Development Of Electrochemical Aptasensors For Ochratoxin A Detection In Medicinal And Edible Malt

Objective:New methods were established for rapid,accurate,economic,and sensitive on-site detection of ochratoxin A(OTA)in medicinal and edible malt,in order to realize the on-site monitoring of OTA contamination,to guarantee the quality and safety of medicinal and edible malt,and to promote the development of international trade of malt and its processed products;Meanwhile,the established methods will provide a theoretical basis for improving the establishment of the limit standard of mycotoxins in traditional Chinese medicine(TCM).Methods:(1)The development of the "Competitive-mode based label-free electrochemical aptasensor" for detection of OTA in malt with Au electrode as the working electrode:The complementary DNA(complementary DNA,cDNA)of the aptamers were immobilized on the surface of Au electrode via Au-S bonds to hybridize with aptamers;The optimal experimental conditions were investigated;The fabrication process of the aptasensor was characterized via electrochemical impedance spectroscopy(EIS);The performance of the aptasensor was evaluated by analyzing the specificity of the sensor,as well as the limit of detection(LOD)and linear detection range of OTA;The accuracy of the aptasensor was investigated by studying the recoveries of OTA in spiked malt samples;The practicability of the aptasensor in actual malt samples was evaluated by detecting the potential OTA in 10 batches of natural contaminated malt samples;The results were validated via ultra-fast liquid chromatography-tamdem mass spectrometry(UFLC-MS/MS).(2)An electrochemical aptasensor for detection of OTA in malt based on functionalized multi-walled carbon nanotubes(f-MWCNTs)was developed by using the screen-printed carbon electrode(SPCE):The chemical groups of MWCNTs were further carboxylated by dealing with the concentrated HNO3;The functional groups of MWCNTs before and after carboxylation were analyzed via Fourier transform infrared spectroscopy(FT-IR);The Nafion-f-MWCNTs were fixed on the surface of the SPCE by drip coating;The morphology of the modified SPCE was characterized via scanning electron microscopy(SEM);The f-MWCNTs were coupled with streptavidin(SA)via 1-ethyl-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide(EDC/NHS)activation method;Aptamers were coupled with SA via biotin-streptavidin system;The optimum experimental conditions were investigated;The fabrication of the aptasensor was characterized via differential pulse voltammetry(DPV);The performance of the aptasensor was evaluated by analyzing the reproducibility,specificity,and stability of the sensor,as well as the LOD and linear detection range of OTA;The accuracy of the aptasensor was investigated by studying the recoveries of OTA in spiked malt samples;The practicability of the aptasensor was evaluated by detecting the potential OTA in 10 batches of actual malt samples;The results were validated via UFLC-MS/MS.(3)A label-free electrochemical aptasensor for detection of OTA in malt based on gold nanoparticles(AuNPs)was developed by using SPCE:AuNPs were in-situ generated via electroreduction of HAuCl4;The aptamer probes were immobilized on the surface of SPCE via Au-S bonds;The morphology of the modified SPCE was characterized via SEM;The elements of the modified SPCE were analyzed via cyclic voltammetry(CV)and X-ray photoelectron spectroscopy(XPS);The optimum experimental conditions were investigated via DPV method;The fabrication of the aptasensor was characterized via EIS;The performance of the aptasensor was evaluated by analyzing the reproducibility and specificity of the sensor,as well as the LOD and linear detection range of OTA;The accuracy of the aptasensor was investigated by studying the recoveries of OTA in spiked malt samples;The practicability of the aptasensor was evaluated by detecting the potential OTA in 10 batches of actual malt samples,and the results were validated via UFLC-MS/MS.(4)An electrochemical aptasensor for detection of OTA in malt based on Nafion-MWCNTs/Au nanopopcorns was developed by using the SPCE:Nafion-MWCNTs were fixed on the surface of SPCE by drip coating;Au nanppopcorns were synthesized via electroreduction of HAuCl4;The aptamer probes were immobilized on the surface of SPCE via Au-S bonds;The morphology of modified SPCE was characterized via SEM;The elements of the modified SPCE were analyzed via XPS;The optimum experimental conditions were investigated via DPV method;The fabrication of the aptasensor was characterized via CV and EIS;The performance of the aptasensor was evaluated by analyzing the reproducibility and specificity of the sensor,as well as the LOD and linear detection range of OTA;The accuracy of the aptasensor was investigated by studying the recoveries of OTA in spiked malt samples;The practicability of the aptasensor was evaluated by detecting the potential OTA in 10 batches of actual malt samples;The results were validated via UFLC-MS/MS.Results:(1)The competitive-mode based label-free electrochemical aptasensor was successfully fabricated.The optimum experimental conditions were as follows:The concentration of cDNA was 2.0 ?μM and the incubation time of cDNA was 2 h.The linear detection range of OTA was 0.05 ng/mL-10 ng/mL,with a LOD of 0.05 ng/mL.The established aptasensor showed high sensitivity and specificity towards OTA.The recoveries of OTA in spiked malt samples were 74.8%-105%,with RSDs of 8.86%-13.09%.One out of ten batches of naturally contaminated malt samples was detected to be positive(containing OTA)by the aptasensor,with the content of OTA 3.18 ng/mL.The results agreed with UFLC-MS/MS.(2)The electrochemical aptasensor based on f-MWCNTs was successfully constructed.The FT-IR spectra of MWCNTs showed that the amounts of COOH-increased after the further carboxylation of MWCNTs.SEM images showed that the carbon material on the surface of the bare SPCE has small protruding particles,which is the amplification of the carbon morphology,without other special structural characteristics.f-MWCNTs evenly distributed on the surface of SPCE and presented a "noodle-like" feature.The optimum experimental conditions were as follows:The concentration of f-MWCNTs was 0.5 mg/mL;The concentration of Nafion was 0.2%;The concentration of aptamers was 2 μM.The linear detection range of OTA was 0.005 ng/mL-10 ng/mL,with a LOD of 0.005 ng/mL.The established aptasensor showed high sensitivity,specificity,and reproducibility towards OTA.The aptasensor also showed good stability.The recoveries of OTA in spiked malt samples were 78.2%-96.8%.Ten batches of actual malt samples were detected with no OTA by the aptasensor,and the results were consistent with UFLC-MS/MS.(3)The label-free electrochemical aptasensor based on AuNPs was successfully constructed.SEM images showed that AuNPs homogeneously distributed on the surface of SPCE.CV curves and Au 4f XPS spectra further verified the generation of AuNPs.The P 2p XPS spectra verified the coupling of aptamers on AuNPs modified electrode.The optimum experimental conditions were as follows:The concentration of HAuCl4 was 6 mM;The electrodeposition time was 300 s;The concentration of aptamers was 4 μM.The linear detection range of OTA was 0.001 ng/mL-5 ng/mL,with a LOD of 0.001 ng/mL.The established aptasensor showed high sensitivity,specificity,and reproducibility towards OTA.The recoveries of OTA in spiked malts were 90.4%-108.7%.Ten batches of actual malt samples were detected with no OTA by the aptasensor,and the results were consistent with UFLC-MS/MS.(4)The electrochemical aptasensor based on N afi on-MW CNT s/Au nanopopcorns was successfully constructed.SEM images showed that MWCNTs evenly distributed on SPCE,and Au nanopopcorns evenly distributed on Nafion-MWCNTs modified SPCE.Au 4f XPS spectra further verified the generation of Au nanopopcorns.The optimum experimental conditions were as follows:The concentration of Nafion was 0.1%;The electrodeposition time was 300 s;The concentration of MWCNTs was 1 mg/mL;The concentration of aptamers was 2 μM.The linear detection range of OTA was 0.001 ng/mL-10 ng/mL,with a LOD of 0.001 ng/mL.The established aptasensor showed high sensitivity,specificity,and reproducibility towards OTA.The recoveries of OTA in spiked malt samples were 89.8%-95.7%.Ten batches of actual malt samples were detected with no OTA by the aptasensor,and the results were consistent with UFLC-MS/MS.Conclusion:In this study,four types of electrochemical aptasensors were developed for rapid,accurate,and ultrasensitive detection of OTA in malt samples.The established four aptasensors demonstrated high specificity,reproducibility,stability,and accuracy.LODs of OTA were 0.05 ng/mL,0.005 ng/mL,0.001 ng/mL,and 0.001 ng/mL,respectively;The linear detection range were 0.05 ng/mL-10 ng/mL,0.005-10 ng/mL,0.001 ng/mL-5 ng/mL,and 0.001 ng/mL-10 ng/mL,respectively.Among them,the Nafion-MWCNTs/Au nanopopcorns based electrochemical aptasensor presented the best sensing performance.The developed novel aptasensors realized the rapid batch detection of OTA in medicinal and edible malts,which can provide theoretical basis for on-site rapid detection of OTA in malt and the establishment of mycotoxin limit standard in traditional Chinese medicine.