A method for chemically modifying chip surface

Abstract

The present invention discloses a method for chemically modifying the surface of a chip, comprising: providing a first solid support including a surface, wherein the surface includes a continuous coating; forming a micrometer-scale or nanometer-scale profile below the surface through the coating; providing a second solid support having the same surface material as the coating, and packaging it and the first solid support into a chip; performing hydrophobic modification on the outside of the coating profile, wherein the modifier reacts with the coating surface but not with the inside of the profile; performing hydrophilic modification on the inside of the micrometer-scale or nanometer-scale profile, wherein the modifier reacts with the inner surface of the profile but not with the coating. The method provided by the present invention is to first package the chip into a flow cell, and then complete the chemical modification of the chip surface, which can avoid the limitation of the surface modification on the packaging process such as pasting and bonding.

Description

Technical Field

[0001] The invention relates to a chip surface chemical modification method and belongs to the field of biochemistry. Background Art

[0002] Gene sequencing is one of the representative technologies of today's scientific and technological development. Next-generation sequencing (NGS) is a very mature sequencing technology. In the second-generation gene sequencing technology, the chip is one of the key factors that determine the quality of sequencing. In many cases, the chip needs to have etched micropits. The main function of these micropits is to provide reaction chambers and separations for the reaction. In particular, when using fluorescence generation technology to achieve high-throughput sequencing, the information of the nucleic acid sequence to be tested is obtained by reading the signal intensity of the fluorescent molecules released in the solution. Since the fluorescent molecules will diffuse, the sequencing chip must use micropits to confine the fluorescent molecules in situ and isolate different micropits. Since the chemical reaction system in the micropit is an aqueous phase, it can be isolated by an oil phase (oil seal). This puts forward high-difficulty requirements for the micro-machining and surface modification of the chip, that is, each micropit needs to meet the requirements that the inner surface of the micropit is hydrophilic and the outer surface of the micropit is hydrophobic. In addition, the size of the micropits in the high-throughput sequencing chip is very small, usually only at the micro-nano level, and it is very difficult to perform differential modification on the inside and outside of such a tiny micropit. Therefore, a chemical modification method that can strictly distinguish the inner and outer surfaces of the modified chip micropits is needed. Summary of the invention

[0003] High-throughput sequencing chips based on fluorescence generation technology must have both hydrophobic and hydrophilic modification layers on the chip surface. The hydrophobic part is a necessary condition for achieving oil sealing, while the hydrophilic part can significantly reduce the nonspecific adsorption of biological macromolecules such as proteins and DNA on the surface. The existing technical route uses chemical vapor deposition (CVD) to introduce silanes containing carbon fluorine chain functional groups to achieve hydrophobic modification, and also uses CVD to introduce silanes with functional groups such as amino, thiol, carboxyl, norbornene, and alkynyl, and further connects hydrophilic macromolecules such as polyethylene glycol, polyvinyl alcohol, hydrogel, and polylactic acid through a coupling reaction to achieve hydrophilic modification. This technical route has the following disadvantages:

[0004] 1. The modification process does not distinguish between the structures inside the micropits (side walls, bottom surface) and outside the micropits (connection areas between micropits) of the microarray, and there is no significant difference in the modification density of each functional group in different structural areas. The connection area between the micropits is the key to achieving oil sealing. In theory, it needs to be as hydrophobic as possible. Indiscriminate modification methods will introduce hydrophilic macromolecules in this area, reducing the success rate of oil sealing during the sequencing process. The inside of the micropit constitutes the microreactor for the sequencing reaction, and it is also the area where the sequence to be tested is fixed. In theory, it needs to be as hydrophilic as possible, and the immobilization of the sequence to be tested should be as stable as possible. Introducing hydrophobic functional groups in these areas will increase the nonspecific adsorption of macromolecules such as proteins and DNA in the sequencing reaction.

[0005] 2. The chip needs to complete several surface modifications such as CVD first, then be packaged into a flow cell, and then complete a series of subsequent surface connection reactions in the flow cell. Therefore, during CVD, it is necessary to consider distinguishing the area of ​​the chip used for packaging to prevent the surface modification from affecting the effect of the packaging process such as pasting and bonding, and to avoid the operating conditions of the pasting and bonding processes from destroying the various silanes and their functional groups that have been introduced. These restrictions restrict the range of options for process means and process conditions.

[0006] 3. In order to complete the modification on a sufficiently clean chip with high silanol activity, the CVD step of hydrophobic silane must be completed before the chemical reaction of connecting hydrophilic molecules and biomacromolecules. Therefore, these macromolecules will inevitably be adsorbed on the hydrophobic layer during the subsequent reaction, further weakening the hydrophobic nature of the area. At the same time, since bubbles in the hydrophobic flow pool are less likely to be pushed away by the aqueous solution, the fluid injection of these subsequent reactions also requires very careful operation to prevent the introduction of bubbles.

[0007] In order to overcome the above disadvantages, the present invention discloses a chip surface chemical modification method, which is characterized by comprising:

[0008] providing a first solid support comprising a surface, the surface comprising a continuous coating thereon;

[0009] forming a subsurface micrometer-scale or nanometer-scale profile through the coating;

[0010] Providing a second solid support having the same surface material as the coating, and packaging the second solid support and the first solid support into a chip;

[0011] Performing a hydrophobic modification on the outside of the coating profile, wherein the modification reacts with the coating surface but does not react with the inside of the profile;

[0012] Performing hydrophilic modification on the inside of a micrometer-scale or nanometer-scale profile, wherein the modifier reacts with the inner surface of the profile but does not react with the coating;

[0013] Wherein, the first solid support comprises glass; and the contour comprises micro-pits.

[0014] The present invention also discloses a method for using a gene sequencing chip, wherein the gene sequencing chip is characterized in that: it comprises a first solid support and a second solid support, wherein the first solid support comprises a surface, the surface comprises a continuous coating, and the surface has a micrometer-scale or nanometer-scale profile; the surface material of the second solid support is the same as the coating; the method for using the gene sequencing chip comprises the following steps:

[0015] 1) performing hydrophobic modification on the outside of the profile, wherein the modification reacts with the coating surface but does not react with the inside of the profile;

[0016] 2) hydrophilic modification is performed on the inside of the micrometer-scale or nanometer-scale profile, wherein the modifier reacts with the inner surface of the profile but does not react with the coating;

[0017] 3) Immobilizing nucleic acid primers or microspheres connected with nucleic acid molecules into the contour;

[0018] Perform amplification and / or sequencing reactions.

[0019] According to a preferred embodiment, the first solid support is one of quartz glass, sapphire glass and ordinary glass.

[0020] According to a preferred embodiment, the coating material is selected from one of gold, aluminum, copper, aluminum oxide, titanium dioxide, zinc oxide, and gallium nitride.

[0021] According to a preferred embodiment, the coating thickness is 10 nm-150 nm, preferably 20 nm-100 nm.

[0022] According to a preferred embodiment, the profile is a discrete micro-pit in the first solid support, which is a cylindrical, truncated cone, groove, quasi-truncated cone, quasi-hexagonal column structure, or a combination thereof; the opening diameter of the profile is 0.2-5 microns, preferably 0.3-3 microns; the depth of the profile is 0.1-5 microns, preferably 0.2-2 microns; the period of the profile is 0.6-8 microns, preferably 1-5 microns.

[0023] According to a preferred embodiment, the microscale or nanoscale contours are separated from each other by hydrophobic interstitial regions.

[0024] According to a preferred embodiment, the coating material is gold, and the hydrophobic modifier can be from the following substances: thiols containing fatty chains such as decanethiol and octanethiol, or thiols containing carbon fluoride chains such as perfluorodecanethiol and perfluorooctanethiol; if the coating material is a metal oxide such as aluminum oxide and zinc oxide, or a substance such as gallium nitride, the hydrophobic modifier is a phosphonic acid with a fatty chain or a carbon fluoride chain.

[0025] According to a preferred embodiment, the surface of the second solid support adjacent to the coating also undergoes the hydrophobic modification reaction.

[0026] According to a preferred embodiment, the hydrophilic modification includes: first modifying silanes with functional groups such as thiol, amino, carboxyl, norbornene, alkynyl, etc., and then using corresponding reactions such as maleimide, isopropyl succinate, EDC chemistry, click chemistry, etc., and then connecting hydrophilic polymers such as polyethylene glycol, polyvinyl alcohol, hydrogel, polylactic acid, etc.

[0027] According to a preferred embodiment, the hydrophilically modified contour includes a fixing point, and a specific nucleic acid primer or a microsphere connected with a nucleic acid molecule can be fixed in the contour.

[0028] The present invention provides a gene sequencing chip, characterized in that the surface of the flow pool of the chip includes repeated characteristic patterns, wherein the first characteristic pattern is a micrometer or nanometer scale profile, the profile includes discrete micro-pits, and the inside of the micro-pits is a hydrophilic modifier; the second characteristic pattern is a profile spacing area, which includes a coating and a hydrophobic modifier on the coating; the coating is one of gold, aluminum, copper, aluminum oxide, titanium dioxide, zinc oxide, and gallium nitride; the opening diameter of the profile is 0.2-5 microns, preferably 0.3-3 microns; the depth of the profile is 0.1-5 microns, preferably 0.2-2 microns; the period of the profile is 0.6-8 microns, preferably 1-5 microns; there are fixed points in the profile, and nucleic acid primers or microspheres connected to nucleic acid molecules can be fixed in the profile for amplification and / or sequencing reactions.

[0029] Beneficial effects of the present invention

[0030] The present invention provides a method for distinguishing the inner and outer surfaces of a modified gene sequencing chip, which has the following beneficial effects compared with the prior art:

[0031] 1. The method provided by the present invention is to first encapsulate the chip into a flow pool and then complete the chemical modification of the chip surface. This order is opposite to the order of first modification and then encapsulation in the traditional method. It can avoid the limitation of surface modification on packaging processes such as pasting and bonding, and expand the optional range of process means and process conditions.

[0032] 2. Due to the presence of the coating, a material that is completely different from the glass surface is introduced. A modification chemistry with sufficient specificity can be selected to modify the two materials inside and outside the micropits separately. The hydrophobic modification outside the micropits ensures the success rate of the oil seal during the sequencing process, and the hydrophilic modification inside the micropits makes the nucleic acid immobilization more stable.

[0033] 3. In actual use, the order of each step of the reaction can be flexibly adjusted according to the material of the selected coating and the corresponding chemical modification method to avoid interference factors such as adsorption and bubbles. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The dimensions and proportions in the drawings are only used to illustrate the method of the present invention and are not necessarily drawn to scale.

[0035] Figure 1 . Schematic diagram of the effect of differential modification of the chip surface using a coating. In the figure, 101 represents the coating on the surface of the first solid support, 102 represents the surface of the second solid support adjacent to the coating, the surface material is the same as the coating, 103 represents a hydrophobic modifier, and 104 represents a hydrophilic modifier within the outline. DETAILED DESCRIPTION

[0036] In order to further illustrate the core content of the present invention, the present invention is now illustrated by the following examples. The examples are for further explaining the content of the invention and do not limit the present invention.

[0037] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources. The sizes and shapes of the parts in the drawings do not represent the actual proportions, but are only used to illustrate the content of the present invention.

[0038] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. The use of the term "comprising" is not restrictive and should be interpreted as having an open meaning, that is, it should be interpreted synonymously with the phrase "at least comprising".

[0039] Specifically, the first aspect of the present invention provides a method for chemically modifying a chip surface using a coating, characterized by comprising:

[0040] providing a first solid support comprising a surface, the surface comprising a continuous coating thereon;

[0041] forming a subsurface micrometer-scale or nanometer-scale profile through the coating;

[0042] Providing a second solid support having the same surface material as the coating, and packaging the second solid support and the first solid support into a chip;

[0043] Performing a hydrophobic modification on the outside of the coating profile, wherein the modification reacts with the coating surface but does not react with the inside of the profile;

[0044] Performing hydrophilic modification on the inside of a micrometer-scale or nanometer-scale profile, wherein the modifier reacts with the inner surface of the profile but does not react with the coating;

[0045] Wherein, the first solid support comprises glass; and the contour comprises micro-pits.

[0046] In the present invention, "profile" refers to a structure formed by a local change in the shape of the surface of a solid support. Exemplary profiles include recessed structures such as holes and micropits, which are the places where sequencing biochemical reactions occur. The profile can be cylindrical, truncated cone, groove, quasi-truncated cone, quasi-hexagonal column structure, or a combination thereof. The present invention has no specific requirements for the shape of the profile. In a preferred embodiment, the profile (or micropit) is provided on the surface of the solid support in a regular or repeated pattern.

[0047] In the present invention, "solid support" refers to a rigid material that is immiscible with aqueous solution. The solid support has a substantially flat or planar surface and is a component that forms a chip flow cell.

[0048] In the present invention, the "surface" of the solid support refers to its exterior, and the surface may be in contact with another material such as a coating, a gas, a liquid, a polymer, etc. The surface may be planar or substantially flat, and the surface may also have contours such as micropits. The contour is generally formed by preparing a concave structure on a planar plate by etching or other means, thereby forming the contour of the array. In particular, some areas of the contour of the array have distinguishable marks, such as areas where micropits are missing, such as micropits of special shapes, etc.

[0049] In the present invention, the position-related words involved, such as "up", "down", "left", "right", "vertical", "horizontal", "X direction", "Y direction", "Z direction", "front", "back", "high", "low", "inside" and "outside" express relative positional relationships and do not represent specific positions. The inside or outside referred to in the present invention is of a general meaning and a common description method. Among them, the "inside" of the contour refers to the space located in the contour. For example, when the contour is a micro-pit, "hydrophilic modification is performed inside the contour" means that the hydrophilic modifier is on the bottom and side walls of the micro-pit. Figure 1 As shown, the inside of the contour is hydrophilically modified 104, and the outside of the contour, that is, the spacing area between the contours, has a hydrophobic surface modification 103, wherein the hydrophobic modification occurs on the surface of the coating shown in 101 and the surface of the second support adjacent to the coating shown in 102.

[0050] The hydrophilic and hydrophobic described in the present invention are also hydrophilic and hydrophobic in the conventional sense. Generally, a material surface is hydrophobic when the contact angle of water is greater than 90 degrees, and is hydrophilic when the contact angle of water is less than 90 degrees.

[0051] According to a preferred embodiment, the first solid support is one of quartz glass, sapphire glass and ordinary glass.

[0052] According to a preferred embodiment, the coating material is selected from one of gold, aluminum, copper, aluminum oxide, titanium dioxide, zinc oxide, and gallium nitride.

[0053] According to a preferred embodiment, the coating thickness is 10 nm-150 nm, preferably 20 nm-100 nm.

[0054] According to a preferred embodiment, the coating can be added to the surface of the first solid support by electroplating or the like.

[0055] According to a preferred embodiment, a photoresist is coated on the coating on the surface of the first solid support, and then exposed, developed, and etched to form a profile below the surface. Afterwards, the first solid support and the second solid support are encapsulated and then chemically modified. The material on the surface of the second solid support is the same as that of the coating.

[0056] According to a preferred embodiment, the profile is a discrete micro-pit in the first solid support, which is a cylindrical, truncated cone, groove, quasi-truncated cone, quasi-hexagonal column structure, or a combination thereof; the opening diameter of the profile is 0.2-5 microns, preferably 0.3-3 microns; the depth of the profile is 0.1-5 microns, preferably 0.2-2 microns; the period of the profile is 0.6-8 microns, preferably 1-5 microns.

[0057] According to a preferred embodiment, the microscale or nanoscale contours are separated from each other by hydrophobic interstitial regions.

[0058] In the present invention, the hydrophobic modification of the interval area between the contours is achieved by using a substance that reacts with the coating but does not react with the inner surface of the contour. For example, by using the specific reaction of thiol and gold, the surface of the gold coating is hydrophobically modified by using thiols containing aliphatic chains such as decanethiol and octanethiol, or thiols containing fluorocarbon chains such as perfluorodecanethiol and perfluorooctanethiol (1H,1H,2H,2H-Perfluorodecanethiol). It is also possible to use the specific reaction of phosphonic acid with aliphatic chains or fluorocarbon chains (1H,1H,2H,2H-Perfluorooctanephosphonic acid) with metal oxides such as aluminum oxide and zinc oxide, and substances such as gallium nitride, to perform hydrophobic modification on the metal oxide or nitride coating.

[0059] According to a preferred embodiment, the second solid support is adjacent to the surface of the coating (i.e. Figure 1 The hydrophobic modification reaction also occurs (shown in 102).

[0060] In the present invention, the hydrophilic modification in the contour is carried out by chemical vapor deposition (CVD), which refers to the process of introducing the vapor of gaseous reactants or liquid reactants containing the elements constituting the thin film and other gases required for the reaction into the reaction chamber, and chemically reacting on the surface of the substrate to form a thin film. In the present invention, various types of silanes are used to introduce functional groups such as thiol, amino, carboxyl, norbornene, and alkynyl, and then corresponding reactions such as maleimide, isopropyl succinate, EDC chemistry, click chemistry, etc. are used, and then hydrophilic polymers such as polyethylene glycol, polyvinyl alcohol, hydrogel, and polylactic acid are connected, thereby achieving hydrophilic modification in the contour.

[0061] In the present invention, the chemical modification inside the contour needs to be determined according to the subsequent application, such as modifying with a specific chemical group for connecting nucleic acid molecules or nucleic acid carriers such as microspheres.

[0062] According to a preferred embodiment, the hydrophilically modified contour includes a fixing point, and a specific nucleic acid primer or a microsphere connected with a nucleic acid molecule can be fixed in the contour.

[0063] According to a preferred embodiment, the inner surface of the contour can be modified with a hydrogel. Hydrogel is a type of extremely hydrophilic three-dimensional network structure gel. Due to inherent cross-linking, the structural integrity of the hydrogel network will not be dissolved by high concentrations of water. Commonly used ones include PAZAM hydrogel, BPAM hydrogel, etc. Reactive groups are reserved on the hydrogel to react with chemical groups at the ends of nucleic acids, thereby fixing nucleic acid fragments into the contour, and the nucleic acid fragments can be primers. Optionally, a hydrogel with bromine atoms is modified on the inner surface of the chip contour, and the bromine atoms thereon can react with nucleic acid primers modified with 5'-end thiophosphates, thereby fixing the primers within the contour of the chip.

[0064] According to a preferred embodiment, the inner surface of the contour can be pre-connected with exposed thiol groups, and through reaction with amino groups, amino groups or solid substances such as microspheres are connected together.

[0065] According to a preferred embodiment, the chemical modifications of the inner surface of the chip outline and its outer spacer area do not crosstalk with each other.

[0066] It should be noted that the contact angle of the hydrophobic modification of the chip described in the present invention refers to the contact angle of the entire surface to water after the entire chip is modified. There may be a situation where the hydrophilic modification inside the contour causes the contact angle of the entire chip to be slightly reduced, and in this case, the requirement for the contact angle of the entire chip does not change.

[0067] The hydrophilic and hydrophobic modifications described in the present invention have good differentiation. Generally, the hydrophilic modification inside the chip contour will not significantly affect the contact angle of the chip.

[0068] According to a preferred embodiment, the chip contour is hydrophobically modified, and the surface contact angle after hydrophobic modification (the inner surface of the chip is chemically modified or not) is greater than 118°, preferably greater than 120°. For the purpose of being beneficial to application, the contact angle of the hydrophobic modification refers to the average contact angle.

[0069] According to a preferred embodiment, for the measurement of the surface properties after hydrophilic modification inside the profile, since the profile is of micrometer or nanometer scale, it is not suitable for contact angle measurement. Therefore, the plane can be modified with the same hydrophilic substance, and then the contact angle of a similar surface can be measured. In the present invention, the surface contact angle after hydrophilic modification is less than 90°.

[0070] According to a preferred embodiment, when selecting the chemical modification group on the inner surface of the contour, it is necessary to consider whether it is conducive to the gene sequencing reaction, for example, it can be carried out in combination with the common SBS gene sequencing technology.

[0071] In the present invention, the first solid support and the second solid support may be rectangular thin sheet structures. The length of the solid support may range from 1 to 10 cm, preferably 3 to 8 cm; the width may range from 1 to 8 cm, preferably 1.5 to 4.5 cm. The thickness may range from 0.1 to 2 mm, preferably 0.4 to 1 mm, and more preferably 0.5 to 0.9 mm.

[0072] According to a preferred embodiment, the gene sequencing chip further comprises a glue layer. The glue layer is a double-sided adhesive with a flow channel shape cut out, which bonds the first solid support and the second solid support together to form a sequencing reaction flow cell, wherein the reaction flow cell comprises a chamber for reaction, an inlet for delivering a reaction reagent to the chamber, and an outlet for removing the reagent from the chamber. The chamber further comprises at least one transparent surface to allow optical detection.

[0073] The second aspect of the present invention discloses a method for using a gene sequencing chip, wherein the gene sequencing chip is characterized in that: it comprises a first solid support and a second solid support, wherein the first solid support comprises a surface, the surface comprises a continuous coating, and the surface has a micrometer-scale or nanometer-scale profile; the surface material of the second solid support is the same as the coating; the method for using the gene sequencing chip comprises the following steps:

[0074] 1) performing hydrophobic modification on the outside of the coating profile, wherein the modifier reacts with the coating surface but does not react with the inside of the profile;

[0075] 2) hydrophilic modification is performed on the inside of the micrometer-scale or nanometer-scale profile, wherein the modifier reacts with the inner surface of the profile but does not react with the coating;

[0076] 3) Immobilizing nucleic acid primers or microspheres connected with nucleic acid molecules into the contour;

[0077] 4) Perform amplification and / or sequencing reactions.

[0078] Each of the features discussed in the first aspect of the present invention's specific implementation section is equally applicable to the specific implementation of the second aspect of the present invention. As shown above, some other features are not repeated here and should be considered to be repeated by reference. Those of ordinary skill in the art will understand how the features identified in these specific implementations can be easily combined with the basic feature groups identified in other specific implementations.

[0079] According to a preferred embodiment, the first solid support is one of quartz glass, sapphire glass and ordinary glass.

[0080] According to a preferred embodiment, the coating material is selected from one of gold, aluminum, copper, aluminum oxide, titanium dioxide, zinc oxide, and gallium nitride; the coating thickness is 10nm-150nm, preferably 20nm-100nm.

[0081] According to a preferred embodiment, the profile is a discrete micro-pit in the first solid support, which is a cylindrical, truncated cone, groove, quasi-truncated cone, quasi-hexagonal column structure, or a combination thereof; the opening diameter of the profile is 0.2-5 microns, preferably 0.3-3 microns; the depth of the profile is 0.1-5 microns, preferably 0.2-2 microns; the period of the profile is 0.6-8 microns, preferably 1-5 microns.

[0082] According to a preferred embodiment, the microscale or nanoscale contours are separated from each other by hydrophobic interstitial regions.

[0083] According to a preferred embodiment, the coating material is gold, and the hydrophobic modifier can be from the following substances: thiols containing fatty chains such as decanethiol and octanethiol, or thiols containing carbon fluoride chains such as perfluorodecanethiol and perfluorooctanethiol; if the coating material is a metal oxide such as aluminum oxide and zinc oxide, or a substance such as gallium nitride, the hydrophobic modifier is a phosphonic acid with a fatty chain or a carbon fluoride chain.

[0084] According to a preferred embodiment, the hydrophilic modification includes: first modifying silanes with functional groups such as thiol, amino, carboxyl, norbornene, alkynyl, etc., and then using corresponding reactions such as maleimide, isopropyl succinate, EDC chemistry, click chemistry, etc., and then connecting hydrophilic polymers such as polyethylene glycol, polyvinyl alcohol, hydrogel, polylactic acid, etc.

[0085] According to a preferred embodiment, the hydrophilically modified contour includes a fixing point, and a specific nucleic acid primer or a microsphere connected with a nucleic acid molecule can be fixed in the contour.

[0086] Optionally, a hydrogel with bromine atoms is modified on the inner surface of the chip contour, and the bromine atoms on the hydrogel can react with the 5'-end thiophosphate-modified nucleic acid primer, thereby fixing the primer within the contour of the chip. After the nucleic acid primer is fixed, the nucleic acid amplification reaction can be continued. In the present invention, a variety of different nucleic acid amplification technologies can be used in combination with the chip described herein, and exemplary technologies include but are not limited to: multiple displacement amplification (MDA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), etc. After the amplification is completed, the nucleic acid within the contour can be sequenced.

[0087] Alternatively, nucleic acid amplification can also be completed in advance outside the chip, for example, by amplifying the nucleic acid molecule to be tested on a carrier such as a microsphere, and then loading the microsphere or other carrier connected with the amplification product into the contour of the chip, reacting with the immobilization point to immobilize the microsphere, and then continuing the sequencing reaction. The microsphere can be a hydrogel microsphere or a magnetic microsphere.

[0088] The third aspect of the present invention also provides a gene sequencing chip, characterized in that the surface of the flow pool of the chip includes a repeated characteristic pattern, wherein the first characteristic pattern is a micrometer or nanometer scale contour, the contour includes discrete micro-pits, and the inside is a hydrophilic modifier; the second characteristic pattern is a contour spacer, which includes a coating and a hydrophobic modifier on the coating; the coating is one of gold, aluminum, copper, aluminum oxide, titanium dioxide, zinc oxide, and gallium nitride; the opening diameter of the contour is 0.2-5 microns, preferably 0.3-3 microns; the depth of the contour is 0.1-5 microns, preferably 0.2-2 microns; the period of the contour is 0.6-8 microns, preferably 1-5 microns; there are fixing points in the contour, and nucleic acid primers or microspheres connected to nucleic acid molecules can be fixed to the contour for amplification and / or sequencing reactions.

[0089] Each of the features discussed in the first aspect of the present invention's specific implementation section is equally applicable to the specific implementation of the third aspect of the present invention. As shown above, all other features are no longer repeated here, and should be considered as repeated by reference. Those of ordinary skill in the art will understand how the features identified in these specific implementations can be easily combined with the basic feature groups identified in other specific implementations.

[0090] In particular, the surface of the flow pool of the present invention includes a gene sequencing chip with a repeated characteristic pattern, which is particularly suitable for the fluorescence sequencing method. This method requires "oil sealing" when performing the sequencing reaction to limit the fluorescent molecules released by the reaction within each micro-reaction chamber (micro-pit), and uses the oil phase fluid to seal the aqueous phase fluid within the micro-reaction chamber of the sequencing chip, and the sequencing reagent in the aqueous phase fluid reacts with the nucleic acid to be tested in the micro-reaction chamber. The specific hydrophobic modification outside the micro-reaction chamber ensures the success rate of the oil seal, allowing hundreds of "oil sealing" and "oil removal" cycles to proceed normally.

[0091] The sequencing method involved in the present invention can be specifically referred to CN201510822361.9 and CN201710630287.X. In particular, the contents of the above two patents can be incorporated into this patent by reference.

[0092] Example 1

[0093] 1. Coating the surface of the first solid support: Rinse the aluminum-plated quartz chip with isopropanol and water respectively, and blow dry with a nitrogen gun. Soak the chip in 98% concentrated sulfuric acid and leave it at room temperature for 10 minutes to passivate a dense aluminum oxide film on the surface of the aluminum. Then take the chip out of the concentrated sulfuric acid, soak it in clean water, quickly rinse the chip with a large amount of clean water, and then blow dry with a nitrogen gun. The aluminum-plated glass bottom plate (i.e., the second solid support) is treated with the same process as above.

[0094] 2. Etching: Coat photoresist on the coating layer, and treat the first solid support with air PLASMA for 10 minutes to form micro-reaction chambers (micro-pits).

[0095] 3. Chip packaging: Use double-sided tape cut into the shape of a flow channel to bond the aluminum-plated glass bottom plate and the first solid support together to form a sequencing reaction flow cell.

[0096] 4. Hydrophobic modification of the outer surface of the microreactor: Dissolve 1H,1H,2H,2H-Perfluorooctanephosphonic acid (PFPOA) dry powder in ethanol to prepare a 5mM solution. Fill the reaction flow cell with the PFPOA solution and react at room temperature for 24 hours, or heat at 70°C for 1 hour. After the reaction, rinse the flow cell with ethanol and then heat at 150°C for 2 hours. After heating, clean the flow cell with a large amount of ethanol and fluorine oil in turn to complete the hydrophobic modification. After surface modification with PFPOA, the contact angle of the aqueous solution on the micropit array chip will increase from 90° before treatment to 130°.

[0097] 5. Hydrophilic modification of the inner surface of the microreactor: A low cross-linked hydrogel was obtained by copolymerization of acrylamide and its derivative (N-(5-(2-bromoacetamido)pentyl)acrylamide (BRAPA). 700 μL of 0.1 mg / μL BRAPA DMF solution was added to 10 mL of 0.28 M acrylamide aqueous solution and argon was blown for 15 minutes. After deoxygenation by blowing, 11.5 μL TEMED and 100 μL of 0.18 M potassium persulfate aqueous solution were added successively, and the mixture was quickly stirred and mixed. Then, the mixture was poured into the reaction flow cell and polymerized at room temperature for 30 minutes under argon and saturated water vapor. After the polymerization, the reaction flow cell was rinsed with 200 mL of deionized water.

[0098] In this way, a sequencing chip with hydrophilic modification inside the micropits and hydrophobic modification outside the micropits can be obtained.

[0099] Example 2

[0100] Immobilization of nucleic acid molecules on the inner surface of micropits.

[0101] The DNA modified with 5'-terminal thiophosphate reacts with the bromine atoms on the surface of the hydrogel in Example 1, thereby fixing the DNA on the hydrogel.

[0102] 1. Prepare DNA solution: DNA with 5'-terminal phosphorothioate modification is prepared with PBS buffer to a final concentration of 20uM.

[0103] 2. Pour the above DNA solution into the reaction flow cell and heat at 65°C for 1 hour.

[0104] 3. Rinse the flow cell with deionized water.

[0105] 4. Pour 10 mM mercaptoethanol PBS buffer solution into the chip and react at room temperature for 30 minutes to replace all the bromine atoms that have not reacted completely on the surface.

[0106] 5. Rinse the chip again with deionized water.

[0107] After completing the above reaction, DNA can be immobilized inside the micropits.

[0108] Although the above several embodiments have been described in detail, it should be understood that the disclosed embodiments can be modified. Therefore, the foregoing description should be regarded as non-restrictive, and any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for chemically modifying a chip surface, characterized in that: include: providing a first solid support comprising a surface, the surface comprising a continuous coating thereon; forming a subsurface micrometer-scale or nanometer-scale profile through the coating; Providing a second solid support having the same surface and coating as the first solid support, and packaging the second solid support and the first solid support into a chip; The outer part of the coating profile is subjected to hydrophobic modification, and the hydrophobic modifier reacts with the coating surface but not with the inner part of the profile; the inner part of the micrometer-scale or nanometer-scale profile is subjected to hydrophilic modification, and the hydrophilic modifier reacts with the inner surface of the profile but not with the coating; in, The first solid support comprises glass; the profile comprises micropits; The coating material is selected from one of gold, aluminum oxide, zinc oxide, and gallium nitride; when the coating material is gold, the hydrophobic modifier can be selected from the following substances: one of decanethiol, octanethiol, perfluorodecanethiol, and perfluorooctanethiol; when the coating material is aluminum oxide, zinc oxide or gallium nitride, the hydrophobic modifier is a phosphonic acid with a fatty chain or a carbon fluoride chain.

2. A method for using a gene sequencing chip, characterized in that: The gene sequencing chip comprises a first solid support and a second solid support, wherein the first solid support comprises a surface, the surface comprises a continuous coating, and the surface has a micrometer-scale or nanometer-scale profile; The surface material of the second solid support is the same as that of the coating, and the second solid support is packaged with the first solid support into a chip; The method for using the gene sequencing chip comprises the following steps: The outer part of the profile is subjected to hydrophobic modification, and the hydrophobic modifier reacts with the coating surface but not with the inner part of the profile; the inner part of the micrometer-scale or nanometer-scale profile is subjected to hydrophilic modification, and the hydrophilic modifier reacts with the inner surface of the profile but not with the coating; nucleic acid primers or microspheres connected with nucleic acid molecules are immobilized in the profile; and amplification and / or sequencing reactions are performed; The coating material is selected from one of gold, aluminum oxide, zinc oxide, and gallium nitride; when the coating material is gold, the hydrophobic modifier can be selected from the following substances: one of decanethiol, octanethiol, perfluorodecanethiol, and perfluorooctanethiol; when the coating material is aluminum oxide, zinc oxide or gallium nitride, the hydrophobic modifier is a phosphonic acid with a fatty chain or a carbon fluoride chain.

3. The method according to claim 1 or 2, characterized in that: The first solid support is quartz glass or sapphire glass.

4. The method according to claim 1 or 2, characterized in that: The coating thickness is 10 nm-150 nm.

5. The method according to claim 1 or 2, characterized in that: The profile is a discrete micro-pit in the first solid support, which is cylindrical, truncated cone, quasi-truncated cone, quasi-hexagonal column structure, or a combination thereof; the opening diameter of the profile is 0.2-5 microns; the depth of the profile is 0.1-5 microns; and the period of the profile is 0.6-8 microns.

6. The method according to claim 1 or 2, characterized in that: The hydrophilic modification includes: first modifying silane with thiol, amino, carboxyl, norbornene or alkynyl functional groups, then using the corresponding maleimide, isopropyl succinate, EDC chemistry or click chemistry reaction, and then connecting any hydrophilic polymer among polyethylene glycol, polyvinyl alcohol, hydrogel and polylactic acid.

7. The method according to claim 1 or 2, characterized in that: The hydrophilically modified contour includes a fixing point, and the nucleic acid primer or the microsphere connected with the nucleic acid molecule is fixed in the contour.

8. A gene sequencing chip, characterized in that: Providing a first solid support including a surface, wherein the surface includes a continuous coating; forming a micrometer-scale or nanometer-scale profile below the surface through the coating; providing a second solid support having a surface material identical to that of the coating, and packaging the second solid support and the first solid support into a chip; The surface of the flow cell of the chip includes a repeating feature pattern, wherein a first feature pattern is a micrometer-scale or nanometer-scale profile, and the profile includes discrete micro-pits, and the hydrophilic modifier is inside the pits; The second characteristic pattern is a contour spacer, which includes a coating and a hydrophobic modifier on the coating, wherein the hydrophobic modifier reacts with the coating but does not react with the inner surface of the contour; the hydrophilic modifier reacts with the inner surface of the contour but does not react with the coating; The coating is one of gold, aluminum oxide, zinc oxide, and gallium nitride; when the coating material is gold, the hydrophobic modifier can be selected from the following substances: one of decanethiol, octanethiol, perfluorodecanethiol, and perfluorooctanethiol; when the coating material is aluminum oxide, zinc oxide, or gallium nitride, the hydrophobic modifier is a phosphonic acid with an aliphatic chain or a carbon fluoride chain; The opening diameter of the contour is 0.2-5 microns; the depth of the contour is 0.1-5 microns; the period of the contour is 0.6-8 microns; there are fixing points in the contour, and nucleic acid primers or microspheres connected to nucleic acid molecules can be fixed in the contour for amplification and / or sequencing reactions.

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