Novel Aptasensors Based On Enzyme-catalyzed Signal Amplification For Visual Detection Of Ochratoxin A

Ochratoxin A（OTA）,being a toxic secondary metabolite,is primarily derived from Aspergillus and Penicillium species.Moreover,it is extensively existed in foods and feeds.As revealed by toxicological research studies,OTA shows strong hepatotoxicity,renal toxicity,teratogenic,carcinogenic,and also mutagenic effect,which seriously endangers the safety and health of humans and animals.Currently,conventional techniques for the detection of OTA mainly include high performance liquid chromatography and high-performance liquid chromatography-tandem mass spectrometry.Despite the fact that these techniques manifest satisfactory sensitivity and repeatability,these techniques still suffer limitations of long analysis times,complicated sample pre-treatment,requirement of large-scale precision instruments and trained personnel.Therefore,the development of sensitive,fast and simple novel detection methods for OTA on-site monitoring and analysis is of great significance for ensuring food safety and human health.In this paper,by using aptamers as recognition molecules,novel aptasensors based on enzyme-catalyzed signal amplification strategy were developed for visual detection of OTA.The research contents are as follows:1.A nanozyme-based cascade colorimetric aptasensor for amplified detection of OTAIn this work,a novel aptasensor based on nanozyme-catalyzed cascade signal amplification strategy was developed for visual detection of OTA.In order to achieve the detection of OTA,the DNA-alkaline phosphatase-immobilized magnetic beads（MBs-DNA-ALP）consisting of MBs,OTA aptamers,complementary DNA（cDNA）,and ALP were designed.With OTA binding to its aptamer,the structural switching of the aptamer resulted in the release of the ALP-labelled cDNA（cDNA-ALP）.Following the magnetic separation,the cascade reaction was initiated through the enzymatic conversion of ascorbic acid-2-phosphate（AAP）into ascorbic acid（AA）.Subsequent to that,the generated AA reduceed MnO₂ nanosheets to Mn²⁺ions,accordingly destroying the oxidase-mimicking activity of MnO₂ nanosheets.In consequence,it was not possible to oxidize 3,3′,5,5′-tetramethylbenzidine,a substrate for oxidase,with Mn²⁺for the production of the blue colour product.With the increasing amount of OTA,a color change occured from blue to colorless.As an efficient signal amplification strategy,the cascade reaction composed of nanozyme and ALP can realize ultra-sensitive visual detection of OTA,and the detection limit reached 0.069 nM（0.028μg/kg）.Moreover,it has also been verified for real sample application by testing 2%grape juices spiked with a scries of concentrations of OTA.The colorimetric aptasensor has the potential to provide a universal and promising platform for the detection and monitoring of mycotoxins in food safety and quality control.2.Multicolor colorimetric aptasensor based on enzyme-induced metallization of gold nanorods for amplified detection of OTAIn this chapter,a multicolor colorimetric aptasensor based on gold nanorods（AuNRs）was constructed to improve the accuracy of visual detection.AuNRs,as a typical plasmonic noble metal nanostructures,possess two localized surface plasmon resonance（LSPR）peaks,namely a transverse LSPR peak and a longitudinal LSPR peak.The latter is highly sensitive to their size,shape,and composition.Small changes in the morphology of AuNRs will lead to evident LSPR peak shifts,exhibiting a range of different colors,as colorful as a rainbow.In the work,the MBs-DNA-ALP were still utilized for the detection of OTA.After the addition of OTA,the cDNA-ALP obtained by magnetic separation catalyzed the decomposition of AAP to AA.The produced AA reduced Ag⁺to Ag⁰,forming an Ag shell on the surface of AuNRs.This caused a blue-shift of the longitudinal LSPR peak of the AuNRs and a naked eye visible multicolor change.Under optimal conditions,the assay exhibited a 9.0 nM（3.63μg/kg）detection limit for OTA,with high specificity.Besides,this method can also be used to detect OTA in 2%grape juices.This high-resolution multicolor colorimetric method not only provides new ideas for the construction of a new visual colorimetric aptasensor,but also is expected to be used for the on-site visual semi-quantitative detection of mycotoxins in food.