CN209098651U - Digital pcr chip based on hydrogel - Google Patents

Abstract

The utility model provides a kind of digital pcr chip based on hydrogel: the chip includes miniature hydrogel array, polymethyl methacrylate cover board, double-sided adhesive middle layer and substrate of glass, the miniature hydrogel array contains 2300 to 46000 microgels, and the single volume that microgel is lyophilized is 0.2~5 nanoliter.It when nucleic acid samples solution flows through miniature hydrogel array, is absorbed, monodisperse and is retained in each microgel, then be passed through Seal Oil and realize the completely isolated of micro- reaction system.Digital pcr chip described in the utility model has the advantages that novel in design, simple for production, low in cost, biocompatibility is good, easy to operate, applied widely.

Description

Digital pcr chip based on hydrogel

Technical field

The utility model belongs to Biochemistry and Molecular Biology technical applications, and in particular to a kind of disperse minor core The device and application method of acid or other biological sample solution.

Background technique

DNA (DNA) carries biological heredity information, plays important work in the storage and transmitting of hereditary information With.The amplification in vitro of DNA is extremely important for the detection specificity and accuracy that improve DNA.Nineteen eighty-three is sent out by K.B.Mullis Bright polymerase chain reaction (Polymerase Chain Reaction, PCR) makes it possible external DNA cloning.PCR skill Art realizes the qualitative analysis of DNA product by the agarose gel electrophoresis after amplification.With the continuous increasing of quantitative DNA detection demand Long, PCR of the tradition based on electrophoresis is gradually replaced quantitative PCR analysis, i.e. real-time fluorescence quantitative PCR.But quantitative fluorescent PCR is adopted With large volume reaction system, it is all it is nucleic acid-templated be present in a system, non-specific amplification will increase false positive results and Background noise, therefore can not still obtain absolute quantitation result.To achieve the purpose that absolute quantitation detects, digital pcr comes into being. In digital pcr (dPCR), DNA sample is dispersed into thousands of a droplets in isolated microcavity or compartment and is used to expand, Ability with low noise and the low jump signal of capture.Compared with real-time fluorescence quantitative PCR, dPCR technology has " unimolecule expansion The advantages of increasing " and " absolute quantitation ", thus it is much sensitive to the minor change of DNA abundance.It is main that digital pcr detects nucleic acid process It is divided into four steps: the extraction of nucleic acid samples, the dispersion of sample, sample amplification and signal detection.Wherein, the dispersion of nucleic acid samples is The most crucial technical step of digital pcr.

Up to the present, the laboratory equipment of commercialized digital pcr instrument and place under study for action is based on four kinds of samples Dispersing method, including Water-In-Oil (W/O) drop, micropore, microfluidic channel, inkjet printing.However these sample dispersion methods are deposited In some similar defects, such as it is at high cost, operation sequence is complicated, needs additional ancillary equipment.For example, by more than ten in droplet distribution Microlitre sample dispersion needs special expensive ancillary equipment at thousands of a microlayer models；Existing digital pcr chip Manufacturing process complexity (such as Chinese patent CN105543064A), when use, need professional to operate；One commercialized number PCR instrument usually requires to spend RMB up to a million.The above reason limits digital pcr technology to a certain extent Further development and application.Core component of the digital pcr chip as digital pcr system, carry sample of nucleic acid dispersion and Amplification needs to have the advantages such as structure is simple, easily use, low cost and good biocompatibility.

Summary of the invention

The utility model aim is to provide a kind of structure and manufacturing process is simple, easy to use, inexpensive, bio-compatible Property it is good, can automatic dispersed nucleic acid and other biological sample, digital pcr chip based on hydrogel.

To achieve the above object, the utility model uses following technical scheme:

A kind of digital pcr chip, including chip housing and the hydrogel array and sample flow that are set in chip housing Road, the hydrogel array include be fixed on chip housing inner wall, by dry multiple microgel columns and for by sample Product diffuse to each microchannel by dry microgel column, and the microchannel is by the gap structure between the microgel column At；Well is provided in the chip housing, well is connected by sample flow channel with the microchannel.

Preferably, the chip housing includes the cover board being cascading, middle layer and substrate, the side of middle layer Surface is connected with cover board, and another side surface of middle layer is connected with substrate；It is provided with hollow out window in middle layer and is engraved with this Two connected hollow out bands of empty window, the hydrogel array are located in hollow out window, well there are two settings on cover board, The connection corresponding with two hollow out bands in middle layer respectively of two wells, the channel that hollow out band and cover board and substrate surround For sample flow channel.

Preferably, the hollow out window include 1~10 round bore region being connected in series (round hole be it is multiple when, specifically Quantity can be increased or decreased according to sample to be tested amount), the edge of hollow out window is in symmetrical wavy (similar sugarcoated haws shape), Sample solution is read in hydrogel array region generation bubble, while with fluorescence during can effectively solving the problems, such as sample-adding Taking the circular visual field of device to match, (multiple round hole series connection, one facilitate sample flow, can flow through all micro- solidifying The region of rubber column gel column guarantees that absorption liquid is abundant；Secondly, facilitate fluorescence to read)；There are two hollow out band and two for setting in middle layer A diameter is not less than the through-hole of well, and two wells pass through two through-holes with corresponding hollow out band far from hollow out window respectively One end be connected, the other end of two hollow out bands phase corresponding with hollow out window outermost two round bore regions are located at respectively Even.

Preferably, the diameter of the round bore region is≤7mm, and the quantity for the microgel column being distributed in round bore region is 2300 to 46000.

Preferably, the cover board is made of PMMA (polymethyl methacrylate) plate, with a thickness of 0.4~2mm, middle layer Using double-sided adhesive (acrylic material), with a thickness of 50~200 μm；Substrate is made of translucent material (such as sheet glass), with a thickness of 0.3~1mm, substrate surface is hydrophilic (having hydrophilic surface layer), and hydrogel array is supported on the water-wetted surface on the inside of substrate, i.e., Water-wetted surface plays the effect of connection glass sheet substrate and hydrogel.Cover board medial surface is directly contacted with microgel column top, but Without physical connection.

Preferably, the diameter of the microgel column is 25~100 μm, and adjacent microgel intercolumniation is 25~120 μm；Sample-adding Hole is the circular hole of 0.5~2mm of diameter, and the length of the sample flow channel is 0.5~1.5cm (for example, 1cm), the width of sample flow channel Degree is 0.2~1mm (for example, 0.5mm), and sample flow channel one end passes through the through-hole (such as diameter 1.5mm) and sample-adding in middle layer Hole is connected.

Preferably, the hydrogel array includes passing through UV crosslinking exposure mask by polyethyleneglycol diacrylate and photosensitizer Method (carries out ultraviolet light to the mixed aqueous solution of polyethyleneglycol diacrylate and photosensitizer under the covering of photomask template) Orderly, spaced multiple microgel columns, the shape (such as cylindric) of single microgel column by certain shapes region formed Corresponding with shape (as round) of the transmission region of photomask template, the diameter and spacing of microgel can be with template used sizes Change, the water suction volume by dry single microgel column is 0.2~5 nanoliter.

The preparation method of above-mentioned digital pcr chip, comprising the following steps:

1) formation of chip housing

Using double-sided adhesive as middle layer, the patterns such as the hollow out window and hollow out band are processed on the intermediate layer；Then, will One side surface of double-sided adhesive and above-mentioned substrate adhesion, when adhesion, avoid the presence of bubble between the two；By the another of cover board and double-sided adhesive Side surfaces stick makes to be machined in the well of two sides and the corresponding connection of two hollow out bands on cover board；

2) formation of hydrogel array

The polyethyleneglycol diacrylate (PEG-DA) and photosensitizer (2- hydroxyl for being 190~630 by Mn (number-average molecular weight) Base -2- methyl phenyl ketone) mixed aqueous solution by side well through sample flow channel (by the hollow out band and the cover board The channel surrounded with substrate) it spreads and fills to the cavity surrounded by hollow out window, cover board and substrate (i.e. in chip housing Entire area of space corresponding to hollow out window), the photomask template with multiple loopholes is then placed on Basolateral And hollow out window region is covered, then carry out ultraviolet light to photomask template, and by through the photomask template Ultraviolet light make to fill mixed aqueous solution in the cavity and crosslink；Wherein, PEG-DA in the mixed aqueous solution Volume fraction is 10%~50% (for example, 20%), and the volume fraction of photosensitizer is 0.2%~1%, in the photomask template Loophole distribution and size determine hydrogel array in microgel column diameter and spacing.After irradiation, it will not hand over The PEG-DA solution of connection is discharged using the form (such as injection pure water or air) of external force by the well of the other side, is then placed (freeze-drying) is freeze-dried into freeze drier, for removing the moisture inside and out cross-linking products, for use.

Preferably, the well is etched by laser cutting machine, and the pierced pattern in middle layer is set by Core DRAW software It counts and etches to be formed using laser cutting machine.

Preferably, the specific preparation flow of the substrate are as follows: surface hydrophilic processing is carried out to sheet glass, i.e., is set sheet glass The immersion treatment in 3- silane propyl methacrylate (TMS-PMA), processing the time be 10~12 hours, treatment temperature be 75~ 85℃。

Preferably, the light intensity of the ultraviolet light is 60~100mW/cm², direction of illumination is to hang down with the photomask template Directly, irradiation time is 100~150 seconds, and environment is room temperature.Light intensity is high or low can not all to obtain ideal microgel structure.It hangs down It is directly irradiated in photomask template (90 degree), can just process the microgel column not interconnected.

Preferably, the time of the freeze-drying is 2~3 days, and temperature is -90 DEG C~-100 DEG C, and pressure is 0.6~1pa.

The application method of above-mentioned digital pcr chip, comprising the following steps:

1) hydrophily sample solution is added drop-wise to side well, the sample solution is automatically along the runner and microchannel It is diffused in the cavity by capillary force, since the hydrogel after freeze-drying has mandruka structure, in the cavity Hydrogel array sample solution is dispersed in microgel column；

2) after step 1), Seal Oil is injected by above-mentioned well, fills hydrogel in the cavity using Seal Oil Part except array, while the intracorporal undispersed solution of the chamber and air is discharged, it is single for being formed with the microgel column Multiple independent closed micro- reaction systems mutually of position.

For nucleic acid samples, after filling Seal Oil, the substrate of glass side of the chip is placed on alternating temperature warm table, The temperature control program that PCR reaction can be run, by nucleic acid amplification reaction, and cooperates fluoroscopic imaging systems, it can realizes monokaryon The PCR of acid molecule reacts and fluorescence detection.

Preferably, the injection uses syringe.

Preferably, the sample solution is solution or pigment solution containing nucleic acid.

Preferably, the concentration of the nucleic acid less than 1063 copy/microlitre.

The beneficial effects of the utility model are embodied in:

The utility model makes full use of capillary possessed by gap between cover board and substrate (i.e. sample flow channel and fluid channel) to make With and the characteristics of hydrogel is soft, good biocompatibility, good hydrophilic property, make hydrophily sample (such as nucleic acid) solution When flowing through hydrogel array, it can be absorbed, monodisperse and be retained in each microgel column, in conjunction with the sealing being passed through Oil can be realized to the completely isolated of micro- reaction system each in microgel column, so that micro- reaction system high reliablity, no intersection Pollution.Chip described in the utility model has novel design, and simple for production, low in cost, biocompatibility is good, operation side Just, advantage applied widely.

Further, the water suction volume of microgel column can be true by the spacing and diameter of microgel column in control array It is fixed, it can be used for accurately controlling the volume of micro- reaction system.

Detailed description of the invention

Fig. 1 is the structural schematic diagram of hydrogel digital pcr chip described in the utility model；

Fig. 2 is the multilayer assembling schematic diagram of hydrogel digital pcr chip part described in the utility model；

Fig. 3 is hydrogel digital pcr chip pictorial diagram described in the utility model；

Fig. 4 a is the scanning electron microscope of microgel array on hydrogel digital pcr chip described in the utility model (passing through drying) Scheme (side view of microgel array)；

Fig. 4 b is the scanning electron microscope of microgel array on hydrogel digital pcr chip described in the utility model (passing through drying) Scheme (top view of microgel array)；

Fig. 5 is diffusion flow chart of the hydrogel digital pcr chip described in the utility model for hydrophily pigment solution；Its In: (a) the microgel array being lyophilized, upper left are the appearance that hydrogel digital pcr chip is lyophilized, and lower-left is the microgel battle array of freeze-drying Column optical microscope photograph (10 times)；(b) load liquid sample, in it is upper for red pigments solution spread appearance in chip, under For the microgel array optical microscope photo for loading red pigments solution；(c) liquid sample dispersed, upper right is red pigments The chip appearance that solution dispersion is completed, bottom right are the microgel array optical microscope photo that the dispersion of red pigments solution is completed.

Fig. 6 is that the optics of the various sizes of microgel array of hydrogel digital pcr chip interior described in the utility model is aobvious Micro mirror photo (20 times), wherein a represents the diameter of microgel column, and d represents the distance between adjacent microgel column；

Fig. 7 a is institute in the diameter and spacing and array of microgel column in hydrogel digital pcr chip described in the utility model The relational graph between microgel column number for including；

Fig. 7 b is the diameter of microgel column and spacing and single freeze-drying in hydrogel digital pcr chip described in the utility model Relational graph between the water absorption of microgel column；

Fig. 8 is the pcr amplification reaction that hydrogel digital pcr chip described in the utility model carries out various concentration nucleic acid samples Fluorescent image (20 times)；Wherein: (a) blank control (0 copy/microlitre)；(b) 70 copy/microlitre sample solution；(c)140 Copy/microlitre sample solution；(d) 280 copy/microlitre sample solution；(e) 550 copy/microlitre sample solution；(f) 1100 copy/microlitre sample solution；

In figure: 1 is PMMA plate, and 2 be well, and 3 be middle layer, and 4 be hydrogel array, and 5 be substrate of glass, and 6 be through-hole, 7 be hollow out band, and 8 be hollow out window.

Specific embodiment

It elaborates with reference to the accompanying drawings and examples to the utility model.

Referring to Fig. 1, Fig. 2 and Fig. 4 a and Fig. 4 b, the utility model provides a kind of hydrogel digital pcr chip, including one It is layer PMMA plate 1 (with a thickness of 1mm, PMMA, that is, polymethyl methacrylate, that is, organic glass), well 2, middle layer 3, miniature Hydrogel array 4 and substrate of glass 5 (sheet glass).The well 2 is by laser cutting machine (U.S. Universal, VLS2.30) It to be formed in being performed etching on PMMA plate 1, well is having a size of diameter 1.5mm.The middle layer 3 is acrylic material double-sided adhesive (50 μm), middle layer 3 connect 1 inside of PMMA plate on one side, and another side connects hydrophilic treated substrate of glass 5.In middle layer 3 by Core DRAW software design and the pierced pattern that sugarcoated haws shape is formed with using laser cutting machine etching, pierced pattern are specifically wrapped The hollow out window 8 for the round hole (i.e. three array regions) being connected containing there are three is included, and positioned at the logical of 8 two sides of hollow out window Hole 6, two through-holes 6 are connect with two round holes for being located at outside in hollow out window 8 by respective side hollow out band 7.Miniature water Gel array 4 is the array of 2300 to the 46000 hydrogel microtrabeculaes (i.e. microgel column) formed using UV crosslinking mask method (micro post diameter is 25~100 μm；The spacing of every two microtrabeculae is 25~120 μm；), therefore miniature hydrogel array is alternatively referred to as micro- Gel array.It (is formed after being closed by PMMA plate 1 and substrate of glass 5) in the corresponding space of above-mentioned array region.Glass base Bottom 5 needs to carry out 80 DEG C of processing overnight by 3- silane propyl methacrylate (TMS-PMA), then uses with a thickness of 0.3cm Ethyl alcohol and pure water rinse 3 times respectively, naturally dry, form water-wetted surface, play the effect of connection glass sheet substrate and hydrogel.

The shell manufacturing process of the hydrogel digital pcr chip is exemplified below: the glass base that will first handle through TMS-PMA Bottom 5 and 3 side adhesion of double-sided adhesive, when adhesion, avoid the presence of bubble between the two；1 side of PMMA plate is sticked in into double-sided adhesive 3 again The other side, wherein 6 center of through-hole at 1 both ends well 2 of PMMA plate and double-sided adhesive middle layer both ends be overlapped, thus by hollow out Band 7 forms the runner in connection array region corresponding space and well.

Referring to Fig. 3, the forming method of the miniature hydrogel array 4 of the hydrogel digital pcr chip is exemplified below: will be matched Polyethyleneglycol diacrylate (PEG-DA, Mw=10kDa, the Mn=575 of system；Sigma Aldrich) and photosensitizer (2- hydroxyl Base -2- methyl phenyl ketone, Tokyo Chemical Industry) mixed solution (PEG-DA and photosensitizer body in mixed solution Fraction is respectively 20% and 0.2%) is filled it up in three array regions by well, then places photomask template In the substrate of glass side for being close to chip, light intensity value 80mW/cm is given²Vertical ultraviolet light, irradiation time are 120 seconds. The light transmission bore dia of photomask template is 50 μm, and adjacent light transmission pitch of holes is 50 μm.The single diameter of array region is 7mm, The quantity for the cylindrical shape hydrogel microtrabeculae being wherein cross-linked to form is 11697.It, need to will be uncrosslinked after aforesaid operations PEG-DA solution is discharged using external force (syringe injects pure water or air) by well, and is placed into freeze drier (temperature is subzero 100 DEG C, pressure 0.7pa), the single water of freeze-drying is lyophilized in freezing 72h in (Power DRY LL1500) The water suction volume of gel microtrabeculae is about 1 nanoliter, for use.

Corresponding to 3 array regions of hollow out window, the volume that can store sample solution is about 8 μ L.Less than 8 μ L, portion Subregion can not absorb sample, and testing result cannot be calculated accurately；More than 8 μ L, redundant sample will not be absorbed again, can only be discharged Chip.

Referring to Fig. 6 and Fig. 7 a and Fig. 7 b, microgel column number can be micro- by adjusting in the hydrogel digital pcr chip The diameter of gel column and spacing change, and the microgel column array of different-diameter and spacing can be by replacement different size (thoroughly Unthreaded hole diameter and spacing) photomask template carry out UV crosslinking.Single microgel column water absorption also with its diameter and spacing and Change.Referring to Fig. 7 a, with the diameter of microgel column and the increase of spacing, total microgel column quantity is reduced therewith；Referring to figure 7b, with the diameter of microgel column and the increase of spacing, the water absorption of single microgel column is increased with it.

The applicable range of hydrogel digital pcr chip described in the utility model is very extensive, with the chip dispersible one Determine the nucleic acid solution in concentration range, carry out PCR amplification, the detection accuracy (detectable limit) of chip is 46000 nucleic acid point A target nucleic acid can be detected in son；With the chip can also the hydrophilic colored samples solution of automatic absorption, carry out such as The dispersion of edible pigment solution.

Pigment solution is added drop-wise to side well 2 by the dispersion for pigment sample, and the PMMA plate 1 is translucency Fabulous organic polymer hydrophobic material, the slight void formed between substrate of glass 5 is (including the stream connecting with well Gap between road and microgel column) capillary force can be generated, so that the pigment solution is automatically along the glass base Gap diffuses to miniature 4 place array region of hydrogel array by capillary force between bottom 5 and PMMA plate 1, at this time due to freezing The water imbibition of the porous structure of dry microgel column, solution are dispersed in each microgel column in array region, microgel column it is more Solution is maintained in gel by pore structure (spongy).Then well is added in Seal Oil (mineral oil), is pushed by syringe Runner flows through miniature 4 surface of hydrogel array, pushes the pigment solution for not absorbing and spreading, and fills up miniature hydrogel array 4 Surrounding space excludes the air in chip, plays sealing function.

Dispersion for the nucleic acid samples within the scope of a certain concentration.By nucleic acid solution (less than 1063 copy/microlitre) be added dropwise To side well 2, the PMMA plate 1 is the fabulous organic polymer hydrophobic material of translucency, with miniature hydrogel battle array The slight void formed between column 4 can generate capillary force, so that the nucleic acid solution is automatically along substrate of glass 5 and PMMA 1 gap of plate diffuses to miniature 4 place array region of hydrogel array by capillary force, at this time since microgel column is lyophilized Porous structure water imbibition, solution is dispersed in each microgel column in array region, the porous structure (sponge of microgel column Shape) solution is maintained in gel, the nucleic acid molecules in solution disperse and enter in the porous structure of each microgel column.

It can be then slowly added to by well 2 Seal Oil (such as mineral oil), for the hydrogel digital pcr to be discharged The nucleic acid solution for not absorbing and spreading in chip runner and around miniature hydrogel array 4, while filling miniature hydrogel battle array Each microgel column of miniature hydrogel array 4 is also isolated entirely from by the space around column 4, Seal Oil, so that each Microgel column is as individual micro- reaction system.

Example 1. disperses pigment solution with hydrogel digital pcr chip

Step 1: the side well in pcr chip is added dropwise in 8 microlitres of pigment solutions, the pigment solution is automatically along described Runner flow to array region where the miniature hydrogel array of hydrogel digital pcr chip, since the hydrogel of freeze-drying is with good Good water imbibition plays the role of dispersing pigment solution so that pigment aqueous solution is dispersed in each microtrabeculae of porous aquagel (see Fig. 5).

Step 2: after step 1), external pressure being applied by well and injects Seal Oil, filled using Seal Oil described miniature Part except hydrogel array, while the air being discharged in the miniature hydrogel array place array region, form with institute 11697 mutual independent closed microbody systems that microgel column is unit are stated, it is just that 8 microlitres of pigment solutions of addition are uniform in this way Ground has been dispersed into the microbody system of about 1 nanoliter of about 11697 each volumes, illustrates that the pcr chip has evenly dispersed micro liquid The ability of body sample (see Fig. 5).

The hydrogel digital pcr chip dispersed nucleic acid solution of example 2., and carry out PCR amplification

Step 1: by the standard nucleic acid solution of the various concentration after dilution and PCR premixed liquid (comprising PCR polymerase, dNTP, PCR probe, primer etc.) mixing (reaching 8 microlitres after mixing), then 8 microlitres of mixed solutions are added dropwise and are added in the side of pcr chip Sample hole, the mixed solution diffuse to miniature hydrogel array place array area by capillary force along the runner automatically Domain, due to the porous structure after hydrogel freeze-drying, the nucleic acid molecules in solution are absorbed and are dispersed into each of porous aquagel In a microtrabeculae.

Step 2: injection Seal Oil is filled the part except the miniature hydrogel microarray using Seal Oil, is arranged simultaneously Air where the miniature hydrogel array in array region out, forms 11697 mutual independent micro- reaction systems of PCR, It in most cases, can include the nucleic acid sequence of at most 1 copy in each micro- reaction system, in this way, effectively preventing Intersect pollution between reaction member.The substrate of glass one of the chip is placed on alternating temperature warm table, PCR can be run The temperature control program of reaction.To the end of PCR, pcr chip is placed under fluorescence microscope, can be obtained the glimmering of the standard nucleic acid sample Light detection image (see Fig. 8).Brighter microgel column represents and can be expanded with the nucleic acid sequence in conjunction with fluorescence probe in Fig. 8 Increase, the purpose fluorescence constantly accumulated in amplification procedure causes microgel column to shine.The quantity of the corresponding microgel column of purpose fluorescence The ratio for accounting for all microgel columns can represent concentration of the target nucleic acid sequence in entire sample.

For the utility model compared with the method for traditional Water-In-Oil monodisperse nucleic acid samples, sample preparation time is short, operates Method is simple, without professional sample adding device；Chip cost is cheap, and cost reduces nearly more than 100 times；It can be according to sample size Size, the quantity by adjusting serial array region flexibly control detection flux.The accuracy of dispersed nucleic acid sample, reaction simultaneously The reliability of system is splendid, and cross contamination is small, biocompatibility is good.

Claims (7)

1. a kind of digital pcr chip, it is characterised in that: including chip housing and the hydrogel array being set in chip housing (4) and sample flow channel, the hydrogel array (4) include the multiple microgels by drying for being fixed on chip housing inner wall Column, there are gaps between the microgel column；It is provided in the chip housing well (2), well (2) passes through sample flow Road is connected with the gap.

2. a kind of digital pcr chip according to claim 1, it is characterised in that: the chip housing includes stacking gradually to set Cover board, middle layer (3) and the substrate set, a side surface of middle layer (3) are connected with cover board, the other side table of middle layer (3) Face is connected with substrate；The two hollow out items for being provided with hollow out window (8) in middle layer (3) and being connected with the hollow out window (8) Band (7), the hydrogel array (4) are located in hollow out window (8), and there are two well (2), two wells for setting on cover board (2) connection corresponding with two hollow out band (7) on middle layer (3) respectively, hollow out band (7) and cover board and substrate surround logical Road is sample flow channel.

3. a kind of digital pcr chip according to claim 2, it is characterised in that: the hollow out window (8) includes 1~10 The round bore region being connected in series, the edge of hollow out window (8) is in symmetrical wavy；There are two through-holes for setting in middle layer (3) (6), two wells (2) pass through one end phase of two through-holes (6) and corresponding hollow out band (7) separate hollow out window (8) respectively Even, the other end of two hollow out bands (7) phase corresponding with hollow out window (8) outermost two round bore region is located at respectively Even.

4. a kind of digital pcr chip according to claim 3, it is characterised in that: it is described circle bore region diameter be≤ The quantity of 7mm, the interior microgel column being distributed of round bore region are 2300~46000.

5. a kind of digital pcr chip according to claim 2, it is characterised in that: the cover board is made of PMMA plate (1), PMMA plate (1) is with a thickness of 0.4~2mm；Middle layer (3) uses double-sided adhesive, and middle layer (3) is with a thickness of 50~200 μm；Substrate uses Translucent material is made, and substrate thickness is 0.3~1mm, and substrate surface is hydrophilic.

6. a kind of digital pcr chip according to claim 1, it is characterised in that: the hydrogel array (4) include orderly, Spaced multiple microgel columns, the microgel column are formed by UV crosslinking mask method, the shape of single microgel column It is corresponding with the shape of the transmission region of photomask template, it is received by the water suction volume of dry single microgel column for 0.2~5 It rises.

7. a kind of digital pcr chip according to claim 1, it is characterised in that: the diameter of the microgel column is 25~100 μm, adjacent microgel intercolumniation is 25~120 μm；Well (2) is the circular hole of 0.5~2mm of diameter, and the length of sample flow channel is 0.5~1.5cm, sample flow channel one end are connected by the through-hole (6) being set on middle layer (3) with well (2).