CN111269799B

CN111269799B - Preparation method of oil-sealed biochemical chip

Info

Publication number: CN111269799B
Application number: CN202010087609.2A
Authority: CN
Inventors: 张晓璐; 刘珊珊; 陈子天; 段海峰
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2020-02-12
Filing date: 2020-02-12
Publication date: 2023-07-04
Anticipated expiration: 2040-02-12
Also published as: CN111269799A

Abstract

The invention discloses a preparation method of an oil-sealed biochemical chip. The inner surface of the biochemical chip or the functional area thereof is subjected to integral hydrophobic modification, hydrophilic microspheres are introduced, and the microspheres are connected in the prefabricated micro pits of the biochemical chip in a chemical or physical mode. The oil-sealed biochemical chip does not need to carry out distinguishing chemical modification on the inner surface of the chip, and the original technology for distinguishing chemical modification is replaced by hydrophilic microspheres, so that the production steps of the chip are simplified, and the yield is improved.

Description

Preparation method of oil-sealed biochemical chip

Technical Field

The invention relates to a preparation method of an oil-sealed biochemical chip, in particular to a preparation method of an oil-sealed gene sequencing chip, and belongs to the field of biochemistry.

Background

Biochemical chips are also called microfluidic chips and the like. The microfluidic chip technology (Microfluidics) integrates basic operation units of sample preparation, reaction, separation, detection and the like in biological, chemical and medical analysis processes on a micron-scale chip, and automatically completes the whole analysis process. Because of its great potential in biological, chemical, medical and other fields, it has been developed into a new research field where the disciplines of biology, chemistry, medicine, fluids, electronics, materials, machinery and the like are crossed. Biochemical chips are mostly used in a sealed manner. The reaction chamber is provided with a fluid inlet and a fluid outlet, a fluid space and a reaction region. The requirements for fluid sealing in biochemical chips are of special application, such as PCR, gene sequencing, etc. The use of oil to seal the aqueous solution in a very small reaction cell has many advantages, such as a throughput of 100M or more, and the separation of each small micro-reaction chamber for reaction and detection. Sealing generally requires a distinguishing hydrophilic-hydrophobic modification on the tiny pits or other structures of the reaction area. The distinguishing hydrophilic and hydrophobic modification process is complex and has high cost, and the single hydrophobic modified chip cannot guarantee good oil seal efficiency. The invention discloses a biochemical chip which is provided with a micro-reaction structure, wherein the inside of a micro-pit is provided with hydrophilic property by means of hydrophilic microspheres such as hydrogel under the condition that the inside and the outside of the micro-pit are not subjected to differential modification on the surface of a chip subjected to hydrophobic modification, and the outside of the micro-pit still keeps hydrophobic, so that the effect that each micro-pit is an independent reactor is achieved through seal.

Disclosure of Invention

The aim of the invention is achieved by the following technical scheme. The invention discloses a preparation method of an oil-sealed biochemical chip, which is characterized by comprising the following steps of performing hydrophobic modification on the inner surface of the biochemical chip; adding the solution mixed with the hydrophilic microspheres to the biochemical chip such that the microspheres enter and are immobilized to the pre-processed microstructures of the inner surface of the biochemical chip; the biochemical chip is provided with a fluid outlet and an inlet and a fluid chamber; the surface of the fluid chamber is the inner surface of the biological chip; at least one surface of the fluid chamber has a prefabricated microstructure.

According to a preferred embodiment, the microstructures are micro-pits of an array, with a size of 0.1-100 micrometers; preferably 0.2-50 microns; more preferably 0.3-30 microns; more preferably 0.5-5 microns.

According to a preferred embodiment, the microspheres have a size of 0.05-10 microns, preferably 0.1-8 microns, more preferably 0.3-5 microns; more preferably 0.5-3 microns.

According to a preferred embodiment, the fluid chamber is flat, with a height of 10-200 micrometers, preferably 15-100 micrometers, more preferably 30-80 micrometers.

According to a preferred embodiment, the hydrophobic modification of the inner surface of the biochemical chip means that the inner surface of the biochemical chip is subjected to an overall hydrophobic modification.

According to a preferred embodiment, the microsphere is attached to the surface of the microstructure of the chip by one of hydrogen bonding, ionic bonding, covalent bonding.

According to a preferred embodiment, the inner surface of the biochemical chip is hydrophobically modified; then modifying the first compound; then adding the solution mixed with the hydrophilic microspheres into the biochemical chip, so that the microspheres enter the prefabricated microstructures on the inner surface of the biochemical chip; the second compound on the microsphere reacts with the first compound such that the microsphere is immobilized to the interior of the microstructure.

The invention discloses an oil-sealed biochemical chip, which is characterized in that the inner surface of a fluid chamber of the biochemical chip is subjected to hydrophobic modification; the inner surface of the biochemical chip is provided with a prefabricated microstructure, and hydrophilic microspheres are arranged in the micropits; after the liquid containing water is introduced into the oil-sealed biochemical chip, the liquid containing water is sealed in the prefabricated microstructure by introducing the oily liquid which is insoluble in water.

An oil-sealed biochemical chip, characterized in that the biochemical chip is provided with a fluid outlet, an inlet and a fluid chamber; at least one inner surface of the biochemical chip is provided with a micro-structure with a concave which is processed in advance; the inner surface of the biochemical chip is integrally hydrophobically modified; the microstructure contains hydrophilic microspheres.

According to a preferred embodiment, the microsphere surface is modified with a first compound, and the microsphere surface is reacted together by a second compound modified on the inner surface of the chip.

The preparation method of the oil-sealed biochemical chip has the advantages that (1) the oil-sealed biochemical chip does not need to be subjected to distinguishing modification and only needs to be subjected to complete hydrophobic modification. The difficulty of chip production is reduced, and the surface of the chip is improved. (2) The interior of the micropit is also hydrophobically modified, and the adsorption of molecules such as proteins is greatly reduced during sequencing or biochemical experiments. (3) Since only hydrophobic modification is needed, no hydrophilic modification is needed in a specific small area, such as the interior of a micro pit, the difficulty of hydrophobic modification is reduced, and the aim can be achieved by directly excessively modifying the hydrophobic. (4) Even though the micro pits may require modification of the surface with a second chemical for fixation reasons, the modification is achieved in a general manner, such as CVD. The amount of the second modification need not be large and may be carried out after the hydrophobic modification. The second material modification is simply to attach to a functional group on the microsphere, thereby immobilizing the microsphere.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. In the drawings:

FIG. 1. Schematic representation of a dimple and an internal microsphere;

FIG. 2 is a fluorescence characterization micrograph of the microspheres after they have entered the chip;

FIG. 3. Dark areas formed after fluorescence bleaching in oil seal test;

fig. 4 diffusion diagram after five minutes in dark area.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure have been described, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

The biochemical chip is a common sealed chip, and the functional reaction area comprises a chip with a prefabricated microstructure. Commonly, biochemical chips are made of glass, silicon wafers, etc., and have a multi-layer structure with a space into which fluid is introduced. Biochemical chips are sometimes also referred to as microfluidic chips or microfluidic chips. Typical biochemical chips, such as CN2017208542125, CN2017208542017, CN2017105741742, CN2017105741441, may consist of three layers of chip structures with pre-fabricated access ports with fluid spaces inside. The contents of the cited partial patents, as a general description of the chips of the present invention, may be incorporated by reference into the present invention.

The biochemical chip according to the present invention refers to a chip having a prefabricated structure on an inner surface thereof. Generally, different processing methods can be used when the materials are different. Micromachining is a common process that uses exposure, etc., to form specific microstructures on one or more interior surfaces of a chip. In particular, the microstructures may be arranged in an array; the microstructures may be pits; the special microstructure is a micro pit arranged in an array; a particular of the microstructures is microfluidic channels. The size of the microstructure is 0.1-100 micrometers; preferably 0.2-50 microns; more preferably 0.3-30 microns; more preferably 0.5-5 microns.

The invention discloses a preparation method of an oil-sealed biochemical chip, which is characterized by comprising the following steps of performing integral hydrophobic modification on the inner surface of the biochemical chip;

adding the solution mixed with the hydrophilic microspheres into a biochemical chip;

a pre-processed microstructure that allows the microspheres to enter the inner surface of the biochemical chip;

the microspheres are immobilized in the microstructure.

According to a preferred embodiment, the inner surface of the biochemical chip is hydrophobically modified, modified during chip preparation, or hydrophobically modified after chip preparation.

According to a preferred embodiment, the inner surface of the chip is chemically modified and reacted with a compound on the microsphere for immobilization of the microsphere before the microsphere is added to the biochemical chip.

According to a preferred embodiment, the microsphere is attached to the chemical group of the surface of the microstructure of the chip by one of hydrogen bonding, ionic bonding, covalent bonding.

The microstructure is a pattern formed by a prefabricated concave-convex structure. Preferably, the microstructure is a pattern of arrays; more preferably, the microstructures are micro-pits of an array.

Generally, the microspheres according to the present invention refer to rounded or quasi-rounded or non-sharp corner polyhedrons.

Generally, the microspheres of the present invention have a size of 0.05 to 10 microns, preferably 0.1 to 8 microns, more preferably 0.3 to 5 microns; more preferably 0.5-3 microns.

Generally, the microspheres of the present invention are hydrogel microspheres, polystyrene microspheres, glass microspheres, and the like.

The microspheres of the present invention are generally transparent or opaque microspheres.

Generally, the internal height of the biochemical chip according to the present invention is 10 to 200 microns, preferably 15 to 100 microns, more preferably 30 to 80 microns, or a range of any two values therein.

Generally, the biochemical chip according to the present invention is a gene sequencing chip.

Generally, the inner surface of the biochemical chip in advance is chemically modified. The chemical modification may be performed in multiple steps, it being understood that the hydrophobic chemical modification is performed first, and then the corresponding modification to the compound match on the microsphere surface is modified to the inner surface.

The method of modification is not particularly limited. Common fluid modification methods, CVD, etc. are possible. Chemical modification as used herein refers to modification that does not require distinction. In the case of chemical modification, it is simple to modify all the modification inside the chip. Or according to the requirement, in the process of preparing the chip, one or two inner surfaces are modified as required, and then the chip is integrally packaged. When the inner surface of the chip is chemically modified, the modified region is not limited, for example, only a region of 4m by 4m is a functional reaction region in the whole chip, and it is also suitable to modify only the region. Thus, the hydrophobic chemical modification described in the present invention is all indifferent hydrophobic chemical modification for the functional region.

The modified compounds may be selected from common hydrophobic agents. Such as the usual trimethylchlorosilanes, trichlorofluorosilanes, and the like. Selection of the reagent according to specific contact angle requirements is possible.

In addition to hydrophobic reagents, other chemical modifications are often required. The function is to fix the microspheres. In the prior art, it is a relatively common technique to attach microspheres to a surface through certain chemical groups. The microspheres can be fixed inside the microstructure as long as different chemical groups are respectively connected on the microspheres and the surfaces of the microspheres and can be chemically connected under the conditions of heating, illumination and the like.

The immobilized microspheres may be attached to the interior of the microstructure using at least one of hydrogen bonding, covalent bonding, and ionic bonding.

The hydrophobic chemical modification may be performed first and then the compound to which the microspheres are immobilized may be attached. Before the hydrophobic chemical modification, the surface of the chip can be cleaned, for example, common plasma cleaning and sulfuric acid cleaning, and the surface of materials such as glass can be cleaned, so that the function of the chip is better stable.

The technique of immobilizing the microspheres within the microstructure is not limited to chemical modification. In addition, for example, a cylindrical pit is produced. The diameter of the opening of the micro pit close to the surface is slightly larger than that of the microsphere, and the diameter of the micro pit far away from the surface is slightly smaller than that of the microsphere. After the microspheres enter the micro-pits, the microspheres can be clamped in the micro-pits only by conventional centrifugation and the like. This approach is a physical limitation.

In general, a chemical group is connected to the microsphere, another chemical group is connected to the surface of the chip, and the two chemical groups are connected together through reaction under the conditions of heating or illumination. It is conceivable to use hydrogen bonding or bonding by covalent bonding or the like.

Generally, when the surface of the biochemical chip may have micro pits arranged in an array. A biochemical chip can be considered to be composed of two substrates plus an intermediate space. The micro-pits may be divided into an inner surface and an outer surface. The inner surface refers to the part of the substrate where the micro pits are located after the micro pits are processed. The outer surface is the surface of the substrate after micro pit processing. The size of the microspheres used is smaller than the diameter and depth of the micropits, so that the microspheres can enter the interior surface of the micropits.

The material of the microspheres is optional. Microspheres of soft materials are relatively readily available. Such as hydrogel microspheres. Rigid microspheres, such as PS microspheres, and the like, are also readily available.

Generally, the size of the microstructures is comparable to the size of the microspheres. For example, the microstructures are cylindrical micro-pits of 3 microns diameter, then 1-2 microns diameter can be used for the microspheres. Generally, the microstructures have a size smaller than the microstructures.

The sealing oil refers to an oily fluid which is not mutually soluble with water. Commonly used are mineral oils, fluorooils such as FC70, the common fluorooils of dakaning, and the like. The method has the function of isolating aqueous solution in microstructures such as micro pits and the like to form a micro reaction chamber taking the microstructures as units.

In the invention, the inner surface or the outer surface is in accordance with the general definition. Micropits are pits "dug" in the surface of a substrate by a certain technique. The inner surface of the dimple refers to the portion of the dimple interior, including the exposed surfaces of the sidewalls and bottom. The outer surface of the micropits refers to the portion of the surface of the substrate between the micropits and the micropits.

In the invention, the inside and the outside of the micro pit also belong to the inside and the outside in the general sense, and accord with the general definition. Micropits are pits "dug" in the surface of a substrate by a certain technique. The inside of the micro pit refers to the inside of the dug pit, and the outside of the micro pit refers to the outside of the dug pit. In general, the portions above the surface of the substrate are outside the micropits.

In the invention, the micro pits are array micro pits. The shape of the micro-pits is not particularly required. The size of the opening of the micro pit is 0.3-5 microns, the depth of the micro pit is 0.3-5 microns, and the period of the micro pit is 0.6-8 microns. Preferably, the micropits have an opening size of 0.6-4.2 microns, more preferably 1.0-3.8 microns, more preferably 1.5-3 microns, more preferably 1.8-2.6 microns. The depth of the micropits is 0.6-4.2 microns, more preferably 1.0-3.8 microns, more preferably 1.5-3 microns, more preferably 1.8-2.6 microns. The period of the micropits is 0.6-8 microns, preferably 0.7-7.5 microns, more preferably 1.0-6 microns, more preferably 1.5-5 microns, more preferably 1.9-4.2 microns, more preferably 2.5-3.5 microns. The size of the opening of the dimple plus the thickness of the outer surface wall of the dimple is the period of the dimple. In the present invention, the micro-pits are array micro-pits. In particular, a partial region of the micro-pits of the array has a distinguishing mark, such as a region where the micro-pits are missing, such as a specially shaped micro-pit, or the like.

The contact angle of the hydrophobic modification is greater than 113 °, preferably greater than 118 °. The hydrophobically modified contact angle refers to the average contact angle for the purpose of facilitating application.

The preceding section is directed to patent CN201811643917.8 by the applicant. However, as a distinct distinction, the outer surface of the dimple in the cited patent is hydrophobically modified and its inner surface is hydrophilically modified or directly hydrophilic. The techniques disclosed herein are different. In the present invention, it is understood that both the outer surface and the inner surface of the micro-pits are hydrophobically modified, however, hydrophilic microspheres are attached to the interior of the micro-pits. The two techniques are significantly different.

From the purpose of being favorable for application, the outer surface of the micro-pit is subjected to hydrophobic modification, and the inner wall of the micro-pit is also subjected to hydrophobic modification.

According to a preferred embodiment, the hydrophobically modified contact angle is greater than 118 degrees. The contact angle refers to the average contact angle after both the inner and outer surfaces of the dimple are modified. In a common measurement method, a hydrophobically modified platelet is placed on a contact angle measuring instrument for measurement.

The meaning of some terms in the present invention, such as inner surface, outer surface, dimples, etc., are the same as patent CN201811643917.8. The content of this patent may be incorporated by reference into the present invention as necessary. Notably, the inner surface of the biochemical chip is actually also the outer surface of the micropits (microstructure).

Illustratively, referring to FIG. 1, the inner surface 101 of a biochemical chip according to the present invention is schematically shown, and the inner surface of the micro-pits is 102. The microspheres are attached or immobilized to the interior surface of the micropits. It should be noted that the microspheres may be provided in the micropits, for example, attached to the sidewall or bottom of the micropits.

Preferably, when the microstructures are micro-pits. The volume ratio of the micro pits to the micro balls is less than or equal to 10 (the volume of the micro balls is more than 10 percent of the volume of the micro pits); more preferably, the volume ratio of micropits to microspheres is less than 8, more preferably less than 6, more preferably less than 5.

Preferably, when the microstructure is a pit, the volume is V; the volume of the microspheres is greater than 0.1V, preferably greater than 0.125V, more preferably greater than 0.2V.

It has been found experimentally that when the volume of the microspheres is too small compared to the volume of the micropits, a small amount of microspheres does not serve the purpose of retaining the reaction solution in the micropits. Alternatively, when the microsphere volume is too small, the success rate of the oil seal will be reduced.

Experimentally, when the volume ratio of the micro pits to the micro balls is less than or equal to 10, the efficiency of the oil seal (success rate, calculated as the ratio of the number of micro pits to the total number of micro pits of the successful oil seal, which can be generally expressed by the ratio of areas) is greater than 99%. The efficiency of repeated oil sealing is lowered.

Preferably, the efficiency of the oil seal according to the invention is higher than 80%, more preferably higher than 85%, more preferably higher than 90%. It is known that the oil sealing efficiency is very low, for example 30%, when the inner surface of the chip is only hydrophobically modified and hydrophilic microspheres are not added. The hydrophilic-hydrophobic differential modification inside and outside the micro pits of the chip, or the microsphere is used for replacing the original hydrophilic modification, is the key for maintaining the oil seal efficiency.

Experimentally, when the volume ratio of the micro-pits to the micro-spheres is greater than 10, for example, the ratio is 100, the oil seal efficiency is obviously reduced by about 80%; the efficiency of repeated oil sealing is further lowered. As an illustration, the method of oil sealing is relatively simple, firstly, introducing aqueous solution, and then introducing FC770 as sealing liquid; photographing is carried out, and fluorescent substances can be added into the aqueous solution for obvious distinction.

Experimentally, when the surface of the micro-pits is subjected to overall hydrophobic modification, and no microspheres enter the micro-pits, the efficiency of the oil seal is further reduced. Much less than 80%, about 30% found by experimentation.

In experimental details, when the filling rate of the microspheres in the micropits is relatively low, the success rate of oil sealing only calculates the micropits containing the microspheres.

Experimentally, when the micro pits are very large. For example, over 100 microns, has a serious impact on the oil seal.

Very flat dimples that can be obtained are detrimental to the oil seal. Applicant discloses the technique of micro-pit preparation several times in the previous patents. Experimentally, when the microstructure is a micro pit, when the height of the micro pit is less than 0.1×the diameter (or length and width) of the micro pit, the micro pit is in a flat structure, which is unfavorable for oil sealing.

Oil sealing refers to the process of sealing an aqueous reaction liquid inside a micro-pit or microstructure using a fluid that is insoluble in water. This process and concept are also disclosed in applicant's previous patents. Such as CN201710630287X, e.g., 201811643917.8. The contents of part of the patent may be incorporated into this patent by reference, if necessary.

Example 1

Preparation of pit raw materials: taking a micro-pit chip quartz plate processed by dry etching; placing into a plasma instrument, vacuumizing, treating for 1min, and taking out the quartz plate to be treated for chlorosilane modification.

(3-mercaptopropyl) trimethoxysilane modification: 20ul of mercaptosilane solution was placed in a 2ml glass vial in a mercapto heated oven and placed for 30min at 130 degrees Celsius under vacuum.

Quality inspection after chip hydrophobic modification: the contact angle of the surface of the quartz plate was measured using a contact angle tester. Each sample takes 5 position contact angle values, contact angle detection results:

the antenna of the micro-pit surface finished by the two silane modification is more than 110 degrees, and the surface of the chip is in a hydrophobic structure.

And packaging the processed quartz plate to form a chip. For the chip structure see patent CN2017105741742 or CN2017105741441 or 2019202627006.

Pretreatment before entering:

1) 100uM biotin maleimide (mal-biotin) solution was prepared:

weighing 0.001g of the dry powder of the polymaleimide, adding 1 XPBS to dissolve the dry powder of the maleimide at the ratio of 400ul/mg, and preparing 100uM of the polymaleimide reagent.

300ul of prepared 100uM substance maleimide reagent is added into the chip, and the reaction is carried out for 10min at normal temperature and in a closed way.

Randomly extracting a chip for subsequent quality inspection verification. The reaction amount of the biotin maleimide can be verified by using streptavidin (streptavidin) having a fluorescent group, cy 5-streptavidin.

2) The modified substance of the maleimide chip is required to be modified by strepavidin.

Configuring 0.1mg/ml strepitavidin; each chip was added with 300ul, and the reaction was performed at room temperature for 5min while being closed. In order to confirm that the modified chip still has hydrophobicity, the chip is disassembled for contact angle detection, and the data is as follows, and both sides are more than 100 degrees and still belong to a hydrophobic structure. The results of multiple experiments are similar, and the step is not repeated in the later stage.

The hydrogel microsphere based on the acrylamide monomer is synthesized by using a dispersion polymerization method, DNA with specific sequence and concentration is immobilized on the surface of the microsphere by click chemistry, and a biotin group is connected to the surface of the microsphere for immobilization on the surface of a chip. Each microsphere is a micro-reactor. The hydrogel microspheres may also be commercially available products. So long as it matches the dimple size, e.g., is slightly smaller than the dimple size.

Immobilization of microspheres on chip surface:

the hydrogel microsphere is used as a hydrophilic substance and is added into a runner to carry out local hydrophilic modification.

And (5) performing quality inspection on microsphere entering results by using a fluorescent probe. The results were seen by fluorescent microscopy, as shown in FIG. 2. Microspheres are entered into the display chip. The number of the microspheres and the number of the pits are 1:1, the entering rate is about 50% -60%, which accords with the expectations of poisson distribution.

The 10uM of fluorescein solution was added to the flow channel and then FC770 fluorooil was added to the flow channel, and since fluorooil would seal the fluorescein solution in the micropits, separate reactors were formed for checking no crosstalk between micropits, and the fluorescein in the micropits in the middle area of the field was bleached (bleached) by high intensity excitation light through the small holes (pin holes), as shown in fig. 3. Dark areas resulting from fluorescent bleaching can be seen.

After waiting for five minutes, it was observed whether the area of the bleachs was contracted to determine whether crosstalk occurred in the fluorescein solution of the unblocked area to compensate for the fluorescein concentration of the bleachs. The results are shown in FIG. 4. As can be seen in the picture, the dark areas are unchanged.

Experimental results prove that crosstalk does not occur among the micro-pits, the micro-pit where each microsphere is located is an independent reactor, and the hydrogel microsphere entering the micro-pits can be used as a method for modifying the hydrophilic-hydrophobic difference inside and outside the micro-pits of the chip.

The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. The preparation method of the oil-sealed biochemical chip is characterized by comprising the following steps of performing hydrophobic modification on the inner surface of the biochemical chip; then modifying the first compound; adding the solution mixed with the hydrophilic microspheres to the biochemical chip so that the microspheres enter a pre-processed microstructure of the inner surface of the biochemical chip; reacting the second compound on the microsphere with the first compound such that the microsphere is immobilized to the interior of the microstructure; wherein the biochemical chip is provided with a fluid outlet and inlet, and a fluid chamber; the surface of the fluid chamber is the inner surface of the biological chip; at least one surface of the fluid chamber has a prefabricated microstructure; the hydrophobic modification of the inner surface of the biochemical chip means that the inner surface of the biochemical chip is subjected to overall hydrophobic modification;

the microstructure is an array of micro pits with the size of 0.1-100 micrometers;

the volume ratio of the micro pits to the micro spheres is less than or equal to 10;

wherein the oil seal refers to a process of sealing an aqueous reaction liquid inside a microstructure by using a fluid insoluble in water; the efficiency of the oil seal is higher than 90%.

2. The method of claim 1, wherein the microspheres have a size of 0.05 to 10 microns.

3. The method of claim 1, wherein the fluid chamber is flat and has a height of 10-200 microns.

4. The method of claim 1, wherein the microsphere is attached to the surface of the microstructure of the chip by one of hydrogen bonding, ionic bonding, and covalent bonding.