Study On Electrochemical Aptasensor Based On Multiple Signal Amplification Strategies For Patulin Detection

Fruits and their derived products are a critical source of human nutrition.Yet adverse production and storage conditions will cause the growth of funguses in fruits,patulin（PAT）is one of the most common mycotoxins.Excessive intake of PAT can cause acute poisoning,and even teratogenicity and carcinogenicity.Besides,PAT cannot be completely removed during fruit processing because of its heat and acid tolerance.Therefore,there is significant research value for developing analytical methods for the detection of PAT.Electrochemical analysis technology has the advantages of simplicity,rapid response,and high sensitivity.Nucleic acid aptamers（Apt）have good selectivity and specific binding ability to target analytes.In this paper,an electrochemical aptasensor for rapid and sensitive detection of PAT was constructed by using Apt as the target recognition element and electrochemical analysis method as the detection method.Aiming at the low detection sensitivity of electrochemical aptamers for small molecule pollutants,improvements were made in terms of electrode modification materials and nucleic acid amplification strategies.The main research contents are as follows:1.An electrochemical aptasensor was proposed for patulin（PAT）based on tetrahedral DNA nanostructures（TDNs）and thionine（Thi）-labeled Fe₃O₄ nanoparticles（Fe₃O₄NPs）/r GO signal amplification strategy.The rigid structure of TDNs could effectively improve the binding efficiency.Fe₃O₄NPs/r GO with excellent electrical conductivity and large specific surface area was used as a label material,which could load more Thi and accelerate electron transfer.Besides,the unique catalytic properties of Fe₃O₄NPs could achieve active signal amplification.Once PAT existed,PAT aptamer was released from the capture probe,thereby introducing Fe₃O₄NPs/r GO with Thi onto the electrode surface.Therefore,a noticeable increase in Thi DPV response was observed after introduction PAT.Under the optimized conditions,the proposed aptasensor showed superior performance with a linear range from 5×10^-8 to 5×10^-1μg·m L^-1 and a detection limit of 30.4 fg·m L^-1.The recoveries of spiked apple juice samples were 96.9%-105.8%.2.In order to improve the utilization of target molecules and further enhance the sensitivity of the sensor,dual-EATR strategy was constructed to amplify the signal.Au NPs/FeMOF/PEI-GO nanocomposites were prepared as electrode substrates,and branched AgPdNPs were used as Thi carriers.Multiple strategies synergistically improve aptasensor sensitivity.In the presence of PAT,HP-S1 containing aptamer sequence was unfolded to hybridize with HP-S2.With the assistance of ExonucleaseⅢ,HP-S1 was re-exposed to participate in the new recycling process and release amounts of T-DNA.Then T-DNA could trigger Mg²⁺-dependent DNAzyme-assisted cyclic amplification,causing a weakening of the thionine electrical signal.The dual-EATR strategy gradually accumulates detection signals and can realize ultra-sensitive detection of PAT.With the optimal conditions,the developed aptasensor showed satisfactory performance with the range from5×10^-7 ng·m L^-1 to 5 ng·m L^-1 and a low detection limit of 0.217 fg·m L^-1.The recoveries of spiked apple juice samples were 91.0%to 103%.3.The research in the previous chapter realized the efficient utilization of the target PAT molecule and effectively improved the sensitivity of the aptasensor,but the complex operation procedures and long sensing time lead to its limitation in practical detection scenarios.One-step dual-target cycling strategy was designed to improve the efficiency of signal amplification,DNAzymes are activated in the presence of PAT,cyclically cleave substrate DNA,and the released sequences can be used as a new trigger to mediate the secondary recirculation.The strategy enables double target recycling without additional procedures and effectively shorten the sensing time.In addition,the branched AgPdNPs prepared in the previous chapter were used as the signal carrier material,and the multifunctional AuNFs/C₃N₄ nanocomposite was prepared as the substrate to increase the loading of the signal label and accelerate the electron conduction.Under the optimal conditions,the electrochemical aptasensor achieved a satisfactory linear range from 5×10^-6ng·m L^-1 to 50 ng·m L^-1 and limit of 0.917 fg·m L^-1 was obtained for the determination of PAT.The recoveries of spiked apple juice samples were 96.9%to 105.8%.