CN118006180A - Protective coating of gene chip and preparation method and application thereof - Google Patents
Protective coating of gene chip and preparation method and application thereof Download PDFInfo
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- CN118006180A CN118006180A CN202311804557.6A CN202311804557A CN118006180A CN 118006180 A CN118006180 A CN 118006180A CN 202311804557 A CN202311804557 A CN 202311804557A CN 118006180 A CN118006180 A CN 118006180A
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 64
- 239000011253 protective coating Substances 0.000 title claims abstract description 29
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 239000011248 coating agent Substances 0.000 claims abstract description 43
- 238000000576 coating method Methods 0.000 claims abstract description 43
- 239000000523 sample Substances 0.000 claims abstract description 29
- 239000003223 protective agent Substances 0.000 claims abstract description 12
- 239000004094 surface-active agent Substances 0.000 claims abstract description 10
- 239000008367 deionised water Substances 0.000 claims abstract description 9
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000002156 mixing Methods 0.000 claims abstract description 7
- 229920002401 polyacrylamide Polymers 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 7
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 6
- 238000003618 dip coating Methods 0.000 claims description 6
- 238000007654 immersion Methods 0.000 claims description 6
- 235000019333 sodium laurylsulphate Nutrition 0.000 claims description 5
- 238000009423 ventilation Methods 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 3
- 239000002202 Polyethylene glycol Substances 0.000 claims description 2
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 2
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 2
- 229920002125 Sokalan® Polymers 0.000 claims description 2
- 239000013504 Triton X-100 Substances 0.000 claims description 2
- 229920004890 Triton X-100 Polymers 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 238000011065 in-situ storage Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 239000004584 polyacrylic acid Substances 0.000 claims description 2
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 2
- 229920000053 polysorbate 80 Polymers 0.000 claims description 2
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 2
- 230000001681 protective effect Effects 0.000 claims description 2
- 238000003786 synthesis reaction Methods 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims 1
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- 238000009396 hybridization Methods 0.000 abstract description 10
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- 201000010099 disease Diseases 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 239000004005 microsphere Substances 0.000 description 7
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
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- 108091034117 Oligonucleotide Proteins 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
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- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a protective coating of a gene chip, a preparation method and application thereof, wherein the protective coating comprises 2 to 8 mass percent of probe protective agent, 0.005 to 0.05 mass percent of surfactant and the balance of deionized water. Adding the probe protective agent into deionized water according to a preset amount, adding the surfactant, vibrating and uniformly mixing, and performing centrifugal defoaming after uniform mixing to obtain a coating solution. The invention further provides a method for applying the protective coating of the gene chip and the gene chip with the protective coating. The comparison experiment result shows that the gray value is obviously less reduced after the chip with the coating protection is hybridized, which indicates that the hybridization activity of the gene recognition probe on the surface of the chip is better preserved, and the coating component plays a role in protecting the probe.
Description
Technical Field
The invention belongs to the technical field of biochip hybridization, relates to a hybridization buffer solution based on a biochip solid-phase hybridization technology, and also relates to a preparation method and application of the hybridization buffer solution as a probe fluorescence protective agent.
Background
The gene chip provides an efficient and convenient means for modern medicine, especially medical diagnostics, and provides a reliable basis for gene diagnosis and gene therapy at the molecular level. The correlation of genes with diseases (such as cancer, infectious disease and genetic disease) can be analyzed by using the gene chip, so that we can deeply recognize the root cause of the disease. The most direct application of gene chip in medical diagnosis is to detect genes related to diseases. Biomedical research shows that the pathogenesis of most diseases of human beings is fundamentally related to genes. Therefore, the gene chip has important significance in medical application, and can rapidly detect genes and mutations related to diseases. The gene chip can not only improve the scientificity of disease diagnosis, but also has guiding significance for treating diseases, and a targeted treatment scheme can be formulated according to the detection result of genes related to the diseases.
The high-density gene chip is a gene detection means with low cost, large detection site quantity, uniform format and quick analysis, which takes the oligonucleotide fixed on the surface of the silicon dioxide microsphere as a gene recognition probe and uses a silicon plate as a main bearing matrix of the gene chip. The detection sensitivity of the gene chip depends on the activity of the recognition probe, so that the activity protection of the surface recognition probe and the stability under long-term storage conditions have important influence on the detection quality of the gene chip. In addition, the storage mode of the gene chip is convenient, low in cost, wide in applicability and the like in consideration of the use cost and the transportation mode of the gene chip.
In the prior art, in order to improve the service life of the chip, the chip is usually stored in a clean container with controlled environment (dry air or nitrogen) in consideration of the storage of the chip, for example, in short-term storage, and the chip is originally packaged as much as possible; when the chip is stored for a long time, the chip is stored under the conditions of 99% nitrogen or inert gas, 17-25 ℃ and 7-25% humidity and gas pressure higher than the ambient atmospheric pressure, and the storage can be carried out for more than 12 months in the undisturbed environment. However, no report on chip protection from the aspect of activity protection of surface recognition probes is known in the prior art.
Disclosure of Invention
The invention aims at the problems, provides a protective coating of a gene chip based on the aspect of maintaining the activity of a probe for a long time, and also provides a preparation method of the chip coating.
The technical scheme of the invention is summarized as follows: hydrophilic polymer with obvious protection effect on the chip probe is selected as a functional component of the coating, and the hydrophilic polymer is prepared into coating liquid with the aid of a surfactant; the coating is then applied to the chip surface by conventional dip coating, spray coating, or the like.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided a protective coating for a gene chip comprising: 2 to 8 percent (preferably 6 percent) of probe protective agent, 0.005 to 0.05 percent (preferably 0.01 percent) of surfactant and the balance of deionized water.
Preferably, the probe protecting agent is selected from any one or more of polyacrylamide, polyvinylpyrrolidone, polyacrylic acid, polyethylene glycol and polyvinyl alcohol; the surfactant is selected from one or more of sodium dodecyl sulfate, tween 20, tween 80 and Triton X-100.
Further preferably, the molecular weight of the probe protecting agent is 10000.
In a second aspect of the present invention, a method for preparing a protective coating for a gene chip as described above is provided, comprising the steps of: adding the probe protective agent into deionized water according to a preset amount, adding the surfactant, vibrating and uniformly mixing, and centrifuging for foam removal 500g for 2min to obtain a coating solution.
In a third aspect of the present invention, there is provided a method for applying a protective coating of a gene chip, wherein the above-mentioned coating solution is applied to the surface of the gene chip to be treated in a conventional manner to a thickness of 800 to 1000nm.
In the specific embodiment of the invention, the application mode is dip-coating, and the method is as follows: preheating the coating solution to 40-60 ℃ (preferably 50 ℃) and then placing the coating solution in a ventilation environment, wherein the wind speed is kept at 0.1-0.7 m/s; immersing the gene chip to be treated into the coating solution in an up-down vertical mode, wherein the immersion rate is 1-10 mm/s; standing for 1min after complete immersion, and then vertically lifting the chip upwards at a speed of 6-15 mm/s until the chip leaves the liquid level for more than 10 mm; the chip was then kept in the vented environment for 20min to complete the complete evaporation and drying of the coating liquid on the chip surface.
In a fourth aspect of the present invention, there is provided a gene chip with a protective coating, which is prepared by the protective coating application method described above. The gene chip is any one of a micropore array chip, a sample application array chip and an in-situ synthesis chip.
Compared with the prior art, the invention has the beneficial effects that:
The invention provides a protective coating of a gene chip, which is additionally subjected to one-step coating treatment after production, and the coating has an effective protective effect on a gene identification probe on the surface of the chip on the premise of not influencing normal use of the gene chip, so that the stability of the gene chip under the condition of long-term storage is ensured. The comparison experiment result shows that the gray value is obviously less reduced after the chip with the coating protection is hybridized, which indicates that the hybridization activity of the gene recognition probe on the surface of the chip is better preserved, and the coating component plays a role in protecting the probe.
Drawings
FIG. 1 is a schematic diagram of the structure of a gene chip with a protective coating prepared by the invention.
1-Gene chip substrate, 2-microsphere with Gene recognition probes on surface, 3-protective coating
Detailed Description
In order to make the objects, 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 with reference to the accompanying drawings of the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without creative efforts, based on the described embodiments of the present invention fall within the protection scope of the present invention.
The following examples illustrate the composition, preparation and technical effects of the protective coating using polyacrylamide.
1. Protective coating formulation and preparation
The protective coating of the gene chip provided in this embodiment comprises 6% by mass of polyacrylamide with molecular weight of 10000, 0.01% Sodium Dodecyl Sulfate (SDS), and the balance of deionized water.
The preparation method of the coating solution comprises the following steps: adding polyacrylamide into deionized water according to a preset amount, adding sodium dodecyl sulfate, uniformly mixing, and centrifuging for foam removal 500g for 2min to obtain a coating solution.
2. Gene chip coating application
Preheating the coating solution to 50 ℃, and then placing the coating solution in a ventilation environment, wherein the wind speed is kept between 0.1 and 0.7m/s; immersing the gene chip to be treated into the coating solution in an up-down vertical mode, wherein the immersion rate is 1-10 mm/s; standing for 1min after complete immersion, and then vertically lifting the chip upwards at a speed of 6-15 mm/s until the chip leaves the liquid level for more than 10 mm; the chip was then kept in the vented environment for 20min to complete the complete evaporation and drying of the coating liquid on the chip surface.
The structure of the gene chip coated with the coating is schematically shown in fig. 1, and the gene chip substrate 1, the microsphere 2 with the gene recognition probe on the surface and the protective coating 3 are sequentially arranged from bottom to top.
3. Polyacrylamide concentration screening
Taking polyacrylamide with molecular weight of 10000, respectively preparing aqueous solutions with concentration of 4%, 6%, 8% and 10%, and then forming a coating on the surface of the gene chip by dip-coating. The coating thickness was measured by randomly selecting different positions of the coating and using the thickness profile CV values as a basis for coating uniformity, the results are shown in table 1 below:
TABLE 1 Polyacrylamide dip coating thickness and distribution CV for different concentrations
The results showed that when the concentration of polyacrylamide was 6%, the uniformity of the thickness of the surface coating of the gene chip was optimal, and the CV value of the thickness distribution was also optimal.
4. Molecular weight screening of polyacrylamide
An aqueous solution with a concentration of 6% (containing 0.01% sodium dodecyl sulfate) was prepared using polyacrylamides with molecular weights of 5000, 10000, 20000, 50000, respectively, and then the solution was dip-coated on the surface of the gene chip. The thickness of the coating is measured by randomly selecting different positions of the coating, and CV values of thickness distribution are used as the basis of uniformity of the coating. The coating thickness uniformity was best at a molecular weight of 10000, the specific results are shown in table 2 below:
TABLE 2 Dip coating thickness and distribution CV for polyacrylamides of different molecular weights
5. Chip preservation stability test
The 4 chips with the coating protection were kept under air at 37 ℃ with a humidity of 30% and a holding time of one month. And taking out the chip, soaking the chip in deionized water, and oscillating for 2min at 1500rpm by using a uniform mixing oscillator. Hybridization treatment was performed using an oligonucleotide corresponding to the sequence of the decoding region of the gene recognition probe, the oligonucleotide being labeled with a Cy3 or Cy5 fluorescent group at 44℃for 30min. After hybridization, fluorescent scanning imaging is carried out on the microsphere area of the chip, scanning images of two different fluorescent channels of Cy3 and Cy5 are respectively obtained, and the images are analyzed to obtain the average gray value GS-Coating of the microsphere. As a control, the average gray value of microspheres of 4 uncoated protected chips was GS-Blank, and the comparison results are shown in table 3:
TABLE 3 average gray scale value comparison of microspheres for coated and uncoated chips
According to the comparison result, the gray value of the chip with the coating protection is obviously less reduced after hybridization, which indicates that the hybridization activity of the gene recognition probe on the surface of the chip is better preserved, and the coating component plays a role in protecting the probe.
While the preferred embodiments of the present application have been described in detail, the present application is not limited to the embodiments, and various equivalent modifications and substitutions can be made by those skilled in the art without departing from the spirit of the present application, and these equivalent modifications and substitutions are intended to be included in the scope of the present application as defined in the appended claims.
Claims (10)
1. A protective coating for a gene chip comprising: 2 to 8 percent of probe protective agent, 0.005 to 0.05 percent of surfactant and the balance of deionized water.
2. The protective coating for a gene chip of claim 1, wherein:
wherein the probe protecting agent is selected from any one or a combination of more of polyacrylamide, polyvinylpyrrolidone, polyacrylic acid, polyethylene glycol and polyvinyl alcohol;
The surfactant is selected from one or more of sodium dodecyl sulfate, tween 20, tween 80 and Triton X-100.
3. The protective coating for a gene chip of claim 1, wherein:
wherein the molecular weight of the polymer selected as the probe protective agent is 10000.
4. The protective coating for a gene chip of claim 1, wherein:
Wherein the mass fraction of the probe protective agent is 6%, and the concentration of the surfactant is 0.01%.
5. The method for producing a protective coating for a gene chip according to any one of claims 1 to 4, comprising the steps of: adding the probe protective agent into deionized water according to a preset amount, adding the surfactant, vibrating and uniformly mixing, and performing centrifugal defoaming after uniform mixing to obtain a coating solution.
6. The method for preparing a protective coating for a gene chip according to claim 5, wherein:
wherein, the centrifugation condition is as follows: 500g,2min.
7. A method for applying a protective coating for a gene chip, characterized in that the coating solution according to claim 5 is applied to the surface of the gene chip to be treated in a conventional manner to a thickness of 800 to 1000nm.
8. The method for applying a protective coating to a gene chip according to claim 7, wherein:
Wherein the application mode is dip coating, the coating solution is preheated to 40-60 ℃ and then placed in a ventilation environment, and the wind speed is kept at 0.1-0.7 m/s; immersing the gene chip to be treated into the coating solution in an up-down vertical mode, wherein the immersion rate is 1-10 mm/s; standing for 1min after complete immersion, and then vertically lifting the chip upwards at a speed of 6-15 mm/s until the chip leaves the liquid level for more than 10 mm; and then keeping the chip in the ventilation environment for 20min, and finishing the complete volatilization and drying of the coating liquid on the surface of the chip.
9. A gene chip with a protective coating, which is prepared by the protective coating application method according to claim 7 or 8.
10. The protective coated gene chip of claim 9, wherein the gene chip is any one of a microwell array chip, a spotting array chip, and an in situ synthesis chip.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311804557.6A CN118006180A (en) | 2023-12-26 | 2023-12-26 | Protective coating of gene chip and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311804557.6A CN118006180A (en) | 2023-12-26 | 2023-12-26 | Protective coating of gene chip and preparation method and application thereof |
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| Publication Number | Publication Date |
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| CN118006180A true CN118006180A (en) | 2024-05-10 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311804557.6A Pending CN118006180A (en) | 2023-12-26 | 2023-12-26 | Protective coating of gene chip and preparation method and application thereof |
Country Status (1)
| Country | Link |
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| CN (1) | CN118006180A (en) |
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- 2023-12-26 CN CN202311804557.6A patent/CN118006180A/en active Pending
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