Construction Of The TiO₂ Inverse Opal Photoelectrochemical Sensing Platform For AFB1 Analysis Application

Aflatoxin B1 is produced by aspergillus flavus and aspergillus parasiticus,it is widely found in corns,wheats,oats,sorghum and other feed and feed raw materials.It not only brings losses to the breeding industry for a serious threat to animal health,but also enters the human body to endanger human health through animal products such as meat,eggs,and milk.For quantitative assessment of AFB1,several methods have been exploited recently,including thin layer chromatographic,enzyme-linked immunosorbent assay,HPLC and HPLC-MS.However,these analytical methods are cumbersome in sample handling,require expensive instruments and skilled professionals,so it greatly limits the application.Photoelectrochemical（PEC）biosensors have been developed into effective analytical tools because of their high sensitivity,low background signal,low cost,simple equipment,good reproducibility and are considered to be a promising mycotoxin detection method.In this dissertation,we construct a PEC platform based on TiO₂ inverse opal structure with excellent photoelectric conversion performance,the design of various PEC sensing strategies and the proposal of optical dual-mode immunoassay are used to achieve the goal of specific and high sensitivity detection of AFB1 in feed and its raw materials.Firstly,In this dissertation a novel protocol of PEC and optical dual-mode immunoassay based on the integration of the enzymatic generation of biocatalytic precipitation into the CdS QDs-sensitized three-dimensional TiO₂ IO network for the simultaneous detection of human Ig G as a model target.The three-dimensional CdS QDs/TiO₂ IO network which possesses the features of high specific surface area and interconnected porosity is immobilized with antibody to lead the subsequent sandwich immunocomplex reaction,and then the BCP is triggered in the pores by ALP.The photocurrent is suppressed and the color of the photoelectrode is changed by catalysis products,based on this,the PEC and optical double signals which are correlated with the concentration of Ig G are outputted simultaneously.This proposed immunosensor exhibits a good linear range 5 pg/m L-100 ng/m L with the detection limit of 0.15 pg/m L（S/N=3）,The photoelectrode with macroporous inverse opal（IO）architecture creates an elegant platform for the design of PEC bioassay,laying a solid foundation for AFB1 detection,holding a great promise for bioanalysis.Secondly,a new PEC biosensor has been constructed for the detection of AFB1 based on the prepared three-dimensional CDs/TiO₂ IO/FTO electrodes with inverse opal structure.Specifically,the heterojunction formation of TiO₂ IO and CDs leads to the enhancement of PEC performance due to the enhanced visible light utilization and the band gap matching effect.After the EDC coupling reations between the carboxyl group of the CDs and the amino group of the AFB1 aptamer,the aptamers were fixed on the surface of CDs/TiO₂ IO.Insulating layer was formed on the biosensoring interface while the target AFB1 was specifically recognized,and then the photocurrent decreased by the impediment of electron transfer.Based on this design,the PEC biosensor has a good linear range for AFB1determination from 0.005-50 ng/m L and the detection limit is 1.27 pg/m L（S/N=3）.In addition,the PEC biosensor has been successfully applied to the detection of real samples of corn,thus it fully demonstrates the great potential of the platform for PEC biosensor in the area of AFB1 detection and feed safety.Lastly,an optical dual-mode PEC immunosensor has been proposed to achieve a sensitive detection of the target AFB1 based on the biological recognition of aptamers and BCP reactions.In detail,CDs/TiO₂ IO/FTO electrodes were fabricated,and then the amino modified c DNA was bound onto the electrode surface by EDC coupling reaction,after the hybridization of c DNA and AFB1 aptamers,the biosensoring platform was constructed.In the absence of AFB1,the ALP-aptamer hybridized with c DNA and acted as a signal quenching element through enzymatic BCP reaction.In the present of AFB1,the ALP-aptamer would competitively combine with AFB1 target to greatly hinder the hybridization with c DNA,leading to the recovery of photocurrent.The principle of the design is that ALP-catalyzed BCIP reaction produces insoluble precipitates which will greatly reduce the photocurrent,when AFB1 is added,the catalytic precipitation process is inhibited,and the photoelectric signal increases in the reverse direction.Under optimal conditions,this photoelectrochemical sensing strategy shows a linear relationship between photocurrent variation and the logarithm of target AFB1 concentration in the range of 5 pg/m L-50ng/m L,the detection limit is 2.38 pg/m L（S/N=3）.In addition,the PEC biosensor had been successfully applied to the detection of AFB1 in actual samples,such as feed and flour.It exhibites enormaous potential in feed safety and other application fields.So herein,various novel photoelectrochemical biosensors based on TiO₂ inverse opal structure have been successfully designed to realize the detection of AFB1 in feed and feed raw materials,it provides new ideas for the detection of other toxins in feed and feed raw materials,and it is also expected to be applied to the analysis of other important targets in the fields of food safety,environmental protection and clinical medicine.