Glass-based nucleic acid aptamer biosensor and preparation method thereof

Abstract

The invention provides a glass-based nucleic acid aptamer biosensor and a preparation method thereof, and belongs to the technical field of analytical chemistry detection. The preparation method provided by the invention comprises the steps of firstly carrying out specific pretreatment on the glass substrate, and then carrying out mixed incubation on the glass substrate and the nucleic acid aptamer to finish modification, so that the problems that in the prior art, the modification steps of the nucleic acid aptamer are complicated, and the material cost is relatively high are solved. The glass-based nucleic acid aptamer biosensor provided by the invention is simple in preparation method, stable, controllable, high in activity, cheap in raw materials, capable of being prepared in batches, good in reproducibility and capable of remarkably improving the nucleic acid immobilization efficiency. Technical support is provided for developing a stable and reliable aptamer colorimetric sensing technology, the anti-interference performance of the colorimetric sensing technology is improved, then the detection accuracy is improved, the reproducibility between detection batches is improved, and meanwhile the detection difficulty is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry detection, and in particular to a glass-based nucleic acid aptamer biosensor and a preparation method thereof. Background Art

[0002] Aptamer biosensors are sensors that use aptamers as probes to detect specific target molecules (such as proteins, small molecules, pathogens, etc.). Aptamers have received extensive attention in the development of biosensors because of their high specificity, high affinity, and good stability. Aptamer biosensors usually work through the following steps: Fixation of aptamers: The selected aptamers are fixed on a solid substrate (such as a metal surface, glass, plastic, etc.). Binding of targets: When the target molecule is present in the sample, it binds to the fixed aptamer to form an aptamer-target complex. Signal detection: Use different signal transduction methods (such as electrochemistry, fluorescence, surface-enhanced Raman scattering, etc.) to detect changes caused by aptamer-target binding, and analyze the concentration of the target through these signals. Currently, aptamer biosensors are widely used in medical diagnosis, food safety, environmental monitoring, etc.

[0003] In recent years, in order to improve the fixation efficiency and binding performance of nucleic acid aptamers on solid surfaces, interface modification technology has received increasing attention. Self-assembled monolayer technology is a method of tightly fixing a monolayer on the surface of a metal material (such as gold or silver) or other solid substrates through a chemical reaction. This technology performs well in the fixation of nucleic acid aptamers. For example, the commonly used SAM components are long-chain thiol molecules, which form a self-assembled monolayer on the metal surface. Introducing molecules with nucleic acid aptamers at the end of the membrane can effectively improve the binding ability and concentration of nucleic acids. Polymer materials (such as polyvinyl alcohol, polyacrylamide, etc.) as carriers can provide a larger fixed surface area for nucleic acid aptamers, making it easier to optimize the fixation efficiency of aptamers. For example, the functional groups on the polymer chain can fix the aptamer on the surface through covalent bonds, for example, through amino or carboxyl groups to the amino or carboxyl groups of the aptamer. Compared with chemical fixation, physical adsorption methods are simple and easy, but due to weak binding strength, they are affected by environmental conditions. In addition, nanomaterials (such as gold nanoparticles, carbon nanotubes, etc.) are widely used in the interface modification of nucleic acid aptamers due to their high specific surface area and good biocompatibility. The surface of gold nanoparticles can bind to nucleic acid aptamers through thiol groups. Carbon nanotubes and graphite: can enhance conductivity and specific surface area, and improve the sensitivity of biosensors. Through chemical oxidation treatment, the surface functionalization of these materials can easily achieve the fixation of aptamers.

[0004] Chinese patent CN118290728A discloses a nanocomposite material, an electrochemical nucleic acid aptamer biosensor, and its preparation method and application. The invention first prepares Au / PANI / MWCNTs-COOH / MoS with good conductivity. 2 The nanocomposite material is then adsorbed on the surface of a conductive substrate, and then the nucleic acid aptamer is modified on the surface of the nanocomposite material by a gold-sulfur bond self-assembly method to obtain an electrochemical nucleic acid aptamer biosensor. The sensor provided by the invention can achieve specific selection of endotoxins, and has good stability, low detection limit, high sensitivity, good reproducibility, and can be used for food endotoxin detection.

[0005] However, the aptamer modification steps in the above scheme are complicated and the material cost is high. Summary of the invention

[0006] The purpose of the present invention is to provide a glass-based nucleic acid aptamer biosensor and a preparation method thereof, which has broader application prospects.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing a glass-based nucleic acid aptamer biosensor, comprising the following steps:

[0009] S1, contacting the glass substrate with reagent A for 3 to 12 hours;

[0010] S2, washing the glass substrate;

[0011] S3, contacting the glass substrate with reagent B for 1 to 10 hours;

[0012] S4, washing and drying the glass substrate;

[0013] S5, contacting the glass substrate with reagent C for 10 to 120 minutes;

[0014] S6, washing and drying the glass substrate to obtain a pretreated glass substrate;

[0015] S6, mixing and incubating the pretreated glass substrate with the nucleic acid aptamer to obtain a glass-based nucleic acid aptamer biosensor;

[0016] The reagent A includes one or more of acetone, hydrochloric acid and ammonia water;

[0017] The reagent B includes one or more of 3-aminopropyltrimethoxysilane, 3-mercaptopropyltrimethoxysilane, toluene, ethanol and acetic acid.

[0018] Preferably, the reagent C contains pentanediol.

[0019] Preferably, the washing reagent comprises water and / or ethanol.

[0020] Preferably, the washing is performed 1 to 10 times.

[0021] Preferably, the drying comprises nitrogen drying and / or vacuum drying.

[0022] Preferably, the vacuum drying time is 1 to 6 hours.

[0023] Preferably, the vacuum drying temperature is 60-160°C.

[0024] Preferably, the incubation time is 6 to 12 hours.

[0025] Preferably, after the incubation, the step further comprises applying an SDS aqueous solution or a sodium bicarbonate solution on the surface of the glass substrate;

[0026] The concentration of the SDS aqueous solution or sodium bicarbonate solution is 0.1% to 0.6%.

[0027] The present invention also provides a glass-based nucleic acid aptamer biosensor prepared by the above preparation method.

[0028] Beneficial effects of the present invention:

[0029] (1) The glass-based nucleic acid aptamer biosensor provided by the present invention is simple to prepare, stable and controllable, and has strong activity.

[0030] (2) The glass-based nucleic acid aptamer biosensor preparation method provided by the present invention has cheap raw materials, can be prepared in batches, has good reproducibility, and improves the nucleic acid immobilization efficiency.

[0031] (3) The preparation method of the glass-based nucleic acid aptamer biosensor provided by the present invention is simple and rapid, and is of great significance for constructing a stable and highly anti-interference colorimetric aptamer sensor. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 for Figure 1 Result I of nucleic acid aptamer modification of glass interface, where: aptamer concentration A: 0 μM, B: 6 μM, C: 10 μM;

[0033] Figure 2 This is the result diagram of nucleic acid aptamer modification kinetics;

[0034] Figure 3 This is a comparison diagram of the results of the physical adsorption method and the method of the present invention. DETAILED DESCRIPTION

[0035] The present invention provides a method for preparing a glass-based nucleic acid aptamer biosensor, comprising the following steps:

[0036] S1, contacting the glass substrate with reagent A for 3 to 12 hours;

[0037] S2, washing the glass substrate;

[0038] S3, contacting the glass substrate with reagent B for 1 to 10 hours;

[0039] S4, washing and drying the glass substrate;

[0040] S5, contacting the glass substrate with reagent C for 10 to 120 minutes;

[0041] S6, washing and drying the glass substrate to obtain a pretreated glass substrate;

[0042] S6, mixing and incubating the pretreated glass substrate with the nucleic acid aptamer to obtain a glass-based nucleic acid aptamer biosensor;

[0043] In the present invention, the reagent A includes one or more of acetone, hydrochloric acid and ammonia water; the reagent B includes one or more of 3-aminopropyltrimethoxysilane, 3-mercaptopropyltrimethoxysilane, toluene, ethanol and acetic acid; and the reagent C contains pentanediol.

[0044] In the present invention, preferably, the reagents used for washing include water and / or ethanol; more preferably, washing is performed with water first and then with ethanol, and the number of washings with each reagent is 2 to 5 times; preferably, the water in the present invention is pure water; preferably, the number of washings is 1 to 10 times.

[0045] In the present invention, preferably, the drying includes nitrogen drying and / or vacuum drying; more preferably, nitrogen drying is performed first and then vacuum drying; or only nitrogen drying is performed; preferably, the vacuum drying time is 1 to 6 hours; preferably, the vacuum drying temperature is 60 to 160°C.

[0046] In the present invention, the pretreated glass substrate is mixed with the nucleic acid aptamer for incubation to obtain a glass-based nucleic acid aptamer biosensor. Preferably, the incubation time is 6 to 12 hours. Preferably, after the incubation, an SDS aqueous solution or a sodium bicarbonate solution is applied to the surface of the glass substrate. The concentration of the SDS aqueous solution or the sodium bicarbonate solution is 0.1% to 0.6%.

[0047] The present invention also provides a glass-based nucleic acid aptamer biosensor prepared by the above preparation method.

[0048] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0049] Example 1

[0050] (1) Soak the glass slide in 5M hydrochloric acid solution overnight, take it out, wash it with pure water 3 times, 5 min each time, wash it with ethanol 3 times, 5 min each time, blow dry it with nitrogen, and vacuum dry it for 1.5 h at 115°C;

[0051] (2) A 3% 3-aminopropyltrimethoxysilane solution was dropped onto the glass, and the glass was allowed to stand for 6 hours. The glass was then washed with toluene for 3 times, each time for 10 minutes; and with ethanol for 3 times, each time for 5 minutes; and dried with nitrogen.

[0052] (3) The prepared glass modified film was placed in a vacuum dryer and allowed to stand for 4 hours at a temperature of 115° C. to obtain an amino-activated glass film.

[0053] (4) Prepare 8% glutaraldehyde solution and drop 50 μL of it onto the glass and incubate for 80 min;

[0054] (5) Take out and wash with purified water three times, 5 min each time, wash with ethanol three times, 5 min each time, and blow dry with nitrogen;

[0055] (6) The glutaraldehyde-modified glass film prepared above was placed in a vacuum dryer and vacuum dried for 4 hours to obtain an aldehyde-activated glass film.

[0056] (7) Prepare 100 μM sodium citrate solution of nucleic acid aptamer (sequence: 5'-NH 2 -TTTTTTTTTTGGTGAACTTATTGCCTAGGTAGGTGGCGGT, as shown in SEQ ID NO.1), vortex to mix, place in a water bath at 85°C for 5 min, take out, and let stand at room temperature for 4 h;

[0057] (8) Dilute the aptamer solution into 6 μM and 10 μM solutions with sodium citrate solution, and use sodium citrate buffer as a control. Take appropriate amounts of each solution and drop them on the surface of the glass electrode, keep the interface moist, and incubate at room temperature overnight.

[0058] (9) Remove and then wash once with sodium citrate buffer, twice with 0.3% SDS aqueous solution, and twice with ultrapure water, each for 5 min, and blow dry with nitrogen;

[0059] (10) Placing the prepared nucleic acid-modified membrane glass in a vacuum dryer to obtain glass interfaces modified with different amounts of nucleic acid, such as Figure 1 .

[0060] from Figure 1 It can be seen that increasing the incubation concentration of the aptamer significantly increases the amount of aptamer modification on the glass interface, indicating that this method can achieve effective fixation of DNA on the glass interface.

[0061] Example 2

[0062] (1) Soak the glass slide in 5M hydrochloric acid solution overnight, take it out, wash it with pure water 3 times, 5 min each time, wash it with ethanol 3 times, 5 min each time, blow dry it with nitrogen, and vacuum dry it for 1.5 h at 115°C;

[0063] (2) Drop a 4% 3-aminopropyltrimethoxysilane solution onto the glass, let it stand for 4 hours, wash it with toluene three times, 5 minutes each time; wash it with ethanol three times, 5 minutes each time; blow it dry with nitrogen gas,

[0064] (3) placing the prepared glass modified film in a vacuum desiccator for 4 h at a temperature of 115°C;

[0065] (4) Prepare 8% glutaraldehyde solution, take an appropriate amount of 50 μL and drop it on the above glass interface, incubate for 90 min, take it out and wash it with pure water 3 times, 5 min each time, wash it with ethanol 3 times, 5 min each time, blow dry with nitrogen, and vacuum dry for 4 h;

[0066] (5) Prepare a 6 μM nucleic acid aptamer solution (sequence: 5'-NH 2 -TTTTTTTTTTGGTGAACTTATTGCCTAGGTAGGTGGCGGT, as shown in SEQ ID NO.2), vortex to mix, place in a water bath at 80°C for 10 min, take out, and let stand at room temperature for 4 h;

[0067] (6) Using sodium citrate buffer as a control, drop an appropriate amount onto the glass surface, keep the interface moist, and incubate at room temperature for t (0, 1, 2, 4, 8, 12 h);

[0068] (7) After removal, wash twice with sodium citrate buffer, wash three times with 0.3% SDS aqueous solution, wash twice with ultrapure water, and blow dry with nitrogen gas;

[0069] (8) Placing the above nucleic acid-modified membrane glass in a vacuum desiccator to obtain glass interfaces modified with different amounts of nucleic acid, such as Figure 2 .

[0070] from Figure 2 It can be seen that increasing the incubation concentration of the aptamer significantly increases the amount of aptamer modification on the glass interface, indicating that the method provided by the present invention can achieve effective fixation of DNA on the glass interface.

[0071] Comparative Example

[0072] Physical adsorption method:

[0073] Use sodium citrate solution to prepare 6 μM nucleic acid aptamer solution (sequence: 5'-NH2-TTTTTTTTTTGGTGAACTTATTGCCTAGGTAGGTGGCGGT, as shown in SEQ ID NO.2), mediate and mix, incubate in 80°C water bath for 10 min, take out, let stand at room temperature for 4 h, take appropriate amount and drop it on the glass surface, keep the interface moist, and incubate at room temperature for 12 h.

[0074] The biosensor obtained by this method was compared with the detection results of Example 1. Figure 3 As shown by Figure 3 It can be seen that the preparation method provided by the present invention can significantly improve the performance of the obtained biosensor and achieve effective fixation.

[0075] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a glass-based nucleic acid aptamer biosensor, characterized in that: The steps include: S1, contacting the glass substrate with reagent A for 3 to 12 hours; S2, washing the glass substrate; S3, contacting the glass substrate with reagent B for 1 to 10 hours; S4, washing and drying the glass substrate; S5, contacting the glass substrate with reagent C for 10 to 120 minutes; S6, washing and drying the glass substrate to obtain a pretreated glass substrate; S6, mixing and incubating the pretreated glass substrate with the nucleic acid aptamer to obtain a glass-based nucleic acid aptamer biosensor; The reagent A includes one or more of acetone, hydrochloric acid and ammonia water; The reagent B includes one or more of 3-aminopropyltrimethoxysilane, 3-mercaptopropyltrimethoxysilane, toluene, ethanol and acetic acid.

2. The method for preparing a glass-based nucleic acid aptamer biosensor according to claim 1, characterized in that: The reagent C contains pentanediol.

3. The method for preparing a glass-based nucleic acid aptamer biosensor according to claim 1, characterized in that: The reagents used for the washing include water and / or ethanol.

4. The method for preparing a glass-based nucleic acid aptamer biosensor according to claim 3, characterized in that: The washing is performed 1 to 10 times.

5. The method for preparing a glass-based nucleic acid aptamer biosensor according to claim 1, characterized in that: The drying includes nitrogen blowing and / or vacuum drying.

6. The method for preparing a glass-based nucleic acid aptamer biosensor according to claim 5, characterized in that: The vacuum drying time is 1 to 6 hours.

7. The method for preparing a glass-based nucleic acid aptamer biosensor according to claim 5, characterized in that: The vacuum drying temperature is 60-160°C.

8. The method for preparing a glass-based nucleic acid aptamer biosensor according to claim 1, characterized in that: The incubation time is 6 to 12 hours.

9. The method for preparing a glass-based nucleic acid aptamer biosensor according to claim 8, characterized in that: After the incubation, the step further includes applying an SDS aqueous solution or a sodium bicarbonate solution to the surface of the glass substrate; The concentration of the SDS aqueous solution or sodium bicarbonate solution is 0.1% to 0.6%.

10. A glass-based nucleic acid aptamer biosensor prepared by the preparation method according to any one of claims 1 to 9.

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