Microfluidic detection chip as well as preparation method and fluid filling method thereof

Abstract

The invention discloses a micro-fluidic detection chip and a preparation method and a fluid filling method thereof, and relates to the technical field of micro-fluidic detection chips, the micro-fluidic detection chip comprises a first slide, a chip body layer and a second slide which are attached, the first slide is provided with a liquid inlet hole and a waste liquid hole, the chip body layer is provided with a containing pool, and the second slide is provided with a liquid outlet hole. The containing pool comprises a liquid inlet area, a waste liquid area and a microcavity array between the liquid inlet area and the waste liquid area, the first slide and the containing pool of the chip body layer form a transverse open flow channel with a height-limited top, so that detection liquid added from a liquid inlet hole reaches the microcavity array through the liquid inlet area under the action of negative pressure through a siphonic effect, the liquid inlet speed is high, and blockage is not easy to occur; the number of microcavities of the microcavity array is large and can reach 10,000-100,000 as required, so that high-flux, high-accuracy and high-sensitivity detection of nucleic acid molecules is realized; and the chip body layer is obtained by pouring a mixed solution of a PDMS material and a curing agent on a prefabricated mold and turning over the mold, so that the preparation is simple, the cost is low, and the chip is suitable for large-scale production.

Description

Technical Field

[0001] The present application relates to the technical field of microfluidic detection chips, and in particular to a microfluidic detection chip and a preparation method and a fluid filling method thereof. Background Art

[0002] At present, nucleic acid molecular diagnostic technology mainly involves PCR (polymerase chain reaction, a molecular biology technology used to amplify specific DNA fragments) technologies such as Sanger sequencing, pyrophosphate sequencing, second-generation sequencing, quantitative PCR, AMRS-PCR and Digital-PCR, but it has the disadvantages of high cost, cumbersome operation, long time, limited throughput and high requirements for instruments and equipment. In the field of medical diagnosis, microfluidic detection chips can integrate the entire process of liquid inlet, amplification and detection of traditional samples to be tested into a very small microfluidic detection chip. Due to its small size and high detection sensitivity, it is widely used in high-throughput detection of nucleic acid molecules. Microfluidic detection chips mainly use their reaction chamber arrays to achieve high-throughput detection of nucleic acid molecules.

[0003] At present, some microfluidic detection chips are made of glass or silicon wafers and use laser etching to obtain reaction chamber arrays, but this method has problems such as cumbersome processing, high production cost, limited number of reaction chambers, insufficient etching depth, and failure to seal the reaction chambers, which may increase the risk of contamination. Some microfluidic detection chips are equipped with several parallel series or parallel microarray channels, so that the samples to be tested enter each reaction chamber along the microarray channels, but this method has problems such as slow liquid inlet rate, easy channel blockage and limited number of microarrays. Summary of the invention

[0004] In order to solve the above-mentioned technical problems, the embodiments of the present application provide a microfluidic detection chip and a preparation method thereof, and a fluid filling method, so that the microfluidic detection chip can achieve high-throughput, high-accuracy and high-sensitivity detection of nucleic acid molecules, while having a fast liquid inlet speed and being not prone to clogging. In addition, the processing and preparation are simple and low-cost, and it is suitable for large-scale industrial production needs.

[0005] To achieve the above objectives, the present application provides the following technical solutions: A microfluidic detection chip comprises a first glass slide and a second glass slide arranged opposite to each other, and a chip body layer attached between the first glass slide and the second glass slide; The first glass slide is provided with a liquid inlet hole and a waste liquid hole; A containing pool is provided on the surface of the chip body layer facing the first glass slide, the containing pool comprising a liquid inlet area and a waste liquid area, and a microcavity array between the liquid inlet area and the waste liquid area; The microcavity array comprises a plurality of microcavities arranged in an array and independent of each other, the microcavity is in a groove shape, the top opening of the microcavity is lower than the top opening of the containing pool, and the microcavity is pre-installed with a detection reagent; The liquid inlet area corresponds to the liquid inlet hole, and the liquid inlet area is divergent in a direction close to the microcavity array; The waste liquid area corresponds to the waste liquid hole, and the waste liquid area is in a convergent shape along a direction away from the microcavity array.

[0006] Optionally, the liquid inlet area includes a liquid inlet cavity, a liquid inlet channel and a liquid inlet drainage area which are sequentially connected along a direction close to the microcavity array, the liquid inlet cavity corresponds to the liquid inlet hole, and the liquid inlet drainage area diverges from the liquid inlet channel toward the microcavity array.

[0007] Optionally, the waste liquid area includes a waste liquid drainage area, a waste liquid flow channel and a waste liquid cavity which are connected in sequence along a direction away from the microcavity array, the waste liquid cavity corresponds to the waste liquid hole, and the waste liquid drainage area converges from the microcavity array to the waste liquid flow channel.

[0008] Optionally, the number of the microcavities in the microcavity array ranges from 10,000 to 100,000, including endpoint values.

[0009] Optionally, the material of the chip body layer includes polydimethylsiloxane material.

[0010] A method for preparing a microfluidic detection chip, comprising: The polydimethylsiloxane material and the curing agent are uniformly mixed according to a preset ratio to obtain a mixed solution; The mixed solution is poured into the groove of the prefabricated mold and solidified, and after solidification, the prefabricated mold is turned over to demould the structure in the groove of the prefabricated mold to obtain a chip body layer; The groove of the prefabricated mold is provided with a first boss portion and a second boss portion, and an intermediate boss portion between the first boss portion and the second boss portion, and a microneedle array is provided on the intermediate boss portion, and the microneedle array includes a plurality of microneedles arranged in an array and independent of each other; the first boss portion is divergent in a direction close to the microneedle array, and the second boss portion is convergent in a direction away from the microneedle array; Placing a detection reagent in each microcavity of the microcavity array of the chip body layer corresponding to the microneedle array; Providing a first glass slide, laminating the first glass slide to the surface of the chip body layer having the microcavity array, and providing a second glass slide, laminating the second glass slide to the surface of the chip body layer facing away from the first glass slide; The first glass slide is provided with a liquid inlet hole and a liquid waste hole, wherein the liquid inlet hole corresponds to the liquid inlet area of ​​the chip body layer corresponding to the first boss portion, and the liquid waste hole corresponds to the liquid waste area of ​​the chip body layer corresponding to the second boss portion.

[0011] Optionally, the first boss portion, the second boss portion and the middle boss portion are integrally formed and have the same height.

[0012] Optionally, the process of pouring the mixed solution into the groove of the prefabricated mold and curing it includes: pouring the mixed solution into the groove of the prefabricated mold until overflowing; The prefabricated mold is placed in a vacuum box, and the vacuum box is evacuated to a preset pressure. After standing for a period of time, bubbles overflow from the surface of the mixed solution in the groove of the prefabricated mold; The prefabricated mold is moved out of the vacuum box, and bubbles on the surface of the mixed solution in the groove of the prefabricated mold are broken; Adding a cover plate on the groove of the prefabricated mold; The cover plate is pressed with a heavy object, and the prefabricated mold is placed in an oven for baking to solidify the mixed solution in the groove of the prefabricated mold.

[0013] A method for filling a microfluidic detection chip with fluid, applied to any of the microfluidic detection chips described above, the method for filling a microfluidic detection chip with fluid comprising: Placing the microfluidic detection chip in a confined space, and subjecting the confined space to negative pressure treatment by a negative pressure machine, so that negative pressure is stored inside the microfluidic detection chip; The microfluidic detection chip is removed from the enclosed space, and a detection liquid is added to the liquid inlet of the microfluidic detection chip, so that the detection liquid reaches each microcavity of the microcavity array through the liquid inlet area by a siphon effect under the action of negative pressure to react with the detection reagent in the microcavity; The oil is pushed into the liquid inlet hole of the microfluidic detection chip, and the excess detection liquid is discharged through the waste liquid area and the waste liquid hole in sequence, and each microcavity of the microcavity array is sealed with the oil.

[0014] Optionally, after adding the detection liquid to the liquid inlet hole of the microfluidic detection chip and before pushing the oil into the liquid inlet hole of the microfluidic detection chip, the fluid filling method of the microfluidic detection chip further includes: The microfluidic detection chip is then placed in a closed space, and a negative pressure machine is used to perform negative pressure treatment on the closed space.

[0015] Compared with the prior art, the above technical solution has the following advantages: The microfluidic detection chip provided in the embodiment of the present application includes a first glass slide and a second glass slide arranged opposite to each other, and a chip body layer bonded between the first glass slide and the second glass slide; a liquid inlet hole and a waste liquid hole are arranged on the first glass slide; a containing pool is arranged on the surface of the chip body layer facing the first glass slide, and the containing pool includes a liquid inlet area and a waste liquid area and a microcavity array between the liquid inlet area and the waste liquid area; the liquid inlet area corresponds to the liquid inlet hole, the waste liquid area corresponds to the waste liquid hole, the microcavity array includes a plurality of microcavities arranged in an array and independent of each other, the microcavity is in the shape of a groove, the top opening of the microcavity is lower than the top opening of the containing pool, and the detection reagent is pre-installed in the microcavity. In this way, the first glass slide and the containing pool of the chip body layer constitute a transverse open flow channel with a top height limit, so that the microfluidic detection chip can be placed in a closed space for negative pressure treatment, so that the negative pressure is stored inside the microfluidic detection chip, and then, the detection liquid added to the liquid inlet hole can reach the microcavity array through the liquid inlet area by the siphon effect under the action of negative pressure, flow into each microcavity to react with the preset detection reagent, the liquid inlet speed is fast, and it is not easy to be blocked; after the detection liquid fills each microcavity of the microcavity array, oil can be pushed into the liquid inlet hole to remove the excess detection liquid through the waste liquid area The oil is used to seal each microcavity of the microcavity array to prevent the microcavity from being contaminated; moreover, the liquid inlet area of ​​the chip body layer is divergent in the direction close to the microcavity array, which is conducive to the inflow of the detection liquid from the liquid inlet area to the microcavity array, and the waste liquid area of ​​the chip body layer is convergent in the direction away from the microcavity array, which is conducive to the detection liquid filling each microcavity and then flowing out from the waste liquid area; the whole detection process is simple to operate and can be applied to various types of nucleic acid sample detection, such as early cancer screening, clinical pathogen molecular detection, epidemiological investigation, food inspection and quarantine and other instant detection fields.

[0016] The number of microcavities of the microcavity array of the chip body layer in the microfluidic detection chip provided in the embodiment of the present application can be very large, ranging from 10,000 to 100,000 as required, thereby achieving high-throughput, high-accuracy and high-sensitivity detection of nucleic acid molecules.

[0017] Furthermore, the microcavity array of the chip body layer in the microfluidic detection chip provided in the embodiment of the present application can be obtained not by laser etching of glass or silicon wafer, but by pouring a mixed solution of polydimethylsiloxane material and curing agent onto a prefabricated mold. The processing and preparation are simple and low-cost, and it is suitable for large-scale industrial production needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 An exploded diagram of a microfluidic detection chip provided in an embodiment of the present application; Figure 2 for Figure 1 Schematic diagram of the structure of the first slide; Figure 3 for Figure 1 Schematic diagram of the structure of the second slide; Figure 4 for Figure 1 Schematic diagram of the structure of the chip body layer; Figure 5 A schematic diagram of the cross-sectional structure of a microfluidic detection chip provided in an embodiment of the present application and a corresponding schematic diagram of a chip body layer in the microfluidic detection chip; Figure 6 for Figure 5 A schematic diagram of the cross-sectional enlarged structure of the microfluidic detection chip shown; Figure 7 A schematic cross-sectional structure diagram of another microfluidic detection chip provided in an embodiment of the present application; Figure 8 A schematic structural diagram of a prefabricated mold for preparing a chip body layer in a microfluidic detection chip provided in an embodiment of the present application; Fig. 9 A schematic top view and a schematic cross-sectional structure diagram of a prefabricated mold for preparing a chip body layer in a microfluidic detection chip provided in an embodiment of the present application; Fig.10 A schematic top view and a schematic cross-sectional view of another prefabricated mold for preparing a chip body layer in another microfluidic detection chip provided in an embodiment of the present application; Fig.11 Schematic diagram of the microcavity array in the chip body layer under a microscope obtained by pouring a mixed solution of PDMS material and curing agent onto a prefabricated mold and then overmolding.

[0020] Reference numerals: 10-first glass slide; 11-liquid inlet hole; 12-waste liquid hole; 20-second glass slide; 30-chip body layer; 300-accommodation pool; 310-liquid inlet area; 311-liquid inlet cavity; 312-liquid inlet channel; 313-liquid inlet drainage area; 320-waste liquid area; 321-waste liquid drainage area; 322-waste liquid channel; 323-waste liquid cavity; 330-microcavity array; 331-microcavity; 100-prefabricated mold; 110-groove; 111-first boss portion; 112-second boss portion; 113-middle boss portion; 120-microneedle array; 121-microneedle. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0022] In the following description, many specific details are set forth to facilitate a full understanding of the present application, but the present application may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present application. Therefore, the present application is not limited to the specific embodiments disclosed below.

[0023] Secondly, the present application is described in detail with reference to the schematic diagram. When describing the embodiments of the present application in detail, for the sake of convenience, the cross-sectional diagrams showing the device structure will not be partially enlarged according to the general scale, and the schematic diagrams are only examples, which should not limit the scope of protection of the present application. In addition, in actual production, the three-dimensional dimensions of length, width and depth should be included.

[0024] The present application embodiment provides a microfluidic detection chip, Figure 1 An exploded view of a microfluidic detection chip provided in an embodiment of the present application is shown, Figure 1 As shown, the microfluidic detection chip includes a first glass slide 10 and a second glass slide 20 that are arranged opposite to each other, and a chip body layer 30 bonded between the first glass slide 10 and the second glass slide 20 .

[0025] Figure 2 Shows Figure 1 The schematic diagram of the structure of the first glass slide 10, combined with Figure 1 and Figure 2 As shown, the first glass slide 10 is provided with a liquid inlet 11 and a waste liquid hole 12 .

[0026] Figure 3 Shows Figure 1 The structural diagram of the second glass slide 20 is combined with Figure 1 and Figure 2 As shown, the second glass slide 20 is used to support the chip body layer 30 .

[0027] In the embodiment of the present application, the first glass slide 10 and the chip body layer 30 , as well as the second glass slide 20 and the chip body layer 30 , can be bonded together by plasma bonding.

[0028] Figure 4 Shows Figure 1 The schematic diagram of the structure of the chip body layer 30 is combined with Figure 1 and Figure 4As shown, a holding pool 300 is provided on the surface of the chip body layer 30 facing the first glass slide 10, and the holding pool 300 includes a liquid inlet area 310 and a waste liquid area 320, and a microcavity array 330 between the liquid inlet area 310 and the waste liquid area 320, wherein the microcavity array 330 includes a plurality of microcavities 331 arranged in an array and independent of each other, the microcavity 331 is in a groove shape, and a detection reagent is pre-installed in the microcavity 331, and the detection reagent can be in the form of a freeze-dried powder.

[0029] It is understandable that each microcavity 331 of the microcavity array 330 is a reaction microchamber independent of each other and having the same structure. The microcavity 331 is in a groove shape, but the present application does not limit the specific shape of the microcavity 331.

[0030] Optionally, the diameter of the microcavity 331 may be 40 μm, and the depth of the microcavity 331 may be 80 μm, but the present application does not limit the diameter and depth of the microcavity 331 .

[0031] like Figure 1 As shown, the liquid inlet area 310 of the containing pool 300 of the chip body layer 30 corresponds to the liquid inlet hole 11, and the liquid inlet area 310 is divergent in the direction close to the microcavity array 330, so that the detection liquid flows from the liquid inlet hole 11 through the liquid inlet area 310 and flows into each microcavity 331 of the microcavity array 330.

[0032] Optional, such as Figure 1 and Figure 4 As shown, the liquid inlet area 310 of the containing pool 300 of the chip body layer 30 includes a liquid inlet cavity 311, a liquid inlet channel 312 and a liquid inlet drainage area 313 which are sequentially connected along the direction close to the microcavity array 330. The liquid inlet cavity 311 corresponds to the liquid inlet hole 11, and the liquid inlet drainage area 313 diverges from the liquid inlet channel 312 to the microcavity array 330. Therefore, the detection liquid can flow from the liquid inlet hole 11 through the liquid inlet cavity 311, the liquid inlet channel 312 and the liquid inlet drainage area 313 in sequence, and then flow into each microcavity 331 of the microcavity array 330.

[0033] like Figure 1 As shown, the waste liquid area 320 of the containing pool 300 of the chip body layer 30 corresponds to the waste liquid hole 12, and the waste liquid area 320 is convergent in the direction away from the microcavity array 330, so that after the detection liquid fills each microcavity 331 of the microcavity array 330, the excess detection liquid flows through the waste liquid area 320 and flows out to the waste liquid hole 12.

[0034] Optional, such as Figure 1 and Figure 4As shown, the waste liquid area 320 of the containing pool 300 of the chip body layer 30 includes a waste liquid drainage area 321, a waste liquid flow channel 322 and a waste liquid cavity 323 which are sequentially connected along a direction away from the microcavity array 330, the waste liquid cavity 323 corresponds to the waste liquid hole 12, and the waste liquid drainage area 321 converges from the microcavity array 330 to the waste liquid flow channel 322, so that after the detection liquid fills each microcavity 331 of the microcavity array 330, the excess detection liquid can flow through the waste liquid drainage area 321, the waste liquid flow channel 322 and the waste liquid cavity 323 in sequence, and flow out from the waste liquid hole 12.

[0035] In order to better understand this application, Figure 5 1 shows a schematic cross-sectional structure diagram of a microfluidic detection chip provided in an embodiment of the present application and a corresponding top view schematic diagram of a chip body layer 30 in the microfluidic chip, Figure 6 It further shows Figure 5 The cross-sectional enlarged structural diagram of the microfluidic detection chip shown in FIG. Figure 1 , Figure 5 and Figure 6 It can be seen that in the embodiment of the present application, the top opening of the microcavity 331 in the microcavity array 330 is lower than the top opening of the containing pool 300. It can be understood that, since the first glass slide 10 and the chip body layer 30 are attached, the top opening of the containing pool 300 is located on the surface of the chip body layer 30 facing the first glass slide 10, that is, located on the surface of the first glass slide 10 facing the chip body layer 30, and the top opening of the microcavity 331 in the microcavity array 330 is lower than the top opening of the containing pool 300, so that the containing pool 300 area of ​​the first glass slide 10 and the chip body layer 30 constitutes a transverse open flow channel with a top limit, which facilitates the detection liquid to flow from the liquid inlet hole 11 through the liquid inlet area 310 into each microcavity 331 of the microcavity array 330, and after the detection liquid fills each microcavity 331 of the microcavity array 330, it is convenient to push the oil from the liquid inlet hole 11, and discharge the excess detection liquid through the waste liquid area 320 and the waste liquid hole 12.

[0036] When the detection liquid enters each microcavity 331 of the microcavity array 330, it reacts with the detection reagent preset in the microcavity 331, and then by observing the number of fluorescent spots after the reaction between the detection liquid and the detection reagent, absolute quantitative detection of nucleic acid molecules can be achieved.

[0037] In the embodiment of the present application, the first glass slide 10, the second glass slide 20 and the chip body layer 30 are all made of transparent materials to facilitate observation of the fluorescence state. The first glass slide 10 and the second glass slide 20 can be made of glass.

[0038] In the embodiment of the present application, the material of the chip body layer 30 may include polydimethylsiloxane (PDMS) material. PDMS material has high transparency, and its transmittance in the optical wavelength range (such as 240nm-1100nm) can reach more than 94%, which is helpful to observe microorganisms, cells and various biochemical reaction changes in microfluidic channels through optical equipment such as microscopes; PDMS material is also biocompatible and can easily pass cytotoxicity, skin irritation and subcutaneous reactivity tests; PDMS material has excellent flexibility and deformability, and can be used to make microfluidic chips with complex channel structures; PDMS material is also breathable and easy to process. In short, PDMS material is widely used in microfluidic detection chips due to its unique physical, chemical and biological properties.

[0039] When the microfluidic detection chip provided in the embodiment of the present application is used, since the area of ​​the accommodating pool 300 of the first glass slide 10 and the chip body layer 30 constitutes a transverse open flow channel with a top height limit, and the liquid inlet area 310 of the accommodating pool 300 corresponds to the liquid inlet hole 11 of the first glass slide 10, the liquid inlet area 310 of the accommodating pool 300 is divergent in the direction close to the microcavity array 330, and the waste liquid area of ​​the accommodating pool 300 corresponds to the waste liquid hole 12 of the first glass slide 10, and the waste liquid area of ​​the accommodating pool 300 is convergent in the direction away from the microcavity array 330, therefore, the microfluidic detection chip can be placed in a confined space for negative pressure treatment, so that negative pressure is stored inside the microfluidic detection chip, thereby The detection liquid added to the liquid inlet hole 11 can flow through the liquid inlet area 310 to the microcavity array 330 through the siphon effect under the action of negative pressure, and flow into each microcavity 331 to react with the preset detection reagent. The liquid inlet speed is fast and it is not easy to be blocked. Then, oil can be pushed into the liquid inlet hole 11 to discharge the excess detection liquid through the waste liquid area 320 and the waste liquid hole 12, and the oil is used to seal each microcavity 331 of the microcavity array 330 to prevent the microcavity 331 from being contaminated. Moreover, the whole detection process is simple to operate and can be applied to various types of nucleic acid sample detection, such as early cancer screening, clinical pathogen molecular detection, epidemiological survey, food inspection and quarantine and other instant detection fields.

[0040] It should be noted that placing the microfluidic detection chip in a confined space for negative pressure treatment can be understood as placing the microfluidic detection chip in a confined space for vacuuming, so that the negative pressure is stored in the transverse open flow channel with a top limit formed by the containing pool 300 area of ​​the first glass slide 10 and the chip body layer 30 inside the microfluidic detection chip, specifically, the negative pressure is stored. Therefore, the detection liquid added to the liquid inlet hole 11 will flow through the liquid inlet area 310 through the siphon effect under the action of negative pressure to reach each microcavity 331 of the microcavity array 330.

[0041] The number of microcavities 331 in the microcavity array 330 of the chip body layer 30 of the microfluidic detection chip provided in the embodiment of the present application can be very large, and the number of microcavities 331 can range from 10,000 to 100,000, including endpoint values, and can be set from 10,000 to 100,000 as required, thereby achieving high-throughput, high-accuracy and high-sensitivity detection of nucleic acid molecules.

[0042] Furthermore, the microcavity array 330 of the chip body layer 30 of the microfluidic detection chip provided in the embodiment of the present application can be obtained not by laser etching of glass or silicon wafer, but by pouring a mixed solution of PDMS material and curing agent onto a prefabricated mold. The processing and preparation are simple and low-cost, and it meets the needs of large-scale industrial production.

[0043] It should be noted that, on the basis of setting the top opening of the microcavity 331 in the microcavity array 330 to be lower than the top opening of the containing pool 300, optionally, Figure 6 As shown, the top opening of the microcavity 331 in the microcavity array 330 can be flush with the bottom of the liquid inlet area 310 and the waste liquid area 320 of the receiving pool 300. In this way, the liquid inlet area 310 and the waste liquid area 320 of the receiving pool 300 will not store the detection liquid. In other words, after the detection liquid flows into the receiving pool from the liquid inlet hole 11, it can diffuse into each microcavity 331 of the microcavity array 330 only through the liquid inlet area 310 of the receiving pool 300, and will not be stored in the liquid inlet area 310 of the receiving pool. The liquid inlet speed is faster and the detection liquid is saved. Similarly, the excess detection liquid will not be stored in the waste liquid area 320 of the receiving pool 300.

[0044] Another option, such as Figure 7 As shown, the top opening of the microcavity 331 in the microcavity array 330 can also be slightly higher than the bottom of the liquid inlet area 310 and the waste liquid area 320 of the containing pool 300. In this way, after the detection liquid flows in from the liquid inlet hole 11, it needs to be slightly stored in the liquid inlet area 310 of the containing pool 300 and then diffused into each microcavity 331 of the microcavity array 330. Similarly, excess detection liquid will also be slightly stored in the waste liquid area 320 of the containing pool 300, but it has little impact on the use of the microfluidic detection chip.

[0045] The microfluidic detection chip provided in the embodiment of the present application has corresponding sizes of the first glass slide 10, the second glass slide 20 and the chip body layer 30. The chip size can be 7.4cm×2.5cm, and the four corners of the chip can be rounded. However, the present application does not limit the chip size and chip shape.

[0046] Accordingly, the present application also provides a method for preparing a microfluidic detection chip, and the method for preparing the microfluidic detection chip includes: S10: Evenly mix the PDMS material and the curing agent according to a preset ratio to obtain a mixed solution.

[0047] Specifically, the PDMS material and the curing agent can be uniformly mixed according to a preset mass ratio of 10:1, and can be stirred manually or by machine to obtain a uniformly mixed solution.

[0048] S20: pouring a mixed solution of PDMS material and curing agent into the groove of the prefabricated mold and curing it. After curing, the prefabricated mold is turned over to demould the structure in the groove of the prefabricated mold to obtain the chip body layer 30.

[0049] Figure 8 A schematic diagram of the structure of a prefabricated mold 100 is shown. Figure 8 As shown, the prefabricated mold 100 has a groove 110. After a mixed solution of PDMS material and curing agent is poured into the groove 110 of the prefabricated mold 100 and cured, the prefabricated mold 100 is turned over to demold the structure in the groove 110 of the prefabricated mold 100, and the chip body layer 30 can be obtained.

[0050] As is known from the above, a containing pool 300 is provided on the surface of the chip body layer 30, and the containing pool 300 includes a liquid inlet area 310 and a waste liquid area 320, and a microcavity array 330 between the liquid inlet area 310 and the waste liquid area 320. In order to realize the above structure of the chip body layer 30, it is necessary to design the interior of the groove 110 of the prefabricated mold 100 accordingly.

[0051] Optional, Fig. 9 A top view schematic diagram of a prefabricated mold 100 and a cross-sectional structure schematic diagram are shown. It can be seen that a first boss portion 111 and a second boss portion 112 are arranged in the groove 110 of the prefabricated mold 100, and an intermediate boss portion 113 between the first boss portion 111 and the second boss portion 112 is provided. A microneedle array 120 is arranged on the intermediate boss portion 113. The microneedle array 120 includes a plurality of microneedles 121 arranged in an array and independent of each other; the first boss portion 111 is divergent in a direction approaching the microneedle array 120, and the second boss portion 112 is convergent in a direction away from the microneedle array 120.

[0052] refer to Fig. 9 It can be understood that in step S20, the mixed solution of PDMS material and curing agent is poured into the groove 110 of the prefabricated mold 100 and cured. After curing, the prefabricated mold 100 is turned over to demould the structure in the groove 110 of the prefabricated mold 100, and the prefabricated mold 100 is obtained. Figure 5 and Figure 6 The chip body layer 30 is shown.

[0053] Among them, the groove 110 of the prefabricated mold 100 corresponds to the containing pool 300 of the chip body layer 30, the microneedle 121 in the prefabricated mold 100 corresponds to the microcavity 331 of the chip body layer 30, the microneedle array 120 corresponds to the microcavity array 330, the first boss portion 111 of the prefabricated mold 100 corresponds to the liquid inlet area 310 of the chip body layer 30, the second boss portion 112 of the prefabricated mold 100 corresponds to the waste liquid area 320 of the chip body layer 30, and the middle boss portion 113 of the prefabricated mold 100 corresponds to the lateral open flow channel between the microcavity array 330 of the chip body layer 30 and the first glass slide 10.

[0054] It can be understood that, since the size of the microcavity 331 is very small, the microneedle 121 corresponding to the microcavity 331 is similar to a needle, but in fact the microneedle 121 is a microcolumn.

[0055] It is also understandable that, combined with Figure 5 and Fig. 9 As shown, the shape of the first boss portion 111 of the prefabricated mold 100 corresponds to the shape of the liquid inlet area 310 of the chip body layer 30 , and the shape of the second boss portion 112 of the prefabricated mold 100 corresponds to the shape of the waste liquid area 320 of the chip body layer 30 .

[0056] Optional, Fig.10 A schematic top view of another prefabricated mold 100 and a schematic cross-sectional view thereof are shown. Fig. 9 The prefabricated mold shown is different in that Fig.10 In the prefabricated mold shown in FIG. 1 , the heights of the first boss portion 111 and the second boss portion 112 are higher than the middle boss portion 113, so that the following Figure 7 The chip body layer 30 shown in the figure makes the top opening of the microcavity 331 in the microcavity array 330 of the chip body layer 30 slightly higher than the bottom of the liquid inlet area 310 and the waste liquid area 320 of the containing pool 300. In this way, the liquid inlet area 310 and the waste liquid area 320 of the containing pool 300 will store a part of the detection liquid. Fig. 9 In the prefabricated mold shown in the figure, the first boss portion 111, the second boss portion 112 and the middle boss portion 113 are integrally formed, and the heights of the three are equal, forming Figure 5 and Figure 6 The chip body layer 30 shown in the figure enables the top opening of the microcavity 331 in the microcavity array 330 of the chip body layer 30 to be flush with the bottom of the liquid inlet area 310 and the waste liquid area 320 of the containing pool 300, so that the liquid inlet area 310 and the waste liquid area 320 of the containing pool 300 will not store the detection liquid.

[0057] Fig.11A schematic diagram of the microcavity array 330 in the chip body layer 30 obtained by pouring a mixed solution of PDMS material and curing agent onto a prefabricated mold under a microscope is shown. It can be seen that each microcavity is independent of each other and has the same structure, the microcavity size can be very small, and the number of microcavities can be very large.

[0058] Optionally, the process of pouring the mixed solution of PDMS material and curing agent into the groove of the prefabricated mold and curing in step S20 includes: S21 : pouring a mixed solution of the PDMS material and the curing agent into the groove 110 of the prefabricated mold 100 until overflowing, ensuring that the mixed solution of the PDMS material and the curing agent fills the groove 110 of the prefabricated mold 100 .

[0059] In practical applications, before pouring the mixed solution of PDMS material and curing agent into the groove 110 of the prefabricated mold 100 , the surface of the prefabricated mold 100 needs to be cleaned first.

[0060] S22: placing the prefabricated mold 100 in a vacuum box, and evacuating the vacuum box to a preset pressure. After standing for a period of time, bubbles overflow from the surface of the mixed solution in the groove 110 of the prefabricated mold 100 .

[0061] S23: The prefabricated mold 100 is moved out of the vacuum box, and the bubbles on the surface of the mixed solution in the groove of the prefabricated mold 100 are broken.

[0062] It is understandable that by placing the prefabricated mold 100 in a vacuum box and applying negative pressure to the vacuum box, the bubbles in the mixed solution can be overflowed, thereby making the quality of the prepared chip body layer 30 better.

[0063] Optionally, step S22 pumps the negative pressure of the vacuum box to -0.08 MPa.

[0064] S24: Adding a cover plate to the groove 110 of the prefabricated mold 100; S25: Press the cover plate with a heavy object, place the prefabricated mold 100 in an oven for baking, and solidify the mixed solution in the groove 110 of the prefabricated mold 100 .

[0065] Optionally, in step S25, the prefabricated mold 100 is placed in an oven for baking at a temperature of 60° C.-70° C., and the baking time may be 60 min-100 min.

[0066] In this way, the mixed solution poured into the groove 110 of the prefabricated mold 100 is solidified, and after cooling to room temperature, the chip body layer 30 can be re-molded.

[0067] S30: placing a detection reagent in each microcavity 331 of the microcavity array 330 of the chip body layer 30 corresponding to the microneedle array 120. The detection reagent may be in the form of a freeze-dried powder.

[0068] S40: Reference Figure 1 As shown, a first glass slide 10 is provided, and the first glass slide 10 is bonded to the surface of the chip body layer 30 having the microcavity array 330, and a second glass slide 20 is provided, and the second glass slide 20 is bonded to the surface of the chip body layer 30 away from the first glass slide 10; The first glass slide 10 is provided with a liquid inlet hole 11 and a waste liquid hole 12 . The liquid inlet hole 11 corresponds to the liquid inlet area 310 of the first boss portion 111 of the chip body layer 30 , and the waste liquid hole 12 corresponds to the waste liquid area 320 of the second boss portion 112 of the chip body layer 30 .

[0069] Furthermore, an embodiment of the present application also provides a fluid filling method for a microfluidic detection chip, which is applied to the microfluidic detection chip provided in any of the above embodiments. The fluid filling method for the microfluidic detection chip includes: S100: placing the microfluidic detection chip in a closed space, and subjecting the closed space to negative pressure treatment by a negative pressure machine, so that negative pressure is stored inside the microfluidic detection chip.

[0070] S110: The microfluidic detection chip is removed from the enclosed space, and detection liquid is added to the liquid inlet hole 11 of the microfluidic detection chip, so that the detection liquid flows through the liquid inlet area 310 through the siphon effect under the action of negative pressure to reach each microcavity 331 of the microcavity array 330 to react with the detection reagent in the microcavity 331.

[0071] It should be noted that placing the microfluidic detection chip in a confined space for negative pressure treatment can be understood as placing the microfluidic detection chip in a confined space for vacuuming, so that the negative pressure is stored in the transverse open flow channel with a top limit formed by the containing pool 300 area of ​​the first glass slide 10 and the chip body layer 30 inside the microfluidic detection chip, specifically, the negative pressure is stored. Therefore, the detection liquid added to the liquid inlet hole 11 will flow through the liquid inlet area 310 through the siphon effect under the action of negative pressure to reach each microcavity 331 of the microcavity array 330.

[0072] S120: Pushing oil into the liquid inlet of the microfluidic detection chip, draining the excess detection liquid through the waste liquid area 320 and the waste liquid hole 12 in sequence, and using the oil to seal each microcavity 331 of the microcavity array 330.

[0073] Based on the above embodiment, optionally, after adding the detection liquid to the liquid inlet hole 11 of the microfluidic detection chip and before pushing the oil into the liquid inlet hole 11 of the microfluidic detection chip, the fluid filling method of the microfluidic detection chip may further include: S130: The microfluidic detection chip is then placed in a closed space, and a negative pressure machine is used to perform negative pressure treatment on the closed space.

[0074] Thus, when the detection liquid flows into each microcavity 331 of the microcavity array 330, the microfluidic detection chip is subjected to negative pressure treatment, which can accelerate the flow of the detection liquid into each microcavity 331 of the microcavity array 330 through the siphon effect under the action of negative pressure.

[0075] In practical applications, isothermal amplification technologies such as recombinase polymerase amplification (RPA) and CRISPR (clustered regularly interspaced short palindromic repeats) detection systems can be transferred to the microfluidic detection chip provided in the embodiments of the present application to achieve absolute quantification of nucleic acid molecules similar to Digital-PCR to detect mutations and methylation, and achieve early screening or rapid diagnosis of cancer. Specifically, a single detection indicator in the form of freeze-dried powder is pre-embedded in the microcavity 331 of the microcavity array 330 of the chip body layer 30 using RPA and CRISPR-Cas detection systems, and the first glass slide 10, the chip body layer 30 and the second glass slide 20 are formed into a microfluidic detection chip by plasma bonding; the microfluidic detection chip is placed in a closed space (such as a closed bag) and negative pressure is pumped to store negative pressure inside the microfluidic detection chip; during detection, the detection liquid is dripped into the liquid inlet 11, and the detection liquid is under the action of negative pressure. The liquid flows through the liquid inlet area 310 through the siphon effect and flows into each microcavity 331 of the microcavity array 330, reacts with the RPA and CRISPR-Cas detection system, and the microfluidic detection chip can be placed in a closed space again to draw negative pressure to make the liquid inlet faster; after the detection liquid fills each microcavity 331 of the microcavity array 330, the microfluidic detection chip can be placed in a matching microfluidic detection box and heated to 37°C-42°C, react for 20 minutes, and then perform luminescence detection through the microfluidic detection box. Since a large number of independent and structurally identical reaction microcavities, such as 100,000, are provided on the chip body layer 30 of the microfluidic detection chip, forming a large independent reaction unit, the absolute quantification of nucleic acid molecules can be achieved by calculating the number of fluorescent spots.

[0076] In summary, the microfluidic detection chip provided in the embodiment of the present application includes a first glass slide and a second glass slide arranged relatively to each other and a chip body layer bonded between the first glass slide and the second glass slide; a liquid inlet hole and a waste liquid hole are arranged on the first glass slide; a containing pool is arranged on the surface of the chip body layer facing the first glass slide, the containing pool includes a liquid inlet area and a waste liquid area and a microcavity array between the liquid inlet area and the waste liquid area; the liquid inlet area corresponds to the liquid inlet hole, the waste liquid area corresponds to the waste liquid hole, the microcavity array includes a plurality of microcavities arranged in an array and independent of each other, the microcavity is in the shape of a groove, the top opening of the microcavity is lower than the top opening of the containing pool, and the detection reagent is pre-installed in the microcavity. In this way, the first glass slide and the containing pool of the chip body layer constitute a transverse open flow channel with a top height limit, so that the microfluidic detection chip can be placed in a closed space for negative pressure treatment, so that the negative pressure is stored inside the microfluidic detection chip, and then, the detection liquid added to the liquid inlet hole can reach the microcavity array through the liquid inlet area by the siphon effect under the action of negative pressure, flow into each microcavity to react with the preset detection reagent, the liquid inlet speed is fast, and it is not easy to be blocked; after the detection liquid fills each microcavity of the microcavity array, oil can be pushed into the liquid inlet hole to remove the excess detection liquid through the waste liquid area The oil is used to seal each microcavity of the microcavity array to prevent the microcavity from being contaminated; moreover, the liquid inlet area of ​​the chip body layer is divergent in the direction close to the microcavity array, which is conducive to the inflow of the detection liquid from the liquid inlet area to the microcavity array, and the waste liquid area of ​​the chip body layer is convergent in the direction away from the microcavity array, which is conducive to the detection liquid filling each microcavity and then flowing out from the waste liquid area; the whole detection process is simple to operate and can be applied to various types of nucleic acid sample detection, such as early cancer screening, clinical pathogen molecular detection, epidemiological investigation, food inspection and quarantine and other instant detection fields.

[0077] The number of microcavities of the microcavity array of the chip body layer in the microfluidic detection chip provided in the embodiment of the present application can be very large, ranging from 10,000 to 100,000 as required, thereby achieving high-throughput, high-accuracy and high-sensitivity detection of nucleic acid molecules.

[0078] Furthermore, the microcavity array of the chip body layer in the microfluidic detection chip provided in the embodiment of the present application can be obtained not by laser etching of glass or silicon wafer, but by pouring a mixed solution of PDMS material and curing agent onto a prefabricated mold. The processing and preparation are simple and low-cost, and it is suitable for large-scale industrial production needs.

[0079] The various parts in this manual are described in a combination of parallel and progressive ways. Each part focuses on the differences from other parts, and the same or similar parts between the various parts can be referenced to each other.

[0080] With respect to the above description of the disclosed embodiments, the features described in the embodiments in this specification may be replaced or combined with each other, so that professionals in the field can implement or use the present application. Various modifications to these embodiments will be apparent to professionals in the field, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A microfluidic detection chip, characterized in that: It includes a first glass slide and a second glass slide that are arranged opposite to each other, and a chip body layer that is attached between the first glass slide and the second glass slide; The first glass slide is provided with a liquid inlet hole and a waste liquid hole; A containing pool is provided on the surface of the chip body layer facing the first glass slide, the containing pool comprising a liquid inlet area and a waste liquid area, and a microcavity array between the liquid inlet area and the waste liquid area; The microcavity array comprises a plurality of microcavities arranged in an array and independent of each other, the microcavity is in a groove shape, the top opening of the microcavity is lower than the top opening of the containing pool, and the microcavity is pre-installed with a detection reagent; The liquid inlet area corresponds to the liquid inlet hole, and the liquid inlet area is divergent in a direction close to the microcavity array; The waste liquid area corresponds to the waste liquid hole, and the waste liquid area is in a convergent shape along a direction away from the microcavity array.

2. The microfluidic detection chip according to claim 1, characterized in that: The liquid inlet area includes a liquid inlet cavity, a liquid inlet channel and a liquid inlet drainage area which are sequentially connected along a direction close to the microcavity array. The liquid inlet cavity corresponds to the liquid inlet hole, and the liquid inlet drainage area diverges from the liquid inlet channel to the microcavity array.

3. The microfluidic detection chip according to claim 1, characterized in that: The waste liquid area includes a waste liquid drainage area, a waste liquid flow channel and a waste liquid cavity which are sequentially connected in a direction away from the microcavity array. The waste liquid cavity corresponds to the waste liquid hole. The waste liquid drainage area converges from the microcavity array to the waste liquid flow channel.

4. The microfluidic detection chip according to claim 1, characterized in that: The number of the microcavities in the microcavity array ranges from 10,000 to 100,000, including endpoint values.

5. The microfluidic detection chip according to claim 1, characterized in that: The material of the chip body layer includes polydimethylsiloxane material.

6. A method for preparing a microfluidic detection chip, characterized in that: include: The polydimethylsiloxane material and the curing agent are uniformly mixed according to a preset ratio to obtain a mixed solution; The mixed solution is poured into the groove of the prefabricated mold and solidified, and after solidification, the prefabricated mold is turned over to demould the structure in the groove of the prefabricated mold to obtain a chip body layer; The groove of the prefabricated mold is provided with a first boss portion and a second boss portion, and an intermediate boss portion between the first boss portion and the second boss portion, and a microneedle array is provided on the intermediate boss portion, and the microneedle array includes a plurality of microneedles arranged in an array and independent of each other; the first boss portion is divergent in a direction close to the microneedle array, and the second boss portion is convergent in a direction away from the microneedle array; Placing a detection reagent in each microcavity of the microcavity array of the chip body layer corresponding to the microneedle array; Providing a first glass slide, laminating the first glass slide to the surface of the chip body layer having the microcavity array, and providing a second glass slide, laminating the second glass slide to the surface of the chip body layer facing away from the first glass slide; The first glass slide is provided with a liquid inlet hole and a liquid waste hole, wherein the liquid inlet hole corresponds to the liquid inlet area of ​​the chip body layer corresponding to the first boss portion, and the liquid waste hole corresponds to the liquid waste area of ​​the chip body layer corresponding to the second boss portion.

7. The method for preparing the microfluidic detection chip according to claim 6, characterized in that: The first boss portion, the second boss portion and the middle boss portion are integrally formed and have the same height.

8. The method for preparing the microfluidic detection chip according to claim 6, characterized in that: The process of pouring the mixed solution into the groove of the prefabricated mold and curing it includes: pouring the mixed solution into the groove of the prefabricated mold until overflowing; The prefabricated mold is placed in a vacuum box, and the vacuum box is evacuated to a preset pressure. After standing for a period of time, bubbles overflow from the surface of the mixed solution in the groove of the prefabricated mold; The prefabricated mold is moved out of the vacuum box, and bubbles on the surface of the mixed solution in the groove of the prefabricated mold are broken; Adding a cover plate on the groove of the prefabricated mold; The cover plate is pressed with a heavy object, and the prefabricated mold is placed in an oven for baking to solidify the mixed solution in the groove of the prefabricated mold.

9. A fluid filling method for a microfluidic detection chip, characterized in that: The microfluidic detection chip according to any one of claims 1 to 5, wherein the fluid filling method of the microfluidic detection chip comprises: Placing the microfluidic detection chip in a confined space, and subjecting the confined space to negative pressure treatment by a negative pressure machine, so that negative pressure is stored inside the microfluidic detection chip; The microfluidic detection chip is removed from the enclosed space, and a detection liquid is added to the liquid inlet of the microfluidic detection chip, so that the detection liquid reaches each microcavity of the microcavity array through the liquid inlet area by a siphon effect under the action of negative pressure to react with the detection reagent in the microcavity; The oil is pushed into the liquid inlet hole of the microfluidic detection chip, and the excess detection liquid is discharged through the waste liquid area and the waste liquid hole in sequence, and each microcavity of the microcavity array is sealed with the oil.

10. The fluid filling method of the microfluidic detection chip according to claim 9, characterized in that: After adding the detection liquid into the liquid inlet hole of the microfluidic detection chip and before pushing the oil into the liquid inlet hole of the microfluidic detection chip, the fluid filling method of the microfluidic detection chip further includes: The microfluidic detection chip is then placed in a closed space, and a negative pressure machine is used to perform negative pressure treatment on the closed space.

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