CN108624478B - Digital PCR chip based on hydrogel and application method thereof - Google Patents
Digital PCR chip based on hydrogel and application method thereof Download PDFInfo
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- CN108624478B CN108624478B CN201810764928.5A CN201810764928A CN108624478B CN 108624478 B CN108624478 B CN 108624478B CN 201810764928 A CN201810764928 A CN 201810764928A CN 108624478 B CN108624478 B CN 108624478B
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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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a digital PCR chip based on hydrogel and a using method thereof, which comprises the following steps: the chip comprises a micro hydrogel array, a polymethyl methacrylate cover plate, a double-sided adhesive tape interlayer and a glass substrate, wherein the micro hydrogel array contains 2300 to 46000 microgels, and the volume of a single freeze-dried microgel is 0.2 to 5 nanoliters. The nucleic acid sample solution is absorbed, monodisperse and retained in each microgel when flowing through the micro hydrogel array, and then the sealing oil is introduced to realize complete isolation of the micro reaction system. The digital PCR chip has the advantages of novel design, simple and convenient manufacture, low cost, good biocompatibility, convenient operation and wide application range.
Description
Technical Field
The invention belongs to the application field of biochemical and molecular biological technologies, and particularly relates to a device for dispersing trace nucleic acid or other biological sample solutions and a use method thereof.
Background
Deoxyribonucleic acid (DNA) carries biological genetic information and plays an important role in the storage and transmission of genetic information. In vitro amplification of DNA is important to improve the specificity and accuracy of DNA detection. In 1983, the polymerase chain reaction (Polymerase Chain Reaction, PCR) invented by K.B.Mullis made possible in vitro DNA amplification. The PCR technology realizes the qualitative analysis of the DNA product by agarose gel electrophoresis after amplification. With the increasing demand for quantitative DNA detection, traditional electrophoresis-based PCR is gradually replaced by quantitative PCR analysis, i.e., real-time fluorescent quantitative PCR. However, fluorescent quantitative PCR uses a large-volume reaction system, and all nucleic acid templates exist in one system, and nonspecific amplification increases false positive results and background noise, so that absolute quantitative results cannot be obtained. For the purpose of absolute quantitative detection, digital PCR has been developed. In digital PCR (dPCR), a DNA sample is dispersed into thousands of droplets in separate microcavities or compartments for amplification, which has low noise and the ability to capture low mutation signals. Compared to real-time fluorescent quantitative PCR, dPCR techniques have the advantages of "single molecule amplification" and "absolute quantification" and are therefore much more sensitive to small changes in DNA abundance. The digital PCR nucleic acid detection process mainly comprises four steps: extraction of nucleic acid samples, dispersion of samples, amplification of samples, and detection of signals. Among them, the dispersion of nucleic acid samples is the most central technical step of digital PCR.
To date, commercial digital PCR instruments and laboratory equipment under investigation are based on four sample dispersion methods, including water-in-oil (W/O) droplets, microwells, microfluidic channels, inkjet printing. However, these sample dispersion methods have some similar drawbacks such as high cost, complicated operation procedures, the need for additional auxiliary equipment, and the like. For example, dispersing tens of microliters of sample into thousands of microdroplets in droplet dispersion requires specialized expensive auxiliary equipment; the existing digital PCR chip has complex manufacturing process (such as China patent CN 105543064A), and needs professional operation during use; a commercial digital PCR instrument typically requires millions of renmins. These reasons have limited to a certain extent the further development and application of digital PCR techniques. The digital PCR chip is used as a core component of the digital PCR system, is used for dispersing and amplifying nucleic acid samples, and needs to have the advantages of simple structure, easy use, low cost, good biocompatibility and the like.
Disclosure of Invention
The invention aims to provide a hydrogel-based digital PCR chip which has simple structure and manufacturing process, convenient use, low cost and good biocompatibility, can automatically disperse nucleic acid and other biological samples, and a use method thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a digital PCR chip, comprising a chip shell, a hydrogel array and a sample runner, wherein the hydrogel array is arranged in the chip shell, the hydrogel array comprises a plurality of dried microgel columns fixed on the inner wall of the chip shell, and micro channels for diffusing samples to the dried microgel columns, and the micro channels are formed by gaps among the microgel columns; the chip shell is provided with a sample adding hole, and the sample adding hole is communicated with the micro-channel through a sample flow channel.
Preferably, the chip shell comprises a cover plate, an intermediate layer and a substrate which are sequentially stacked, wherein one side surface of the intermediate layer is connected with the cover plate, and the other side surface of the intermediate layer is connected with the substrate; the hydrogel array is positioned in the hollow window, two sample adding holes are formed in the cover plate, the two sample adding holes are respectively communicated with the two hollow strips on the middle layer, and a channel surrounded by the hollow strips, the cover plate and the substrate is a sample flow channel.
Preferably, the hollow window comprises 1-10 circular hole areas (when the number of the circular holes is multiple, the specific number can be increased or decreased according to the amount of the sample to be measured), and the edge of the hollow window is in a symmetrical wavy shape (similar to a sugar gourd shape), so that the problem that a sample solution generates bubbles in the area where the hydrogel array is positioned in the sample adding process can be effectively solved, and the hollow window is matched with the circular imaging field of view of the fluorescence reading device (the circular holes are connected in series, so that the sample can flow conveniently, and can flow through the areas of all microgel columns, thereby ensuring sufficient absorption liquid; the middle layer is provided with two hollowed-out strips and two through holes with diameters not smaller than that of the sample adding holes, the two sample adding holes are respectively connected with one ends, far away from the hollowed-out window, of the corresponding hollowed-out strips through the two through holes, and the other ends of the two hollowed-out strips are respectively correspondingly connected with two circular hole areas located at the outermost sides of the hollowed-out window.
Preferably, the diameter of the circular hole area is less than or equal to 7mm, and the number of microgel columns distributed in the circular hole area is 2300 to 46000.
Preferably, the cover plate is made of PMMA (polymethyl methacrylate) plate, the thickness is 0.4-2 mm, the middle layer is made of double faced adhesive tape (acrylic material), and the thickness is 50-200 mu m; the substrate is made of a light-transmitting material (such as a glass sheet) with the thickness of 0.3-1 mm, the surface of the substrate is hydrophilic (provided with a hydrophilic surface layer), and the hydrogel array is loaded on the hydrophilic surface on the inner side of the substrate, namely the hydrophilic surface plays a role in connecting the glass sheet substrate and the hydrogel. The inner side of the cover plate is in direct contact with the top of the microgel column, but no physical connection exists.
Preferably, the diameter of the microgel column is 25-100 μm, and the distance between adjacent microgel columns is 25-120 μm; the sample adding hole is a round hole with the diameter of 0.5-2 mm, the length of the sample flow channel is 0.5-1.5 cm (for example, 1 cm), the width of the sample flow channel is 0.2-1 mm (for example, 0.5 mm), and one end of the sample flow channel is connected with the sample adding hole through a through hole (for example, the diameter of 1.5 mm) on the middle layer.
Preferably, the hydrogel array comprises a plurality of microgel columns which are formed by polyethylene glycol diacrylate and a photosensitizer through an ultraviolet cross-linking mask method (ultraviolet irradiation is carried out on a mixed aqueous solution of the polyethylene glycol diacrylate and the photosensitizer under the coverage of a photomask template) and are orderly and alternately arranged according to a certain shape area, the shape (such as a cylinder) of each microgel column corresponds to the shape (such as a circle) of a light-transmitting area of the photomask template, the diameter and the distance of microgel can be changed along with the size of the used template, and the water absorption volume of each dried microgel column is 0.2-5 nanoliter.
The preparation method of the digital PCR chip comprises the following steps:
1) Formation of chip housing
The double faced adhesive tape is used as an intermediate layer, and patterns such as the hollowed-out window, the hollowed-out strip and the like are processed on the intermediate layer; then, adhering one side surface of the double-sided adhesive to the substrate, and avoiding bubbles between the double-sided adhesive and the substrate during adhesion; adhering the cover plate to the surface of the other side of the double-sided adhesive tape to ensure that the sample adding holes processed on the two sides of the cover plate are correspondingly communicated with the two hollowed-out strips;
2) Formation of hydrogel arrays
A mixed aqueous solution of polyethylene glycol diacrylate (PEG-DA) with Mn (number average molecular weight) of 190-630 and a photosensitizer (2-hydroxy-2-methyl propiophenone) is diffused through a sample flow channel (a channel surrounded by the hollowed-out strip, the cover plate and the substrate) at one side through a sample adding hole and filled into a cavity (namely, the whole space area corresponding to the hollowed-out window in the chip shell) surrounded by the hollowed-out window, the cover plate and the substrate, then a photomask template with a plurality of light holes is placed outside the substrate and covers the area where the hollowed-out window is located, ultraviolet irradiation is carried out on the photomask template, and the mixed aqueous solution filled in the cavity is crosslinked by ultraviolet light penetrating through the photomask template; wherein the volume fraction of PEG-DA in the mixed aqueous solution is 10% -50% (e.g. 20%), the volume fraction of photosensitizer is 0.2% -1%, and the distribution and size of the light holes on the photomask template determine the diameter and spacing of the microgel columns in the hydrogel array. After the irradiation is finished, the uncrosslinked PEG-DA solution is discharged through a sample-adding hole on the other side in the form of external force (such as injection of pure water or air), and then is placed into a freeze dryer for freeze drying (freeze drying) to remove the moisture in and outside the crosslinked product for later use.
Preferably, the sample adding hole is etched by a laser cutting machine, and the hollowed pattern on the intermediate layer is designed by Core DRAW software and is etched by the laser cutting machine.
Preferably, the specific preparation flow of the substrate is as follows: the surface hydrophilic treatment is carried out on the glass sheet, namely, the glass sheet is soaked in 3-silane propyl methacrylate (TMS-PMA) for 10 to 12 hours at the temperature of 75 to 85 ℃.
Preferably, the light intensity of the ultraviolet light is 60-100 mW/cm 2 The irradiation direction is perpendicular to the photomask template, the irradiation time is 100-150 seconds, and the environment is room temperature. Neither the high nor low light intensity results in the desired microgel structure. Irradiation perpendicular to the photomask template (90 degrees) was performed to produce non-interconnected microgel columns.
Preferably, the freeze drying time is 2-3 days, the temperature is-90 ℃ to-100 ℃, and the pressure is 0.6-1 pa.
The use method of the digital PCR chip comprises the following steps:
1) Dropwise adding a hydrophilic sample solution into a sample adding hole at one side, automatically diffusing the sample solution into the cavity along the flow channel and the micro channel by capillary force, and dispersing the sample solution into a micro gel column by a hydrogel array in the cavity due to the porous sponge structure of the freeze-dried hydrogel;
2) After the step 1), injecting sealing oil into the sample adding hole, filling the part outside the hydrogel array in the cavity by using the sealing oil, and discharging undispersed solution and air in the cavity to form a plurality of independent closed micro-reaction systems taking the micro-gel column as a unit.
And after the nucleic acid sample is filled with sealing oil, placing one side of a glass substrate of the chip on a variable-temperature heating table, and running a temperature control program of the PCR reaction.
Preferably, the injection is by means of a syringe.
Preferably, the sample solution is a solution containing nucleic acid or a pigment solution.
Preferably, the nucleic acid concentration is less than 1063 copies/microliter.
The beneficial effects of the invention are as follows:
the invention fully utilizes the capillary action of the gap between the cover plate and the substrate (namely the sample flow channel and the micro flow channel), and the characteristics of soft texture, good biocompatibility and good hydrophilicity of the hydrogel, so that the hydrophilic sample (such as nucleic acid) solution can be absorbed, monodisperse and remain in each micro gel column when flowing through the hydrogel array, and then the introduced sealing oil is combined, thus the complete isolation of each micro reaction system in the micro gel column can be realized, the reliability of the micro reaction system is high, and no cross contamination exists. The chip has the advantages of novel design, simple and convenient manufacture, low cost, good biocompatibility, convenient operation and wide application range.
Further, the water absorption volume of the microgel columns can be determined by controlling the distance and the diameter of the microgel columns in the array, and can be used for accurately controlling the volume of a micro-reaction system.
Drawings
FIG. 1 is a schematic diagram of the structure of a hydrogel digital PCR chip according to the present invention;
FIG. 2 is a schematic diagram of the multi-layer assembly of hydrogel digital PCR chip components according to the present invention;
FIG. 3 is a diagram of a hydrogel digital PCR chip according to the present invention;
FIG. 4a is a scanning electron microscope image (side view of a microgel array) of a hydrogel digital PCR chip according to the present invention (dried);
FIG. 4b is a scanning electron microscope image (top view of the microgel array) of the hydrogel digital PCR chip according to the invention (dried);
FIG. 5 is a flow chart showing the diffusion of the hydrogel digital PCR chip to the hydrophilic dye solution according to the present invention; wherein: (a) The lyophilized microgel array, the upper left is the appearance of the lyophilized hydrogel digital PCR chip, the lower left is the lyophilized microgel array optical micrograph (10 x); (b) Loading a liquid sample, wherein the middle upper part is the diffusion appearance of the red pigment solution in the chip, and the middle lower part is a microgel array optical microscope photo loaded with the red pigment solution; (c) The dispersed liquid sample has the appearance of a chip with the red pigment solution dispersed, and has the optical microscopic photograph of the microgel array with the red pigment solution dispersed.
FIG. 6 is an optical micrograph (20 Xof magnification) of a microgel array of different sizes inside a hydrogel digital PCR chip according to the invention, wherein a represents the diameter of the microgel columns and d represents the distance between adjacent microgel columns;
FIG. 7a is a graph showing the relationship between the diameter and spacing of the microgel columns in the hydrogel digital PCR chip and the number of microgel columns contained in the array according to the present invention;
FIG. 7b is a graph showing the relationship between the diameter and spacing of the microgel columns in the hydrogel digital PCR chip and the water absorption capacity of a single lyophilized microgel column according to the present invention;
FIG. 8 is a fluorescence image (20 times) of the hydrogel digital PCR chip of the present invention performing PCR amplification reactions of nucleic acid samples of different concentrations; wherein: (a) a blank (0 copies/microliter); (b) 70 copies/microliter of the sample solution; (c) 140 copies/microliter of the sample solution; (d) 280 copies/microliter of the sample solution; (e) 550 copies/microliter of the sample solution; (f) 1100 copies/microliter of the sample solution;
in the figure: 1 is a PMMA plate, 2 is a sample adding hole, 3 is an intermediate layer, 4 is a hydrogel array, 5 is a glass substrate, 6 is a through hole, 7 is a hollowed-out strip, and 8 is a hollowed-out window.
Detailed Description
The invention will now be described in detail with reference to the drawings and examples.
Referring to fig. 1, 2 and 4a and 4b, the present invention provides a hydrogel digital PCR chip, which comprises a PMMA plate 1 (1 mm thick, PMMA is polymethyl methacrylate, i.e. organic glass), a sample well 2, an intermediate layer 3, a micro hydrogel array 4 and a glass substrate 5 (glass sheet). The loading well 2 was etched on the PMMA plate 1 by a laser cutter (U.S. Universal, VLS 2.30), and the size of the loading well was 1.5mm in diameter. The middle layer 3 is made of acrylic double faced adhesive tape (50 mu m), one surface of the middle layer 3 is connected with the inner side of the PMMA plate 1, and the other surface of the middle layer 3 is connected with the hydrophilic treated glass substrate 5. The middle layer 3 is designed by Core DRAW software and is etched by a laser cutting machine to form a hollow pattern in the shape of a sugarcoated haw, and the hollow pattern specifically comprises a hollow window 8 containing three communicated circular holes (namely three array areas) and through holes 6 positioned at two sides of the hollow window 8, wherein the two through holes 6 are connected with two circular holes positioned at the outer side of the hollow window 8 through corresponding side hollow strips 7. The micro hydrogel array 4 is an array of 2300 to 46000 hydrogel microcolumns (i.e., microgel columns) formed by an ultraviolet crosslinking mask method (the diameter of the microcolumns is 25 to 100 μm; the distance between every two microcolumns is 25 to 120 μm; and) so that the micro hydrogel array may also be referred to as a microgel array. Is located in the space corresponding to the array region (formed by sealing the PMMA plate 1 and the glass substrate 5). The glass substrate 5, which has a thickness of 0.3cm, is subjected to 3-silane propyl methacrylate (TMS-PMA) overnight at 80 ℃, and then is washed 3 times with ethanol and pure water respectively, and naturally dried to form a hydrophilic surface, which plays a role in connecting the glass sheet substrate and the hydrogel.
The manufacturing process of the shell of the hydrogel digital PCR chip is exemplified as follows: firstly, adhering a TMS-PMA treated glass substrate 5 to one side of a double-sided adhesive tape 3, and avoiding bubbles between the two when adhering; and then one side of the PMMA plate 1 is adhered to the other side of the double faced adhesive tape 3, wherein the sample adding holes 2 at the two ends of the PMMA plate 1 are overlapped with the centers of the through holes 6 at the two ends of the double faced adhesive tape intermediate layer, so that a flow channel which is communicated with a space corresponding to the array area and the sample adding holes is formed by means of the hollowed-out strip 7.
Referring to fig. 3, the method for forming the micro hydrogel array 4 of the hydrogel digital PCR chip is exemplified as follows: a mixed solution (the volume fractions of the PEG-DA and the photosensitizer in the mixed solution are respectively 20 percent and 0.2 percent) of the prepared polyethylene glycol diacrylate (PEG-DA, mw=10 kDa and Mn= 575;Sigma Aldrich) and the photosensitizer (2-hydroxy-2-methyl propiophenone, tokyo Chemical Industry) is added in the three array areas by a sample Kong Jiaman, and then a photomask template is placed on the glass substrate side which is closely attached to a chip, and the light intensity value of 80mW/cm is given 2 The vertical irradiation of the ultraviolet light is carried out,the irradiation time was 120 seconds. The diameter of the light transmission hole of the photomask template is 50 μm, and the distance between the adjacent light transmission holes is 50 μm. The array region had a single diameter of 7mm, with the number of cylindrical hydrogel microcolumns formed by crosslinking being 11697. After the above operation, the uncrosslinked PEG-DA solution was discharged through the sample-loading hole by external force (injection of pure water or air by syringe), and was frozen in a freeze-dryer (Power DRY LL 1500) for 72 hours for freeze-drying (temperature of minus 100 ℃ and pressure of 0.7 pa), and the water absorption volume of the single lyophilized hydrogel microcolumn was about 1 nanoliter for use.
The volume of storable sample solution is approximately 8 μl corresponding to the 3 array regions of the hollowed-out window. Less than 8 mu L, part of the area can not absorb the sample, and the detection result can not be accurately calculated; more than 8. Mu.L, the excess sample is no longer absorbed and can only be discharged from the chip.
Referring to fig. 6 and fig. 7a and 7b, the number of microgel columns in the hydrogel digital PCR chip can be changed by adjusting the diameter and the spacing of the microgel columns, and the array of the microgel columns with different diameters and spacing can be subjected to ultraviolet crosslinking by replacing photomask templates with different specifications (light hole diameters and spacing). The water uptake of an individual microgel column also varies with its diameter and spacing. Referring to fig. 7a, as the diameter and spacing of the microgel columns increases, the total number of microgel columns decreases; referring to fig. 7b, as the diameter and spacing of the microgel columns increases, the water uptake of the individual microgel columns increases.
The hydrogel digital PCR chip has a very wide application range, can be used for dispersing nucleic acid solution within a certain concentration range for PCR amplification, and can detect one target nucleic acid in 46000 nucleic acid molecules with detection accuracy (detection limit); the chip can also automatically absorb hydrophilic colored sample solution for dispersion such as food coloring solution.
For dispersion of pigment sample, the pigment solution is dripped into the sample adding hole 2 at one side, the PMMA plate 1 is an organic polymer hydrophobic material with excellent light transmittance, and tiny gaps (comprising a runner connected with the sample adding hole and gaps between microgel columns) formed between the PMMA plate and the glass substrate 5 can generate capillary force, so that the pigment solution automatically diffuses to an array area of the microgel array 4 along the gaps between the glass substrate 5 and the PMMA plate 1 by the capillary force, at the moment, the solution is dispersed into each microgel column in the array area due to water absorption of a porous structure of the freeze-dried microgel column, and the porous structure (sponginess) of the microgel column keeps the solution in gel. And then adding sealing oil (mineral oil) into the sample adding hole, pushing the sealing oil into a flow channel by a syringe, flowing through the surface of the micro hydrogel array 4, pushing away unabsorbed and diffused pigment solution, filling the space around the micro hydrogel array 4, and removing air in a chip to play a role in sealing.
Dispersion of nucleic acid samples over a range of concentrations. The nucleic acid solution (less than 1063 copies/microliter) is dripped into the sample adding hole 2 at one side, the PMMA plate 1 is an organic polymer hydrophobic material with excellent light transmittance, and tiny gaps formed between the PMMA plate and the micro hydrogel array 4 can generate capillary force, so that the nucleic acid solution automatically diffuses to the array area of the micro hydrogel array 4 along the gap between the glass substrate 5 and the PMMA plate 1 by the capillary force, at the moment, the solution is dispersed into each micro gel column in the array area due to the water absorption of the porous structure of the freeze-dried micro gel column, the porous structure (spongy) of the micro gel column keeps the solution in gel, and the nucleic acid molecules in the solution are dispersed and enter the porous structure of each micro gel column.
A sealing oil (e.g., mineral oil) may then be slowly added through the loading well 2 to drain the non-absorbed and diffused nucleic acid solution within the hydrogel digital PCR chip flow channel and around the micro hydrogel array 4 while filling the space around the micro hydrogel array 4, the sealing oil also completely isolating each micro gel column of the micro hydrogel array 4 such that each micro gel column acts as a separate micro reaction system.
EXAMPLE 1 pigment solution was dispersed using hydrogel digital PCR chip
Step 1: and 8 microliters of pigment solution is dripped into a sample adding hole at one side of the PCR chip, and the pigment solution automatically flows to an array area of a miniature hydrogel array of the hydrogel digital PCR chip along the flow channel, and the lyophilized hydrogel has good water absorption, so that the pigment solution is dispersed into each microcolumn of the porous hydrogel to play a role in dispersing the pigment solution (see figure 5).
Step 2: after the step 1), external pressure is applied to the sample adding hole to inject sealing oil, the sealing oil is used for filling the part outside the micro hydrogel array, and meanwhile, air in the array area where the micro hydrogel array is positioned is discharged to form 11697 independent closed microsystems taking the micro gel column as a unit, so that the added 8 microliter pigment solution is uniformly dispersed into about 11697 microsystems with each volume of about 1 nanoliter, and the PCR chip has the capability of uniformly dispersing trace liquid samples (see figure 5).
EXAMPLE 2 dispersing nucleic acid solution with hydrogel digital PCR chip and performing PCR amplification
Step 1: the diluted standard nucleic acid solutions with different concentrations are mixed with PCR premix (comprising PCR polymerase, dNTP, PCR probes, primers and the like) (8 microliter after mixing), then 8 microliter of mixed solution is dripped into a sample adding hole at one side of a PCR chip, the mixed solution automatically diffuses to an array area where a micro hydrogel array is located along the flow channel by capillary force, and nucleic acid molecules in the solution are absorbed and dispersed into each microcolumn of the porous hydrogel due to the porous structure of the hydrogel after freeze-drying.
Step 2: injecting sealing oil, filling the part outside the micro hydrogel microarray by using the sealing oil, and discharging air in the array area where the micro hydrogel array is positioned to form 11697 mutually independent PCR micro-reaction systems, wherein in most cases, each micro-reaction system contains at most 1 copy of nucleic acid sequences, so that the mutual cross contamination among reaction units is effectively avoided. And placing one side of the glass substrate of the chip on a variable-temperature heating table, and running a temperature control program of the PCR reaction. After the PCR is finished, the PCR chip is placed under a fluorescence microscope, and a fluorescence detection image of the standard nucleic acid sample can be obtained (see FIG. 8). The brighter microgel column in FIG. 8 represents the amplification of nucleic acid sequences that bind to fluorescent probes, and the accumulation of fluorescence of interest during the amplification process causes the microgel column to illuminate. The number of the microgel columns corresponding to the target fluorescence is the proportion of all the microgel columns, and the concentration of the target nucleic acid sequence in the whole sample can be represented.
Compared with the traditional method for preparing the water-in-oil monodisperse nucleic acid sample, the method has the advantages of short sample preparation time, simple operation method and no need of a special sample adding device; the chip has low cost, and the manufacturing cost is reduced by more than 100 times; the detection flux can be flexibly controlled by adjusting the number of the serial array areas according to the size of the sample size. Meanwhile, the accuracy of the dispersed nucleic acid sample and the reliability of a reaction system are excellent, the cross contamination is small, and the biocompatibility is good.
Claims (9)
1. A digital PCR chip, characterized in that: the device comprises a chip shell, a hydrogel array (4) and a sample runner, wherein the hydrogel array (4) and the sample runner are arranged in the chip shell, the hydrogel array (4) comprises a plurality of dried microgel columns fixed on the inner wall of the chip shell, and gaps are reserved among the microgel columns; the chip shell is provided with a sample adding hole (2), the sample adding hole (2) is communicated with the gap through a sample flow channel, and hydrophilic sample solution automatically diffuses into the gap between the microgel columns along the sample flow channel communicated with the sample adding hole (2) by capillary force and disperses into the microgel columns;
the hydrogel array (4) comprises a plurality of orderly and alternately arranged microgel columns formed by polyethylene glycol diacrylate and a photosensitizer through an ultraviolet cross-linking mask method, the shape of each microgel column corresponds to the shape of a light-transmitting area of a photomask template, and the water absorption volume of each dried microgel column is 0.2-5 nanoliters;
the number of microgel columns distributed in the array area where the hydrogel array (4) is positioned is 2300-46000;
the diameter of the microgel column is 25-100 mu m, and the distance between adjacent microgel columns is 25-120 mu m.
2. The digital PCR chip as set forth in claim 1, wherein: the chip shell comprises a cover plate, an intermediate layer (3) and a substrate which are sequentially stacked, wherein one side surface of the intermediate layer (3) is connected with the cover plate, and the other side surface of the intermediate layer (3) is connected with the substrate; be provided with fretwork window (8) and two fretwork strips (7) that link to each other with this fretwork window (8) on intermediate level (3), hydrogel array (4) are located fretwork window (8), are provided with two application of sample holes (2) on the apron, and two application of sample holes (2) correspond the intercommunication with two fretwork strips (7) on intermediate level (3) respectively, and the passageway that fretwork strips (7) and apron and basement enclose is the sample runner.
3. The digital PCR chip as set forth in claim 2, wherein: the hollow window (8) comprises 1-10 circular hole areas which are connected in series, and the edge of the hollow window (8) is in a symmetrical wavy shape; two through holes (6) are formed in the middle layer (3), the two sample adding holes (2) are connected with one ends, far away from the hollow window (8), of the corresponding hollow strips (7) through the two through holes (6), and the other ends of the two hollow strips (7) are correspondingly connected with two circular hole areas located at the outermost sides of the hollow window (8) respectively.
4. A digital PCR chip according to claim 3, wherein: the diameter of the circular hole area is less than or equal to 7mm, and the number of microgel columns distributed in the circular hole area is 2300-46000.
5. The digital PCR chip as set forth in claim 2, wherein: the cover plate is made of a PMMA plate (1), and the thickness of the PMMA plate (1) is 0.4-2 mm; the middle layer (3) adopts double-sided adhesive tape, and the thickness of the middle layer (3) is 50-200 mu m; the substrate is made of light-transmitting materials, the thickness of the substrate is 0.3-1 mm, and the surface of the substrate is hydrophilic.
6. The digital PCR chip as set forth in claim 1, wherein: the sample adding hole (2) is a round hole with the diameter of 0.5-2 mm, the length of the sample flow channel is 0.5-1.5 cm, and one end of the sample flow channel is connected with the sample adding hole (2) through a through hole (6) arranged on the middle layer (3).
7. A method of preparing a digital PCR chip according to claim 2, wherein: the method comprises the following steps:
1) Formation of chip housing
Processing a hollowed pattern comprising the hollowed window (8) and the hollowed strip (7) on the middle layer (3); then, adhering one side surface of the intermediate layer (3) to a light-transmitting substrate with a hydrophilic surface; adhering the cover plate to the surface of the other side of the middle layer (3) to ensure that two sample adding holes (2) processed on the cover plate are correspondingly communicated with two hollowed-out strips (7);
2) Formation of hydrogel arrays
The method comprises the steps of diffusing and filling a mixed aqueous solution of polyethylene glycol diacrylate and a photosensitizer into a cavity surrounded by a hollowed-out window (8), a cover plate and a substrate through one sample adding hole through a sample runner communicated with the sample adding hole, then placing a photomask template with a plurality of light holes outside the substrate and covering the area where the hollowed-out window (8) is located, then irradiating ultraviolet light on the photomask template, and crosslinking the polyethylene glycol diacrylate positioned in the cavity by means of the ultraviolet light penetrating through the photomask template; wherein the volume fraction of polyethylene glycol diacrylate in the mixed aqueous solution is 10-50%, and the volume fraction of the photosensitizer is 0.2-1%; after the irradiation is finished, the uncrosslinked polyethylene glycol diacrylate is discharged through another sample-adding hole, and then the moisture in the crosslinked product and the moisture in the crosslinked product are removed through drying, so that an internal porous sponge structure is formed.
8. The method according to claim 7, wherein: the light intensity of the ultraviolet light is 60-100 mW/cm 2 The irradiation direction is perpendicular to the photomask template, and the irradiation time is 100-150 seconds.
9. A method of using the digital PCR chip as claimed in claim 2, wherein: the method comprises the following steps:
1) Dripping hydrophilic sample solution into a sample adding hole, and automatically diffusing the sample solution into gaps between the microgel columns along a sample flow channel communicated with the sample adding hole by capillary force and dispersing the sample solution into a porous sponge structure in the microgel columns;
2) After the step 1), injecting sealing oil into the sample adding holes, filling gaps among the microgel columns by using the sealing oil, and discharging air and undispersed hydrophilic sample solution in a cavity surrounded by the hollow window (8), the cover plate and the substrate through the other sample adding holes to form a mutually independent closed micro-reaction system taking the microgel columns as units.
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| CN113308351B (en) * | 2020-02-26 | 2022-12-27 | 京东方科技集团股份有限公司 | Detection chip, preparation method thereof and reaction system |
| CN113070113B (en) * | 2021-06-03 | 2021-08-20 | 成都齐碳科技有限公司 | Chip structure, film forming method, nanopore sequencing device and application |
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