A Study Of The Construction Of Biosensor Interface Based On Novel Nanomaterials Signal Amplification For Detection Of Ochratoxin A

Ochratoxin A has potently nephrotoxic,carcinogenic and teratogenic,and regarded as an immunosuppressive agent.It has attracted increasing attention due to its highly hepatotoxicand mutagenic to most mammalian species and crops.Hence,sensitive and reliable methods for OTA monitoring in food sample are of crucial importance for food safety.Biosensor,combined biotechnology with sensing technology,is an analytical method,which taken the advantages of good selectivity,high sensitivity,simple operation,and so on.Recently,Novel biosensor,based on the nanomaterials signal amplification technique,has become one of the predominant analytical methods for the detection of mycotoxins.This research focuses on the preparation of functionalized nanocomposites,the construction of sensitive interface,the application of novel signal amplification strategies.Three new kinds of biosensors are constructed in this paper,the major contents are summarized as follows:1.A new impedimetric immunosensor for the fast determination of ochratoxin A?OTA?in food samples was developed based on the instant catalyst as enhancer.Initially,the signal tags were prepared via coimmobilization of anti-OTA antibody and amine-terminated dendrimer?PAMAM?on the graphene oxide nanosheets through the covalent interaction,which were utilized as a good platform for combining manganese ion(anti-OTA-GO-PAMAM-Mn²⁺).Upon target OTA introduction,a competitive-type immunoreaction was implemented between the analyte and the immobilized OTA-BSA on the electrode for the anti-OTA antibody on the graphene oxide nanosheets labels.After a competitive immunoassay format,the anti-OTA-GO-PAMAM-Mn²⁺were captured onto the electrode surface,which could induce the formation of MnO₂ via classical redox reaction between Mn²⁺and KMnO₄on the immunesensing platform.Moreover,the generated MnO₂ nanoparticles acting as efficient catalyst could catalyze the 4-chloro-1-naphthol?4-CN?oxidation without H₂O₂ to generate an insoluble precipitation on the platform.Under the optimal conditions,the instant catalyst based impedimetric immunosensor displayed a wide dynamic working range between 0.1 pg m L^-1 and 30 ng mL^-1.The detection limit?LOD?of the assay was 0.055 pg m L^-1.The developed method exhibited high selectivity and can be used for the determination of OTA in real red wine samples.2.This paper constructed a label free aptasensor by taking advantages of the specificity of the OTA aptamer and the catalytic reduction of MoS₂.Firstly,the OTA aptamer would combine with the complementary DNA probe which had been modified on the gold electrode surface to form dsDNA.The target OTA competed with the DNA probe to combine with the OTA aptamer to break away from the electrode surface to produce free ssDNA probe.Then the MoS₂ nanoparticles adsorbed onto the DNA probe.Finally,the electrode was measured in a PBF buffer solution containing a certain amount of hydrogen peroxide.The electrochemical aptasensor was provided with a good reproducibility and stability and low detection limit by the experimental results.Under the optimal experimental conditions,the between peak current value of the response and the concentration of OTA(0.5 pg m L^-1-1.0 ng m L^-1)showed a good linear relationship,the LOD of 0.23 pg m L^-1.3.A sensitive photoelectrochemical?PEC?aptasensor has been successfully constructed for Ochratoxin A?OTA?biosensing based on the dual signal amplification strategy.Initially,the GO/CdS/MoS₂/AuNPs was employed as a matrix to immobilize the auxiliary DNA.Then the OTA aptamer would combine with the auxiliary DNA on the electrode surface to form dsDNA.Porphyrin?TMPyP?would combine dsDNA through the intercalation mode to sensitize the optoelectronic materials,leading to the relatively strong photocurrent.In the presence of the target OTA,the SiO₂-labeled DNA,as signal amplification elements,can be introduced by hybridization with auxiliary DNA on the surface of sensing platform.The ultrahigh sensitivity of this immunoassay derived from the two major reasons as below.First,since the affinity of TMPyP for dsDNA is considerably larger than that for ssDNA,so TMPyP would remove from the electrode surface,leading to obviously decreased photocurrent intensity;The second is that the immobilized SiO₂@DNA conjugates could evidently increase the steric hindrance of the sensing electrode and effectively depress the electron transfer,leading to further decreased photocurrent intensity.Accordingly,the well-designed photoelectrochemical aptasensor exhibited good sensitivity,specificity and stability.The method was applied to the determination of OTA in spiked red wine and might open a new promising platform for the detection of other important mycotoxins.