CN111562721A - Preparation method of micro-reaction pool array for high-throughput pyrosequencing chip - Google Patents
Preparation method of micro-reaction pool array for high-throughput pyrosequencing chip Download PDFInfo
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- 238000006243 chemical reaction Methods 0.000 title claims abstract description 93
- 238000012175 pyrosequencing Methods 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 239000013307 optical fiber Substances 0.000 claims abstract description 132
- 229920001486 SU-8 photoresist Polymers 0.000 claims abstract description 63
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 32
- 238000004140 cleaning Methods 0.000 claims abstract description 32
- 229920002120 photoresistant polymer Polymers 0.000 claims abstract description 32
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims description 43
- 230000008569 process Effects 0.000 claims description 29
- 238000012163 sequencing technique Methods 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 238000001816 cooling Methods 0.000 claims description 15
- 238000001035 drying Methods 0.000 claims description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 12
- 239000008367 deionised water Substances 0.000 claims description 12
- 229910021641 deionized water Inorganic materials 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 9
- 238000000576 coating method Methods 0.000 claims description 8
- 239000000835 fiber Substances 0.000 claims description 7
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- XSTXAVWGXDQKEL-UHFFFAOYSA-N Trichloroethylene Chemical group ClC=C(Cl)Cl XSTXAVWGXDQKEL-UHFFFAOYSA-N 0.000 claims description 6
- 239000011248 coating agent Substances 0.000 claims description 6
- 238000007599 discharging Methods 0.000 claims description 6
- UBOXGVDOUJQMTN-UHFFFAOYSA-N trichloroethylene Natural products ClCC(Cl)Cl UBOXGVDOUJQMTN-UHFFFAOYSA-N 0.000 claims description 6
- 238000004506 ultrasonic cleaning Methods 0.000 claims description 6
- 239000011521 glass Substances 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- 238000002791 soaking Methods 0.000 claims description 3
- 238000002493 microarray Methods 0.000 claims 8
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 claims 6
- 235000011180 diphosphates Nutrition 0.000 claims 6
- 238000000861 blow drying Methods 0.000 claims 1
- 230000008646 thermal stress Effects 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 4
- 230000005540 biological transmission Effects 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 239000004005 microsphere Substances 0.000 description 3
- 238000007664 blowing Methods 0.000 description 2
- XPPKVPWEQAFLFU-UHFFFAOYSA-N diphosphoric acid Chemical compound OP(O)(=O)OP(O)(O)=O XPPKVPWEQAFLFU-UHFFFAOYSA-N 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- LLHKCFNBLRBOGN-UHFFFAOYSA-N propylene glycol methyl ether acetate Chemical compound COCC(C)OC(C)=O LLHKCFNBLRBOGN-UHFFFAOYSA-N 0.000 description 2
- 229940005657 pyrophosphoric acid Drugs 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 239000004593 Epoxy Substances 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003822 epoxy resin Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000003365 glass fiber Substances 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 238000001459 lithography Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229920000647 polyepoxide Polymers 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
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- 238000000926 separation method Methods 0.000 description 1
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- G—PHYSICS
- G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
- G03F—PHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR
- G03F7/00—Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
- G03F7/16—Coating processes; Apparatus therefor
- G03F7/168—Finishing the coated layer, e.g. drying, baking, soaking
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B08—CLEANING
- B08B—CLEANING IN GENERAL; PREVENTION OF FOULING IN GENERAL
- B08B3/00—Cleaning by methods involving the use or presence of liquid or steam
- B08B3/04—Cleaning involving contact with liquid
- B08B3/08—Cleaning involving contact with liquid the liquid having chemical or dissolving effect
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B08—CLEANING
- B08B—CLEANING IN GENERAL; PREVENTION OF FOULING IN GENERAL
- B08B3/00—Cleaning by methods involving the use or presence of liquid or steam
- B08B3/04—Cleaning involving contact with liquid
- B08B3/10—Cleaning involving contact with liquid with additional treatment of the liquid or of the object being cleaned, e.g. by heat, by electricity or by vibration
- B08B3/12—Cleaning involving contact with liquid with additional treatment of the liquid or of the object being cleaned, e.g. by heat, by electricity or by vibration by sonic or ultrasonic vibrations
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
- C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
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- G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
- G03F—PHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR
- G03F7/00—Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
- G03F7/16—Coating processes; Apparatus therefor
- G03F7/162—Coating on a rotating support, e.g. using a whirler or a spinner
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- G—PHYSICS
- G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
- G03F—PHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR
- G03F7/00—Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
- G03F7/20—Exposure; Apparatus therefor
- G03F7/2002—Exposure; Apparatus therefor with visible light or UV light, through an original having an opaque pattern on a transparent support, e.g. film printing, projection printing; by reflection of visible or UV light from an original such as a printed image
- G03F7/2004—Exposure; Apparatus therefor with visible light or UV light, through an original having an opaque pattern on a transparent support, e.g. film printing, projection printing; by reflection of visible or UV light from an original such as a printed image characterised by the use of a particular light source, e.g. fluorescent lamps or deep UV light
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- G03F7/00—Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
- G03F7/26—Processing photosensitive materials; Apparatus therefor
- G03F7/30—Imagewise removal using liquid means
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- G03F7/26—Processing photosensitive materials; Apparatus therefor
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- G03F7/00—Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
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Abstract
The invention relates to a preparation method of a micro reaction pool array for a high-throughput pyrosequencing chip, which comprises the following steps: step 1, cleaning an optical fiber panel; step 2, obtaining an SU8 photoresist film; step 3, carrying out heat baking on the optical fiber panel and the SU8 photoresist film, and step 4, exposing the SU8 photoresist film; step 5, re-baking the optical fiber panel and the graphical photoresist film, and placing the optical fiber panel and the graphical photoresist film in a nitrogen environment to cool to room temperature after baking; and 6, developing the patterned photoresist film to obtain a required micro-reaction cell array on the optical fiber panel. The invention can effectively prepare the micro-reaction tank, is compatible with the prior art, and is safe and reliable.
Description
Technical Field
The invention relates to a preparation method, in particular to a preparation method of a micro-reaction cell array for a high-throughput pyrosequencing chip, belonging to the technical field of pyrosequencing.
Background
Among the existing 2 nd generation sequencers, the high-throughput gene sequencer based on the pyrosequencing principle has the advantages of high sequencing speed, long reading length and the like, and has irreplaceable effects on de novo sequencing and metagenome sequencing. The high-throughput gene sequencer completes the sequencing process through an array type micro-reaction pool sequencing chip on the basis of the pyrosequencing principle, and the basic working principle is as follows: filling the microspheres coated with the amplified DNA library and enzyme molecules into array type micro reaction pools of a sequencing chip, so that the pyrosequencing reaction is independently carried out in each micro reaction pool, and the sequencing flux is determined by the number of the array type micro reaction pools. The back of the sequencing chip is closely attached to a CCD (Charge-coupled device), and optical signals released by sequencing reaction are transmitted to the CCD from the bottom of the micro reaction pool in parallel and are output in an image form. And analyzing the images generated in the whole sequencing process, and then aligning and superposing to obtain the base sequence of the DNA library to be detected.
At present, a high-throughput gene sequencer based on a pyrophosphoric acid sequencing principle mainly comprises a 454 sequencer and a BIGIS-4 sequencer, wherein the used sequencing chips are PTP (Pico Titer plate) developed by Roche company, only the Roche company has the capacity of producing PTP at present, the price is high, about $ 1000/piece, and the high-throughput gene sequencer cannot be reused; in addition, the PTP preparation process is complicated and protected by the related patent art.
Therefore, how to prepare a micro-reaction cell for a high-throughput pyrosequencing chip is a current problem.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a preparation method for a high-throughput pyrosequencing chip micro-reaction cell array, which can effectively prepare a micro-reaction cell, is compatible with the prior art, can effectively reduce the cost, and is safe and reliable.
According to the technical scheme provided by the invention, the preparation method of the micro reaction cell array for the high-throughput pyrosequencing chip comprises the following steps:
and 6, developing the graphical photoresist film to obtain a required micro-reaction tank array on the optical fiber panel, wherein the micro-reaction tank array comprises a plurality of micro-reaction tanks, each micro-reaction tank comprises a reaction tank body vertical to the optical fiber panel and a tank body groove positioned in the central area in the reaction tank body, and the tank body groove penetrates through the reaction tank body.
After obtaining the micro-reaction cell array on the optical fiber panel, placing the optical fiber panel with the micro-reaction cell array in an oven, wherein the temperature of the oven is set to be 180-210 ℃ and kept for 2h, and then gradually cooling to the normal temperature.
In the gradual cooling process, the temperature in the oven is sequentially reduced to 150 ℃, 100 ℃ and 50 ℃, and finally the temperature in the oven is reduced to normal temperature; wherein, after the temperature in the oven is reduced to 150 ℃, the temperature in the oven is kept at 150 ℃ for 10min to 15min, and after the temperature in the oven is reduced to 100 ℃, the temperature in the oven is kept at 100 ℃ for 10min to 15 min; and after the temperature in the oven is reduced to 50 ℃, keeping the temperature in the oven at 50 ℃ for 10-15 min.
The depth of the reaction tank body is 30 μm, the thickness of the side wall of the reaction tank body is 6 μm, and the width of the tank body groove is 28 μm.
In step 1, the cleaning process for the optical fiber panel comprises the following steps:
step 1.1, placing the optical fiber panel in glass water, cleaning the optical fiber panel by using deionized water after ultrasonic cleaning, and drying the optical fiber panel by using nitrogen after cleaning the optical fiber panel by using the deionized water;
step 1.2, cleaning the optical fiber panel by using plasma, after cleaning, sequentially adopting trichloroethylene, acetone and ethanol to carry out organic cleaning on the optical fiber panel, and after organic cleaning, cleaning the optical fiber panel by using deionized water;
step 1.3, drying the cleaned optical fiber panel by adopting nitrogen, and placing the dried optical fiber panel at 110-130 ℃ for heat drying for 20-40 min; after the heat drying, the optical fiber panel is cooled to normal temperature and then placed in a nitrogen cabinet.
In the step 2, the coating process for obtaining the SU8 photoresist film comprises the following steps:
step 2.1, providing SU8 photoresist liquid, standing and discharging bubbles, taking 10ml of SU8 photoresist liquid after standing and discharging bubbles, and dripping the SU8 photoresist liquid on an optical fiber panel arranged on a spin coater tray;
2.2, rotating the optical fiber panel by using a spin coater, wherein during rotation, the optical fiber panel rotates for 10 to 20 seconds at 600 to 700r/min, then rotates for 30 to 40 seconds at 2000 to 2500r/min, and finally rotates for 10 to 20 seconds at 800 to 1000 r/min; after the rotation is finished, SU8 photoresist film with 25-30 μm can be obtained on the optical fiber panel.
In step 4, the SU8 photoresist film is exposed under 365nm ultraviolet rays, and the exposure dose is 170mJ/cm2～200mJ/cm2。
In step 6, the developing process includes the following steps:
6.1, soaking the optical fiber panel and the graphical photoresist film in a developing solution for 1-2 min, then shaking for 20-30 s, and finally developing the optical fiber panel and the graphical photoresist film in the developing solution for 10-30 min;
and 6.2, developing the optical fiber panel and the patterned photoresist film for 6-12 min by using a new developing solution, and rinsing the optical fiber panel and the patterned photoresist film for 2-10 min by using isopropanol after the development so as to obtain the required micro-reaction cell array on the optical fiber panel.
The cross section of the tank body groove is hexagonal.
The invention has the advantages that: coating an SU8 photoresist film on the optical fiber panel, and performing pre-baking, exposure, post-baking, development and other processes on the SU8 photoresist film to prepare a micro-reaction cell array on the optical fiber panel; in the glue homogenizing process, the gradient acceleration and deceleration is adopted for the glue homogenizing speed of the optical fiber panel, so that the influence of quick termination on the surface flatness of the SU8 photoresist film is avoided; because the SU8 thermal expansion coefficient is higher, the pre-baking process and the post-baking process can bring thermal stress to the SU8 photoresist film, and simultaneously the SU8 is easy to fall off from the surface of the optical fiber panel due to the thermal expansion performance difference of the SU8 and the optical fiber panel, in order to effectively relieve the SU8 thermal stress and increase the adhesion of the SU8 thermal stress and the substrate, the gradient temperature rising and falling mode is adopted to control the thermal baking process in the pre-baking process and the post-baking process, so that the expansion deformation of the SU8 photoresist can be avoided, the micro-reaction tank can be effectively prepared, the micro-reaction tank is compatible with the existing process, the sequencing of high-flux pyrophosphoric acid can be realized, the cost can be effectively reduced.
Drawings
FIGS. 1-4 are cross-sectional views of process steps in accordance with an embodiment of the present invention, wherein
FIG. 1 is a cross-sectional view of a fiber optic faceplate of the present invention.
FIG. 2 is a cross-sectional view of a fiber optic faceplate having a SU8 photoresist film according to the present invention.
FIG. 3 is a cross-sectional view of a SU8 photoresist film patterned using a reticle in accordance with the present invention.
FIG. 4 is a cross-sectional view of a micro reaction cell array formed on a fiber optic faceplate according to the present invention.
FIG. 5 is a top view of the micro reaction cell array of the present invention on a fiber optic faceplate and an enlarged schematic view of the micro reaction cell.
Description of reference numerals: 1-optical fiber panel, 2-SU8 photoresist film, 3-mask, 4-patterned photoresist film, 5-micro reaction tank, 6-tank and 7-reaction tank.
Detailed Description
The invention is further illustrated by the following specific figures and examples.
As shown in fig. 1 to 5: in order to effectively prepare and obtain the micro-reaction tank, the preparation method of the micro-reaction tank array comprises the following steps:
as shown in fig. 1, which is a cross-sectional view of a fiber optic faceplate 1, in the embodiment of the present invention, the fiber optic faceplate 1 is used as a substrate material; the optical fiber panel 1 has the characteristics of high light transmission efficiency, small interstage coupling loss, clear and real image transmission, zero thickness in optics and the like, and is suitable for weak light signals or image transmission, and specifically, the cleaning process of the optical fiber panel 1 comprises the following steps:
step 1.1, placing the optical fiber panel 1 in glass water, cleaning with deionized water after ultrasonic cleaning, and drying the optical fiber panel 1 with nitrogen after cleaning with deionized water;
specifically, the concentration of the glass water can be 1%, in the ultrasonic cleaning, the ultrasonic power can be 100W-1000W, the ultrasonic cleaning is carried out for 10min, the glass fiber panel 1 is placed for more than 2 hours after the ultrasonic cleaning, then the optical fiber panel is cleaned for 10min under the ultrasonic with the power of 100W-1000W, and the ultrasonic vibration frequency is 50 KHz-150 KHz during the ultrasonic vibration. The process of cleaning with deionized water and blowing the optical fiber panel 1 with nitrogen is the same as the conventional cleaning and blowing processes, and is well known to those skilled in the art, and will not be described herein again.
Step 1.2, cleaning the optical fiber panel 1 by using plasma, after cleaning, sequentially adopting trichloroethylene, acetone and ethanol to carry out organic cleaning on the optical fiber panel 1, and after organic cleaning, cleaning the optical fiber panel 1 by using deionized water;
in the embodiment of the invention, in the process of carrying out organic cleaning by using trichloroethylene, acetone and ethanol, deionized water is not needed for washing the optical fiber panel 1, and after the ethanol cleaning is finished, the optical fiber panel 1 is cleaned by using the deionized water, so that the optical fiber panel 1 can be cleaned for more than 20 times. The selectable concentration of trichloroethylene is 99.5%, the selectable concentration of acetone is 99.5%, and the selectable concentration of ethanol is 99.5%, of course, trichloroethylene, acetone and ethanol may also be selected from other required concentrations, which may be specifically selected as required, and are specifically known to those skilled in the art, and will not be described herein again.
Step 1.3, drying the cleaned optical fiber panel 1 by adopting nitrogen, and placing the dried optical fiber panel 1 at the temperature of 110-130 ℃ for heating and drying for 20-40 min; after the heat-baking, the optical fiber panel 1 is cooled to room temperature and then placed in a nitrogen cabinet.
In the embodiment of the invention, the conventional common high-purity nitrogen is adopted to blow the optical fiber panel 1, then the optical fiber panel 1 is placed in an oven at the temperature of 110-130 ℃ for heating for 20-40 min, and the temperature is reduced to the normal temperature in the oven, and then the optical fiber panel 1 is placed in a nitrogen cabinet for standby. After heating, the moisture on the optical fiber panel 1 can be removed.
specifically, the coating process of obtaining the SU8 photoresist film 2 comprises the following steps:
step 2.1, providing SU8 photoresist liquid, standing and discharging bubbles, taking 10ml of SU8 photoresist liquid after standing and discharging bubbles, and dripping the SU8 photoresist liquid on the optical fiber panel 1 arranged on the spin coater tray;
in the embodiment of the invention, SU8 photoresist can be SU-82025 photoresist. The SU8 photoresist has excellent mechanical properties and better biocompatibility. SU8 is a negative, epoxy, near-uv photoresist with a high aspect ratio >5:1 and is biocompatible. SU8 can be applied to thick resist lithography processes.
The SU8 photoresist needs to be left to stand before coating to expel air bubbles in the SU8 photoresist. In order to uniformly coat SU8 photoresist solution on the optical fiber panel 1, place the optical fiber panel 1 on the tray of the spin coater, open the vacuum adsorption button of the spin coater, ensure that the optical fiber panel 1 can be firmly adsorbed on the tray of the spin coater, wherein the spin coater can adopt the equipment commonly used in the technical field, the processes of specifically utilizing the spin coater to adsorb the optical fiber panel 1 and driving the optical fiber panel 1 to rotate are all consistent with the prior art, and are specifically known to those skilled in the art, and the process is not repeated herein.
2.2, rotating the optical fiber panel 1 by using a spin coater, wherein during rotation, the optical fiber panel 1 rotates for 10 to 20 seconds at 600 to 700r/min, then rotates for 30 to 40 seconds at 2000 to 2500r/min, and finally rotates for 10 to 20 seconds at 800 to 1000 r/min; after the rotation, the motor of the spin coater is turned off, the optical fiber panel 1 rotates under inertia until the rotation is stopped, and after the rotation is finished, the SU8 photoresist film 2 with the thickness of 25-30 μm can be obtained on the optical fiber panel 1.
As shown in fig. 2, a cross-sectional view of a photoresist film 2 is obtained 8 on an optical fiber panel 1. When the process is adopted to homogenize the glue on the optical fiber panel 1, the influence of quick termination on the surface flatness of the SU8 photoresist film 2 is avoided due to the adoption of gradient speed reduction.
specifically, the optical fiber panel 1 is placed on a hot plate to realize the thermal baking of the optical fiber panel 1 and the SU8 photoresist film 2, i.e., the pre-baking process. In specific implementation, due to the fact that the thermal expansion coefficient of the SU8 photoresist is high, thermal stress is brought to the SU8 photoresist film 2 in the hot baking process, and meanwhile due to the difference of the thermal expansion performance of the SU8 photoresist film 2 and the optical fiber panel 1, the SU8 photoresist film 2 is easy to fall off from the surface of the optical fiber panel 1. In order to effectively relieve the thermal stress of the SU8 photoresist film 2 and increase the adhesion of the SU8 photoresist film to the optical fiber panel 1, in the embodiment of the invention, the pre-baking is performed in a gradient temperature rise and drop manner, so that the problem caused by expansion and deformation of the SU8 photoresist in the pre-baking process can be effectively avoided.
as shown in FIG. 3, the SU8 photoresist film 2 was exposed by using a reticle 3, and the SU8 photoresist film 2 was exposed by 365nm ultraviolet rays at an exposure dose of 170mJ/cm2～200mJ/cm2。
specifically, the optical fiber panel 1 and the patterned photoresist film 4 are placed on a hot plate for thermal baking, i.e., a post-baking process is performed. In the embodiment of the invention, due to the difference between the thermal expansion coefficients of the optical fiber panel 1 and the SU8, a large thermal stress is generated, which is consistent with the thermal stress in the pre-baking process, and besides the thermal stress, epoxy resin molecules in the SU8 are further crosslinked under thermal catalysis, and a large internal stress is generated, so that the expansion deformation of the optical fiber panel is also avoided by adopting a gradient temperature rise and decrease mode.
And 6, developing the patterned photoresist film to obtain a required micro-reaction tank array on the optical fiber panel 1, wherein the micro-reaction tank array comprises a plurality of micro-reaction tanks 5, the micro-reaction tanks 5 comprise reaction tank bodies 7 vertical to the optical fiber panel 1 and tank body grooves 6 located in the central area of the reaction tank bodies 7, and the tank body grooves 6 penetrate through the reaction tank bodies 7.
Specifically, the developing method comprises the following steps:
6.1, soaking the optical fiber panel 1 and the patterned photoresist film in a developing solution for 1-2 min, then shaking for 20-30 s, and finally developing the optical fiber panel 1 and the patterned photoresist film in the developing solution for 10-30 min;
and 6.2, developing the optical fiber panel 1 and the patterned photoresist film for 6-12 min by using a new developing solution, and rinsing the optical fiber panel 1 and the patterned photoresist film for 2-10 min by using isopropanol after the development so as to obtain the required micro-reaction cell array on the optical fiber panel 1.
In the embodiment of the invention, the developing solution is a PGMEA solution, the selectable concentration is 99.5%, and the total developing time is 20-30 min. The specific development process using PGMEA is consistent with the prior art, and is well known in the art, and will not be described herein.
As shown in fig. 5, after development, a multi-micro reaction cell array is formed on the optical fiber panel 1, wherein the micro reaction cell array includes a plurality of micro reaction cells 5, and each micro reaction cell 5 is independent of each other. For each micro reaction tank 5, a reaction tank body 7 is included, namely the reaction tank body 7 is prepared from the SU8 photoresist film 2. The reaction tank body 7 is vertically distributed on the optical fiber panel 1, the reaction tank body 7 is in a hexahedron shape, and the cross section of the tank body groove 6 is in a hexagon shape. In specific implementation, the depth h of the reaction tank body 7 is 30 μm, the thickness w of the side wall of the reaction tank body 7 is 6 μm, and the width l of the tank body groove 6 is 28 μm. In addition, the size of the reaction tank body 7 can be specifically selected according to actual needs, for example, the depth of the tank body can be adjusted according to the size of the DNA cells, but the thickness of the side wall of the reaction tank body 7 cannot be too thin, otherwise, the reaction tank bodies 7 are interfered with each other.
In the embodiment of the present invention, the number of the micro reaction wells 5 in the micro reaction well array determines the sequencing flux, and the micro reaction wells 5 having the honeycomb structure can maximize the number of the micro reaction wells 5 in the micro reaction well array in the same area, thereby providing a guarantee for high throughput sequencing, as shown in fig. 5. The side wall thickness of the reaction tank body 7 is reduced as much as possible to increase the density of the micro-reaction tanks 5 in the micro-reaction tank array, the depth of the tank body groove 8 in the reaction tank body 7 is slightly larger than the diameter of the microspheres coated with the DNA library, and at most one DNA microsphere is arranged in each micro-reaction tank 5 on the premise of ensuring the independent separation of the sequencing reaction in the micro-reaction tank.
Further, after obtaining the micro reaction cell array on the optical fiber panel 1, placing the optical fiber panel 1 with the micro reaction cell array in an oven, wherein the temperature of the oven is set to 180-210 ℃ and kept for 2h (of course, the specific keeping time can be adjusted according to actual needs), and then gradually cooling to normal temperature. In the embodiment of the invention, the optical fiber panel 1 and the micro-reaction cell array are placed in the oven at 210 ℃, so that the mechanical strength of the micro-reaction cell 5 in the micro-reaction cell array and the adhesion of the micro-reaction cell 5 and the optical fiber panel 1 can be improved, and the stability and the reliability of the micro-reaction cell array on the optical fiber panel 1 can be improved.
Specifically, in the gradual cooling process, the temperature in the oven is sequentially reduced to 150 ℃, 100 ℃ and 50 ℃, and finally the temperature in the oven is reduced to the normal temperature; wherein, after the temperature in the oven is reduced to 150 ℃, the temperature in the oven is kept at 150 ℃ for 10min to 15min, and after the temperature in the oven is reduced to 100 ℃, the temperature in the oven is kept at 100 ℃ for 10min to 15 min; and after the temperature in the oven is reduced to 50 ℃, keeping the temperature in the oven at 50 ℃ for 10-15 min.
Claims (9)
1. A preparation method of a micro reaction cell array for a high-throughput pyrosequencing chip is characterized by comprising the following steps:
step 1, providing an optical fiber panel (1), and cleaning the optical fiber panel (1) as required;
step 2, coating the cleaned optical fiber panel (1) to obtain an SU8 photoresist film (2);
step 3, carrying out hot baking on the optical fiber panel (1) and the SU8 photoresist film (2), wherein during hot baking, the optical fiber panel is firstly baked at 65-85 ℃ for 5-15 min, and then baked at 95-105 ℃ for 10-20 min; after the baking, cooling to 45-65 ℃, baking for 10-20 min, and finally naturally cooling to normal temperature;
step 4, exposing the SU8 photoresist film (2) to obtain a patterned photoresist film on the optical fiber panel (1);
step 5, carrying out hot baking on the optical fiber panel (1) and the graphical photoresist film again, and placing the optical fiber panel and the graphical photoresist film in a nitrogen environment to cool to room temperature after the hot baking; wherein, during the hot drying, firstly hot drying at 65-85 ℃ for 10-20 min, then hot drying at 90-100 ℃ for 15-25 min, then cooling to 45-65 ℃ for 10-20 min, and finally naturally cooling to normal temperature;
and 6, developing the patterned photoresist film to obtain a required micro-reaction tank array on the optical fiber panel (1), wherein the micro-reaction tank array comprises a plurality of micro-reaction tanks (5), each micro-reaction tank (5) comprises a reaction tank body (7) perpendicular to the optical fiber panel (1) and a tank body groove (6) located in the central area of the reaction tank body (7), and the tank body groove (6) penetrates through the reaction tank body (7).
2. The method for preparing the microarray of high-throughput pyrophosphate sequencing chip according to claim 1, wherein the method comprises the following steps: after obtaining the micro-reaction cell array on the optical fiber panel (1), placing the optical fiber panel (1) with the micro-reaction cell array in an oven, wherein the temperature of the oven is set to be 180-210 ℃ and kept for 2h, and then gradually cooled to the normal temperature.
3. The method for preparing the microarray of high-throughput pyrophosphate sequencing chip according to claim 2, wherein the method comprises the following steps: in the gradual cooling process, the temperature in the oven is sequentially reduced to 150 ℃, 100 ℃ and 50 ℃, and finally the temperature in the oven is reduced to normal temperature; wherein, after the temperature in the oven is reduced to 150 ℃, the temperature in the oven is kept at 150 ℃ for 10min to 15min, and after the temperature in the oven is reduced to 100 ℃, the temperature in the oven is kept at 100 ℃ for 10min to 15 min; and after the temperature in the oven is reduced to 50 ℃, keeping the temperature in the oven at 50 ℃ for 10-15 min.
4. The method for preparing the microarray of high throughput pyrosequencing chips according to claim 1, 2 or 3, wherein the method comprises the steps of: the depth of the reaction tank body (7) is 30 μm, the thickness of the side wall of the reaction tank body (7) is 6 μm, and the width of the tank body groove (6) is 28 μm.
5. The method for preparing the microarray of high-throughput pyrophosphate sequencing chip according to claim 1, wherein the step 1 of cleaning the fiber optic faceplate (1) comprises the following steps:
step 1.1, placing the optical fiber panel (1) in glass water, cleaning with deionized water after ultrasonic cleaning, and blow-drying the optical fiber panel (1) with nitrogen after cleaning with deionized water;
step 1.2, cleaning the optical fiber panel (1) by using plasma, after cleaning, sequentially adopting trichloroethylene, acetone and ethanol to carry out organic cleaning on the optical fiber panel (1), and after organic cleaning, cleaning the optical fiber panel (1) by using deionized water;
step 1.3, drying the cleaned optical fiber panel (1) by adopting nitrogen, and placing the dried optical fiber panel (1) at 110-130 ℃ for baking for 20-40 min; after the heating and drying, the optical fiber panel (1) is cooled to the normal temperature and then is placed in a nitrogen cabinet.
6. The method for preparing the microarray of high-throughput pyrophosphate sequencing chip according to claim 1, wherein the step 2 comprises the following steps when coating SU8 photoresist film (2):
step 2.1, providing SU8 photoresist liquid, standing and discharging bubbles, taking 10ml of SU8 photoresist liquid after standing and discharging bubbles, and dripping the SU8 photoresist liquid on an optical fiber panel (1) arranged on a spin coater tray;
2.2, rotating the optical fiber panel (1) by using a spin coater, wherein during rotation, the optical fiber panel (1) rotates for 10 to 20 seconds at 600 to 700r/min, then rotates for 30 to 40 seconds at 2000 to 2500r/min, and finally rotates for 10 to 20 seconds at 800 to 1000 r/min; after the rotation is finished, SU8 photoresist film (2) with 25-30 μm can be obtained on the optical fiber panel (1).
7. The method for preparing the microarray of high-throughput pyrophosphate sequencing chip of claim 1 wherein in step 4, 365nm UV is usedThe SU8 photoresist film (2) is exposed at an exposure dose of 170mJ/cm2～200mJ/cm2。
8. The method for preparing the microarray of high-throughput pyrophosphate sequencing chip according to claim 1, wherein the step 6 comprises the following steps:
6.1, soaking the optical fiber panel (1) and the graphical photoresist film in a developing solution for 1-2 min, then shaking for 20-30 s, and finally developing the optical fiber panel (1) and the graphical photoresist film in the developing solution for 10-30 min;
and 6.2, developing the optical fiber panel (1) and the patterned photoresist film for 6-12 min by using a new developing solution, and rinsing the optical fiber panel (1) and the patterned photoresist film for 2-10 min by using isopropanol after the development so as to obtain the required micro-reaction cell array on the optical fiber panel (1).
9. The method for preparing the microarray of high throughput pyrosequencing chips according to claim 1, 2 or 3, wherein the method comprises the steps of: the cross section of the tank body groove (6) is hexagonal.
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Application publication date: 20200821 |