Determination Of Ochratoxin A Based On Electrochemical DNA Sensor

Nowadays,the issue of mycotoxin contamination of food has received widespread attention to the world.Among them,Ochratoxin A（OTA）is a secondary metabolite produced by Penicillium and Aspergillus that is widely found in food ingredients and processed products,posing a serious threat to human health and food safety.Hence,there is an urgent need to establish a sensitive detection method for OTA.The method of the electochemical aptasensor,which combines an electrochemical method with aptamer,has the advantages of simple operation,good specificity and easy on-site identification.To further improve the sensitivity and specificity of electrochemical detection,three innovative electrochemical aptasensors were designed to detect OTA using rolling circle amplification（RCA）,DNAzyme,gold nanowires（Au NWs）,DNA nanoflowers,and silver nanoclusters（Ag NCs）signal amplification strategies.The details are as follows.1.A novel electrochemical aptasensor was prepared by designing OTA-mediated formation of DNA ring probes to initiate rolling circle amplification（RCA）reaction,RCA-driven Ag⁺-DNAzyme as signal amplification strategy,simultaneously.The sensor used methylene blue（MB）as the signal tag,which when not present OTA could not trigger the formation of DNA ring probes and thus the RCA reaction,and therefore could not generate Ag⁺-DNAzyme cleaved,MB-labeled hairpin DNA with r A sites（HP-MB）,resulting in a large initial current（I₀）;When OTA was present,it triggered the formation of a DNA ring probe,which in turn triggered the RCA reaction,generating long-stranded DNA with a repetitive Ag⁺-DNAzyme sequence that cleaves HP-MB,leading to a decrease in the MB electrical signal（I）and ultimately the detection of OTA by the MB signal response.The gel electrophoresis characterization results verified that the RCA reaction was successfully performed.By optimizing the conditions that affect the performance of the sensor,the optimal ratio of C_HP-MB:C_{c DNA}is 10:1,the optimal ratio of C_Bio-Apt:C_Padlock:C_Primeris 1:1:1,the reaction time of RCA is 60 min,and the optimal dosage of Phi29 polymerase is 5 U.Under the above optimal conditions,the prepared sensor showed a good linear relationship between（I₀-I）/I₀and Lg C_OTAin the range of 5×10^-4ng/m L～50 ng/m L,and the detection limit was 38 fg/m L.It was found that the sensor also exhibited better results in the detection of corn flour and red wine in real samples with average recoveries of 86.2%～113%and 116%～12%,respectively.The sensor also exhibited high specificity,acceptable repeatability,reproducibility,and stability.2.Previous experiments found that signal-reducing sensors were affected by background currents and that labeling MB signal tags on DNA strands was expensive.Therefore,in this chapter,a bifunctional DNA nanoflower containing aptamer and MB is induced by RCA reaction with Au NWs as the electrode modification material for OTA detection.when OTA was not present,it could not form a sandwich composite structure on the electrode surface,leading to a smaller MB current;When OTA was present,the bifunctional DNA nanoflower formed a sandwich composite structure with the aptamer and OTA on the electrode surface,leading to a higher MB signal on the electrode surface,and and finally the detection of OTA was achieved by the change of MB signal.By comparing the electroactive surface area of Au NWs before and after modification,the results showed that the electroactive surface area increased by 0.34 times.In addition,electrochemical measurements were performed to verify that when using MB and OTA concentrations of 8 m M and 1 ng/m L,respectively,the MB current without modification of Au NWs was 5.9μA,while the MB current after modification was 9.7μA,and the signal increased by 0.64 times,indicating a significant effect of Au NWs on accelerating electron transfer at the electrode surface.The DNA nanoflowers were characterized by SEM,TEM and gel electrophoresis,and the results showed the successful preparation of DNA nanoflowers.It was also characterized by gel electrophoresis,and the results also indicated the successful generation of DNA nanoflowers.By exploring the conditions affecting the sensor performance,the current value exhibited a good linear relationship with Lg C_OTAin the range of 1×10^-4ng/m L～10 ng/m L with a detection limit of 1.12 fg/m L under the optimal conditions.The detection studies on spiked wheat flour and oilseed bean samples showed average recoveries of 96.6%～98.1%and 99%～113%,respectively.The quality control samples of OTA corn flour processed by HPLC method were tested and the test results were analyzed by t-test using SPSS software,and the p-values of 0.68 and 0.36 for corn flour 1 and corn flour2,respectively,were greater than 0.05 at 95%confidence interval,indicating that the differences between this method and HPLC were not significant,indicating that the prepared sensors were accurate.Besides,the sensor also showed good specificity,stability,reproducibility and repeatability.3.After a previous study,it was found that methods of electrochemical aptasensors could achieve highly sensitive detection of OTA.However,most sensors focused on a single-mode output signal,and these single-mode methods could not eliminate the effects of operating conditions,biological environment,and other factors,thus inevitably leading to false positive results.In contrast,sensors based on a dual-mode strategy not only provided a variety of methods for practical detection requirements,but also enabled quantitative determination of results through mutual verification of the two modes,further improving the reliability of the sensors.However,in recent years,most of the dual-mode sensors relied on the signal response of complex nanomaterials,leading to complex and time-consuming preparation processes.Therefore,this chapter designed a simple and efficient electrochemical-fluorescent dual-mode sensor based on magnetic separation technique combined with DNA-silver nanoclusters（Ag NCs）probes.Using DNA-Ag NCs as fluorescent and electrochemical signal probes,when OTA was absent,DNA-Ag NCs probes could not be synthesized in the system,resulting in lower fluorescence and electrochemical signals in the system;When OTA was present,it led to the shedding of sulfhydryl-modified and cytosine-rich DNA（C-DNA）,which reacted with the added silver nitrate（Ag NO₃）and sodium borohydride（Na BH₄）after magnetic separation and formed DNA-Ag NCs probes with higher fluorescence and electrochemical signals,and finally achieved bimodal detection of OTA through the signal response to fluorescence and electrochemistry.The experimental parameters of the sensor were optimized,and the optimal amount of magnetic beads for this experiment was 2.5μL,the optimal time for OTA incubation was 30 min,the optimal placement time of Ag NCs was 6 h,and the optimal time of DNA-Ag NCs immobilization onto the electrode surface was 60 min.The generated Ag NCs were characterized by TEM and high-resolution TEM,and the results showed that most of the Ag NCs were nearly spherical and uniformly dispersed with an average particle size of 3～5 nm.Under the above optimal conditions,in the fluorescence mode,the sensor exhibited a good linear relationship between fluorescence intensity and Lg C_OTAin the range of 2.5×10^-4ng/m L～50 ng/m L,with a detection limit of 0.11 pg/m L;in the electrochemical mode,the current value had a good linear relationship with Lg C_OTAin the range of 2.5×10^-4ng/m L～25 ng/m L,with a detection limit of 0.025pg/m L.It was found that the dual-mode sensors exhibited good anti-interference capability against AFB1,AFB2,AFG1,FB1,ZEN,and OTB with satisfactory repeatability and reproducibility.For the detection of spiked peanut and wheat flour,the average recoveries were found to be 94.5%～106%and 101%～111%in the fluorescence mode and 91.8%～96.7%and 96.5%～98.8%in the electrochemical mode,respectively.The OTA quality control samples of corn flour processed by HPLC method were tested and the results were analyzed by t-test using SPSS software,and the p-values of cornmeal 1 in electrochemical and fluorescence modes were 0.11 and 0.27 with RSDs of 6.1%and 3.7%,respectively,at 95%confidence intervals;The p-values for the electrochemical and fluorescence modes of maize flour2 were 0.14 and 0.76,respectively,with RSDs of 10.1%and 5.3%;the p-values of the dual-mode sensor for both maize flour 1 and 2 were greater than 0.05,indicating that the differences between this method and HPLC were not significant,indicating that the prepared sensor was accurate.It was found that the dual-mode sensor exhibited better interference immunity with satisfactory repeatability and reproducibility.