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Method for preparing label-free portable aptamer sensor for detecting AFB1

A kind of aptamer sensor, portable technology, applied in the direction of instruments, measuring devices, scientific instruments, etc., can solve the problems of limited application of immunoassay methods, complex antibody preparation process, expensive synthesis, etc., to achieve low detection cost, avoid electrode The effect of simple pretreatment procedure and preparation process

Active Publication Date: 2018-07-31
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complicated preparation process of antibodies, expensive synthesis, poor stability of antibodies, easy to be denatured by external environment, difficult to store, etc., the popularization and application of immunoassay methods are limited.

Method used

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  • Method for preparing label-free portable aptamer sensor for detecting AFB1

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Experimental program
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Effect test

Embodiment 1

[0036] Magnetic nanoparticles Fe 3 o 4 preparation of

[0037] (1) Magnetic nanoparticles Fe 3 o 4 The preparation method of NPs is as follows: 2mmol of FeCl 3 ·6H 2 O was dissolved in a mixed solution of 10mL ethylene glycol (EG) and 10mL ethylene glycol (DEG), and ultrasonically reacted for 30min; then, 20mmol sodium acetate and 2mmol polyethylene glycol (PEG) were added in sequence; the reaction was stirred for 30min, The mixture was moved to a reaction kettle and reacted at 200°C for 3 hours; after the reaction was completed, it was cooled to room temperature, washed several times with ethanol and water, redispersed in 50 mL of ethanol, ultrasonicated for 5 minutes, and set aside.

Embodiment 2

[0039] Magnetic core-shell nanoparticles Fe 3 o 4 Preparation of @Au NPs

[0040] (2) Magnetic core-shell nanoparticles Fe 3 o 4 The preparation method of @Au NPs is: take the Fe in step 1 3 o 4 Magnetic nanoparticle dispersion 40mL, add 400μL propylaminotriethoxysilane (APTS) mechanical stirring reaction at room temperature for 7h, magnetically separate and wash with ethanol several times, redisperse the product in 40mL of water; 4mL of HAuCl 4 Solution (1wt%) was added dropwise to the above dispersion liquid, reflux reaction at 100°C for 30min, then 8mL sodium citrate solution (1wt%) was added dropwise, and after reflux reaction at 100°C for 3h, the obtained product Magnetically separated and washed several times with water, redispersed in 40mL water for later use.

Embodiment 3

[0042] aptamer-modified Fc 3 o 4 @Au nanobiological probe (Fe 3 o 4 Preparation of @Au-apt)

[0043] (3) Aptamer-modified Fc 3 o 4 The preparation method of @Au nanobiological probe is as follows: firstly, take 500 μL of fresh AFB1 (10 μM) nucleic acid aptamer solution activated by tris(2-ethylcarboxy) phosphine hydrochloride (TCEP) and add it to 35 mL Fe 3 o 4 @AuNPs (1mg mL –1 ) in a Tris-HCl buffer solution (50 mM, pH 7.4), and shaken at 37° C. for 1 h. Using an external magnetic field separation method, the magnetron product was repeatedly washed several times with Tris-HCl buffer solution to remove unreacted aptamer. It was redispersed in 35 mL of 2 μM 6-mercaptohexanol (MCH) solution, and reacted for 1 h at 37 ° C to obtain MCH-blocked nanobiological probes (Fe 3 o 4 @Au-apt), washed several times with 10mM Tris-HCl buffer solution to remove unreacted MCH, redispersed it in 35mL Tris-HCl buffer solution (50mM, pH 7.4), and stored at 4°C for later use.

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Abstract

The invention provides a method for preparing a label-free portable aptamer sensor for detecting AFB1, and belongs to the field of electrochemical analysis. The method comprises the following steps: preparation of magnetic ferroferric oxide nanoparticles (Fe3O4); preparation of gold-coated ferroferric oxide core-shell magnetic nanoparticles (Fe3O4@Au); preparation of a nucleic acid aptamer-modified Fe3O4@Au nano-bioprobe; pretreatment of a screen printed electrode; construction of the label-free portable aptamer sensor; and detection of the AFB1 by electrochemical ac impedance spectroscopy. The method adopts the nucleic acid aptamer-modified magnetic core-shell material Fe3O4@Au to modify the surface of the screen printed electrode, the material is quickly modified on the surface of the electrode by the action of an external magnetic field, the operation is simple, and the use time is short; Au-S bonds can be used to fix more thiolated aptamers on the surface of the electrode more firmly, and the sensitivity of the sensor is effectively improved. The method has the advantages of simple preparation process, short time consumption, less reagent dosage and low detection cost and the like.

Description

technical field [0001] The invention belongs to the field of electrochemical analysis, and is characterized in that the rapid and sensitive detection of aflatoxin B1 is realized through a label-free method. It is technically involved in the fields of nanomaterial science and biosensing. Background technique [0002] Aflatoxin (AF for short) is a class of metabolites produced by Aspergillus flavus and Aspergillus parasiticus, more than 20 species have been isolated and identified, mainly including aflatoxin B1 (AFB1), B2 (AFB2), M1 ( AFM1), M2 (AFM2) and other subtypes. Among them, AFB1 is the most toxic mycotoxin found so far. It has strong carcinogenic, teratogenic and mutagenic properties. Its toxicity is 10 times that of potassium cyanide and 68 times that of arsenic. Its carcinogenicity is dimethylnitrosamine 75 times. Therefore, many countries and organizations in the world have set maximum limits for aflatoxins in agricultural products and food. The Food and Agricu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327G01N27/416B82Y5/00B82Y30/00
CPCB82Y5/00B82Y30/00G01N27/3278G01N27/416
Inventor 王成全任婵婵钱静王坤黄星奕
Owner JIANGSU UNIV
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