Design And Research Of Novel Electrochemical Aptasensors For Ochratoxin A Based On Hairpin Aptamer

Objective Based on the problems of single sensing configuration,poor specificity,complex detection and low sensitivity faced by electrochemical aptasensors for ochratoxin A(OTA),this paper started from the sensor configuration design and the synergistic amplification strategy of electrochemical detection signals to establish a series of electrochemical aptasensing system for simple and rapid detection of OTA with high sensitivity and specificity.It provides an efficient and precise methodological basis for the identification and quantitative detection of trace substances in food,and new ideas for the detection of low concentration substances.Methods In this paper,the hairpin aptamer(HA,hairpin type oligonucleotide chain containing the sequence of aptamer)was designed as the recognition element.Compared with the traditional linear aptamer,the hybridization specificity of hairpin structure and the bonding affinity of aptamer can significantly improve the selectivity of the sensor.At the same time,the aptamer without modification can retain its unique structure and activity to the greatest extent,further improving the specificity of the sensor.Bare gold electrode,multi-walled carbon nanotube-gold nanoparticles(MWCNTs-AuNPs)and poly(3,4-ethylenedioxythiophene)(PEDOT)-AuNPs modified electrodes were used as sensing interfaces to immobilize HA.Aiming at the special configuration of the HA,three novel OTA electrochemical aptasensors were designed respectively based on auxiliary dual-signal probe/TCA auxiliary signal amplification system,poly-o-phenylenediamine(POPD)-AuNPs as signal marker/AuNPs-based dual synergistic signal amplification system and peroxidase-like active PtPd nanoalloy-electron mediator catalysis system.The modified electrodes and signal markers were characterized by scanning electron microscopy,transmission electron microscopy,ultraviolet-visible spectroscopy and energy dispersive X-ray spectrometer.Cyclic voltammetry(CV)and electrochemical impedance spectroscopy(EIS)were used to monitor the process of sensor construction.Differential pulse voltammetry(DPV)was used to detect OTA sensitively.The analytical performances of the three sensors are systematically studied through methodological research and feasibility validation.Results Quantitative detection ranges of the three electrochemical aptasensors for OTA were 1 pg/mL~1 ng/mL,2 pg/mL~1 ng/mL and 0.1 pg/mL~0.1 ng/mL,with detection limits of 0.5 pg/mL,1 pg/mL and 0.05 pg/mL,respectively.Systematic methodological validation and feasibility analysis confirmed that the three aptasensors had good stability,reproducibility,selectivity and regeneration.They were applied to the detection of standard-added coffee samples and wine samples,respectively.The recoveries ranged from 95.8% to 102.3% with RSD less than 4.71%,and the results of wine sample detection were well correlated with those of high performance liquid chromatography(HPLC).Conclusions A series of novel electrochemical aptasensors using HA as recognition elements,based on symmetrical dual-signal probe,AuNPs-based dual-synergistic signal amplification technology,peroxidase-like active nanoalloy-electronic mediator catalytic system,had shown comparable or even better analytical performance than those reported.The purpose of signal multi-amplification can be achieved by simple DNA hybridization or redox reaction.OTA can be detected specifically and conveniently while ensuring sensitivity,which provides a new idea for detecting trace substances.They have satisfactory recovery rate in OTA detection of various standard samples and good correlation with HPLC,which proves that the constructed series of electrochemical aptasensors have good application potential in practical detection.