Construction And Application Of Visualized Biosensing Platform For Graphene-based Mycotoxins

Mycotoxin contamination in food can seriously endanger human health.Therefore,the development of a simple,fast,sensitive,and on-site method for the detection of mycotoxins has become an important issue of common concern for the food safety sector and related scientific researchers.In this dissertation,several graphene-based visualized biosensing platforms with low cost and no complicated instruments are designed and constructed,and their application in the detection of mycotoxins is investigated.The details are as follows:?1?The designed single-stranded DNA₁ and the photochromic dye phenolphthalein?PP?were simultaneously loaded on the GO surface by?-?adsorption to obtain the probe PP-DNA₁-GO;The magnetic oxide graphene nanomaterial?Fe₃O₄/GO?prepared by co-precipitation method was used as the carrier to support the designed single-stranded DNA₂and obtained the probe DNA₂-Fe₃O₄/GO.Further use of ochratoxin A aptamer?OTA aptamer?will hybridize with DNA₁ and DNA₂ parts separately to construct a PP-DNA₁-GO-OTA aptamer-DNA₂-Fe₃O₄/GO colorimetric sensing platform for the detection of OTA;Based on OTA aptamer-specific recognition,magnetic separation and regulation of supernatant pH release PP to detect OTA.Under optimized conditions,the linear range of OTA 10-250 ng·mL^-11 and color changes can be visually identified.According to the design principle of OTA colorimetric sensing platform,such as aflatoxin B1?AFB1?aptamer and its semi-complementary strands?DNA₃ and DNA₄?as probes,thymidine?TP?is a signal molecule,The same principle can be used to achieve AFB1 visual detection.More importantly,it combines the two colorimetric sensing platforms of OTA and AFB1,the simultaneous visual detection of OTA and AFB1 in the same sample can be achieved under alkaline conditions.Therefore,this colorimetric sensing platform design principle is universal,the detection of other corresponding mycotoxins can be achieved by replacing the discoloration dye,mycotoxins aptamer and its semi-complementary strands.?2?According to the design principle and construction method of universal colorimetric sensing platform in?1?,increased pH parameters as a control condition,and further constructed a pH-resolved visualization sensing platform that can simultaneously detect four targets.Four kinds of commercially available color-shifting dye molecules PP,TP,malachite green?MGCB?and methyl violet?MV?,which have large differences in pH sensitivity range,were selected as signal molecules.By adjusting the pH value of the sensing system to control the mutual interference of coloration between different dyes,releasing and collecting MGCB and MV under acidic conditions,releasing and collecting PP and TP under alkaline conditions;For example,releasing and collecting MGCB for detecting AFB1 under acidic conditions and releasing and collecting PP for detecting OTA under alkaline conditions,the linear range is 10-200ng·m L^-11 and5-250 ng·mL^-1,respectively.More importantly,the constructed pH-resolved colorimetric sensing platform enables simultaneous visualization of four target OTA and fumonisin B1?FB1?,AFB1and microcystin LR?MC-LR?.?3?Using TP as a signal molecule,a novel colorimetric sensing platform for TP-DNA₁-GO-AFB₁ aptamer-DNA₂-Fe₃O₄/GO was developed to detect AFB1;The prepared gold nanoparticles?Au NPs?are peroxidase-like,Fe₃O₄@Au is a carrier and a magnetic separation agent,and TMB is a signal molecule.The Au NPs-OTA aptamer-Fe₃O₄@Au-COTA aptamer colorimetric detection platform for detecting OTA was constructed using OTA aptamer and its complementary strand?C OTA aptamer?.After magnetic separation,the magnetically separated solids released TP to detect AFB1 under alkaline conditions and the Au NPs in the supernatant catalyzed the TMB for the colorimetric detection of OTA under acidic conditions.Because the two sensing systems do not interfere with each other,simultaneous visual detection of AFB1 and OTA can be achieved.Under optimized conditions,the linear range of AFB1 is5-250 ng·mL^-1,and the linear range of OTA is 0.5-80 ng·m L^-1.The detection method can be used to detect the actual samples of peanuts.