CN114950583A

CN114950583A - Preparation method of enhanced fluorescence epoxy modified substrate, microarray chip and application of microarray chip

Info

Publication number: CN114950583A
Application number: CN202210447714.1A
Authority: CN
Inventors: 张大霄; 陈玉珠; 刘亚飞; 徐红星
Original assignee: Beijing Kangmin Biotechnology Co ltd
Current assignee: Beijing Kangmin Biotechnology Co ltd
Priority date: 2022-04-26
Filing date: 2022-04-26
Publication date: 2022-08-30

Abstract

The invention discloses a preparation method of an enhanced fluorescence epoxy modified substrate, a microarray chip and application thereof, wherein the preparation method comprises the following steps: s1, sequentially placing a substrate in a titanium dioxide sol-gel solution for dipping, pulling and drying to form a film; s2, placing the film in a silica sol-gel solution for dipping, pulling and drying the film; s3, alternately repeating the steps S1 and S2, and then dipping and drying in the sol-gel of the silicon oxide and the epoxy active groups to obtain the enhanced fluorescent epoxy modified substrate; compared with the existing preparation method, the preparation method has the advantages of low cost, simple process, obvious fluorescence enhancement, stability and reliability, is suitable for large-scale production of fluorescence-enhanced epoxy microarray biochips, and further promotes the application of microarray technology in clinical detection.

Description

Preparation method of enhanced fluorescence epoxy modified substrate, microarray chip and application of microarray chip

Technical Field

The invention relates to the technical field of microarray chip preparation, in particular to a preparation method of an enhanced fluorescent epoxy modified substrate, a microarray chip and application thereof.

Background

The microarray technology is used as a high-throughput and low-dosage multi-index protein detection means, and has wide application in the fields of disease diagnosis, curative effect evaluation, pathological research and the like. However, the sensitivity of the traditional microarray technology is insufficient, and the application and popularization of the technology are limited.

To compensate for this, various enhancements have been investigated in the field. Mainly divided into chemical biological enhancement (mainly relying on the catalytic amplification effect of enzymes) and physical enhancement. Compared with chemical biological enhancement, the physical enhancement has the characteristics of simple operation steps, instant enhancement, less time consumption and the like. The physical enhancement is mainly realized by processing a special microarray substrate, the common means is to process metal nano structures such as gold, silver and the like on the substrate, and the plasmon effect generated by the metal nano structures is used for enhancing the fluorescent molecules adsorbed on the substrate. However, this approach involves large-area fabrication of micro-nano structures, and a series of semiconductor-related processes such as photolithography, etching, deposition, etc. and a matched clean room or workshop are usually used, which can dramatically increase the production cost. On the other hand, the enhancement of the metal micro-nano structure is not uniform enhancement of the whole substrate surface, and only fluorescent molecules adsorbed in the vicinity of the micro-nano structure can be enhanced, which makes reproducibility very poor when detecting low-concentration substances.

While another enhancement, reflective interferometric enhancement can also serve the same enhancement. Regardless of the gold nanostructure or the reflection interference type enhancement, the fluorescence molecular signal is enhanced by regulating and controlling the intensity of the electromagnetic field. The difference is that the enhancement of the metal nano structure is local, the regulation and control of the electromagnetic field only occur around the metal nano structure, and molecules adsorbed on a non-structural area cannot enjoy the enhancement effect. The reflection interference type enhancement is that the whole plane is enhanced, so the uniformity is better and the reproducibility is better. The reflection interference type enhancement is mainly realized by a Bragg mirror which is mainly composed of a periodic thin film structure formed by alternately depositing high-refractive-index media and low-refractive-index media.

Disclosure of Invention

The invention aims to provide a preparation method of an enhanced fluorescence epoxy modified substrate, a microarray chip and application thereof, so as to at least solve one of the defects in the prior art.

In view of this, the scheme of the invention is as follows:

a preparation method of an enhanced fluorescence epoxy modified substrate is characterized by comprising the following steps:

s1, sequentially placing a substrate in a titanium dioxide sol-gel solution for dipping, pulling and drying to form a film;

s2, placing the film in a silica sol-gel solution for dipping, pulling and drying the film;

and S3, alternately repeating the steps S1 and S2, and then dipping and drying in the silica and the sol-gel of the epoxy active groups to obtain the enhanced fluorescent epoxy modified substrate.

In the invention, the titanium dioxide sol-gel solution is prepared by dropwise adding an acidic catalyst and water into tetrabutyl titanate in a solvent, and carrying out hydrolytic polymerization under stirring; the silica sol-gel solution silica is prepared by dropping an acidic catalyst and water into tetrahexyl orthosilicate in a solvent, and performing hydrolytic polymerization under stirring.

Further, in the preparation method of the titanium dioxide sol-gel solution, the dosage range of the tetrabutyl titanate is 5-15% by volume ratio of the total system; the dosage range of the water is 0.1-5%, the dosage of the acid catalyst is 0.5-5%, and the concentration of the acid catalyst is 1 mol/L.

Further, in the preparation method of the silica sol-gel solution, the dosage range of the tetrahexyl orthosilicate is 5-15% in terms of the volume ratio of the total system; the dosage range of the water is 2-15%, the dosage of the acid catalyst is 0.1-5%, and the concentration of the acid catalyst is 1 mol/L.

In the present invention, the pulling speed of step S1 is 200-3000 μm/S.

In the present invention, the pulling speed of step S2 is 200-4000 μm/S.

A fluorescence-enhanced microarray chip comprises the enhanced fluorescence epoxy modified substrate obtained by the preparation method and biomolecules which are adsorbed on the substrate and distributed in an array.

In the fluorescence enhancement microarray chip, the surface of the fluorescence enhancement epoxy modified substrate is coated with the titanium dioxide film and the silicon dioxide film which are alternately arranged, the thickness of the titanium dioxide film is 50-90nm, and the thickness of the silicon dioxide film is 70-120 nm.

Preferably, in the fluorescence-enhanced microarray chip, the titanium dioxide film comprises 4 layers, and the silicon dioxide film comprises 3 layers; or the titanium dioxide film is 5 layers, and the silicon dioxide film is 4 layers; or the titanium dioxide film is 6 layers, and the silicon dioxide film is 5 layers.

The invention also provides the application of the fluorescence-enhanced microarray chip in fluorescence scanning quantitative detection of target molecules, such as quantitative detection of target proteins and nucleic acid molecules based on the affinity acting force between biomolecules such as the combination of antigens and antibodies and the base complementary action of nucleic acid molecules.

Compared with the prior art, the beneficial effects of the invention include but are not limited to:

1. compared with the prior art, the fluorescence-enhanced epoxy substrate has the advantages of low preparation cost, simple process, stability and reliability, is suitable for large-scale production of fluorescence-enhanced epoxy microarray biochips, and further promotes the application of microarray technology in clinical detection.

2. The fluorescence-enhanced epoxy substrate disclosed by the invention enables the surface of the substrate to be uniformly coated in a dipping and pulling manner; in addition, the performance of the obtained fluorescence-enhanced microarray chip for quantitative detection is remarkably higher than that of a soaking-modified epoxy substrate, and is 10 times as high as that of a commercial chip, so that excellent detection sensitivity and lower detection limit can be provided.

Drawings

FIG. 1 is a schematic diagram of a process for preparing an enhanced fluorescent epoxy modified substrate according to the present invention.

FIG. 2 is an SEM image of an enhanced fluorescence epoxy modified substrate according to the present invention.

FIG. 3 shows the fluorescence scanning results of the enhanced fluorescence epoxy-based modified substrates 1-8 of the present invention when applied to antibody detection.

FIG. 4 shows the fluorescence intensity results of the enhanced fluorescence epoxy-based modified substrates 1-8 of the present invention when applied to antibody detection.

FIG. 5 shows the fluorescence scanning contrast results of the enhanced fluorescence epoxy-modified substrate 4 of the present invention and the commercial substrate when applied to antibody detection.

FIG. 6 shows the fluorescence intensity control results of the enhanced fluorescence epoxy-modified substrates 4, 8-10 of the present invention when applied to antibody detection with commercial substrates.

FIG. 7 shows the comparison of the sensitivity of the enhanced fluorescent epoxy-modified substrate 4 and the commercial substrate when applied to VEGF antigen detection.

Detailed Description

In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, the present invention is further described in detail with reference to the following detailed description. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The invention provides a preparation method of a multilayer film Bragg mirror microarray substrate capable of enhancing a fluorescence signal, which aims to overcome the defects of chemical and physical methods adopted in the prior art. The high-refractive-index film is titanium oxide, and the low-refractive-index film is silicon oxide; compared with a gold nanostructure method processed by a semiconductor process, the method greatly reduces the cost, the manufacturing steps and the time, and simultaneously enhances the reproducibility of the fluorescent substrate to be better.

The high-refractive-index film is coated on the substrate by adopting titanium dioxide sol-gel solution for dip-coating; the low-refractive-index film is coated on the high-refractive-index film layer by dipping and pulling through a silica sol-gel solution; preferably, the high refractive index thin films and the low refractive index thin films are alternately coated and formed on a substrate including five high refractive index thin films and four low refractive index thin films.

The titanium dioxide sol-gel solution (A sol-gel) is obtained by dropwise adding an acidic catalyst and water into a solvent through tetrabutyl titanate, and carrying out hydrolytic polymerization under stirring; the dosage range of the tetrabutyl titanate is 5-15% by volume ratio of the total system; the dosage range of the water is 0.1-5%, the dosage of the acid catalyst is 0.5-5%, and the concentration of the acid catalyst is 1 mol/L.

The silica sol-gel solution (B sol-gel) is prepared by adding an acidic catalyst and water dropwise into tetraethoxysilane in a solvent, and carrying out hydrolytic polymerization under stirring; the dosage range of the tetrahexyl orthosilicate is 5-15% in terms of the volume ratio of the total system; the dosage range of the water is 2-15%, the dosage of the acid catalyst is 0.1-5%, and the concentration of the acid catalyst is 1 mol/L.

The preparation method of the sol-gel (C sol-gel) of the silicon oxide and the epoxy active groups comprises the following steps: dissolving tetraethyl orthosilicate and gamma-glycidyl ether oxypropyl trimethoxy silane, dropwise adding hydrochloric acid and water, and stirring to obtain a sol-gel solution; the concentration of the hydrochloric acid is 1mol/L, and the hydrochloric acid accounts for 0.05-5% of the total reaction system according to the volume ratio; the water accounts for 2-20% of the total reaction system.

The process of preparing the substrate by adopting the dip-coating method on the fluorescence enhancement substrate is as follows: immersing a substrate into titanium dioxide sol-gel (A sol-gel) (filtering the sol-gel by a microporous filter membrane), pulling at the speed of 200-3000 μm/s, standing, drying and forming a film; then immersing into silica sol-gel (B sol-gel), pulling at the speed of 200-4000 μm/s, standing, drying, and alternately dipping and pulling to form film. The above processes are alternately carried out to reach the required number of layers, and then the materials are placed in the sol-gel (C sol-gel) of the silicon oxide and the epoxy active group for soaking, drying and film forming to obtain the epoxy resin. The preparation process of the substrate is shown in FIG. 1.

The invention obtains the enhanced fluorescence epoxy modified substrate coated with alternate titanium dioxide films and silicon dioxide films by the dipping and pulling method, wherein the thickness of the titanium dioxide film is 50-90nm, and the thickness of the silicon dioxide film is 70-120 nm. Preferably, the titanium dioxide layer has a film thickness of 50nm, 55nm, 60nm, 68nm, 69nm, 90 nm; the thickness of the silicon dioxide film is 70nm, 73nm, 80nm, 90nm, 91nm, 92nm and 120 nm. Preferably, the titanium dioxide film is 4 layers, and the silicon dioxide film is 3 layers; or the titanium dioxide film is 5 layers, and the silicon dioxide film is 4 layers; or the titanium dioxide film is 6 layers, the silicon dioxide film is 5 layers, the performance of the fluorescence enhancement microarray chip under the combined structure for quantitative detection is obviously higher than that of a soaking modified epoxy substrate, and the performance is 10 times as high as that of a commercial chip.

The fluorescence-enhanced epoxy modified microarray chip provided by the invention is obtained by modifying biomolecules in a dot matrix form on the substrate subjected to reduction modification, wherein the biomolecules comprise oligonucleotides, proteins and the like. Blocking the molecule array points by using a silica gel fence to form independent detection holes, wherein the biomolecules comprise but are not limited to oligonucleotides, proteins and the like; the microarray chip is suitable for large-scale production, and can improve excellent detection sensitivity.

The substrate material applied to the microarray chip can be glass, and can also be a high molecular sheet material, such as common PMMA, PS and the like.

The principle of the fluorescence enhancement epoxy modified microarray chip provided by the invention is as follows: through designing many kinds of membranes and multilayer structure, can reflect specific wave band for specific wave band's light wave produces the interference at the substrate surface and adds, and then improves surperficial electromagnetic field intensity, can improve the absorptivity and the radiance of the fluorescence molecule of adsorbing on the surface, finally reaches the effect that improves fluorescence signal. The multilayer film is obtained by alternately depositing dielectric films with different refractive indexes. And at the interface with the changed refractive index, the light wave can be reflected, so after the light wave vertically enters the multilayer film, the light wave can be reflected and interfered at each interface with the changed refractive index, namely the interfaces of different film layers, and finally the light wave in a specific wave band can be mostly reflected to the surface through the thickness regulation and control of the film layers and is added at the surface through interference, so that the electromagnetic field is enhanced, and the effect of enhancing the fluorescent signal is further achieved.

The following are preferred experimental examples of the present invention for illustrating and verifying the above-mentioned scheme and technical effects of the present invention, and the selected examples are only preferred experimental examples and are not intended to limit the present invention.

The following are examples of the preparation of sol-gel solutions:

preparation example 1

1. Preparation of sol-gel solution:

100mL of isopropyl alcohol (IPA) was weighed into a 250mL Erlenmeyer flask, stirred electromagnetically at room temperature, 10% i.e., 11.08mL of tetrabutyl titanate was added, stirred at room temperature for 10 minutes, then a mixed solution of hydrochloric acid (HCl) and water, specifically 3% i.e., 3.32mL of a 1mol/L hydrochloric acid aqueous solution and 3% i.e., 3.32mL of water, was added by a syringe pump at 100. mu.L/min, and stirred in a water bath at 25 ℃ for 12 hours. Thus, a sol-gel solution 1 was obtained.

2. B preparation of sol-gel solution:

102mL of Isopropanol (IPA) is weighed into a 250mL conical flask, stirred electromagnetically, added with 10 percent of tetraethoxysilane (12 mL), stirred for 10 minutes, added with a mixed solution of 1mol/L hydrochloric acid and water by a syringe pump at 1ML/min, and stirred for 12 hours in a water bath at 25 ℃ after the specific components are 2 percent of 2.4mL of 1mol/L hydrochloric acid aqueous solution and 10 percent of water at 12 mL. Thus, a B sol-gel solution 1 was obtained.

Preparation example 2

1. Preparation of sol-gel solution:

100mL of isopropyl alcohol (IPA) is weighed into a 250mL conical flask, stirred electromagnetically, 5% of tetrabutyl titanate, namely 5.54mL is added, after stirring for 10 minutes, a mixed solution of 1mol/L hydrochloric acid and water is added into a syringe pump at 100 mu L/min, the specific components of the mixed solution are 5% of hydrochloric acid aqueous solution, namely 5.54mL of 1mol/L hydrochloric acid aqueous solution and 5% of water, namely 5.54mL of water, and the mixed solution is stirred for 12 hours in a water bath at 25 ℃. Thus, a sol-gel solution 2 was obtained.

2. B preparation of sol-gel solution:

102mL of Isopropanol (IPA) is weighed into a 250mL conical flask, stirred electromagnetically, 5% of tetraethoxysilane (6 mL) is added, after stirring for 10 minutes, a mixed solution of hydrochloric acid and water is added by a syringe pump at 1ML/min, the specific components of the mixed solution are 5% of hydrochloric acid solution at 6mL of 1mol/L and 15% of water at 18mL, and the mixed solution is stirred for 12 hours in a water bath at 25 ℃. Thus, a B sol-gel solution 2 was obtained.

Preparation example 3

1. Preparation of sol-gel solution:

100mL of isopropyl alcohol (IPA) was weighed into a 250mL Erlenmeyer flask, stirred electromagnetically, 15% i.e., 16.62mL of tetrabutyl titanate was added, and after stirring for 10 minutes, a mixed solution of 1mol/L hydrochloric acid and water, specifically comprising 0.5% i.e., 0.55mL of 1mol/L hydrochloric acid aqueous solution and 0.2% i.e., 0.22mL of water, was added by a syringe pump at 100. mu.L/min, and stirred for 12 hours in a water bath at 25 ℃. Thus, a sol-gel solution 3 was obtained.

2. B preparation of sol-gel solution:

102mL of Isopropanol (IPA) is weighed into a 250mL conical flask, stirred electromagnetically, added with 15 percent of tetraethoxysilane (18 mL), stirred for 10 minutes, added with a mixed solution of hydrochloric acid and water at a rate of 1ML/min by a syringe pump, and stirred for 12 hours in a water bath at 25 ℃ after the specific components of the mixed solution are 1 percent of 1.2mL of 1mol/L hydrochloric acid aqueous solution and 5 percent of 6mL of water. Thus, a B sol-gel solution 3 was obtained.

Preparation example 4

1. Preparation of sol-gel solution:

100mL of isopropyl alcohol (IPA) is weighed into a 250mL conical flask, stirred electromagnetically, 10% namely 10mL of tetrabutyl titanate is added, after stirring for 10 minutes, a mixed solution of 1mol/L hydrochloric acid and water is added by a syringe pump at 100 mu L/min, the specific components of the mixed solution are 0.2% namely 0.22mL of 1mol/L hydrochloric acid aqueous solution and 0.6% namely 0.66mL of water, and stirring is carried out in a water bath at 25 ℃ for 12 hours. Thus, a sol-gel solution 4 was obtained.

2. B, preparation of sol-gel solution:

102mL of Isopropanol (IPA) is weighed into a 250mL conical flask, stirred electromagnetically, added with 10 percent of tetraethoxysilane (12 mL), stirred for 10 minutes, added with a mixed solution of hydrochloric acid and water at a concentration of 1ML/min by a syringe pump, and stirred for 12 hours in a water bath at 25 ℃ after the specific components of the mixed solution are 0.1 percent of 0.12mL of 1mol/L hydrochloric acid aqueous solution and 4 percent of 4.8mL of water. Thus, a B sol-gel solution 4 was obtained.

Preparation example 5

102mL of isopropyl alcohol (IPA) is measured in a 250mL conical flask, stirred electromagnetically at room temperature, 5% tetraethyl orthosilicate (TEOS) is added, stirred at room temperature for 10 minutes, and then a mixed solution of hydrochloric acid (HCl) and water, which is 0.05% of 0.075mL of 1M aqueous hydrochloric acid (HCl) and 2% of 2.4mL of water, is added by a syringe pump at 1mL/min, and stirred at room temperature for 12 hours. Then 1% of gamma-Glycidoxypropyltrimethoxysilane (GPTS), i.e., 1.2mL, is added, and then a mixture of hydrochloric acid and water, the specific components of which are 0.15mL of 1M hydrochloric acid (HCl) aqueous solution and 5.1mL of water, is added at a rate of 1mL/min by a syringe pump, and the mixture is stirred at room temperature for 12 hours, thus obtaining a C sol-gel solution.

The sol-gel solution obtained in preparation examples 1 to 5 was dip-pulled on a substrate to obtain a fluorescence-enhanced epoxylated glass slide as in examples 1 to 4.

Example 1

50mL of A, B sol-gel solution 1 from preparation example 1 were each filtered through a 0.22 μm microfiltration membrane and then each filtered in a quartz square cylinder of 7 cm. times.5 cm. times.2 cm. Dip-pulling was performed in the A sol-gel solution 1 at a rate of 200 μm/s using a dip-puller. Standing for 1min after the process is finished, standing for 4min at normal temperature after the process is taken down, baking for 5min in an oven at 100 ℃, cooling to room temperature, then performing impregnation and lifting in the B sol-gel solution 1 at 200 mu m/s, standing for 1min after the process is finished, standing for 4min at normal temperature after the process is taken down, and baking for 5min in an oven at 100 ℃. This operation was performed alternately in the order of 5 layers by solution A and 4 layers by solution B. After the glass slide is baked, the glass slide is aired to the normal temperature and soaked in the C sol-gel containing the silicon oxide and the epoxy active groups for 10 min. And (3) after soaking, airing, baking in an oven at 100 ℃ for 5min, and cooling to room temperature to obtain the enhanced fluorescent epoxy modified substrate 1.

Example 2

50mL of A, B sol-gel solution 2 from preparation example 2 were each filtered through a 0.22 μm microfiltration membrane and then each filtered in a quartz square cylinder of 7 cm. times.5 cm. times.2 cm. Dip-pulling was performed in the A sol-gel solution 2 at a rate of 3000 μm/s using a dip-puller. Standing for 1min after the process is finished, standing for 4min at normal temperature after the process is taken down, baking for 5min in an oven at 100 ℃, cooling to room temperature, then performing impregnation and lifting at 4000 mu m/s in the B sol-gel solution 2, standing for 1min after the process is finished, standing for 4min at normal temperature after the process is taken down, and baking for 5min in an oven at 100 ℃. This operation was performed alternately in the order of 5 layers by solution A and 4 layers by solution B. After the glass slide is baked, the glass slide is aired to the normal temperature and soaked in the C sol-gel containing the silicon oxide and the epoxy active groups for 10 min. And (3) after soaking, airing, baking in an oven at 100 ℃ for 5min, and cooling to room temperature to obtain the enhanced fluorescent epoxy modified substrate 2.

Example 3

50mL of A, B sol-gel solution 3 from preparation example 3 were each filtered through a 0.22 μm microfiltration membrane and then each filtered in a quartz square cylinder of 7 cm. times.5 cm. times.2 cm. Dip-pulling was performed in the A sol-gel solution 3 at a rate of 1500 μm/s using a dip-puller. Standing for 1min after the process is finished, standing for 4min at normal temperature after the process is taken down, baking for 5min in an oven at 100 ℃, cooling to room temperature, then performing impregnation and lifting at 1500 mu m/s in the sol-gel solution B3, standing for 1min after the process is finished, standing for 4min at normal temperature after the process is taken down, and baking for 5min in an oven at 100 ℃. This operation was performed alternately in the order of 5 layers by solution A and 4 layers by solution B. After the glass slide is baked, the glass slide is aired to the normal temperature and soaked in the C sol-gel containing the silicon oxide and the epoxy active groups for 10 min. And (3) after soaking, airing, baking in an oven at 100 ℃ for 5min, and cooling to room temperature to obtain the enhanced fluorescent epoxy modified substrate 3.

Example 4

50mL of A, B sol-gel solution 4 from preparation example 4 were each filtered through a 0.22 μm microfiltration membrane and then each filtered in a quartz square cylinder of 7 cm. times.5 cm. times.2 cm. Dip-pulling was performed in the A sol-gel solution 1 at a rate of 1800 μm/s using a dip-puller. Standing for 1min after the process is finished, standing for 4min at normal temperature after the process is taken down, baking for 5min in an oven at 100 ℃, cooling to room temperature, then performing dip-coating and lifting in the B sol-gel solution at 1800 mu m/s, standing for 1min after the process is finished, standing for 4min at normal temperature after the process is taken down, and baking for 5min in an oven at 100 ℃. This operation was performed alternately in the order of 5 layers by solution A and 4 layers by solution B. After the glass slide is baked, the glass slide is aired to normal temperature and is soaked in C sol-gel containing silicon oxide and epoxy active groups for 10 min. After soaking, drying in the air, and then baking in an oven at 100 ℃ for 5 min. And cooling to room temperature after baking to obtain the enhanced fluorescent epoxy modified substrate 4.

Example 5

50mL of A, B sol-gel solution 1 of preparation example 1 was taken, and dipping and pulling were alternately performed by the method of example 4 to pull 5 layers of solution A and 4 layers of solution B. After the glass slide is baked, the glass slide is aired to the normal temperature and soaked in the C sol-gel containing the silicon oxide and the epoxy active groups for 10 min. After soaking, drying in the air, and then baking in an oven at 100 ℃ for 5 min. And cooling to room temperature after baking to obtain the enhanced fluorescent epoxy modified substrate 5.

Example 6

50mL of A, B sol-gel solution 2 of preparation example 2 was taken, and dipping and pulling were alternately performed by the method of example 4 to pull 5 layers of solution A and 4 layers of solution B. After the glass slide is baked, the glass slide is aired to the normal temperature and soaked in the C sol-gel containing the silicon oxide and the epoxy active groups for 10 min. After soaking, drying in the air, and then baking in an oven at 100 ℃ for 5 min. And cooling to room temperature after baking to obtain the enhanced fluorescent epoxy modified substrate 6.

Example 7

50mL of A, B sol-gel solution 3 of preparation example 3 was taken, and dipping and pulling were alternately performed by the method of example 4 to pull 5 layers of solution A and 4 layers of solution B. After the glass slide is baked, the glass slide is aired to the normal temperature and soaked in the C sol-gel containing the silicon oxide and the epoxy active groups for 10 min. After soaking, drying in the air, and baking in an oven at 100 ℃ for 5min after drying in the air. And cooling to room temperature after baking to obtain the enhanced fluorescent epoxy modified substrate 7.

Example 8

50mL of A, B sol-gel solution 1 of preparation example 1 was taken, and dipping and pulling were alternately performed by the method of example 4 to pull 6 layers of solution A and 5 layers of solution B. After the glass slide is baked, the glass slide is aired to the normal temperature and soaked in the C sol-gel containing the silicon oxide and the epoxy active groups for 10 min. After soaking, drying in the air, and then baking in an oven at 100 ℃ for 5 min. And cooling to room temperature after baking to obtain the enhanced fluorescent epoxy modified substrate 8.

Example 9

Adopting the method of embodiment 3, A, B sol-gel solution 3 is taken, and the substrate is respectively and alternately dipped and pulled, the pulling speed is 1500 μm/s, wherein solution A pulls 4 layers, and solution B pulls 3 layers; after the glass slide is baked, the glass slide is aired to the normal temperature and soaked in the C sol-gel containing the silicon oxide and the epoxy active groups for 10 min. After soaking, drying in the air, and baking in an oven at 100 ℃ for 5min after drying in the air. And cooling to room temperature after baking to obtain the fluorescence enhanced epoxy modified contrast substrate 9.

Example 10

Adopting the method of the embodiment 4, taking A, B sol-gel solution 4, respectively and alternately dipping and pulling the substrate, wherein the pulling speed is 1800 mu m/s, the A solution pulls 4 layers, and the B solution pulls 3 layers; after the glass slide is baked, the glass slide is aired to the normal temperature and soaked in the C sol-gel containing the silicon oxide and the epoxy active groups for 10 min. After soaking, drying in the air, and then baking in an oven at 100 ℃ for 5 min. And cooling to room temperature after baking to obtain the fluorescence enhanced epoxy modified contrast substrate 10.

Comparative example 1

Adopting the method of embodiment 3, A, B sol-gel solution 3 is taken, and the substrate is respectively and alternately dipped and pulled, the pulling speed is 1500 μm/s, wherein, solution A pulls 7 layers, solution B pulls 6 layers; after the glass slide is baked, the glass slide is aired to the normal temperature and soaked in the C sol-gel containing the silicon oxide and the epoxy active groups for 10 min. After soaking, drying in the air, and then baking in an oven at 100 ℃ for 5 min; after the baking, the substrate was cooled to room temperature, and the film on the surface of the substrate was found to crack.

Comparative example 2

Adopting the method of the embodiment 4, taking A, B sol-gel solution 4, respectively and alternately dipping and pulling the substrate, wherein the pulling speed is 1800 mu m/s, wherein solution A pulls 7 layers, and solution B pulls 6 layers; after the glass slide is baked, the glass slide is aired to the normal temperature and soaked in the C sol-gel containing the silicon oxide and the epoxy active groups for 10 min. After soaking, drying in the air, and then baking in an oven at 100 ℃ for 5 min. After the baking, the substrate was cooled to room temperature, and the film on the surface of the substrate was found to crack.

Comparative example 3

1) mu.L of 2mg/mL IgG antibody mouse was placed in a 200. mu.L centrifuge tube, and 5. mu.L of 0.5M (NH) was added ₄ ) ₂ SO ₄ Adding 5 mu L of glycerol and 10 mu L of LPBS, and fully and uniformly mixing to obtain the sampling solution.

2) Using a spotting instrument produced by Beijing Boao crystal classics Biotechnology Limited to spot on a commercial epoxy modified glass slide (prepared by a chemical soaking method in CN1588006A literature), adding PBS + 1% BSA into the glass slide at a rate of 20mL/min by using an injection pump for cleaning, drying the glass slide, and attaching a self-made silica gel fence to the glass slide to obtain the epoxy substrate.

Experimental example 1 characterization of fluorescence-enhanced epoxy substrate

1) The average thickness of the titanium oxide film and the silica film of examples 1 to 8 and comparative examples 1 to 2 above (thickness measured after single layer dip-pull baking) was measured by X-ray diffraction method, and the results are shown in Table 1.

Table 1:

average film thickness nm	Example 1	Example 2	Example 3	Example 4	Example 5
						Titanium dioxide film	55	90	60	68	69
Silicon dioxide film	73	120	80	90	90
						Average film thickness nm	Example 6	Example 7	Example 8	Example 9	Example 10
Titanium dioxide film	68	69	68	61	68
						Silicon dioxide film	91	91	90	80	90

As is clear from Table 1, the use of the sol-gel solutions A and B obtained in preparation examples 1 to 4 tended to result in a positive correlation between the thickness of the single layer obtained by dip coating and the coating rate; whereas the dipping and pulling speeds of examples 4 to 8 and comparative example 2, and example 3 and comparative example 1 were the same, and the single-layer film thickness was not greatly changed.

2) The enhanced fluorescent epoxy modified substrates obtained in examples 1-7 were observed by Scanning Electron Microscope (SEM), as shown in FIGS. 2a and 2b, and were found to have distinct multi-layer structures, with the bottom layer being the substrate. It can be easily seen from fig. 2b that the four white light bands from bottom to top are silicon dioxide film layers, the middle darker area is a titanium dioxide film layer, and the uppermost white light band is a silicon oxide film layer and an epoxy film layer. As can be seen from fig. 2b, the thicknesses of the titanium dioxide film and the silicon dioxide film are within the film thickness interval of table 1, and the uppermost silicon oxide film and the uppermost epoxy film are relatively thin.

Experimental example 2 preparation of microarray chip and application to antibody detection

2) The fluorescence-enhanced epoxy group prepared in examples 1 to 8 was decorated by spotting using a spotting instrument produced by Beijing Boo classical biotechnology, Inc., and PBS + 1% BSA was added at 20mL/min by a syringe pump for washing, and after drying, a self-made silica gel fence was attached to obtain the fluorescence-enhanced epoxy substrate.

3) 40 μ L of 20ng/mL goat anti-mouse IgG conjugated CY3 was incubated for 2h, washed with pure water, and scanned with a scanner manufactured by Beijing Boo Athens Biotechnology Ltd to obtain a fluorescence scan image as shown in FIG. 3

The LuxScan TM10K series of microarray software gave intensity values as shown in FIG. 4.

The above examples 1-4 differ in the sharpness (i.e., fluorescence signal intensity) of the enhanced fluorescence epoxy-modified substrate detection scans obtained at different dip pull rates when applied to antibody detection. However, as is evident from FIG. 3, the fluorescence scans of examples 1-4 gradually brighten, and the sharpness of examples 4-7 hardly changes; correspondingly, the fluorescence intensity of the examples 1-4 in FIG. 4 gradually becomes stronger, and the intensities of the examples 4-7 are equivalent, thereby illustrating that the pulling speed affects the change of the film thickness based on the combination of the experimental example 1, and the fluorescence signal enhancement is more remarkable under the condition of proper film thickness (such as the film thickness of titanium dioxide is 60-69nm, and the film thickness of silicon dioxide is 80-92 nm). In example 8, when the number of the titanium dioxide film layer and the number of the silicon dioxide film layer are increased to 6 and 5, respectively, the fluorescence signal is further enhanced relative to the chip with the 5 + 4-layer structure, which is consistent with the principle of realizing fluorescence enhancement.

4) The fluorescence-enhanced epoxy group prepared in examples 9-10 and comparative example 3 was spotted using a spotting instrument produced by Beijing Boao crystal Biotechnology Limited, and PBS + 1% BSA was added at 20mL/min by a syringe pump for cleaning, and after drying, a self-made silica gel fence was attached to obtain the fluorescence-enhanced epoxy substrate. The fluorescence scans obtained from examples 9-10 and comparative example 3 were performed using 40 μ L of 20ng/mL goat anti-mouse IgG coupled to CY3 for 2h, washed with pure water, and then scanned using a scanner manufactured by beijing boao crystal biotechnology limited, wherein the results of comparative example 3 and example 4 are shown in fig. 5, fig. 5a is the fluorescence scan result of example 4, and fig. 5b is the fluorescence scan result of comparative example 5, i.e., a conventional commercial chip. Fluorescence scanning results of examples 9-10, comparative example 5

The intensity values obtained by the LuxScan TM10K series microarray software are shown in FIG. 6 in comparison with the results of examples 4 and 8.

It can be seen that the fluorescence of example 8 in FIG. 6 was enhanced by nearly 10-fold compared to the commercial chip. In contrast, in fig. 6, it is apparent that in examples 9 to 10, in the case where the numbers of the titania film and the silica film were decreased by one layer in each of the same dip-coating methods, the fluorescence enhancement effect was reduced to some extent, but the fluorescence enhancement effect was still remarkable as compared with the commercial chips. In contrast, in comparative examples 1 and 2, in the case where the dipping and drawing methods were the same, the numbers of the titanium dioxide film and the silicon dioxide film were 7 and 6, respectively, although theoretically, there was a possibility of fluorescence enhancement, the number of layers was large, and the stress between the films and between the single layers was large, and therefore, the bursting occurred, and further, the situation could not be realized.

Experimental example 3 preparation of microarray chip and detection of cytokine VEGF

1) mu.L of 2mg/mLVEGF antibody was placed in a 200. mu.L centrifuge tube and 5. mu.L of 0.5M (NH) was added ₄ ) ₂ SO ₄ Adding 5 mu L of glycerol, adding 10 mu L of LPBS and 0.1% SDS, and fully and uniformly mixing to obtain a sampling solution.

2) Spotting the epoxy-modified glass slide surface prepared in the example 4 by using a spotting instrument produced by Beijing Boo classical biotechnology, Inc., adding PBS + 1% BSA at 20mL/min of a syringe pump for cleaning, drying by blowing, and attaching a self-made silica gel fence to obtain the epoxy substrate.

3) Adding VEGF antigen with different concentrations into independent detection holes in a glass slide, incubating for 2h, cleaning, adding 40 μ LVEGF detection antibody, incubating for 2h, cleaning with pure water, scanning with scanner produced by Beijing Boao Crystal classic biotechnology, and scanning with scanner

LuxScan ^TM The intensity values obtained by 10K series microarray software are fitted to obtain a standard curve as shown in FIG. 7, and the minimum detection limit of the substrate obtained in example 4 read from FIG. 7 when applied to microarray fluorescence detection is 0.8pg/mL, compared with 2pg/mL using a common commercially available epoxy chip under the same conditions.

Therefore, when the fluorescent detection chip is applied to fluorescent detection, the fluorescent enhancement layer is formed by alternately coating the titanium dioxide film and the silicon dioxide film, and the silicon oxide and epoxy active film layers are carried out on the upper surface of the fluorescent enhancement layer, compared with a commercial chip, the fluorescent detection chip can effectively improve the fluorescent signal intensity, and has better fluorescent enhancement effect when the titanium dioxide film and the silicon dioxide film layer are respectively 4 layers, 3 layers, 5 layers, 4 layers, 6 layers and 5 layers, and the highest value is equivalent to the fluorescent signal of the commercial chip about 10 times. According to the common knowledge of the skilled person, in case of applying to fluorescence detection the signal is significantly enhanced, an excellent detection sensitivity and a lower detection limit can be provided.

The invention is not limited solely to that described in the specification and embodiments, and additional advantages and modifications will readily occur to those skilled in the art, so that the invention is not limited to the specific details, representative embodiments and examples described herein, without departing from the spirit and scope of the general concept as defined by the appended claims and their equivalents.

Claims

1. A preparation method of an enhanced fluorescence epoxy modified substrate is characterized by comprising the following steps:

2. The preparation method according to claim 1, wherein the titania sol-gel solution is obtained by adding an acidic catalyst and water dropwise to tetrabutyl titanate in a solvent, and performing hydrolytic polymerization with stirring; the silicon dioxide sol-gel solution is prepared by dropwise adding an acidic catalyst and water into tetrahexyl orthosilicate in a solvent, and performing hydrolytic polymerization under stirring.

3. The method according to claim 2, wherein the tetrabutyl titanate is used in an amount ranging from 5 to 15% by volume of the total system; the dosage range of the water is 0.1-5%, the dosage of the acid catalyst is 0.5-5%, and the concentration of the acid catalyst is 1 mol/L.

4. The method according to claim 2, wherein the amount of tetrahexyl orthosilicate is in the range of 5-15% by volume of the total system; the dosage range of the water is 2-15%, the dosage of the acid catalyst is 0.1-5%, and the concentration of the acid catalyst is 1 mol/L.

5. The method of claim 1, wherein: the pulling speed of step S1 is 200-3000 μm/S.

6. The method of claim 1, wherein: the step S2 is performed at a pulling speed of 200-4000 μm/S.

7. A fluorescence-enhanced microarray chip comprising the fluorescence-enhanced epoxy-modified substrate obtained by the preparation method of any one of claims 1 to 6, and biomolecules adsorbed on the substrate and distributed in an array.

8. The fluorescence-enhanced microarray chip of claim 7, wherein the surface of the fluorescence-enhanced epoxy modified substrate is coated with alternating titanium dioxide films and silicon dioxide films, the thickness of the titanium dioxide film is 50-90nm, and the thickness of the silicon dioxide film is 70-120 nm.

9. The fluorescence-enhanced microarray chip according to claim 7, wherein the titanium dioxide film comprises 4 layers, and the silicon dioxide film comprises 3 layers; or the titanium dioxide film is 5 layers, and the silicon dioxide film is 4 layers; or the titanium dioxide film is 6 layers, and the silicon dioxide film is 5 layers.

10. Use of the fluorescence-enhanced microarray chip of any one of claims 7 to 9 for the quantitative detection of target molecules by fluorescence scanning.