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Preparation method of jettisonable aptamer sensor for OTA sensitivity detection

An aptamer sensor and sensitive detection technology, which is applied in the field of electrochemical detection, can solve the problems of troublesome and time-consuming processing steps, cumbersome operation steps, and high detection cost, and achieve the advantages of avoiding pretreatment procedures, simple and flexible operation, and simple equipment Effect

Inactive Publication Date: 2017-04-26
JIANGSU UNIV
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

Although these methods generally can more sensitively determine the content of mycotoxin in the sample, often there are many deficiencies, for example, (1) need large-scale expensive instrument, as gas / liquid chromatograph, mass spectrometer etc.; (2) The sample consumption is large and the operation steps are cumbersome; a large amount of waste organic solvents are likely to be generated during the analysis process, which pollutes the environment; (3) Relatively specialized experimenters are required; (4) The detection cost is high and it is not convenient for widespread use to promote
In reality, food is often contaminated by multiple mycotoxins at the same time. Although the problem of multiple mycotoxin residues has attracted attention, the research focus in recent years has focused on the single detection of mycotoxins, and there are few research reports on the simultaneous detection of multiple mycotoxins.

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  • Preparation method of jettisonable aptamer sensor for OTA sensitivity detection

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Embodiment 1

[0031] Preparation of Nano Colloidal Gold Solution Modified by MPA

[0032] (1) Take HAuCl with a mass concentration of 0.01% 4 Solution 100mL, heated to boiling, mass concentration 1% trisodium citrate solution 3mL was added rapidly under stirring condition, and continued to boil for about 15 minutes. After the reaction was completed, the solution was cooled to room temperature, and 10 mL of the colloidal gold prepared above was taken, and 60 μL of MPA was added dropwise with a pipette while shaking, and the shaking was continued for 5 hours after the dropwise addition was completed. After the reaction, the colloid was centrifuged at 13000rpm for 10min to obtain a purple-black precipitate. After washing with water for 3 times, 5mL of 50mM Tris-HCl buffer solution (pH 7.4) was added to the purple-black precipitate, and the precipitate was immediately uniformly dispersed into a purple-red colloidal solution. , which are AuNPs protected by MPA.

[0033] Preparation of PDDA-mod...

Embodiment 2

[0038] Construction of electrochemical aptasensors

[0039] (4) Preparation of gold nanoparticle-modified screen-printed electrode (Au NPs-SPCE): 10 μL of the MPA-modified colloidal gold solution prepared in step (1) was drip-coated on the surface of the screen-printed electrode prepared in step (3), React at room temperature for 2 hours, and seal and store at 4°C for later use.

[0040] (5) Preparation of aptamer-modified Au NPs-SPCE electrode: First, take 10 μL of 100 μM OTA aptamer standard droplet and apply it on the surface of the working electrode prepared in step (4), react at room temperature for 12 hours, and use Rinse with 50mM Tris-HCl buffer solution (pH 7.4), blow dry with nitrogen; then immerse the electrode in 5mL 1mM 6-mercaptohexanol (MCH), react at room temperature for 1 hour, seal the electrode and buffer with Tris-HCl rinse with liquid, blow dry with nitrogen, and store in a sealed container at 4°C for later use.

Embodiment 4

[0042] Establishment of standard curve for ochratoxin (OTA) detection

[0043] (6) Detect the OTA standard and establish a standard curve: drip-coat the OTA standard on the electrochemical sensing interface prepared in step (5), incubate at 37°C for 30 minutes, and then use 50mM Tris-HCl Rinse with buffer solution (pH 7.4), dry with nitrogen gas; finally place the electrode in impedance solution, scan electrochemical impedance spectroscopy (EIS), and establish a standard curve based on the impedance value and the corresponding OTA standard concentration. figure 1 Among them, Figure A is at different OTA concentrations (from a to i are: 0, 0.01, 0.05, 0.1, 0.05, 0.1, 5, 10, 50, 100ng mL -1 ), it can be seen from the figure that with the increase of the concentration, the semicircular arc presented in the impedance spectrum gradually increases, indicating that the addition of OTA has a significant hindrance to the charge transfer in the sensing system ; Figure B is the correspo...

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Abstract

The invention provides a preparation method of a jettisonable aptamer sensor for OTA (ochratoxin) sensitivity detection. The method includes the steps of: S1. preparation of an MPA modified nano colloidal gold solution; S2. activation of an aptamer; S3. preparation of a PDDA modified screen-printed electrode; S4. preparation of a gold nanoparticle modified screen-printed electrode; and S5. preparation of aptamer modified Au NPs-SPCE. Compared with the traditional detection method, the label-free impedance detection method of OTA proposed in the invention has the advantages of simpler and more flexible operation, simpler instrument and equipment, small reagent dosage, and low detection cost, etc.

Description

technical field [0001] The invention belongs to the field of electrochemical detection, in particular to a construction method and application of an electrochemical aptamer sensor based on a screen-printed electrode modified by gold nanoparticles for detecting ochratoxin A. Background technique [0002] Ochratoxin A (OTA) is a fungal nephrotoxin produced by Ochratoxin and Penicillium puree, which is a typical food-borne mycotoxin. OTA can exist stably in polar organic solvents. For example, the ethanol solution of OTA can exist stably for more than one year under refrigerated conditions, but OTA will decompose soon under ultraviolet irradiation. In animals, OTA is very stable and not easily degraded by metabolism, and OTA is toxic in many kinds of animals. Studies have found that its toxicity mainly manifests as renal toxicity, liver toxicity, immunotoxicity, teratogenic toxicity and carcinogenicity. In 1993, OTA was classified as a Class 2B carcinogen by the International ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327
CPCG01N27/3277G01N27/3278
Inventor 钱静安克奇王成全赵路芳任婵婵王坤
Owner JIANGSU UNIV
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