A multi-layer three-dimensional digital nucleic acid amplification chip and its manufacturing and use method

Abstract

The present invention relates to the field of digital PCR, and specifically to a multi-layer three-dimensional digital nucleic acid amplification chip and a method for making and using the same. In the multi-layer three-dimensional digital nucleic acid amplification chip of the present invention, multi-layer superposition of microarray chips can be achieved, greatly increasing the number of reaction units for digital nucleic acid amplification, so that the dynamic range of nucleic acid quantification and the accuracy of the lower limit of detection are greatly improved, which is conducive to greatly increasing the reaction flux in actual detection. In addition, the present invention also provides a method for making and using the multi-layer three-dimensional digital nucleic acid amplification, which can further improve the accuracy of the quantitative results.

Description

Technical Field

[0001] The present invention relates to the field of digital PCR, and in particular to a multi-layer three-dimensional digital nucleic acid amplification chip and a method for making and using the same. Background Art

[0002] Since the invention of PCR in 1983, nucleic acid quantification has developed from qualitative technology (PCR post-electrophoresis) to semi-quantitative (fluorescent quantitative PCR, qPCR), and with the emergence of digital PCR (digital PCR, dPCR), it has developed to absolute quantification. Digital PCR technology randomly distributes nucleic acid templates to a large number of reaction units for amplification reaction. After the amplification, the fluorescence signal of each reaction unit is collected. Since the random distribution of nucleic acid molecules conforms to the Poisson distribution, the original concentration or content of the sample can be calculated by the Poisson distribution formula. The larger the number of partitions, the larger the nucleic acid quantification range and the higher the accuracy; the larger the total reaction volume, the lower the detection limit.

[0003] Digital PCR quantitative detection of nucleic acid molecules is divided into three steps: partitioning, nucleic acid amplification, and fluorescence detection. According to different allocation strategies, the current dPCR technology is divided into two categories, namely chip-based digital PCR (cdPCR) based on physical separation and droplet-based digital PCR (ddPCR) based on droplet emulsification. So far, ddPCR has become the most commonly used method in the biological and medical fields due to its advantages. First, the design and manufacture of droplet generation chips are relatively easy. Second, the size and number of droplets are flexible, so it is easy to generate millions of droplets. Finally, the droplet amplification products can be easily recovered, which is important in the application of second-generation sequencing library quantification. However, it has some limitations. During the PCR process, some droplets will merge due to thermal motion or oil evaporation, which will lead to differences in droplet volume, resulting in differences in the probability of nucleic acid molecule distribution, which does not conform to the Poisson distribution and has a significant impact on accuracy. In addition, droplet dPCR also requires many facilities, including droplet generators, droplet transfer systems, and droplet counting systems, which increases the initial cost of the equipment and requires continuous consumption of expensive materials. These shortcomings hinder its widespread clinical application. Compared with ddPCR, cdPCR divides samples by making physical micropores, which is cheaper and easier to detect, especially in point-of-care testing. The results of chip-based digital PCR can be easily detected using a fluorescence microscope. However, due to its small number of micropores and low throughput, its application is not very widespread.

[0004] At present, most of the commercialized digital PCR platforms in China are ddPCR. Although major scientific research units and institutions have also conducted research on lower-cost chip digital PCR, these studies have mostly focused on basic issues such as sample injection methods and preventing evaporation of reaction solutions. They have not broken the rules in terms of the volume, number, and arrangement of reaction units. Therefore, it is difficult to make significant progress and breakthroughs in key scientific issues such as sensitivity and throughput. Although some foreign studies have reported digital PCR chips that can produce millions of reaction partitions, they use a strategy to increase the surface density of chip micropores. Although this approach increases the number of reaction units, it causes a sharp drop in the volume of the reaction units, improves accuracy but cannot take into account sensitivity. The scope of application is narrow and cannot meet the needs of molecular diagnosis such as early cancer screening and new coronavirus diagnosis. In addition, the increase in surface density leads to higher chip manufacturing requirements and finished product loss rate, which is more expensive. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a multi-layer three-dimensional digital nucleic acid amplification chip and a method for making and using the same.

[0006] Specifically, the present invention first provides a multi-layer three-dimensional digital nucleic acid amplification chip, which comprises:

[0007] A plurality of microarray chips and a water reservoir chip arranged in a stacked manner;

[0008] A liquid inlet, a first branch-type distribution channel, a plurality of microporous arrays, an oil-through hole, a water storage circle and a liquid inlet and outlet of the water storage circle are provided on each of the microarray chips; wherein the liquid inlet and the microporous array are connected via the first branch-type distribution channel; the oil-through hole and the microporous array are connected via the first branch-type distribution channel; the liquid inlet and outlet of the water storage circle are connected via the water storage circle; the water storage circle is provided between the microporous array and the edge of the microarray chip and is not connected to the microporous array; the height of the first branch-type distribution channel, the microporous array and the water storage circle is less than the thickness of the microarray chip; and between the microarray chips of different layers, the liquid inlets are connected to each other, the oil-through holes are connected to each other, and the liquid inlet and outlet of the water storage circle are connected to each other;

[0009] The water storage layer chip is provided with a water inlet and outlet, a water storage channel and a second branch type distribution channel; the water inlet and outlet and the water storage channel are connected through the second branch type distribution channel;

[0010] The water storage channel on the water storage layer chip is arranged in contact with the micropore array on the uppermost microarray chip, and the water storage channel covers the micropore array.

[0011] The present invention has found that stacking multiple microarray chips can effectively increase the number of reaction units. However, compared with single-layer microarray chips, multi-layer microarray chip structures have higher requirements for the sealing, uniformity and structural stability of reaction units during heat treatment. The above structure is conducive to meeting the performance requirements of isothermal amplification reactions for multi-layer three-dimensional digital nucleic acid amplification chips, and effectively increases the number of reaction units.

[0012] Preferably, when the micropores in the micropore array are arranged along each branch channel of the first branch-type distribution channel, the adjacent micropores along the axial direction of the branch channel are staggered on both sides of the branch channel in sequence, and each micropore is connected to the channel, cross contamination can be further prevented, which is more conducive to improving the sealing of a single reaction unit in a long-term amplification reaction.

[0013] Preferably, the material of the microarray chip and the water storage layer chip is polydimethylsiloxane (PDMS).

[0014] Preferably, the height of the micropore array is 20-200 microns, and the ratio of the height of the first branch distribution channel to the height of the micropore array is 1:3-10. By setting the height difference between the micropores and the channel according to the above ratio, it is further beneficial to improve the sealing effect of the reaction unit, and at the same time, it is also beneficial to alleviate the deformation pressure on the chip caused by filling liquid (especially oil substances), thereby ensuring the feasibility of alternating positive pressure and negative pressure to pass the oil, so that the quantitative result is more accurate.

[0015] More preferably, the pore size of a single micropore in the micropore array is larger than the width of a single channel in the first branch distribution channel.

[0016] Preferably, the ratio of the height of the water storage circle to the height of the micropore array is 1:0.5-2.

[0017] Preferably, the height of the water storage channel and the second branch distribution channel are both 50-100 microns, the width of the water storage channel is 200-500 microns, and the interval between two adjacent water storage channels is smaller than the height of the water storage channel. This arrangement is more conducive to reducing possible water evaporation of the microarray chip.

[0018] Preferably, the top view shape of the micropores in the micropore array is square, circular or other polygonal, and a square shape is more conducive to imaging effect.

[0019] Preferably, the multi-layer three-dimensional digital nucleic acid amplification chip further comprises:

[0020] A glass slide is fixedly arranged below the bottom microarray chip and covers the bottom surface of the microarray chip.

[0021] The multi-layer three-dimensional digital nucleic acid amplification chip provided by the present invention greatly increases the number of reaction units for digital nucleic acid amplification by superimposing microarray chip layers, thereby improving the dynamic range and accuracy of nucleic acid quantification; in addition, the microwell array can be expanded or contracted horizontally or vertically, or liquid inlets, oil holes and branch distribution channels matching the microwell array can be added simultaneously to form an independent reaction liquid addition passage, thereby realizing simultaneous reaction of multiple samples on a single chip, and realizing high-throughput nucleic acid quantification.

[0022] The "uppermost layer" and "lowermost layer" mentioned in the present invention are relative, and the judgment standard of their orientation is that the orientation of the side of the microarray chip with the microwell array is upper. In the actual scheme, the statements of "uppermost layer" and "lowermost layer" can be adjusted according to other orientation judgment standards (such as the orientation of the side of the microarray chip without the microwell array is upper), which all belong to the protection scope of the present invention.

[0023] Those skilled in the art can combine the above solutions to obtain a better embodiment of the present invention.

[0024] The present invention further provides a method for preparing the multi-layer three-dimensional digital nucleic acid amplification chip, comprising:

[0025] preparing molds of a water storage layer chip and a plurality of microarray chips respectively;

[0026] coating the prepared material on the mold and performing a curing process;

[0027] The water storage layer chip on the mold and a layer of microarray chip are plasma treated, bonded and baked, and then bonded and baked with the remaining microarray chips.

[0028] When the multi-layer three-dimensional digital nucleic acid amplification chip further includes a glass slide, the preparation method further includes:

[0029] Glue the bottom microarray chip onto the glass slide.

[0030] The present invention also provides the use of the multi-layer three-dimensional digital nucleic acid amplification chip in PCR; preferably, the use of the chip in nucleic acid quantitative PCR.

[0031] After the multi-layer chips are bonded, the multi-layer three-dimensional digital nucleic acid amplification chip can be subjected to surface plasma treatment and packaged with a glass slide.

[0032] In order to smoothly deliver the sample into the microwell array, it is preferred that the structure of the multi-layer three-dimensional digital nucleic acid amplification chip be vacuumed and then sealed and packaged.

[0033] The present invention also provides a method for using the multi-layer three-dimensional digital nucleic acid amplification chip, comprising:

[0034] Introducing the nucleic acid amplification reaction solution into the multilayer three-dimensional digital nucleic acid amplification chip through the liquid inlet on the microarray chip; before or after the nucleic acid amplification reaction solution is introduced, injecting water into the water storage circle through the liquid inlet and outlet of the water storage circle;

[0035] storing water in the aquifer through a water inlet in the aquifer chip;

[0036] Alternately passing oil under negative pressure and positive pressure to the multi-layer three-dimensional digital nucleic acid amplification chip through the oil holes and the liquid inlet;

[0037] The negative pressure oil passage specifically includes: sucking out the residual nucleic acid amplification reaction liquid in the oil passage hole and then introducing the oil phase substance, and applying negative pressure to the liquid inlet; the positive pressure oil passage specifically includes: introducing the oil phase substance from the liquid inlet with positive pressure;

[0038] The chip is packaged, and after a constant temperature amplification reaction is performed, the reaction result is detected.

[0039] The oil can fill the channel with oil phase material, so that the reaction liquid between the micropores is not connected. By alternately passing oil with negative pressure and positive pressure, the block expansion caused by the elasticity of the material can be avoided, the volume uniformity of the reaction unit is better, and the quantitative results are more accurate.

[0040] In fact, the alternating flow of oil under negative and positive pressure also places higher requirements on the structure of the multi-layer three-dimensional digital nucleic acid amplification chip. Only the multi-layer three-dimensional digital nucleic acid amplification chip optimized by the present invention can meet the above requirements.

[0041] If the multi-layer three-dimensional digital nucleic acid amplification chip is not vacuum packaged, or has been unpacked for a long time, it is preferred to vacuum the multi-layer three-dimensional digital nucleic acid amplification chip before sampling. It is more preferred to complete the sampling within 30 minutes after unpacking the vacuum package or vacuum treatment to further improve the sampling effect.

[0042] Preferably, the oil phase material used in the negative pressure oil passing and the positive pressure oil passing is a mixture of HFE7500 and FC40 in a volume ratio of 1-5:1 (more preferably 2-4:1).

[0043] The present invention finds that HFE7500 (fluorinated ether) is easy to evaporate, but has a low viscosity and is easier to push and suck out by low pressure, while FC40 (electronic fluorinated liquid) basically does not evaporate, but has a high viscosity and is not easy to use. After mixing in the above proportion, the actual requirements of the present invention for low viscosity performance and non-evaporation effect can be achieved at the same time.

[0044] Based on the above technical solution, the beneficial effects of the present invention are as follows:

[0045] In the multi-layer three-dimensional digital nucleic acid amplification chip of the present invention, multi-layer superposition of microarray chips can be achieved, which greatly increases the number of reaction units of digital nucleic acid amplification, so that the dynamic range of nucleic acid quantification and the detection limit accuracy are greatly improved. At the same time, in the multi-layer digital nucleic acid amplification chip provided by the present invention, the structural design of the microarray chip can be expanded horizontally or vertically, and the number of single-layer chips, the volume of reaction units, and the number of liquid inlets can be flexibly adjusted, so as to achieve simultaneous quantification of multiple samples on a single chip, which is conducive to greatly increasing the reaction throughput.

[0046] In addition, the method for using the multi-layer three-dimensional digital nucleic acid amplification chip provided by the present invention can avoid the problem of micro-block swelling caused by chip materials by passing oil through negative pressure and positive pressure, improve the uniformity of reaction volume, and make quantitative results more accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is a conceptual schematic diagram of a multi-layer three-dimensional digital nucleic acid amplification chip provided by an embodiment of the present invention;

[0048] Figure 2 is a schematic diagram of the structure of a multi-layer three-dimensional digital nucleic acid amplification chip provided by an embodiment of the present invention;

[0049] Figure 3 It is a schematic diagram of a process for manufacturing a multi-layer three-dimensional digital nucleic acid amplification chip provided by an embodiment of the present invention;

[0050] Figure 4 It is a schematic diagram of a method for manufacturing a multi-layer three-dimensional digital nucleic acid amplification chip provided by an embodiment of the present invention;

[0051] Figure 5 It is a schematic flow chart of a method for using a multi-layer three-dimensional digital nucleic acid amplification chip provided by an embodiment of the present invention;

[0052] Figure 6 is a schematic diagram of a method for using a multi-layer three-dimensional digital nucleic acid amplification chip provided by an embodiment of the present invention;

[0053] Figure 7 This is a physical picture of a multi-layer three-dimensional digital nucleic acid amplification chip provided by an embodiment of the present invention;

[0054] Figure 8 This is a side-cut micrograph of a multi-layer three-dimensional digital nucleic acid amplification chip provided by an embodiment of the present invention;

[0055] Fig. 9 This is a single-layer fluorescein injection diagram of a multi-layer three-dimensional digital nucleic acid amplification chip provided by an embodiment of the present invention;

[0056] Fig.10It is a fluorescence image of a single-layer isothermal amplification reaction of a multi-layer three-dimensional digital nucleic acid amplification chip provided by an embodiment of the present invention;

[0057] Fig.11 It is a fluorescence image of the isothermal amplification reaction result of the three-layer digital nucleic acid amplification chip provided by an embodiment of the present invention;

[0058] Fig.12 It is a statistical graph of nucleic acid quantitative data of a three-layer digital nucleic acid amplification chip provided by an embodiment of the present invention;

[0059] Reference numerals:

[0060] 1. Microarray chip; 2. Water storage layer chip; 3. Glass slide; 11. Micropore array; 12. First branch-type distribution channel; 13. Liquid inlet; 14. Oil hole; 15. Water storage ring; 16. Liquid inlet and outlet of the water storage ring; 21. Water storage channel; 22. Second branch-type distribution channel; 23. Water inlet and outlet. DETAILED DESCRIPTION

[0061] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0062] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0063] Example 1

[0064] This embodiment provides a multi-layer three-dimensional digital nucleic acid amplification chip, the structure of which is as follows: Figure 1 , Figure 2 As shown, including:

[0065] A plurality of stacked microarray chips 1, a water storage layer chip 2 and a glass slide 3;

[0066] Each of the microarray chips 1 is provided with a liquid inlet 13 , a first branch distribution channel 12 , a plurality of micropore arrays 11 , an oil through hole 14 , a water storage circle 15 and a liquid inlet and outlet 16 of the water storage circle.

[0067] Among them, the liquid inlet 13 and the micropore array 11 are connected through the first branch-type distribution channel 12; the oil hole 14 is connected to the micropore array 11 through the first branch-type distribution channel 12; the water storage circle liquid inlet and outlet 16 are connected through the water storage circle 15; the water storage circle 15 is arranged between the micropore array 11 and the edge of the microarray chip 1, and is not connected to the micropore array 11.

[0068] The micropores in the micropore array 11 are arranged along each branch channel of the first branch distribution channel 12, and the adjacent micropores along the axial direction of the branch channel are staggered and distributed on both sides of the branch channel in sequence, and each micropore is connected to the channel.

[0069] The height of the first branch-type distribution channel 12, the micropore array 11 and the water storage circle 15 is less than the thickness of the microarray chip 1; and between the microarray chips 1 on different layers, the liquid inlets 13 are interconnected, the oil holes 14 are interconnected, and the liquid inlet and outlet ports 16 of the water storage circle are interconnected; specifically, the micropores in the micropore array 11 are cube-shaped holes with a length, width and height of 50 microns; the width of a single channel in the first branch-type distribution channel 12 is 20 microns and the height is 15 microns; the water storage circle 15 is 200 microns wide and 50 microns high; the first branch-type distribution channel 12 has a total of 128 branches, and the micropore array 11 has 256 rows and 256 columns, with a total of 65,536 micropores, and the size and number of branches can be adjusted according to experimental requirements.

[0070] The water storage layer chip 2 is provided with a water inlet and outlet 23, a water storage channel 21 and a second branch type distribution channel 22; the water inlet and outlet 23 and the water storage channel 21 are connected through the second branch type distribution channel 22; the water inlet and outlet 23 are symmetrical and interchangeable.

[0071] The height of the water storage channel 21 and the second branch distribution channel 22 are both 75 microns, the width of the water storage channel 21 is 420 microns, and the interval between two adjacent water storage channels 21 is 40 microns.

[0072] The water storage channel 21 on the water storage layer chip 2 is arranged in contact with the microwell array 11 on the uppermost microarray chip 1 , and the water storage channel 21 covers the microwell array 11 .

[0073] The glass slide 3 is fixedly disposed below the bottom microarray chip 1 and covers the bottom surface of the microarray chip 1 .

[0074] The materials of the microarray chip 1 and the water storage layer chip 2 are both polydimethylsiloxane (PDMS).

[0075] Example 2

[0076] This embodiment provides a method for preparing a multi-layer three-dimensional digital nucleic acid amplification chip, such as Figure 3 , Figure 4 As shown, the steps are as follows:

[0077] (1) Preparation of molds. Use L-Edit to draw and print it on a plastic film or optical mask as an exposure mask for subsequent experiments. A total of two silicon wafer molds are required (at least, there can be multiple microarray layer molds, depending on the experimental requirements), one for making microarray chips and one for making water storage layer chips. The preparation process of the two molds is basically the same, except that the masks are different. Use SU8 photoresist and pre-spin at 500 rpm for 10 seconds when spreading the glue, and then set different speeds for 30 seconds according to different height requirements. After baking on a 95-degree hot plate for 15 minutes, put it into the exposure machine for exposure. The exposure time needs to be determined by referring to the help document of the exposure machine and the actual light intensity. After that, bake it on a 95-degree hot plate for another 15 minutes, and develop it to get the mold.

[0078] (2) Injection molding and curing of the chip. Both the microarray chip and the water reservoir chip are obtained by PDMS injection molding. The microarray chip requires a ratio of 7:1 between glue A (monomer) and glue B (cross-linking agent). Mix them evenly, remove the bubbles by vacuuming, and then spin-coat PDMS on the mold using a glue spreader (1000rpm, 30s) to obtain a PDMS film with a thickness of about 100 microns. The water reservoir chip requires a ratio of 7:1 between glue A (monomer) and glue B (cross-linking agent). After vacuuming, the thickness is more than 3mm. When curing, first put the two molds into a 70-degree oven and bake for 45 minutes, then take out the two layers of chips. At this time, the PDMS has been initially cured.

[0079] (3) Chip bonding. Remove the water reservoir layer PDMS chip from the mold, and perform surface plasma treatment together with the mold with the microarray layer PDMS chip. Use a microscope to align (the water reservoir layer water storage channel can cover the microarray), and bake in a 70-degree oven for 15 minutes. Remove the two-layer chip from the mold, and perform surface plasma treatment together with another mold PDMS with a microarray layer PDMS chip. Use a microscope to align through the liquid inlet and oil hole of the microarray layer, and repeat (N-1) times. And encapsulate with glass.

[0080] (4) Chip modification: Before adding the reaction solution, a vacuum treatment of about 20 minutes is required so that the reaction solution can be smoothly loaded into the microwell array.

[0081] It should be noted that after vacuum treatment, the internal pressure of the chip will slowly balance with the external air pressure, so the injection operation needs to be completed within 30 minutes after vacuum treatment.

[0082] Example 3

[0083] This embodiment provides a method for using a multi-layer three-dimensional digital nucleic acid amplification chip, such as Figure 5 , Figure 6As shown, a three-layer digital nucleic acid amplification chip is used to achieve molecular quantification of the internal reference gene β-actin plasmid standard, and the nucleic acid amplification method used is loop-mediated isothermal amplification (LAMP). It mainly includes the following steps:

[0084] (1) Sample injection. Prepare the isothermal nucleic acid amplification reaction solution according to the NEB Bst3.0 instructions. Use tape to seal the oil hole, use a pipette to draw 35 μl of the reaction solution and insert it into the liquid inlet, then use the syringe to provide pressure so that the reaction solution enters the microarray under the external pressure.

[0085] (2) Water storage in the aquifer. Use a 1 ml syringe to draw distilled water, tap the syringe tube to remove bubbles, and connect a hose. Insert the hose into the water inlet of the aquifer, pressurize the syringe, and push the distilled water into the aquifer.

[0086] (3) Oil passing. In order to prevent the reaction liquid between the micro-cubes from being connected, the channels need to be filled with oil phase material. The oil phase material used in this embodiment is a 3:1 (volume ratio) mixed oil of HFE7500 and FC40. The oil passing step includes negative pressure oil passing and positive pressure oil passing:

[0087] Negative pressure oiling: Drain the residual reaction liquid in the oil hole and fill it with mixed oil, insert a hose into the liquid inlet, and apply negative pressure through a syringe or other methods to fill the chip channel with mixed oil;

[0088] Positive pressure oil flow: Use a pipette or other method to draw 50 μl of mixed oil and insert it into the liquid inlet, then provide pressure through the syringe to allow the mixture to enter the chip channel under the applied pressure.

[0089] By passing oil alternately with negative pressure and positive pressure, the block expansion caused by the elasticity of the material can be avoided, the volume uniformity of the reaction unit can be better, and the quantitative results can be more accurate.

[0090] (4) Encapsulate the chip and perform nucleic acid amplification reaction. Place a cover glass on the chip and place it in a culture dish. Add distilled water to the culture dish until half of the chip is covered. Place the culture dish in a 65°C oven and incubate for 45 minutes.

[0091] (5) Result photography and processing. Fluorescence scanning photography was performed using a Ti-E microscope; fluorescence unit counting was performed using imageJ; and the number of nucleic acid molecules in the sample was calculated using the Poisson distribution formula.

[0092] The experimental images obtained are as follows Figure 7 , Figure 8 , Fig. 9 , Fig.10 , Fig.11 and Fig.12 shown. Figure 7This is a physical picture of a multi-layer three-dimensional digital nucleic acid amplification chip provided by an embodiment of the present invention; Figure 8 It is a side-cut view of a multi-layer three-dimensional digital nucleic acid amplification chip provided by an embodiment of the present invention. Fig. 9 This is a fluorescein oil-permeated image of a single-layer microarray chip in a multi-layer three-dimensional digital nucleic acid amplification chip provided by an embodiment of the present invention, indicating that the micropores can be separated by oil. Fig.10 This is a result diagram of the isothermal amplification reaction of β-actin using a single-layer microarray chip in the multi-layer three-dimensional digital nucleic acid amplification chip provided by an embodiment of the present invention. It can be seen that the positive reaction units and the negative reaction units can be clearly distinguished; and the positive reaction units exist independently without cross contamination; the evaporation of the reaction units in the middle and edge of the chip is very weak, which will not affect the nucleic acid amplification reaction. Fig.11 This is a result diagram of isothermal amplification reaction using three layers of microarray chips in the multi-layer three-dimensional digital nucleic acid amplification chip provided by an embodiment of the present invention. All three layers have positive signals and there is no cross contamination. Fig.12 It is the DNA molecular weight detected after nucleic acid quantification of a diluted template using a three-layer microarray chip in the multi-layer three-dimensional digital nucleic acid amplification chip provided by an embodiment of the present invention, and a good linear relationship is shown after taking the logarithm.

[0093] The method for using the multi-layer three-dimensional digital nucleic acid amplification chip provided in this embodiment uses a multi-layer three-dimensional digital nucleic acid amplification chip, and by stacking three layers of microarray chips, the number of reaction units is increased by three times. In fact, in many attempts by the inventors, the number of reaction units can be increased by more times, and high-precision and high-throughput nucleic acid quantification can be achieved.

[0094] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A multi-layer three-dimensional digital nucleic acid amplification chip, characterized in that: It includes: A plurality of microarray chips and a water reservoir chip arranged in a stacked manner; Each of the microarray chips is provided with a liquid inlet, a first branch-type distribution channel, a plurality of micropore arrays, an oil-through hole, a water storage circle and a liquid inlet and outlet of the water storage circle; wherein the liquid inlet and the micropore array are connected via the first branch-type distribution channel; the oil-through hole and the micropore array are connected via the first branch-type distribution channel; the liquid inlet and outlet of the water storage circle are connected via the water storage circle; the water storage circle is arranged between the micropore array and the edge of the microarray chip and is not connected to the micropore array; the height of the first branch-type distribution channel, the micropore array and the water storage circle is less than the thickness of the microarray chip; and between the microarray chips of different layers, the liquid inlets are connected to each other, the oil-through holes are connected to each other, and the liquid inlet and outlet of the water storage circle are connected to each other; The water storage layer chip is provided with a water inlet and outlet, a water storage channel and a second branch type distribution channel; the water inlet and outlet and the water storage channel are connected through the second branch type distribution channel; The water storage channel on the water storage layer chip is arranged in contact with the micropore array on the uppermost microarray chip, and the water storage channel covers the micropore array; The top view shape of the micropores in the micropore array is a square.

2. The multi-layer three-dimensional digital nucleic acid amplification chip according to claim 1, characterized in that: The micropores in the micropore array are arranged along each branch channel of the first branch distribution channel, and the adjacent micropores along the axial direction of the branch channel are staggered and distributed on both sides of the branch channel in sequence, and each micropore is connected to the channel.

3. The multi-layer three-dimensional digital nucleic acid amplification chip according to claim 1, characterized in that: The materials of the microarray chip and the water storage layer chip are polydimethylsiloxane.

4. The multi-layer three-dimensional digital nucleic acid amplification chip according to any one of claims 1 to 3, characterized in that: The height of the microwell array is 20-200 micrometers, and the ratio of the height of the first branch distribution channel to the height of the microwell array is 1:3-10.

5. The multi-layer three-dimensional digital nucleic acid amplification chip according to claim 4, characterized in that: The pore diameter of a single micropore in the micropore array is greater than the width of a single channel in the first branch distribution channel.

6. The multi-layer three-dimensional digital nucleic acid amplification chip according to claim 4, characterized in that: The ratio of the height of the water storage circle to the height of the micropore array is 1:0.5-2.

7. The multi-layer three-dimensional digital nucleic acid amplification chip according to any one of claims 1-3, 5, and 6, characterized in that: The heights of the water storage channel and the second branch distribution channel are both 50-100 microns, the width of the water storage channel is 200-500 microns, and the interval between two adjacent water storage channels is smaller than the height of the water storage channel.

8. The multi-layer three-dimensional digital nucleic acid amplification chip according to claim 4, characterized in that: The heights of the water storage channel and the second branch distribution channel are both 50-100 microns, the width of the water storage channel is 200-500 microns, and the interval between two adjacent water storage channels is smaller than the height of the water storage channel.

9. The multi-layer three-dimensional digital nucleic acid amplification chip according to any one of claims 1-3, 5, 6, and 8, characterized in that: The multi-layer three-dimensional digital nucleic acid amplification chip also includes: A glass slide is fixedly arranged below the bottom microarray chip and covers the bottom surface of the microarray chip.

10. The multi-layer three-dimensional digital nucleic acid amplification chip according to claim 4, characterized in that: The multi-layer three-dimensional digital nucleic acid amplification chip also includes: A glass slide is fixedly arranged below the bottom microarray chip and covers the bottom surface of the microarray chip.

11. The multi-layer three-dimensional digital nucleic acid amplification chip according to claim 7, characterized in that: The multi-layer three-dimensional digital nucleic acid amplification chip also includes: A glass slide is fixedly arranged below the bottom microarray chip and covers the bottom surface of the microarray chip.

12. The method for preparing a multi-layer three-dimensional digital nucleic acid amplification chip according to any one of claims 1 to 11, characterized in that: include: preparing molds of a water storage layer chip and a plurality of microarray chips respectively; coating the prepared material on the mold and performing a curing process; The water storage layer chip on the mold and a layer of microarray chip are plasma treated, bonded and baked, and then bonded and baked with the remaining microarray chips.

13. Use of the multi-layer three-dimensional digital nucleic acid amplification chip according to any one of claims 1 to 11 in PCR for purposes other than disease diagnosis or treatment.

14. The use according to claim 13, characterized in that The PCR is nucleic acid quantitative PCR.

15. A method for using the multilayer three-dimensional digital nucleic acid amplification chip according to any one of claims 1 to 11 for non-disease diagnosis or treatment purposes, characterized in that: include: Introducing the nucleic acid amplification reaction solution into the multi-layer three-dimensional digital nucleic acid amplification chip through the liquid inlet on the microarray chip; Before or after the nucleic acid amplification reaction solution is introduced, water is injected into the water storage circle through the liquid inlet and outlet of the water storage circle; storing water in the aquifer through a water inlet in the aquifer chip; Alternately passing oil under negative pressure and positive pressure to the multi-layer three-dimensional digital nucleic acid amplification chip through the oil holes and the liquid inlet; The negative pressure oil passage specifically includes: sucking out the residual nucleic acid amplification reaction liquid in the oil passage hole and then introducing the oil phase substance, and applying negative pressure to the liquid inlet; the positive pressure oil passage specifically includes: introducing the oil phase substance from the liquid inlet with positive pressure; The chip is packaged, and after a constant temperature amplification reaction is performed, the reaction result is detected.

16. The method of use according to claim 15, characterized in that: The oil phase material used in the negative pressure oil passing and the positive pressure oil passing is a mixture of HFE7500 and FC40 in a volume ratio of 1-5:1.

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