CN108624478A - A kind of digital pcr chip and its application method based on hydrogel - Google Patents
A kind of digital pcr chip and its application method based on hydrogel Download PDFInfo
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- CN108624478A CN108624478A CN201810764928.5A CN201810764928A CN108624478A CN 108624478 A CN108624478 A CN 108624478A CN 201810764928 A CN201810764928 A CN 201810764928A CN 108624478 A CN108624478 A CN 108624478A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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Abstract
The present invention provides a kind of digital pcr chip and its application method based on hydrogel：The chip includes miniature hydrogel array, polymethyl methacrylate cover board, double faced adhesive tape middle layer and substrate of glass, and 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, by absorption, monodisperse and is retained in each microgel, then is passed through Seal Oil and realizes the completely isolated of micro- reaction system.Digital pcr chip of the present invention has the advantages that novel in design, simple for production, of low cost, biocompatibility is good, easy to operate, applied widely.
Description
Technical field
The invention belongs to Biochemistry and Molecular Biology technical applications, and in particular to a kind of disperse minor nucleic acid or
The device and application method of other biological sample solution.
Background technology
DNA (DNA) carries biological heredity information, and important work is played in the storage and transmission 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 PCR (Polymerase Chain Reaction, PCR) makes it possible external DNA cloning.PCR skills
Art realizes the qualitative analysis of DNA product by the agarose gel electrophoresis after amplification.With the continuous increasing of quantitative DNA detection demands
Long, PCR of the tradition based on electrophoresis is gradually replaced by 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 can 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 the microcavity or compartment of separation and is used to expand,
Ability with low noise and the low jump signal of capture.Compared with real-time fluorescence quantitative PCR, dPCR technologies have " 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
At some similar to defect, such as it is of 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 simple in structure, easy use, low cost and good biocompatibility.
Invention content
Present invention aims at a kind of structure of offer and manufacturing process is simple, easy to use, inexpensive, biocompatibility is good
It is good, can automatic dispersed nucleic acid and other biological sample, digital pcr chip and its application method based on hydrogel.
To achieve the above object, present invention employs following technical schemes：
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 is surrounded with cover board and substrate
For sample flow channel.
Preferably, the hollow out window include 1~10 round bore region being 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 generates bubble in hydrogel array region during can effectively solving the problems, such as sample-adding, while being read with fluorescence
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 ensures 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 be not less than well through-hole, two wells respectively by two through-holes with corresponding hollow out band far from hollow out window
One end be connected, the other ends of two hollow out bands respectively with positioned at the corresponding phase of the round bore region of outermost two of hollow out window
Even.
Preferably, the quantity of a diameter of≤7mm of the round bore region, the microgel column being distributed in round bore region is
2300 to 46000.
Preferably, the cover board is made of PMMA (polymethyl methacrylate) plate, and thickness is 0.4~2mm, middle layer
Using double faced adhesive tape (acrylic material), thickness is 50~200 μm；Substrate is made of translucent material (such as sheet glass), and thickness is
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 at connection glass chip bottom and hydrogel.Cover board medial surface is in direct contact with microgel column top, but
Without physical connection.
Preferably, a diameter of 25~100 μm of the microgel column, adjacent microgel intercolumniation are 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 mask by polyethyleneglycol diacrylate and photosensitizer
Method (carries out ultraviolet light) under the covering of photomask template to the mixed aqueous solution of polyethyleneglycol diacrylate and photosensitizer
Orderly, spaced multiple microgel columns, the shape (such as cylindric) of single microgel column by definite shape region formed
Corresponding (as round) with the shape of the transmission region of photomask template, diameter and the 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, includes the following steps：
1) formation of chip housing
Using double faced adhesive tape 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 faced adhesive tape and above-mentioned substrate adhesion, when adhesion, avoid the presence of bubble between the two；By the another of cover board and double faced adhesive tape
Side surfaces stick makes to be machined in the well of both sides and two hollow out bands on cover board and corresponds to connection；
2) formation of hydrogel array
By Mn (number-average molecular weight) polyethyleneglycol diacrylates (PEG-DA) for being 190~630 and photosensitizer (2- hydroxyls
Base -2- methyl phenyl ketones) 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 ultraviolet light is carried out to photomask template, and by through the photomask template
Ultraviolet light so that the mixed aqueous solution of filling in the cavity is crosslinked；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
To (freeze-drying) is freeze-dried in 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 softwares
It counts and etches to be formed using laser cutting machine.
Preferably, the specific preparation flow of the substrate is：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 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, includes 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, hydrogel in the cavity is filled using Seal Oil
Part except array, while undispersed solution and air in the cavity is discharged, it is single to be 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 reactions can be run, by nucleic acid amplification reaction, and coordinates fluoroscopic imaging systems, you can to realize monokaryon
The PCR of acid molecule reacts and fluoroscopic examination.
Preferably, the injection uses syringe.
Preferably, the sample solution is solution or pigment solution containing nucleic acid.
Preferably, the concentration of the nucleic acid be less than 1063 copy/microlitre.
Beneficial effects of the present invention are embodied in：
The present invention makes full use of capillarity possessed by gap between cover board and substrate (i.e. sample flow channel and fluid channel), with
And the characteristics of hydrogel soft texture, good biocompatibility, good hydrophilic property, so that hydrophily sample (such as nucleic acid) solution is being flowed through
When hydrogel array, by absorption, monodisperse and it can be retained in each microgel column, in conjunction with the Seal Oil being passed through, you can
Realize in microgel column each micro- reaction system it is completely isolated so that micro- reaction system reliability height, no cross contamination.This
The invention chip has novel design, and simple for production, of low cost, biocompatibility is good, easy to operate, the scope of application
Extensive advantage.
Further, the water suction volume of microgel column can be true by controlling the spacing and diameter of microgel column in array
It is fixed, it can be used for accurately controlling the volume of micro- reaction system.
Description of the drawings
Fig. 1 is the structural schematic diagram of hydrogel digital pcr chip of the present invention；
Fig. 2 is the multilayer assembling schematic diagram of hydrogel digital pcr chip part of the present invention；
Fig. 3 is hydrogel digital pcr chip pictorial diagram of the present invention；
Fig. 4 a are the scanning electron microscope (SEM) photograph of microgel array on hydrogel digital pcr chip of the present invention (passing through drying)
(side view of microgel array)；
Fig. 4 b are the scanning electron microscope (SEM) photograph of microgel array on hydrogel digital pcr chip of the present invention (passing through drying)
(vertical view of microgel array)；
Fig. 5 is diffusion flow chart of the hydrogel digital pcr chip of the present invention for hydrophily pigment solution；Wherein：
(a) the microgel array being lyophilized, upper left are the appearance that hydrogel digital pcr chip is lyophilized, and lower-left is the microgel array of freeze-drying
Optical microscope photograph (10 times)；(b) load liquid sample, in it is upper for red pigments solution spread appearance in chip, under be
Load the microgel array optical microscope photo of red pigments solution；(c) liquid sample disperseed, upper right are that red pigments are molten
The chip appearance that liquid dispersion is completed, bottom right are the microgel array optical microscope photo that the dispersion of red pigments solution is completed.
Fig. 6 is the light microscope of the various sizes of microgel array of hydrogel digital pcr chip interior of the present invention
Photo (20 times), wherein a represent the diameter of microgel column, and d represents the distance between adjacent microgel column；
Included in diameters and spacing and array of Fig. 7 a for microgel column in hydrogel digital pcr chip of the present invention
Microgel column number between relational graph；
Fig. 7 b are the diameter of microgel column and spacing and to be individually lyophilized micro- solidifying in hydrogel digital pcr chip of the present invention
Relational graph between the water absorption of rubber column gel column；
Fig. 8 is the glimmering of the pcr amplification reaction that hydrogel digital pcr chip of the present invention carries out various concentration nucleic acid samples
Light image (20 times)；Wherein：(a) blank control (0 copy/microlitre)；(b) 70 copy/microlitre sample solution；(c) it 140 copies
Shellfish/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 plates, 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 implementation mode
It elaborates with reference to the accompanying drawings and examples to the present invention.
Referring to Fig. 1, Fig. 2 and Fig. 4 a and Fig. 4 b, the present invention provides a kind of hydrogel digital pcr chip, including one layer
PMMA plates 1 (thickness 1mm, PMMA, that is, polymethyl methacrylate, that is, organic glass), well 2, middle layer 3, Miniature water
Gel array 4 and substrate of glass 5 (sheet glass).The well 2 by laser cutting machine (U.S. Universal, VLS2.30) in
It performs etching to be formed on PMMA plates 1, sample-adding pore size is diameter 1.5mm.The middle layer 3 is acrylic material double faced adhesive tape (50 μ
M), middle layer 3 connects 1 inside of PMMA plates on one side, and another side connects hydrophilic treated substrate of glass 5.By Core in middle layer 3
DRAW Software for Design is simultaneously formed with the pierced pattern of sugarcoated haws shape using laser cutting machine etching, pierced pattern specifically include containing
The hollow out window 8 of three round holes (i.e. three array regions) being connected, and the through-hole 6 positioned at 8 both sides of hollow out window, two
A through-hole 6 is connect with two round holes for being located at outside in hollow out window 8 by respective side hollow out band 7.Miniature hydrogel battle array
Row 4 are that (microtrabeculae is straight for the array of 2300 to 46000 hydrogel microtrabeculaes (i.e. microgel column) that is formed using UV crosslinking mask method
Diameter is 25~100 μm；The spacing of each two microtrabeculae is 25~120 μm；), therefore miniature hydrogel array is alternatively referred to as microgel battle array
Row.It (is formed after being closed by PMMA plates 1 and substrate of glass 5) in the corresponding space of above-mentioned array region.5 thickness of substrate of glass
For 0.3cm, need to carry out 80 DEG C of processing overnight by 3- silane propyl methacrylate (TMS-PMA), then with ethyl alcohol and
Pure water rinses 3 times respectively, naturally dry, forms water-wetted surface, plays the effect at connection glass chip bottom and hydrogel.
The shell manufacturing process of the hydrogel digital pcr chip is exemplified below：The glass base that will first be handled through TMS-PMA
Bottom 5 and 3 side adhesion of double faced adhesive tape, when adhesion, avoid the presence of bubble between the two；1 side of PMMA plates is sticked in into double faced adhesive tape 3 again
The other side, wherein 6 center of through-hole at 1 both ends well 2 of PMMA plates and double faced adhesive tape middle layer both ends overlaps, to 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：It will match
Polyethyleneglycol diacrylate (PEG-DA, Mw=10kDa, the Mn=575 of system；Sigma Aldrich) and photosensitizer (2- hydroxyls
Base -2- methyl phenyl ketones, 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.
A diameter of 50 μm of the loophole of photomask template, adjacent light transmission pitch of holes are 50 μm.The single a diameter of 7mm of the array region,
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 replacing 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 increases therewith.
The applicable range of hydrogel digital pcr chip of the present invention is very extensive, dispersible certain dense with the chip
The nucleic acid solution in range is spent, carries out PCR amplification, the detection accuracy (detectable limit) of chip is in 46000 nucleic acid molecules
It can detect a target nucleic acid；With the chip can also the hydrophilic colored samples solution of automatic absorption, carry out such as edible
The dispersion of pigment solution.
Pigment solution is added drop-wise to side well 2 by the dispersion for pigment sample, and the PMMA plates 1 are translucency
Fabulous organic polymer hydrophobic material, the slight void formed between substrate of glass 5 (include the stream being connect with well
Gap between road and microgel column) capillary force can be generated so that and the pigment solution is automatically along the glass base
Gap diffuses to 4 place array region of miniature hydrogel array by capillary force between bottom 5 and PMMA plates 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 4 surface of miniature 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.Nucleic acid solution (be less than 1063 copy/microlitre) is added dropwise
To side well 2, the PMMA plates 1 are the fabulous organic polymer hydrophobic material of translucency, with miniature hydrogel battle array
The slight void formed between row 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 4 place array region of miniature 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.
Seal Oil (such as mineral oil) can be then slowly added to by well 2, 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 row 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 so that pigment aqueous solution is dispersed in each microtrabeculae of porous aquagel, plays the role of disperseing pigment solution
(see Fig. 5).
Step 2：After step 1), external pressure is applied by well and injects Seal Oil, is 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 11697 each about 1 nanoliter of 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 liquids (comprising PCR polymerases, dNTP,
PCR probes, 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 along the runner by capillary force 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：Seal Oil is injected, the part except the miniature hydrogel microarray is filled using Seal Oil, arranges simultaneously
Air where going out the miniature hydrogel array in array region, forms 11697 mutual independent micro- reaction systems of PCR,
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, you can operation PCR
The temperature control program of reaction.To the end of PCR, pcr chip is placed under fluorescence microscope, you can obtain the glimmering of the standard nucleic acid sample
Light detection image (see Fig. 8).Brighter microgel column represents the nucleic acid sequence that can be combined with fluorescence probe and is expanded 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
Concentration of the target nucleic acid sequence in entire sample can be represented by accounting for the ratio of all microgel columns.
The present invention is compared with the method for traditional Water-In-Oil monodisperse nucleic acid samples, and sample preparation time is short, operating method
Simply, it is not necessarily to professional sample adding device；Chip cost is cheap, and cost reduces nearly more than 100 times；It can be according to the big of sample size
Small, the quantity by adjusting serial array region flexibly controls detection flux.The accuracy of dispersed nucleic acid sample, reactant simultaneously
The reliability of system is splendid, and cross contamination is small, biocompatibility is good.
Claims (10)
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；Well (2) is provided in the chip housing, 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) in middle layer (3) respectively, hollow out band (7) surround logical with cover board and substrate
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 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 two through-holes (6) and one end phase of the corresponding hollow out band (7) far from hollow out window (8) respectively
Even, the other ends of two hollow out bands (7) respectively with positioned at the corresponding phase of the round bore region of outermost two of hollow out window (8)
Even.
4. a kind of digital pcr chip according to claim 3, it is characterised in that：It is described circle bore region it is a diameter of≤
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 plates (1),
PMMA plates (1) thickness is 0.4~2mm；It is 50~200 μm that middle layer (3), which uses double faced adhesive tape, middle layer (3) thickness,；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) includes by poly- second
Orderly, spaced multiple microgel columns that omega-diol diacrylate and photosensitizer are formed by UV crosslinking mask method, it is single
The shape of a microgel column is corresponding with the shape of the transmission region of photomask template, by the water suction of dry single microgel column
Volume is 0.2~5 nanoliter.
7. a kind of digital pcr chip according to claim 1, it is characterised in that：A diameter of the 25~100 of the microgel column
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 in middle layer (3) with well (2).
8. a kind of preparation method of digital pcr chip as claimed in claim 2, it is characterised in that：Include the following steps：
1) formation of chip housing
Processing includes the pierced pattern of the hollow out window (8) and hollow out band (7) in middle layer (3)；It then, will be intermediate
One side surface of layer (3) and the light-transparent substrate adhesion with water-wetted surface；Another side surface of cover board and middle layer (3) is viscous
Even, make to be machined in two wells (2) connection corresponding with two hollow out band (7) on cover board；
2) formation of hydrogel array
The mixed aqueous solution of polyethyleneglycol diacrylate and photosensitizer is passed through and the well phase by one of well
The sample flow channel of connection spreads and fills to the cavity surrounded by hollow out window (8), cover board and substrate, then will have more
The photomask template of a loophole is placed on Basolateral and covers hollow out window (8) region, then to photomask template into
Row ultraviolet light, and the polyethyleneglycol diacrylate for making to be located in the cavity by the ultraviolet light through photomask template
It crosslinks；Wherein, the volume fraction of polyethyleneglycol diacrylate is 10%~50% in the mixed aqueous solution, photosensitizer
Volume fraction be 0.2%~1%；After irradiation, uncrosslinked polyethyleneglycol diacrylate is loaded by another
Hole is discharged, and then by the dry moisture removed inside and out cross-linking products, forms internal porous sponge structure.
9. according to the method described in claim 8, it is characterized in that：The light intensity of the ultraviolet light is 60~100mW/cm2, irradiation
Direction is vertical with the photomask template, and irradiation time is 100~150 seconds.
10. a kind of application method of digital pcr chip as claimed in claim 2, it is characterised in that：Include the following steps：
1) hydrophily sample solution is added drop-wise to a well, edge adds the sample solution with this automatically by capillary force
The sample flow channel of sample hole connection diffuses in the gap between the microgel column, and is dispersed to porous in the microgel column
In sponge structure；
2) after step 1), Seal Oil is injected by above-mentioned well, between being filled between the microgel column using Seal Oil
Gap, while air and undispersed hydrophily sample solution in the cavity is discharged through another well, it is formed with described
Microgel column is mutual independent closed micro- reaction system of unit.
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