, 23-27 or Controllable Monodisperse Multiple Emulsions, Angew. Chem. 2007, 119, 9128-9132.

[0061] The embodiments of the present disclosure also provide applications of the microdroplets as described above in RAA amplification, RPA amplification, RCA amplification, RCT amplification, NASBA amplification, HAD amplification, ERA isothermal amplification, flow cytometry analysis, or CRISPR molecular detection.

[0062] The present disclosure also provides a microfluidic chip including a flow channel, wherein the flow channel may include a main channel and a narrowing portion, and the diameter of the main channel, the diameter of the narrowing portion, and the diameter of the third layer of droplets of the micro-droplets provided in the present disclosure may satisfy:

[0063] R 2min <R3≤R 1min ;

[0064] Among them, R 1min is the minimum diameter of the main channel; R 2min is the minimum diameter of the narrowed portion; R3 is the diameter of the third layer of droplets.

[0065] In an exemplary embodiment of the present disclosure, the narrowing portion may be provided at a corner of the flow channel, or at a non-corner portion of the flow channel.

[0066] Figure 2 Schematic diagram of the structure of the flow channel of the microfluidic chip according to an exemplary embodiment of the present disclosure; Figure 3 FIG. 1 is a schematic structural diagram of a flow channel of a microfluidic chip according to another exemplary embodiment of the present disclosure. Figure 2 and Figure 3 As shown, in an exemplary embodiment of the present disclosure, the flow channel 10 of the microfluidic chip may include a main channel 11 and a narrowing portion 12. The narrowing portion 12 may be provided at a corner of the flow channel 10 or at a non-corner portion of the flow channel 10. Furthermore, the narrowing portion may be provided at a corner or a non-corner portion of the flow channel. When the narrowing portion is provided at a non-corner portion of the flow channel, multiple narrowing portions may be provided at intervals.

[0067] When the microdroplets flow to the position where the narrowing portion is located, the narrowing portion will exert pressure on the microdroplets. In addition, the structural stability of the microdroplets is reduced at the second temperature, which accelerates the fusion of the first layer of droplets and the third layer of droplets in the microdroplets, thereby converting the microdroplets into single-aqueous phase droplets.

[0068] The diameter of the main channel may not be uniform, and the diameter of the narrowing portion may not be uniform, as long as R 2min <R3≤R 1min When the micro-droplets flow through the narrowing portion, the narrowing portion can effectively squeeze the micro-droplets, causing the micro-droplets of the three-layer structure to be transformed into single-water phase droplets. Figure 2 and Figure 3 As shown, R 1min Can be 100μm, R 2min It can be 50μm, and R3 can be 60.5μm, 70μm, 80μm, 90μm, or 100μm.

[0069] In an example embodiment of the present disclosure, the narrowing portion can be formed by disposing an expansion unit in the flow channel, the expansion unit being configured to expand when the temperature of the microfluidic chip reaches the second temperature, where the second temperature is the same as the definition of the second temperature in the microdroplet provided by the example embodiment of the present disclosure.

[0070] When the temperature of the microfluidic chip reaches the second temperature, the expansion unit expands, so that the diameter of the flow channel of the microfluidic chip at the position where the expansion unit is disposed becomes smaller. When the microdroplet flows to the position where the expansion unit is located, the expanded expansion unit exerts pressure on the microdroplet, and in addition, the structural stability of the microdroplet decreases at the second temperature, so that the first layer of droplets and the third layer of droplets in the microdroplet accelerate to fuse together, thereby converting the microdroplet into a single-aqueous-phase droplet.

[0071] In an example embodiment of the present disclosure, the expansion unit can be disposed at a corner of the flow channel.

[0072] Figure 4 A structural schematic diagram of a flow channel of a microfluidic chip according to another example embodiment of the present disclosure; Figure 5 A structural schematic diagram of a flow channel of a microfluidic chip according to another example embodiment of the present disclosure. As shown in Figure 4 In an example embodiment of the present disclosure, an expansion unit 20 can be disposed in the flow channel 10 of the microfluidic chip, and the expansion unit 20 can be disposed at a corner of the flow channel 10 of the microfluidic chip. As shown in Figure 5 In an example embodiment of the present disclosure, the expansion unit 20 can be disposed at a corner and a non-corner of the flow channel 10 of the microfluidic chip. In addition, in an example embodiment of the present disclosure, the expansion unit can also be disposed only at a non-corner of the flow channel of the microfluidic chip. When the expansion unit is disposed at a corner of the flow channel of the microfluidic chip, the expansion unit is more conducive to the conversion of the microdroplet with a three-layer structure into a single-aqueous-phase droplet after thermal expansion.

[0073] In an example embodiment of the present disclosure, the expansion unit can include any one or more of a polydimethylsiloxane-wrapped hydrogel and an aluminum-wrapped silicon dioxide.

[0074] In an example embodiment of the present disclosure, the microfluidic chip can include a heating layer, for example, an indium tin oxide (ITO) heating layer. At this time, the temperature of the microfluidic chip can be converted from the first temperature to the second temperature by using the heating layer.

[0075] In the exemplary embodiments of the present disclosure, the microfluidic chip can further include a chip body, a plurality of CRISPR detection chambers, a centrifugal interface, a sample loading cavity, and a plurality of RAA or RPA constant temperature amplification cavities.

[0076] For example, the microfluidic chip can include a chip body, which can include an upper layer chip and a lower layer chip, and the upper layer chip and the lower layer chip are fixedly connected;

[0077] The lower layer chip is provided with a plurality of CRISPR detection chambers, a centrifugal interface, a sample loading cavity, and a plurality of RAA or RPA constant temperature amplification cavities;

[0078] The centrifugal interface is arranged at the middle position of the upper layer chip, the sample loading cavity is arranged circumferentially outside the centrifugal interface, the plurality of RAA or RPA constant temperature amplification cavities are arranged circumferentially outside the sample loading cavity, the sample loading cavity and the plurality of RAA or RPA constant temperature amplification cavities are in communication respectively, and the plurality of CRISPR detection chambers are arranged circumferentially outside the plurality of RAA or RPA constant temperature amplification cavities, and the plurality of CRISPR detection chambers are in communication with the corresponding RAA or RPA constant temperature amplification cavities respectively;

[0079] A plurality of sample loading holes are formed in the upper layer chip and in communication with the sample loading cavity.

[0080] The present disclosure also provides a CRISPR molecular detection method, which includes using the microdroplet as described above to continuously perform the amplification stage and the detection stage in the same microfluidic chip.

[0081] The CRISPR molecular detection method of the present disclosure can integrate the entire detection into one system by combining the characteristics of CRISPR molecular detection and multiple droplets, so that the amplification stage and the detection stage are performed in the same droplet (here, the three-layer structure microdroplet and the single aqueous phase droplet converted therefrom are regarded as the same droplet system), which reduces the pipetting steps and manual operations, and is conducive to the simplification and automation of the process; at the same time, the entire process is performed in the droplet of the closed microfluidic chip, which eliminates the possibility of aerosol pollution and can further improve the accuracy of detection; the conversion of multiple droplets to single aqueous phase droplets is quickly realized through temperature change and flow channel structure change, which is conducive to further reducing manual operation and reducing the size of the chip, improving the portability of the product, and improving the application potential of the CRISPR-Cas platform in molecular diagnosis point-of-care testing (POCT).

[0082] Figure 6 A flowchart of the CRISPR molecular detection method of the exemplary embodiments of the present disclosure is shown in FIG. 1. Figure 6As shown, in the exemplary embodiments of the present disclosure, the CRISPR molecular detection method can comprise:

[0083] S10: performing an isothermal amplification reaction in the microfluidic chip using the microdroplet at a constant first temperature, to form an amplification product in the first layer of droplets;

[0084] S20: adjusting the temperature of the microfluidic chip to a second temperature, and applying pressure to the microdroplet, to fuse the first layer of droplets and the third layer of droplets together, thereby converting the microdroplet into a single-aqueous-phase droplet;

[0085] S30: adjusting and maintaining the temperature of the microfluidic chip at a constant first temperature, and performing a detection stage in the microfluidic chip using the single-aqueous-phase droplet.

[0086] In the exemplary embodiments of the present disclosure, the isothermal amplification reaction in the CRISPR molecular detection method can be RAA amplification, RPA amplification, RCA amplification, RCT amplification, NASBA amplification, HAD amplification, or ERA isothermal amplification.

[0087] When the microdroplet is used to perform the CRISPR molecular detection method of the present disclosure, the first layer droplet of the microdroplet contains the reaction components required for the isothermal amplification reaction (for example, RAA amplification or RPA amplification) and the sample to be detected, and the third layer droplet of the microdroplet contains the reaction components required for the CRISPR molecular detection. The microdroplet enters the microfluidic chip from the channel inlet on one side of the microfluidic chip (can enter by pneumatic) and moves in the channel, and the temperature of the microfluidic chip is maintained at a first temperature (for example, can be 37°C). At this time, the first layer droplet and the third layer droplet in the microdroplet tend to be layered and separated into a double water phase, so it can be a relatively stable microdroplet with a three-layer structure. The isothermal amplification reaction is carried out in the first layer droplet, and the amplification product is obtained. After a period of time (for example, 10 min) of isothermal amplification reaction, the temperature of the microfluidic chip is adjusted to a second temperature (above 50°C, for example, 55°C). At this time, the structural stability of the microdroplet is reduced, and the first layer droplet and the third layer droplet in the microdroplet tend to and can fuse into a single water phase within a certain period of time. At this time, applying pressure to the microdroplet can accelerate the conversion of the microdroplet into a single water phase droplet, and then the amplification product is released and mixed with the reaction components required for the CRISPR molecular detection. Then the temperature of the microfluidic chip is adjusted and maintained at the first temperature (for example, can be 37°C), and the CRISPR reaction is carried out in the single water phase droplet. Therefore, using the microdroplet and the CRISPR molecular detection method of the present disclosure, the amplification stage and the detection stage can be carried out in the same droplet of a closed microfluidic chip, reducing the pipetting steps and manual operation, and also reducing the risk of environmental pollution, improving the detection efficiency and accuracy.

[0088] Although the embodiments disclosed in the present disclosure are as described above, the content described is only the embodiments adopted for the purpose of understanding the present disclosure, and is not intended to limit the present disclosure. Any person skilled in the art can make any modification and change in the form and details without departing from the spirit and scope of the present disclosure. The patent protection scope of the present application shall be subject to the scope defined by the appended claims.

Claims

1. A microdroplet, characterized in that, The microdroplet comprises, from inside to outside, a first layer of droplets, a second layer of droplets and a third layer of droplets, wherein the first layer of droplets and the third layer of droplets are both aqueous droplets, and the second layer of droplets is an oil droplet; the first layer of droplets and the third layer of droplets are configured to be separated by the second layer of droplets into a double aqueous phase to form the microdroplet at a first temperature, and to fuse together to convert the microdroplet into a single aqueous phase droplet at a second temperature; wherein the first temperature is 37-44°C, and the second temperature is 55-65°C; the material of the first layer of droplets comprises polyethylene glycol, and the material of the third layer of droplets comprises dextran.

2. The microdroplet of claim 1, wherein, The diameter of the first layer of droplets is 50-100μm, the diameter of the third layer of droplets is 60.5-150μm, and the diameter of the third layer of droplets is not more than 2-3 times the diameter of the first layer of droplets.

3. The microdroplet of claim 1, wherein, The average molecular weight of the polyethylene glycol is 6400-12800 g / mol, and the average molecular weight of the dextran is 470000-500000 g / mol.

4. The microdroplet of claim 1 or 3, wherein, In the first layer of droplets, the mass percentage of polyethylene glycol is w1, and w1 is 3.5-3.85%; in the third layer of droplets, the mass percentage of dextran is w2, and w2 is 2-7%.

5. The microdroplet of claim 4, wherein, w1 is 3.5-3.85%, and w2 is 4-5.5%.

6. The microdroplet of claim 4, wherein, w1 and w2 satisfy: w1=-0.34×w2+5.38, wherein 2 7. The microdroplet of any one of claims 1 to 3, 5, 6, wherein, The first layer of droplets contains reaction components and a sample to be tested required for RAA amplification, RPA amplification, RCA amplification, RCT amplification, NASBA amplification, HAD amplification, ERA isothermal amplification or flow cytometry analysis, and the third layer of droplets contains reaction components required for CRISPR molecular detection.

8. The microdroplet of claim 4, wherein, The first layer of droplets contains reaction components and a sample to be tested required for RAA amplification, RPA amplification, RCA amplification, RCT amplification, NASBA amplification, HAD amplification, ERA isothermal amplification or flow cytometry analysis, and the third layer of droplets contains reaction components required for CRISPR molecular detection.

9. The microdroplet of any one of claims 1-8 for use in RAA amplification, RPA amplification, RCA amplification, RCT amplification, NASBA amplification, HAD amplification, ERA isothermal amplification, flow cytometry analysis or CRISPR molecular detection.

10. A microfluidic chip for use in the detection of a microdroplet according to any one of claims 1 to 8; the microfluidic chip comprising a flow channel, characterised in that, The flow channel comprises a main flow channel and a narrowing portion, and the diameter of the main flow channel, the diameter of the narrowing portion and the diameter of the third layer of droplets of the microdroplet satisfy: R 2min ≤ R 1min ; wherein R 1min is the minimum value of the diameter of the main flow channel; R 2min is the minimum value of the diameter of the constriction; R3 is the diameter of the third layer of droplets.

11. The microfluidic chip of claim 10, wherein, The narrowing portion is arranged at a corner of the flow channel, or at a non-corner of the flow channel.

12. The microfluidic chip of claim 10 or 11, wherein, The narrowing portion is formed by an expansion unit arranged in the flow channel, and the expansion unit is configured to expand when the temperature of the microfluidic chip reaches the second temperature.

13. The microfluidic chip of claim 12, wherein, The expansion unit is arranged at a corner of the flow channel.

14. The microfluidic chip of claim 12, wherein, The expansion unit comprises any one or more of polydimethylsiloxane-coated hydrogel and aluminum-coated silicon dioxide.

15. The microfluidic chip of claim 10, 11, 13 or 14, further comprising an indium tin oxide heating layer.

16. The microfluidic chip of claim 12, further comprising an indium tin oxide heating layer.

17. A CRISPR molecular detection method, characterized in that, The amplification phase and the detection phase are performed consecutively in the same microfluidic chip with the microdroplet according to claim 7 or 8.

18. The CRISPR molecular detection method of claim 17, comprising: S10: performing a constant temperature amplification reaction in the microfluidic chip with the microdroplet at a constant first temperature, forming an amplification product in the first layer droplet; S20: adjusting the temperature of the microfluidic chip to a second temperature, and applying pressure to the microdroplet, so that the first layer droplet and the third layer droplet fuse together, thereby converting the microdroplet into a single aqueous phase droplet; S30: adjusting and maintaining the temperature of the microfluidic chip at a constant first temperature, performing a detection phase in the microfluidic chip with the single aqueous phase droplet.

19. The CRISPR molecular detection method of claim 17 or 18, wherein, The microfluidic chip is according to any one of claims 10 to 16.

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