A protective coating for gene chip and its preparation method and application

Abstract

The present invention discloses a protective coating for a gene chip, and a preparation method and application thereof, comprising a probe protective agent with a mass fraction of 2% to 8%, a surfactant of 0.005% to 0.05%, and the balance being deionized water. The probe protective agent is added to deionized water in a predetermined amount, and then the surfactant is added and shaken to mix evenly. After mixing evenly, centrifugation is performed to remove foam to obtain a coating solution. The present invention further provides a method for applying a protective coating for a gene chip and a gene chip with a protective coating. Comparative experimental results show that the grayscale value of the chip with coating protection decreases significantly less after hybridization, indicating that the hybridization activity of the gene recognition probe on the chip surface is better preserved and the coating components play a role in protecting the probe.

Description

Technical Field

[0001] The invention belongs to the technical field of biochip hybridization, relates to a hybridization buffer based on biochip solid-phase hybridization technology, and also relates to a preparation method thereof and application as a probe fluorescence protective agent. Background Art

[0002] Gene chips provide an efficient and convenient tool for modern medicine, especially medical diagnostics, and offer a reliable basis for molecular-level genetic diagnosis and therapy. Gene chips can be used to analyze the correlation between genes and diseases (such as cancer, infectious diseases, and genetic disorders), providing a deeper understanding of the root causes of disease. The most direct application of gene chips in medical diagnostics is the detection of disease-related genes. Biomedical research has shown that the pathogenesis of most human diseases is fundamentally linked to genes. Therefore, gene chips hold significant medical significance, enabling rapid detection of disease-related genes and mutations. Gene chips not only enhance the scientific accuracy of disease diagnosis but also provide guidance for treatment. Targeted treatment plans can be developed based on the detection results of disease-related genes.

[0003] High-density gene chips are a low-cost, high-capacity, uniformly formatted, and fast-analysis genetic testing method. They utilize oligonucleotides immobilized on the surface of silica microspheres as gene recognition probes, and a silicon substrate as the primary substrate for the gene chip. The sensitivity of gene chips depends on the activity of the recognition probes, so maintaining the activity of the surface recognition probes and their stability under long-term storage conditions significantly impact the detection quality of the gene chips. Furthermore, considering the cost and transportation of gene chips, their storage method should be convenient, inexpensive, and widely applicable.

[0004] In the prior art, chip protection is often considered from the perspective of chip storage to extend its lifespan. For example, for short-term storage, chips should be stored in a clean container with a controlled environment (dry air or nitrogen), preferably in their original packaging. For long-term storage, chips should be placed in an atmosphere of 99% nitrogen or inert gas, at a temperature of 17°C to 25°C, with a humidity of 7% to 25%, and a gas pressure higher than the ambient atmospheric pressure. Storage in this undisturbed environment can last for more than 12 months. However, there are no reports of chip protection that specifically protects the activity of surface recognition probes. Summary of the Invention

[0005] The present invention is directed to the above-mentioned problems, and based on the perspective of maintaining the activity of the probe for a long time, provides a protective coating for a gene chip, and also provides a method for preparing the chip coating.

[0006] The technical solution of the present invention is summarized as follows: a hydrophilic polymer with a significant protective effect on chip probes is selected as the functional component of the coating, which is prepared into a coating liquid with the assistance of a surfactant; then the coating is set on the chip surface by conventional dipping, spraying, etc.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a protective coating for a gene chip, comprising: a probe protective agent with a mass fraction of 2% to 8% (preferably 6%), a surfactant of 0.005% to 0.05% (preferably 0.01%), and the balance being deionized water.

[0009] Preferably, the probe protectant is selected from any one or more combinations of polyacrylamide, polyvinyl pyrrolidone, polyacrylic acid, polyethylene glycol, and polyvinyl alcohol; and the surfactant is selected from any one or more combinations of sodium lauryl sulfate, Tween 20, Tween 80, and Triton X-100.

[0010] More preferably, the probe protecting agent uses a polymer with a molecular weight of 10,000.

[0011] In a second aspect, the present invention provides a method for preparing the protective coating of the gene chip described above, comprising the following steps: adding a probe protective agent to deionized water in a predetermined amount, then adding a surfactant and shaking to mix, and after mixing evenly, centrifuging at 500g for 2 minutes to defoam to obtain a coating solution.

[0012] In a third aspect, the present invention provides a method for applying a protective coating to a gene chip, wherein the coating solution is applied to the surface of the gene chip to be processed in a conventional manner, with a thickness of 800 to 1000 nm.

[0013] In a specific embodiment of the present invention, the application method adopted is dip coating, and the method is as follows: the coating solution is preheated to 40-60°C (preferably 50°C) and placed in a ventilated environment with a wind speed maintained at 0.1-0.7 m / s; the gene chip to be treated is immersed in the coating solution in an up-and-down vertical manner at an immersion rate of 1-10 mm / s; after complete immersion, it is kept still for 1 minute, and then the chip is vertically pulled upward at a rate of 6-15 mm / s until the chip is more than 10 mm away from the liquid surface; then the chip is kept in the ventilated environment for 20 minutes to complete the complete volatilization and drying of the coating liquid on the chip surface.

[0014] In a fourth aspect, the present invention provides a gene chip with a protective coating, which is prepared using the above-mentioned method for applying the protective coating. The gene chip is any one of a microwell array chip, a spot array chip, and an in situ synthesis chip.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This invention provides a protective coating for gene chips. This coating undergoes an additional post-production step. Without affecting the normal operation of the gene chip, the coating effectively protects the gene recognition probes on the chip surface, ensuring the stability of the gene chip under long-term storage conditions. Comparative experimental results show that the grayscale value of the chip with the protective coating decreases significantly less after hybridization, indicating that the hybridization activity of the gene recognition probes on the chip surface is better preserved and that the coating components effectively protect the probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the structure of the gene chip with protective coating prepared in the present invention.

[0018] 1-Gene chip substrate, 2-Microspheres with gene recognition probes on the surface, 3-Protective coating DETAILED DESCRIPTION

[0019] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] The following examples take polyacrylamide as an example to describe the composition, preparation and technical effects of the protective coating.

[0021] 1. Protective coating formulation and preparation

[0022] The protective coating of the gene chip provided in this embodiment includes 6% by mass of polyacrylamide with a molecular weight of 10,000, 0.01% sodium dodecyl sulfate (SDS), and the balance is deionized water.

[0023] The coating solution was prepared as follows: polyacrylamide was added to deionized water according to a predetermined amount, and then sodium lauryl sulfate was added. After mixing evenly, the mixture was centrifuged at 500 g for 2 minutes to remove foam and obtain the coating solution.

[0024] 2. Gene chip coating application

[0025] After preheating the coating solution to 50°C, place it in a ventilated environment with a wind speed maintained at 0.1-0.7m / s; immerse the gene chip to be treated vertically in the coating solution at an immersion rate of 1-10mm / s; after complete immersion, let it stand for 1 minute, then pull the chip vertically upward at a rate of 6-15mm / s until the chip is more than 10mm away from the liquid surface; then keep the chip in the ventilated environment for 20 minutes to complete the complete volatilization and drying of the coating liquid on the chip surface.

[0026] The schematic diagram of the structure of the gene chip coated with the coating is shown in Figure 1 , from bottom to top are the gene chip substrate 1, the microspheres 2 with gene recognition probes on the surface, and the protective coating 3.

[0027] 3. Polyacrylamide concentration screening

[0028] Polyacrylamide (MW 10,000) was prepared in aqueous solutions at concentrations of 4%, 6%, 8%, and 10%. These solutions were then applied to the surface of the gene chip by dip coating. The coating thickness was measured at random locations, and the thickness distribution (CV) value was used as a guide to coating uniformity. The results are shown in Table 1 below:

[0029] Table 1 Thickness and distribution CV of polyacrylamide coating at different concentrations

[0030]

[0031]

[0032] The results showed that when the concentration of polyacrylamide was 6%, the thickness uniformity of the gene chip surface coating was optimal and the thickness distribution CV value was also optimal.

[0033] 4. Polyacrylamide molecular weight screening

[0034] Polyacrylamide with molecular weights of 5,000, 10,000, 20,000, and 50,000 was used to prepare a 6% aqueous solution (containing 0.01% sodium lauryl sulfate). This solution was then applied to the surface of the gene chip by dip coating. The coating thickness was measured at random locations, and the thickness distribution (CV) value was used as a measure of coating uniformity. The coating with a molecular weight of 10,000 achieved the best thickness uniformity, as shown in Table 2 below.

[0035] Table 2 Dip coating thickness and distribution CV of different molecular weight polyacrylamide

[0036]

[0037] 5. Chip Storage Stability Test

[0038] The four chips with coating protection were stored in air at 37°C and 30% humidity for one month. The chips were taken out, immersed in deionized water, and shaken at 1500rpm for 2 minutes using a mixing shaker. Hybridization was performed using oligonucleotides corresponding to the decoding region sequence of the gene recognition probe. The oligonucleotides were labeled with Cy3 or Cy5 fluorescent groups, and the hybridization conditions were 44°C for 30 minutes. After hybridization, fluorescence scanning imaging was performed on the microsphere area of ​​the chip, and scanning images of two different fluorescence channels, Cy3 and Cy5, were obtained respectively. The images were analyzed to obtain the average grayscale value of the microspheres, GS-Coating. As a control, the average grayscale value of the microspheres of the four uncoated chips was GS-Blank. The comparison results are shown in Table 3:

[0039] Table 3 Comparison of average grayscale values ​​of microspheres with coated protection chips and uncoated chips

[0040]

[0041]

[0042] According to the above comparison results, the grayscale value of the chip with coating protection decreased significantly less after hybridization, indicating that the hybridization activity of the gene recognition probe on the chip surface was well preserved and the coating component played a role in protecting the probe.

[0043] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A protective coating for a gene chip, characterized in that: include: The mass fraction is 2% to 8% of probe protective agent, 0.005% to 0.05% of surfactant, and the balance is deionized water. The probe protective agent is selected from any one or more combinations of polyacrylamide, polyvinyl pyrrolidone, polyacrylic acid, polyethylene glycol, and polyvinyl alcohol. The protective coating is applied to the gene chip by dip coating. The coating solution is preheated to 40-60° C. and placed in a ventilated environment with a wind speed maintained at 0.1-0.7 m / s. The gene chip to be treated is immersed in the coating solution in a vertical manner at an immersion rate of 1-10 mm / s. After being fully immersed, the chip is kept still for 1 minute, and then the chip is vertically pulled upward at a rate of 6-15 mm / s until the chip is more than 10 mm away from the liquid surface. The chip is then kept in the ventilated environment for 20 minutes to complete the complete volatilization and drying of the coating solution on the chip surface. The molecular weight of the polymer selected for the probe protective agent is 10,000.

2. The protective coating for a gene chip according to claim 1, wherein: in, The surfactant is selected from any one or more combinations of sodium lauryl sulfate, Tween 20, Tween 80, and Triton X-100.

3. The protective coating for a gene chip according to claim 1, wherein: in, The mass fraction of the probe protective agent is 6%, and the concentration of the surfactant is 0.01%.

4. The protective coating for a gene chip according to claim 1, characterized in that: The preparation of the coating solution includes the following steps: adding a probe protective agent into deionized water according to a predetermined amount, then adding a surfactant and shaking and mixing, and after mixing evenly, centrifuging and defoaming to obtain a coating solution.

5. The protective coating for a gene chip according to claim 4, characterized in that: in, The centrifugation conditions were as follows: 500 g, 2 min.

6. The protective coating for a gene chip according to claim 1, characterized in that: The coating thickness is 800-1000nm.

7. A gene chip with a protective coating, characterized in that: The gene chip is prepared by the protective coating application method described in claim 1.

8. The gene chip with a protective coating according to claim 7, characterized in that: The gene chip is any one of a microwell array chip, a spot array chip, and an in situ synthesis chip.

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