Maple Leaf-Shaped Gold Nanocrystals and Their Application in Detecting α-Amanitin

Abstract

The present invention discloses a maple leaf-shaped gold nanocrystal and its application in detecting α-amanitin, belonging to the field of molecular biology detection. In the present invention, L-glutathione is used to promote the asymmetric evolution of gold seeds to prepare a maple leaf-shaped gold nanocrystal (ML-Au), and the aptamer is optimized and truncated to obtain a 20-base short aptamer for α-amanitin, and a biosensor for detecting α-amanitin is prepared. The linear range of the biosensor B1 of the present invention for detecting α-amanitin is 1×10<supgt;−9< / supgt> to 1×10<supgt;−14< / supgt> M (0.9 to 9×10<supgt;5< / supgt> ng / mL), the detection limit is 2.9×10<supgt;−15< / supgt> M (2.61×10<supgt;−3< / supgt> ng / mL), and the detection time only needs 25 min, having the advantages of high sensitivity and rapid detection.

Claims

1. A biosensor, characterized in that: The preparation method of the biosensor comprises the following steps: A maple leaf-shaped gold nanocrystal solution prepared by using L-glutathione as a shape inducer is added dropwise to the surface of a glassy carbon electrode, and dried with nitrogen to obtain a glassy carbon electrode carrying maple leaf-shaped gold nanocrystals; an aptamer stock solution is added dropwise to the surface of a glassy carbon electrode carrying maple leaf-shaped gold nanocrystals, incubated, washed, and dried with nitrogen to obtain a glassy carbon electrode carrying aptamers and maple leaf-shaped gold nanocrystals; a mercaptoethanol solution is added dropwise to the surface of a glassy carbon electrode carrying aptamers and maple leaf-shaped gold nanocrystals, incubated, washed with water, and dried with nitrogen to prepare a thiol-blocked glassy carbon electrode biosensor carrying aptamers and maple leaf-shaped gold nanocrystals; The aptamer stock solution contains an aptamer for α-amanitin, and the aptamer is based on the nucleotide sequence shown in SEQ ID NO.15, and a thiol group -SH-(CH2)6- is connected to the 5' end of the nucleotide sequence.

2. The biosensor according to claim 1, characterized in that The concentration of the maple leaf gold nanocrystal solution is 1-10 mmol / L, the concentration of the aptamer stock solution is 5-20 μM, and the concentration of the mercaptoethanol solution is 0.5-8 mM; the volume ratio of the maple leaf gold nanocrystal solution, the aptamer stock solution and the mercaptoethanol solution is 1-8:1-8:8-15.

3. The biosensor according to claim 1, characterized in that The preparation method of the maple leaf-shaped gold nanocrystal solution comprises: (1) adding a 5-15 mM HAuCl4 solution to a growth solution I, incubating, and then adding an ascorbic acid solution to obtain a gold seed solution; wherein the growth solution I is composed of 10-20 mM hexadecyltrimethylammonium chloride, 2-10 μM potassium bromide, and 0.1-1.5 μM potassium iodide; and the volume ratio of the HAuCl4 solution to the growth solution I is 1-8:15-20; (2) Mixing a 5-15 mM HAuCl4 solution with a growth solution II to obtain a mixed solution, incubating, adding ascorbic acid after incubation, and shaking; collecting the precipitate by centrifugation after shaking to prepare maple leaf-shaped gold nanocrystals; dispersing the collected maple leaf-shaped gold nanocrystals in water to obtain a maple leaf-shaped gold nanocrystal stock solution; the growth solution II is composed of 90-110 mM hexadecyltrimethylammonium chloride, 0.5-1.5 mM potassium bromide, 0.1-1.5 mM potassium iodide, 0.1-1.5 mM L-glutathione and the gold seed solution prepared in step (1); the volume ratio of HAuCl4 solution to growth solution II is 1-3:8-15; the volume ratio of gold seed solution to growth solution II is 0.05-0.2:0.8-1.2; (3) adding chitosan in an amount of 0.1 to 1.5% by mass of the maple leaf-shaped gold nanocrystal stock solution to the maple leaf-shaped gold nanocrystal stock solution, and mixing the mixture to obtain a maple leaf-shaped gold nanocrystal solution.

4. Use of the biosensor according to any one of claims 1 to 3 in the preparation of a product for detecting α-amanitin.

5. The use according to claim 4, characterized in that: The products include nanocrystals, probes or kits.

6. An α-amanitin detection kit, characterized in that: The kit comprises the biosensor according to any one of claims 1 to 3, PBS buffer and potassium ferrocyanide solution.

7. A method for detecting α-amanitin, characterized in that: The biosensor according to any one of claims 1 to 3 is used for detection, and the method is for non-disease diagnosis and treatment purposes, and the method is as follows: 1-8 μL of the sample to be tested is dropped onto the surface of the biosensor, incubated for 25-30 minutes, washed, and the differential pulse voltammetry standard curve is measured in a PBS solution containing 0.1-1.5 mM K4Fe(CN)6 to obtain the current value, which is then inserted into the standard curve to calculate the α-amanitin concentration.

8. The method according to claim 7, characterized in that The standard curve is Ip(μA)=-5.8532×log[Cα-amanitin,fM]+65.505; wherein, Ip(μA) is the current value, and Cα-amanitin,fM is the concentration of α-amanitin.

9. A method for improving the sensitivity and detection limit of a biosensor for detecting α-amanitin, characterized in that: The detection is performed using the biosensor according to any one of claims 1 to 3.

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