WO2019091208A1

WO2019091208A1 - Surface chemical modification method, chip preparation method, and chip

Info

Publication number: WO2019091208A1
Application number: PCT/CN2018/105475
Authority: WO
Inventors: 赵�智; 黄天逊; 赵陆洋; 颜钦
Original assignee: 深圳市瀚海基因生物科技有限公司
Priority date: 2017-11-10
Filing date: 2018-09-13
Publication date: 2019-05-16
Also published as: CN112326949A; CN112326949B

Abstract

The present application provides a chemical modification method for a solid-phase substrate, a chip preparation method, and a chip. The chemical modification method for a solid-phase substrate in the present application comprises: carrying out surface modification on a solid-phase substrate, the surface modification comprising treatment of the surface of the solid-phase substrate by using a mixture, and the mixture comprising epoxy groups and hydrophilic group tail ends whose molar ratio is 1:1 to 1:1000. In the method in the present application, by controlling the molar ratio of the epoxy groups to the hydrophilic groups, the density of the epoxy groups and the hydrophilic groups on the surface can be conveniently regulated according to actual requirements, so that the number of loaded probes can be controlled. In addition, by introducing the hydrophilic groups on the surface, the hydrophobicity of the surface can be adjusted, the non-specific adsorption of the surface of the chip can be reduced, and performance of the chip can be improved.

Description

Surface chemical modification method, chip preparation method and chip

Technical field

The present application relates to the field of biotechnology, and in particular, the present application relates to a chip and a method of preparing the same.

Background technique

In recent years, major developed countries including the United States, the United Kingdom, and France have successively adopted genetic technology as a national strategy, and the development of genetic testing technology has attracted attention.

The use of chips for nucleic acid detection, including the use of chips for target nucleic acid capture, nucleic acid sequencing on the chip, etc., chip performance, including surface affinity / hydrophobic properties, non-specific adsorption of nucleic acid properties, probe / primer amount, probe / Primer fixation density, etc., plays a key role in the detection or measurement results.

The chip still needs to be further developed or improved, especially for specific situations or chips that can meet specific detection requirements.

Summary of the invention

A first aspect of the present application provides a method of chemically modifying a solid phase substrate, the method comprising: surface modifying a solid phase substrate, the surface modification comprising treating the surface of the solid phase substrate with a mixture, the mixture comprising : 1 to 1: 1000 molar ratio of epoxy groups and hydrophilic group ends.

The method of the present application can conveniently adjust the epoxy group on the surface of the solid phase substrate (reactive groups, for example, can be linked to a probe) and hydrophilic groups by controlling the ratio of the epoxy group to the hydrophilic group. Proportion to adjust the load capacity and performance of the solid phase substrate.

A second aspect of the present application provides a method of preparing a chip, the chip comprising a solid phase substrate. In one embodiment of the present application, the method comprises: surface modification of the solid phase substrate, the surface modification comprising using the mixture to the solid phase The surface of the substrate is treated, and the mixture contains an epoxy group and a hydrophilic group terminal in a molar ratio of 1:1 to 1:1000.

The method for preparing a chip of the present application can conveniently adjust the epoxy group on the surface of the solid phase substrate (reactive group, for example, can be linked to a probe) and the hydrophilic group by controlling the ratio of the epoxy group to the hydrophilic group. The ratio of the group to regulate the loading capacity and performance of the solid phase substrate, such as hydrophobicity, which is beneficial to reduce the non-specific adsorption of the solid substrate/chip surface, improve the nucleic acid detection performance of the obtained chip, and is particularly advantageous for high precision and/or Preparation of a single molecule level detection chip.

It should be noted that the “solid phase substrate” of the present application may be any solid support that can be used for immobilizing a nucleic acid sequence, such as a nylon membrane, a glass sheet, a plastic, a silicon wafer, a magnetic bead, etc., unless otherwise specified, the surface of the chip and The substrate surfaces are used interchangeably.

In an embodiment of the present application, the method for preparing a chip of the present application further comprises: immobilizing the nucleic acid probe molecule on the surface-modified solid phase substrate.

In an embodiment of the present application, the epoxy group is derived from an epoxy silane having a silane group end and at least one epoxy group end.

In an embodiment of the present application, the hydrophilic group end is derived from a first silane, and the first silane has a silane group end and at least one hydrophilic group end.

In an embodiment of the present application, the silane group of the epoxy silane and/or the first silane is selected from the group consisting of monomethylsilane, dimethylsilane, trimethylsilane, monomethyloxysilane, dimethoxysilane, and trimethyl At least one of oxysilanes.

In the examples of the present application, the epoxy silane and/or the first silane have a linking group, the linking group and the silane group are bonded, and the linking group is 1 to 12 alkane chains or 1 to 8 ethoxy chains. The above-mentioned linking group is advantageous for forming a monolayer having a relatively uniform surface height on the surface of the solid phase substrate and functioning to isolate the surface of the substrate.

In an embodiment of the present application, the epoxy silane is selected from the group consisting of 3-glycidoxypropyltrimethoxysilane, glycidyloxypropylethoxysilane, glycidyloxypropylmethyldiethoxylate Silane, glycidyloxypropylmethyldimethoxysilane, 2-(3,4-epoxycyclohexane)-ethyltrimethoxysilane and 2-(3,4-epoxy ring At least one of alk)-ethyltriethoxysilane.

In an embodiment of the present application, the first silane is selected from the group consisting of hydroxypropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-ureidopropyltriethoxylate Silane, 3-ureidopropyltrimethoxysilane, (3,3,3-trifluoropropyl)methyldimethoxysilane, (3,3,3-trifluoropropyl)methyldiethoxy At least one of silane, 3-methacryloxypropyltrimethoxysilane, and methacryloxypropyltris(trimethylsiloxy)silane.

In the examples of the present application, the surface modification is carried out in the liquid phase and/or the gas phase. For example, surface modification can be performed by chemical vapor deposition (CVD).

In an embodiment of the present application, the epoxy group is derived from an epoxy silane, the hydrophilic group end is derived from the first silane, and the total content of the epoxy silane and the first silane in the mixture is from 1% to 3%. Further, the silanization efficiency is further improved.

In the embodiment of the present application, before the surface modification is performed, the surface activation of the solid phase substrate is further included to bring the surface of the solid phase substrate with a hydroxyl group.

In an embodiment of the present application, the nucleic acid probe molecule is a nucleic acid molecule modified by terminal amination. The amino group is attached to the modified solid phase substrate via a 1-8 alkyl chain or a 1-4 ethoxy chain.

In the examples of the present application, the fixed density of the nucleic acid probe molecule has a controllable correspondence with the molar ratio of the epoxy group to the hydrophilic group, specifically, the molar ratio of the epoxy group to the hydrophilic group is 1:1000, the nucleic acid probe molecule has a fixed density of 0.6 / μM ² . The molar ratio of the epoxy group to the hydrophilic group was 1:500, and the fixed density of the nucleic acid probe molecule was 1.2 / μM ² . The molar ratio of the epoxy group to the hydrophilic group was 1:250, and the nucleic acid probe molecule had a fixed density of 2.4 / μM ² . The molar ratio of the epoxy group to the hydrophilic group is 1:1 to 1:250, and the nucleic acid probe molecule has a fixed density of more than 2.4 / μM ² .

Since the specific data involved in the embodiment of the present invention is mostly statistically significant, any numerical value expressed in an accurate manner represents a range, that is, an interval including plus or minus 10% of the numerical value, unless otherwise specified. .

In an embodiment of the present application, the activated solid phase substrate is obtained by contacting a solid phase substrate with ethanol or isopropanol and a Piranha solution. This results in a sufficient amount of reactive hydroxyl groups on the surface of the solid phase substrate.

In the examples of the present application, the epoxy silane is 3-glycidoxypropyltrimethoxysilane. Further, the silanization efficiency is further improved.

In the examples of the present application, the hydroxysilane is hydroxypropyltrimethoxysilane. Further, the silanization efficiency is further improved.

In the examples of the present application, the amino group is modified at the 5' end of the nucleic acid probe molecule.

In the examples of the present application, the nucleic acid probe molecule is a primer containing polyT, polyA.

In the examples of the present application, the length of the nucleic acid probe molecule is 10 to 30 bp.

In the examples of the present application, the epoxy ring opening reaction is carried out in a phosphate solution. Further, the reaction pH is effectively controlled to 8 to 10.

In the examples of the present application, the phosphate is a 0.1 M to 2 M K ₂ HPO ₄ solution. Further, the efficiency of the epoxy ring opening reaction is further improved.

In the examples of the present application, the epoxy ring-opening reaction is carried out at 25 to 60 ° C for 4 to 24 hours. Further, the epoxy ring-opening reaction is sufficient, and the nucleic acid probe molecules can be maximally immobilized on the silanized solid phase substrate.

In an embodiment of the present application, after the surface modification, before the immobilizing the nucleic acid probe molecule, the method further comprises: performing a first washing on the silanized solid phase substrate. Further, the excess epoxy silane and hydroxysilane are washed away from the surface of the solid phase substrate, thereby further improving the non-specific adsorption ability of the chip.

In an embodiment of the present application, the immobilizing the nucleic acid probe molecule further comprises: performing a second washing on the solid phase substrate to which the nucleic acid probe molecule is immobilized. Further, excess unfixed nucleic acid probe molecules or non-specific binding are washed away from the surface of the solid phase substrate, thereby further improving the non-specific adsorption ability of the chip.

In the examples of the present application, the first wash is alternately washed with ethanol or isopropanol, water.

In the examples of the present application, the second wash is followed by washing with a mixture of 3 x SSC and 0.1% Triton, 0.1 M to 2 M K ₂ HPO ₄ , 150 mM HEPES and 150 mM NaCl solution and ultrapure water.

A third aspect of the present application proposes a chip prepared by the method of preparing a chip of the present application.

The chip prepared by the invention has superior surface properties, low non-specific adsorption to nucleic acid, facilitates immobilization and uniform distribution of a large number of probes, and is applied to nucleic acid capture detection, and is advantageous for obtaining large-volume, high-quality detection results, and is particularly suitable for use. For highly accurate nucleic acid detection, such as nucleic acid detection at the single molecule level.

DRAWINGS

1 is a schematic view showing a process of a sequencing chip in an embodiment of the present application;

2 is a graph showing the results of changes in surface hybridization fluorescence dots at different hybridization concentrations in the examples of the present application;

3 is a graph showing the results of changes in surface hybridization fluorescence dots at different mixing ratios in the examples of the present application;

Fig. 4 is a graph showing the results of changes in the number of surface non-specifically adsorbed fluorescent dots at different mixing ratios in the examples of the present application.

Detailed ways

This application is based on the discovery and recognition of the following facts and issues by the inventors:

Currently commercially available chips or chips prepared by known methods, the surface resistance of the chip to non-specific adsorption and the like can not meet the requirements of chip-based single molecule detection. For example, when sequencing on a chip, the test found that the surface properties of the chip include non-specific adsorption, etc., and it is important for the immobilization, immobilization and distribution of surface primers/probes, the capture of template nucleic acids, and the biochemical reactions of subsequent sequencing processes. Impact, directly affecting the final sequencing results.

The present application claims priority to Chinese Patent Application No. JP-A No. No. No. No. No. No. No. No.

In the present application, CY3 and CY5 are nucleic acid labeling molecules, and both Cy ⁵ and Cy ³ are water-soluble 3H-phthalocyanine type bioluminescent labeling dyes, which are capable of emitting red color fluorescence and green fluorescence under laser irradiation of 650 nm and 550 nm, respectively. .

The base chip is an empty chip and does not contain a probe.

The epoxy silane may be 3-glycidoxypropyltrimethoxysilane, glycidyloxypropylethoxysilane, glycidyloxypropylmethyldiethoxysilane, glycidyl ether oxygen Propylmethyldimethoxysilane, 2-(3,4-epoxycyclohexane)-ethyltrimethoxysilane, 2-(3,4-epoxycyclohexane)-ethyltriethyl Oxysilane or the like. In the examples of the present application, 3-glycidoxypropyltrimethoxysilane is exemplified.

The first silane may be hydroxypropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-ureidopropyltriethoxysilane, 3-ureidopropyl Trimethoxysilane, (3,3,3-trifluoropropyl)methyldimethoxysilane, (3,3,3-trifluoropropyl)methyldiethoxysilane, 3-methylpropene Acyloxypropyltrimethoxysilane, methacryloxypropyltris(trimethylsiloxy)silane, and the like. In the examples of the present application, hydroxypropyltrimethoxysilane is exemplified.

The present application is further described in detail below by way of specific embodiments. The following examples are only intended to further illustrate the present application and are not to be construed as limiting the invention. The reagents, detection instruments, and the like in the examples can be obtained by themselves or by a commercially available route unless otherwise specified.

Example 1

(1) cleaning, activating the surface of the glass: alternately cleaning the surface of the glass with a diameter of 40 mm with ethanol or isopropanol, water, and activating the glass with a Piranha solution to produce sufficient active hydroxyl groups on the surface;

(2) Surface grafting: a 1:100 mixture of 3-glycidoxypropyltrimethoxysilane and hydroxypropyltrimethoxysilane was prepared, and the glass piece was adjusted to pH=5.5, and the glass piece was immersed in the solution. The reaction was carried out for 12 hours at room temperature, and after completion of the reaction, it was washed three times with ethanol or isopropanol and water.

Example 2

(1) the same as step (1) of the embodiment 1;

(2) the same as step (2) of the embodiment 1;

(3) fixing primer: After drying the grafted glass, placed in a 1.0M concentration of amino-modified nucleic acid primer PolyT K ₂ HPO ₄ solution, the concentration of K ₂ HPO ₄ solution was 150 mM, the reaction 1 After the hour, sequentially using 3×SSC solution (containing 0.1% Triton), 0.2MK ₂ HPO ₄ solution, 150 mM HEPES and 150 mM NaCl mixed solution, and ultrapure water, the substrate chip containing PolyT primer (probe) was completed. . The preparation process of this example is simply shown in Figure 1.

Example 3

After drying the substrate chip prepared in Example 2, different concentrations of PolyA-CY3 were hybridized to the above substrate chip at a hybridization concentration of 0.2 nM, 0.4 nM, 0.6 nM, and 0.8 nM, and repeated three times, and the field of view per square micrometer was counted under a microscope. The average number of fluorescent dots within. The results of the fluorescence points of different hybridization concentrations are shown in Fig. 2. It can be seen from Fig. 2 that: 1) there are fluorescent spots at different hybridization concentrations, indicating that the primers are successfully immobilized on the surface of the base glass; 2) after 0.6 nM, the hybridization concentration is increased, and the number of hybridization points is not significantly changed, indicating that the number of surface-fixed primers is constant. The number of hybridization points does not change with the number of points after a certain concentration.

Example 4

In the step (2) of the second embodiment, the surface grafting is a 1:100 mixture of 3-glycidoxypropyltrimethoxysilane and hydroxypropyltrimethoxysilane, and the mixture is separately adjusted in this example. The ratio of 3-glycidoxypropyltrimethoxysilane and hydroxypropyltrimethoxysilane was 1:1, 1:10, 1:100, 1:1000, and different ratios were prepared according to the method of Example 2. Three sheets of the substrate chip were each mixed with PolyA-CY3 at the same concentration of 0.6 nM, and the average number of fluorescent dots per square micrometer of field of view was counted under a fluorescence microscope. The results are shown in FIG. The results in Figure 3 show that: 1) surface primers are successfully immobilized on the base glass at different ratios; 2) the surface fixation density can be effectively adjusted by adjusting the proportion of the silane mixture.

Example 5

The substrate chips of different proportions in Example 4 were immersed in a solution containing 5% C-base containing CY5, reacted at room temperature for 5 minutes, and then washed with 2×PBS solution and ultrapure water in order to obtain a certain adsorption. For the base chip, the average number of fluorescent dots per square micrometer of field of view was counted under a microscope, and the results are shown in FIG. The results in Figure 4 show that: 1) the base wafer with a mixing ratio of 1:1000 has the least number of fluorescent dots, indicating that the anti-base non-specific adsorption of this surface is strong; 2) as the ratio becomes smaller, the number of fluorescent dots decreases. It is indicated that the anti-base non-specific adsorption of the substrate chip can be achieved by adjusting the mixing ratio.

In the description of the present specification, the description of the terms "one embodiment", "an embodiment", and the like means that the specific features, structures, materials or features described in connection with the embodiments are included in at least one embodiment of the present application. In the present specification, the schematic representation of the above terms is not necessarily directed to the same embodiment. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments. Moreover, those skilled in the art can combine and combine the various embodiments described in the specification and the features of the various embodiments without departing from each other.

The present application has been described with reference to the specific examples, which are used to help the understanding of the application and are not intended to limit the application. Variations to the above-described embodiments may be made by those skilled in the art in light of the teachings herein.

Claims

A method for chemically modifying a solid phase substrate, comprising:

Surface modification of the solid phase substrate, the surface modification comprising treating the surface of the solid phase substrate with a mixture comprising an epoxy group and a hydrophilic group end in a molar ratio of 1:1 to 1:1000 .
A method of preparing a chip, the chip comprising a solid phase substrate, comprising: chemically modifying the solid phase substrate using the method of claim 1.
The method of claim 2, wherein the method further comprises:

The nucleic acid probe molecule is immobilized on a surface modified solid phase substrate.
The method according to claim 3, wherein the nucleic acid probe molecule is a nucleic acid molecule modified by terminal amination.
The method according to claim 3, wherein after the surface modification, before immobilizing the nucleic acid probe molecule, the method further comprises: performing a first washing on the surface modified solid phase substrate.
The method according to claim 5, wherein the immobilizing the nucleic acid probe molecule further comprises: performing a second washing on the solid phase substrate to which the nucleic acid probe molecule is immobilized.
The method according to claim 1 or 2, wherein the epoxy group is derived from an epoxy silane having a silane group terminal and at least one epoxy group terminal; and/or

The hydrophilic group end is derived from a first silane having a silane group end and at least one hydrophilic group end.
The method according to claim 7, wherein the epoxy silane and/or the silane group of the first silane is selected from the group consisting of monomethylsilane, dimethylsilane, trimethylsilane, monomethyl At least one of oxysilane, dimethoxysilane, and trimethoxysilane.
The method according to claim 7 or 8, wherein the epoxy silane and/or the first silane has a linking group, the linking group and the silane group are linked, the linking group The group is 1 to 12 alkane chains or 1 to 8 ethoxy chains.
The method according to claim 7, wherein said epoxy silane is selected from the group consisting of 3-glycidoxypropyltrimethoxysilane, glycidyloxypropylethoxysilane, glycidyl ether oxygen Propylmethyldiethoxysilane, glycidoxypropylmethyldimethoxysilane, 2-(3,4-epoxycyclohexane)-ethyltrimethoxysilane and 2-( At least one of 3,4-epoxycyclohexane)-ethyltriethoxysilane.
The method of claim 7 wherein said first silane is selected from the group consisting of hydroxypropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3 -Ureapropyltriethoxysilane, 3-ureidopropyltrimethoxysilane, (3,3,3-trifluoropropyl)methyldimethoxysilane, (3,3,3-trifluoropropane At least one of methyldiethoxysilane, 3-methacryloxypropyltrimethoxysilane, and methacryloxypropyltris(trimethylsiloxy)silane.
The method of claim 7 wherein said epoxy silane is 3'-glycidoxypropyltrimethoxysilane and said first silane is hydroxypropyltrimethoxysilane.
Process according to claim 1 or 2, characterized in that the surface modification is carried out in the liquid phase and/or in the gas phase.
The method according to claim 1 or 2, wherein the epoxy group is derived from an epoxy silane, the terminal end of the hydrophilic group is derived from a first silane, and the epoxy silane and the first silane are in the mixture. The total content is from 1% to 3%.
The method according to claim 1 or 2, wherein the solid phase substrate is surface-activated before the surface modification to impart a hydroxyl group to the surface of the solid phase substrate.
A chip characterized in that the chip is a chip prepared by the method according to any one of claims 2 to 15.