CN215799530U - Multilayer three-dimensional digital nucleic acid amplification chip - Google Patents

Abstract

The utility model relates to the field of digital PCR, in particular to a multilayer three-dimensional digital nucleic acid amplification chip. In the multilayer three-dimensional digital nucleic acid amplification chip, the multilayer superposition of the microarray chip can be realized, the number of reaction units for the digital nucleic acid amplification is greatly increased, the dynamic range of nucleic acid quantification and the detection lower limit precision are greatly improved, and the reaction flux is favorably and greatly increased in actual detection. In addition, the utility model also provides a manufacturing method and a using method of multilayer three-dimensional digital nucleic acid amplification, which can further improve the accuracy of a quantitative result.

Description

Multilayer three-dimensional digital nucleic acid amplification chip

Technical Field

The utility model relates to the field of digital PCR, in particular to a multilayer three-dimensional digital nucleic acid amplification chip.

Background

Since the 1983 utility model of PCR, nucleic acid quantification has progressed from qualitative techniques (post-PCR electrophoresis) to semi-quantitative (fluorescent quantitative PCR, qPCR), with the advent of digital PCR (dPCR), to absolute quantification. The digital PCR technology randomly distributes the nucleic acid templates to a large number of reaction units for amplification reaction, collects the fluorescence signal of each reaction unit after the amplification is finished, and finally obtains the original concentration or content of the sample through calculation of a Poisson distribution formula because the random distribution of the nucleic acid molecules conforms to the Poisson distribution. The larger the number of partitions, the larger the nucleic acid quantification range and the higher the precision; the larger the total reaction volume, the lower the limit of detection.

The quantitative detection of nucleic acid molecules by digital PCR is divided into three steps: distribution of a substitution sample (partition), nucleic acid amplification, and fluorescence detection. Current dPCR techniques are divided into two broad categories, based on physically separate chip-based digital PCR (cdPCR) and droplet emulsion-based droplet digital PCR (ddPCR), depending on the partitioning strategy. Up to now, ddPCR has become the most commonly used method in the biological and medical fields due to its advantages. First, the design and manufacture of the droplet generating chip is relatively easy. Second, the size and number of droplets is flexible, and thus millions of droplets are easily produced. Finally, the droplet amplification product can be easily recovered, which is important in applications for quantification of second generation sequencing libraries. However, it has some limitations, and during PCR some droplets will merge due to thermal motion or evaporation of oil, which will result in differences in droplet volumes, differences in nucleic acid molecule assignment probabilities, non-poisson distribution, and significant impact on accuracy. In addition, droplet dPCR requires many facilities, including droplet generators, droplet delivery systems, and droplet counting systems, which increase the initial cost of the device and require the continued consumption of expensive materials. These disadvantages prevent their widespread clinical use. In contrast to ddPCR, cdPCR partitions samples by making physical microwells, is less costly and easier to detect, especially in point-of-care assays where the results of chip-based digital PCR can be easily detected using fluorescence microscopy. But due to its small number of pores and low flux, it is not very widely used.

At present, most of domestic commercialized digital PCR platforms are ddPCR, and although various scientific research units and institutions research on chip digital PCR with lower cost, the research focuses on the basic problems of sample introduction mode, prevention of reaction liquid evaporation and the like, and does not break the specifications in terms of volume, quantity and arrangement of reaction units, so that great progress and breakthrough are difficult to be made on key scientific problems such as sensitivity, flux and the like. Although some foreign researches report that a digital PCR chip capable of generating million-level reaction partitions is adopted, a strategy for improving the micropore surface density of the chip is adopted, the method improves the number of reaction units, but the volume of the reaction units is sharply reduced, the precision is improved, but the sensitivity cannot be considered, the application range is narrow, and the molecular diagnosis requirements of early cancer screening, new coronavirus diagnosis and the like cannot be met. And the increase of the surface density leads to the increase of the chip manufacturing requirement and the finished product loss rate, and the cost is higher.

SUMMERY OF THE UTILITY MODEL

In order to solve the technical problems, the utility model provides a multilayer three-dimensional digital nucleic acid amplification chip.

Specifically, the present invention provides a multilayer three-dimensional digital nucleic acid amplification chip, which comprises:

a plurality of microarray chips and a water storage layer chip which are arranged in a stacked manner;

each micro-array chip is provided with a liquid inlet, a first branch type distribution channel, a plurality of micro-pore arrays, an oil through hole, a water storage ring and a water storage ring liquid inlet and outlet; wherein the liquid inlet and the micropore array are communicated through the first branch type distribution channel; the oil through hole and the micropore array are connected through a first branch type distribution channel; the liquid inlet and outlet of the water storage ring are communicated through the water storage ring; the water storage ring is arranged between the micropore array and the edge of the microarray chip and is not communicated with the micropore array; the heights of the first branch-type distribution channel, the micropore array and the water storage ring are smaller than the thickness of the microarray chip; the liquid inlets are communicated with each other, the oil through holes are communicated with each other, and the liquid inlet and the liquid outlet of the water storage ring are communicated with each other;

a water inlet and outlet, a water storage channel and a second branch type distribution channel are arranged on the water storage layer chip; the water inlet and outlet and the water storage channel are communicated through the second branch type distribution channel;

the water storage channel on the water storage layer chip is in contact with the micropore array on the uppermost layer of the microarray chip, and the water storage channel covers the micropore array.

The present inventors have found that stacking and combining a plurality of microarray chips can effectively increase the number of reaction units, but the multi-layer microarray chip structure has higher requirements for the sealing property and uniformity of the reaction units, the structural stability during heat treatment, and the like when used, as compared to a single-layer microarray chip. The structure is favorable for meeting the performance requirements of isothermal amplification reaction on the multilayer three-dimensional digital nucleic acid amplification chip, and the number of reaction units is effectively increased.

Preferably, when the micropores in the micropore array are arranged along each branch channel of the first branch type distribution channel, the micropores adjacent to each other along the axial direction of the branch channel are sequentially distributed on two sides of the branch channel in a staggered manner, and each micropore is communicated with the channel, cross contamination can be further prevented, thereby being more beneficial to improving the sealing performance of a single reaction unit in a long-time amplification reaction.

Preferably, the material of the microarray chip and the water storage layer chip is Polydimethylsiloxane (PDMS).

Preferably, the height of the microwell array is 20 to 200 micrometers, and a ratio of the height of the first branched distribution channel to the height of the microwell array is 1: 3-10. The height difference of the micropores and the channels is set according to the proportion, so that the sealing effect of the reaction unit is further improved, meanwhile, the deformation pressure on the chip caused by filling liquid (especially oil substances) is relieved, the feasibility of alternately filling oil under positive pressure and negative pressure is further ensured, and the quantitative result is more accurate.

More preferably, the pore size of a single microwell in the microwell array is larger than the width of a single channel in the first branched distribution channel.

Preferably, the ratio of the height of the water retention ring to the height of the micro-pore array is 1: 0.5-2.

Preferably, the height of the water storage channel and the height of the second branch-type distribution channel are both 50-100 micrometers, the width of the water storage channel is 200-500 micrometers, and the interval between two adjacent water storage channels is smaller than the height of the water storage channel. This arrangement is more advantageous in reducing possible water evaporation of the microarray chip.

Preferably, the top view shape of the microwells in the microwell array is square, circular or other polygons, and the square shape is more favorable for the imaging effect.

Preferably, the multilayer three-dimensional digital nucleic acid amplification chip further comprises:

and a slide fixedly disposed under the lowermost microarray chip and covering a bottom surface of the microarray chip.

The multilayer three-dimensional digital nucleic acid amplification chip greatly increases the number of reaction units for digital nucleic acid amplification by overlapping the microarray chip layers, so that the dynamic range and the precision of nucleic acid quantification are improved; in addition, the micropore array can be transversely or longitudinally stretched, or a liquid inlet, an oil through hole and a branch-type distribution channel which are matched with the micropore array are added simultaneously to form an independent reaction liquid adding passage, so that simultaneous reaction of a plurality of samples on a single chip is realized, and the nucleic acid quantification can be realized at high flux.

The "uppermost layer" and "lowermost layer" referred to in the present invention are relative terms, and the judgment criterion of the orientation is that the side of the microarray chip having the microwell array is oriented upward. In practical embodiments, the expressions "top layer" and "bottom layer" may be adjusted according to other orientation criteria (e.g., the orientation of the side of the microarray chip without the microwell array is upward), and all of them fall within the scope of the present invention.

The above solutions can be combined by those skilled in the art to obtain preferred embodiments of the present invention.

The utility model further provides a preparation method of the multilayer three-dimensional digital nucleic acid amplification chip, which comprises the following steps:

respectively preparing a water storage layer chip and a plurality of micro-array chips;

coating the preparation material on a mould and carrying out curing treatment;

and carrying out plasma treatment on the water storage layer chip on the die and a layer of microarray chip, then, carrying out bonding baking, and then, continuing to bond and bake the chip with the rest microarray chips.

When the multilayer three-dimensional digital nucleic acid amplification chip further comprises a glass slide, the preparation method further comprises:

the bottom-most microarray chip was adhered to a glass slide.

The utility model also provides the application of the multilayer three-dimensional digital nucleic acid amplification chip in PCR; preferably in nucleic acid quantitative PCR application.

After the multilayer chip is adhered, the multilayer three-dimensional digital nucleic acid amplification chip can be subjected to surface plasma treatment and packaged with a glass slide.

In order to smoothly transfer the sample into the microwell array, it is preferable that the structure of the multilayer three-dimensional digital nucleic acid amplification chip is subjected to a vacuum treatment and then hermetically packaged.

The utility model also provides a using method of the multilayer three-dimensional digital nucleic acid amplification chip, which comprises the following steps:

introducing a nucleic acid amplification reaction solution into the multilayer three-dimensional digital nucleic acid amplification chip through a liquid inlet on the microarray chip; before or after the nucleic acid amplification reaction solution is introduced, injecting water into the water storage ring through the liquid inlet and outlet of the water storage ring;

water is stored in the water storage layer through a water inlet in the water storage layer chip;

carrying out alternate negative pressure oil and positive pressure oil through the oil through hole and the liquid inlet on the multilayer three-dimensional digital nucleic acid amplification chip;

wherein, the negative pressure oil introduction specifically comprises the following steps: sucking up the residual nucleic acid amplification reaction solution in the oil through hole, introducing the nucleic acid amplification reaction solution into an oil phase substance, and applying negative pressure to a liquid inlet; the positive pressure oil supply is specifically as follows: introducing the oil phase substance from the liquid inlet under positive pressure;

and packaging the chip, and detecting a reaction result after carrying out constant-temperature amplification reaction.

The oil can be introduced to fill the oil phase substance in the channel, so that the reaction liquid in the micropores is not communicated. By alternately introducing oil under negative pressure and positive pressure, the square expansion caused by the scalability of the material can be avoided, the volume uniformity of the reaction unit is better, and the quantitative result is more accurate.

In fact, the alternating oil feeding of the negative pressure and the positive pressure also puts higher requirements on the structure of the multilayer three-dimensional digital nucleic acid amplification chip, and the multilayer three-dimensional digital nucleic acid amplification chip optimized by the utility model can meet the requirements.

If the multilayer three-dimensional digital nucleic acid amplification chip is not vacuum-packed or is unpacked for a long time, it is preferable to perform a vacuum-pumping process on the multilayer three-dimensional digital nucleic acid amplification chip before sample injection. More preferably, the sample injection is completed within 30min after the vacuum packaging or vacuumizing treatment is disassembled, so as to further improve the sample injection effect.

Preferably, the oil phase used in the negative pressure oil passing and the positive pressure oil passing is a mixture of oil phase substances in a volume ratio of 1-5: 1 (more preferably 2-4: 1) of a mixture of HFE7500 and FC 40.

The utility model finds that HFE7500 (fluorinated ether) is easy to evaporate, but has low viscosity, is easier to push and suck through low pressure, and FC40 (electronic fluorinated liquid) basically does not evaporate, but has high viscosity, and is difficult to use. The practical requirements of the utility model on low viscosity performance and non-evaporation effect can be simultaneously realized after mixing according to the proportion.

Based on the technical scheme, the utility model has the following beneficial effects:

in the multilayer three-dimensional digital nucleic acid amplification chip, the multilayer superposition of the microarray chip can be realized, the number of reaction units for the digital nucleic acid amplification is greatly increased, and the dynamic range of nucleic acid quantification and the lower limit detection precision are greatly improved. Meanwhile, in the multilayer digital nucleic acid amplification chip provided by the utility model, the structural design of the microarray chip can be transversely or longitudinally stretched, and the number of single-layer chips, the volume of reaction units, the number of liquid inlets and the like can be flexibly adjusted, so that simultaneous quantification of multiple samples on a single chip is realized, and the reaction flux is favorably and greatly increased.

In addition, the application method of the multilayer three-dimensional digital nucleic acid amplification chip provided by the utility model can avoid the problem of micro-block swelling caused by chip materials by introducing oil under negative pressure and positive pressure, improve the uniformity of reaction volume and enable the quantitative result to be more accurate.

Drawings

FIG. 1 is a conceptual diagram of a multilayer three-dimensional digital nucleic acid amplification chip according to an embodiment of the present invention;

FIG. 2 is a schematic structural diagram of a multilayer three-dimensional digital nucleic acid amplification chip according to an embodiment of the present invention;

FIG. 3 is a schematic flow chart of a method for fabricating a multilayer three-dimensional digital nucleic acid amplification chip according to an embodiment of the present invention;

FIG. 4 is a schematic diagram of a method for fabricating a multilayer three-dimensional digital nucleic acid amplification chip according to an embodiment of the present invention;

FIG. 5 is a schematic flow chart of a method for using a multilayer three-dimensional digital nucleic acid amplification chip according to an embodiment of the present invention;

FIG. 6 is a side-cut micrograph of a multilayer three-dimensional digital nucleic acid amplification chip according to an embodiment of the present invention;

FIG. 7 is a sample of a monolayer fluorescein from a multilayer three-dimensional digital nucleic acid amplification chip provided in an embodiment of the present invention;

FIG. 8 is a fluorescence diagram of a single-layer isothermal amplification reaction of a multilayer three-dimensional digital nucleic acid amplification chip according to an embodiment of the present invention;

FIG. 9 is a fluorescence diagram showing isothermal amplification reaction results of a three-layered digital nucleic acid amplification chip according to an embodiment of the present invention;

FIG. 10 is a nucleic acid quantitative data statistical chart of a three-layered digital nucleic acid amplification chip according to an embodiment of the present invention;

reference numerals:

1. a microarray chip; 2. a water storage layer chip; 3. glass slide; 11. an array of microwells; 12. a first branched distribution channel; 13. a liquid inlet; 14. an oil through hole; 15. a water storage ring; 16. a liquid inlet and outlet of the water storage ring; 21. a water storage channel; 22. a second branching distribution channel; 23. a water inlet and a water outlet.

Detailed Description

The following examples are intended to illustrate the utility model but are not intended to limit the scope of the utility model.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

Example 1

This example provides a multilayer three-dimensional digital nucleic acid amplification chip, the structure of which is shown in fig. 1 and 2, and which includes:

a plurality of microarray chips 1, a water storage layer chip 2 and a glass slide 3 which are arranged in a stacked manner;

each micro-array chip 1 is provided with a liquid inlet 13, a first branch distribution channel 12, a plurality of micro-pore arrays 11, oil through holes 14, a water storage ring 15 and a water storage ring liquid inlet and outlet 16.

Wherein the liquid inlet 13 and the micro-pore array 11 are communicated through the first branch type distribution channel 12; the oil through holes 14 are connected with the micropore array 11 through a first branch type distribution channel 12; the water storage ring liquid inlet and outlet 16 is communicated with the water storage ring 15; the water storage ring 15 is arranged between the micropore array 11 and the edge of the microarray chip 1, and is not communicated with the micropore array 11.

The micropores in the micropore array 11 are arranged along each branch channel of the first branch type distribution channel 12, the micropores adjacent to each other along the axis direction of the branch channel are sequentially distributed on two sides of the branch channel in a staggered manner, and each micropore is communicated with the channel.

The height of the first branched distribution channel 12, the micro-pore array 11 and the water trap 15 is smaller than the thickness of the micro-array chip 1; in the microarray chips 1 of different layers, the liquid inlets 13 are communicated with each other, the oil through holes 14 are communicated with each other, and the liquid inlet and outlet 16 of the water storage ring are communicated with each other; specifically, the micropores in the micropore array 11 are square holes each having a length, width, and height of 50 micrometers; the width of a single channel in the first branched distribution channel 12 is 20 micrometers, and the height thereof is 15 micrometers; the width of the water storage ring 15 is 200 micrometers, and the height is 50 micrometers; the first branched distribution channel 12 has 128 branches, the microwell array 11 has 256 rows and 256 columns and 65536 microwells, and the size and the number of branches can be adjusted according to the experimental requirements.

A water inlet and outlet 23, a water storage channel 21 and a second branch type distribution channel 22 are arranged on the water storage layer chip 2; the water inlet and outlet 23 and the water storage channel 21 are communicated through the second branch distribution channel 22; the water inlet and outlet 23 is symmetrically replaceable.

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

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

The glass slide 3 is fixedly arranged below the microarray chip 1 at the bottommost layer and covers the bottom surface of the microarray chip 1.

The materials of the microarray chip 1 and the water storage layer chip 2 are Polydimethylsiloxane (PDMS).

Example 2

This example provides a method for preparing a multilayer three-dimensional digital nucleic acid amplification chip, as shown in fig. 3 and 4, comprising the following steps:

(1) and (5) preparing a mould. The film was printed on plastic film or optical mask as exposure mask for subsequent experiments using L-Edit mapping. A total of two silicon die (at least, there may be multiple microarray layer dies depending on the experimental requirements), one for making microarray chips and one for making water storage layer chips. The preparation processes of the two molds are basically consistent, only the masks are different, SU8 photoresist is used, the photoresist is pre-spun for 10s at the rotating speed of 500rpm during photoresist homogenizing, and then different rotating speed spinners for 30s are set according to different height requirements. And (4) after baking for 15min on a 95-DEG electric hot plate, putting the plate into an exposure machine for exposure, wherein the exposure time needs to be determined by referring to a help document and actual light intensity of the exposure machine. And then baking the die on a 95-DEG C electric heating plate for 15min, and developing to obtain the die.

(2) And injection molding and curing the chip. The microarray chip and the water storage layer chip are obtained by PDMS injection molding, the microarray chip needs glue A (monomer) and glue B (cross-linking agent) in a ratio of 7:1, the mixture is uniform, after air bubbles are removed by vacuumizing, PDMS is spin-coated on a mold through a spin coater (1000rpm, 30s), and a PDMS film with the thickness of about 100 micrometers can be obtained. The water storage layer chip needs to mix glue A (monomer) and glue B (cross-linking agent) in a ratio of 7:1, and after bubbles are removed in vacuum, the thickness is more than 3 mm. During curing, the two molds are placed into a 70-degree oven to be dried for 45min, and then the two layers of chips are taken out, wherein PDMS is primarily cured.

(3) And (5) bonding the chips. And (3) taking off the water storage layer PDMS chip from the mold, carrying out surface plasma treatment together with the mold with the micro-array layer PDMS chip, carrying out alignment (only the water storage layer water storage channel covers the micro-array) by using a microscope, and placing the mold into a 70-degree oven to bake for 15 min. And (3) removing the two layers of chips from the mold, carrying out surface plasma treatment together with another mold PDMS with the micro-array layer PDMS chip, carrying out alignment through a micro-array layer liquid inlet and an oil through hole by using a microscope, and repeating the operation for (N-1) times. And encapsulated with glass.

(4) And (5) modifying the chip. Before the reaction solution is fed, it is necessary to perform a vacuum process for about 20min first, so that the reaction solution can be smoothly loaded into the microwell array.

It should be noted that after the vacuum treatment, the interior of the chip will slowly equilibrate with the external air pressure, so the sample injection operation needs to be completed within 30 minutes after the vacuum treatment.

Example 3

This example provides a method for using a multilayer three-dimensional digital nucleic acid amplification chip, as shown in fig. 5, to quantify molecules of a reference gene β -actin plasmid standard by using a three-layer digital nucleic acid amplification chip, wherein the nucleic acid amplification method used is loop-mediated isothermal amplification (LAMP). The method mainly comprises the following steps:

(1) and (6) sample injection. Isothermal nucleic acid amplification reaction solution was prepared according to the NEB Bst3.0 protocol. The oil through hole is blocked by an adhesive tape, 35 microliter of reaction liquid is absorbed by a liquid transfer gun and inserted into the liquid inlet, and then pressure is provided by an injector, so that the reaction liquid enters the microarray under the action of external pressurization.

(2) The water storage layer stores water. A1 ml syringe is used for sucking distilled water, the wall of the syringe tube is flicked by hand to remove bubbles in the tube, and a hose is connected. The hose port is inserted into the water inlet of the water storage layer, and the distilled water is pushed into the water storage layer by pressurizing through the injector.

(3) And (6) introducing oil. In order to prevent the communication of the reaction solution between the micro blocks, the channels need to be filled with oil phase substances. The oil phase used in this example was 3 for HFE7500 and FC 40: 1 (volume ratio) mixed oil. The oil introducing step comprises negative pressure oil introduction and positive pressure oil introduction:

negative pressure oil filling: completely sucking residual reaction liquid in the oil through hole, filling mixed oil, inserting a hose into the liquid inlet, and applying negative pressure through an injector or other methods to fill the chip channel with the mixed oil;

positive pressure oil filling: a pipette or other means is used to aspirate 50 microliters of the mixed oil into the loading port and then pressure is applied via the syringe to force the mixture under the applied pressure into the channels of the chip.

By alternately introducing oil under negative pressure and positive pressure, the square expansion caused by the scalability of the material can be avoided, the volume uniformity of the reaction unit is better, and the quantitative result is more accurate.

(4) Packaging the chip, and performing nucleic acid amplification reaction. The plate was covered with a glass slide and placed in a petri dish, and distilled water was added to the petri dish until half of the chip was submerged. The petri dish was placed in a 65 ℃ oven for incubation for 45 min.

(5) And (5) shooting and processing the result. Performing fluorescence scanning shooting by using a Ti-E microscope; fluorescence unit counting with imageJ; and obtaining the calculated value of the number of the nucleic acid molecules in the sample through a Poisson distribution formula.

The obtained experimental images are shown in fig. 6, 7, 8, 9 and 10. FIG. 6 is a side-cut view of a multilayer three-dimensional digital nucleic acid amplification chip according to an embodiment of the present invention. FIG. 7 is a fluorescein flux map of a single-layer microarray chip in a multi-layer three-dimensional digital nucleic acid amplification chip according to an embodiment of the present invention, illustrating that microwells may be separated by oil. FIG. 8 is a graph showing the results of beta-actin isothermal amplification reaction using a single-layer microarray chip in a multilayer three-dimensional digital nucleic acid amplification chip according to an embodiment of the present invention, in which positive reaction units and negative reaction units can be clearly distinguished; the positive reaction units exist independently, and the cross contamination phenomenon is avoided; the evaporation of the reaction units in the middle and at the edge of the chip is weak, and the progress of the nucleic acid amplification reaction is not influenced. FIG. 9 is a diagram showing the results of isothermal amplification reactions using three layers of microarray chips in a multilayer three-dimensional digital nucleic acid amplification chip according to an embodiment of the present invention, wherein the three layers have positive signals and no cross contamination. FIG. 10 shows the logarithmic linear relationship of the DNA molecular weights of the template diluted in multiple times by the three-layer microarray chip in the multilayer three-dimensional digital nucleic acid amplification chip according to the present invention.

The use method of multilayer three-dimensional digital nucleic acid amplification chip that this embodiment provided, owing to adopted multilayer three-dimensional digital nucleic acid amplification chip, through stack three-layer microarray chip, improved reaction unit number triple, in fact, in utility model people's many times of attempts, reaction unit number can also improve more multifold, and can realize the high-pass nucleic acid ration of high accuracy.

Although the utility model has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the utility model. Accordingly, such modifications and improvements are intended to be within the scope of the utility model as claimed.

Claims (9)

1. A multilayer three-dimensional digital nucleic acid amplification chip, comprising:

a plurality of microarray chips and a water storage layer chip which are arranged in a stacked manner;

each micro-array chip is provided with a liquid inlet, a first branch type distribution channel, a plurality of micro-pore arrays, an oil through hole, a water storage ring and a water storage ring liquid inlet and outlet; wherein the liquid inlet and the micropore array are communicated through the first branch type distribution channel; the oil through hole and the micropore array are connected through the first branch type distribution channel; the liquid inlet and outlet of the water storage ring are communicated through the water storage ring; the water storage ring is arranged between the micropore array and the edge of the microarray chip and is not communicated with the micropore array; the heights of the first branch-type distribution channel, the micropore array and the water storage ring are smaller than the thickness of the microarray chip; the liquid inlets are communicated with each other, the oil through holes are communicated with each other, and the liquid inlet and the liquid outlet of the water storage ring are communicated with each other;

a water inlet and outlet, a water storage channel and a second branch type distribution channel are arranged on the water storage layer chip; the water inlet and outlet and the water storage channel are communicated through the second branch type distribution channel;

the water storage channel on the water storage layer chip is in contact with the micropore array on the uppermost layer of the microarray chip, and the water storage channel covers the micropore array.

2. The multilayer three-dimensional digital nucleic acid amplification chip of claim 1, wherein the microwells in the microwell array are arranged along each branch channel of the first branch type distribution channel, and the microwells adjacent to each other along the axial direction of the branch channel are sequentially distributed on two sides of the branch channel in a staggered manner, and each microwell is communicated with the channel.

3. The multi-layered three-dimensional digital nucleic acid amplification chip of claim 1, wherein the microarray chip and the water storage layer chip are made of polydimethylsiloxane.

4. The multi-layered three-dimensional digital nucleic acid amplification chip according to any one of claims 1 to 3, wherein the height of the microwell array is 20 to 200 μm, and the ratio of the height of the first branched distribution channel to the height of the microwell array is 1: 3-10.

5. The multi-layered three-dimensional digital nucleic acid amplification chip of claim 4, wherein the pore size of an individual microwell in the microwell array is larger than the width of an individual channel in the first branched distribution channel.

6. The multi-layered three-dimensional digital nucleic acid amplification chip of claim 4, wherein the ratio of the height of the water retention ring to the height of the microwell array is 1: 0.5-2.

7. The chip of any one of claims 1 to 3, wherein the height of the water storage channel and the second branched distribution channel is 50 to 100 microns, the width of the water storage channel is 200 and 500 microns, and the distance between two adjacent water storage channels is smaller than the height of the water storage channel.

8. The multi-layered three-dimensional digital nucleic acid amplification chip of claim 1, wherein the microwells in the microwell array have a circular or polygonal shape in a plan view.

9. The multilayer three-dimensional digital nucleic acid amplification chip of claim 1, further comprising:

and a slide fixedly disposed under the lowermost microarray chip and covering a bottom surface of the microarray chip.

Images