Research On Detection Of Aflatoxin In Peanuts Based On Ratiometric Electrochemical Sensors

The quality of agricultural products is related to the national economy and the people's livelihood.Due to improper storage methods in production,transportation,and processing,peanuts are prone to fungal contamination in high-temperature and high-humidity environments.Among them,Aspergillus flavus infection is the most common,which not only causes great economic losses and endangers development of agricultural,but also seriously threatens human health.Therefore,the realization of accurate analysis of aflatoxin can guarantee the quality of peanuts from the source,and facilitate the dynamic monitoring of the moldy process,which provide technical support for the quality control of agricultural products.There are many existing detection methods for aflatoxin,among which chromatography-mass spectrometry is accurate and reliable but rely on expensive instruments and professional operation.The immunoassay method has a fast response speed,but the stability of antibody is insufficient.Based on this,to realize high-precision and rapid analysis of aflatoxin B1 and B2(AFB1 and AFB2)in peanuts,this paper took advantages of the sensitivity and rapid response of electrochemical method,combined with the aptamer with specific recognition ability,and proposed a verity of high-performance ratiometric electrochemical aptasensor,which realized the rapid and accurate detection of aflatoxins in peanuts.This paper synthesized thionine-graphene composite,and proposed a ratiometric electrochemical aptasensor,which solved the problem of environmental factors'disturbance of detection.Further,a ratiometric electrochemical sensing system based on signal on/off ratiometric strategy was developed,increasing the sensitivity of the sensor;On this basis,a ratiometric electrochemical sensing system based on dual-signal amplification strategy was established,which clarified the interaction mechanism of probe molecules at the sensing interface,and realized high-precision analysis of aflatoxins in peanuts.The content of this thesis includes the following three parts:1)The thionine-reduced graphene oxide(THI-r GO)composite material was modified to the surface of the glassy carbon electrode as a substrate.Then,chitosan and ferrocene-labeled aptamer(Fc-apt)were sequentially modified to construct AFB2ratiometric electrochemical sensor.Among them,the Fc-apt specifically bound to AFB2,which lead to the bending of Fc-apt,and Fc approaches the electrode,causing the Fc current signal(I_Fc)to increase.But the reference signal I_THI derived from THI-r GO was constant.Thus,an AFB2 ratiometric electrochemical sensor was constructed based on the linear relationship between I_THI/I_Fc and concentration of AFB2,with a linear range of 0.001-10 ng m L^-1 and the limit of detection(LOD)was 0.191 pg m L^-1.This sensor was used for the detection of AFB2 in peanut and peanut oil,with a recovery rate of 99.7%to 108%.Meanwhile,its analytical reliability had been verified by high performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS).2)In order to improve the sensitivity of the sensor to detect aflatoxin,a ratiometric electrochemical sensors based on signal on/off was proposed,and a signal on/reference signal ratiometric sensor was developed for comparison.For the signal on/off response strategy,the Fc-apt was combined with the complementary DNA(c DNA)on the electrode surface to form a double strand DNA(ds DNA).After the Fc-apt was combined with AFB1 and peeling off,the c DNA combined with single-strand DNA through hybridization chain reaction(HCR),which formed ds DNA to adsorb methylene blue(MB).With the increasing of concentration of AFB1,the I_Fc decreased,and the MB current signal(I_MB)increased.The linear range of I_MB/I_Fc to AFB1 is 0.1-100 pg m L^-1,and the LOD is 0.065 pg m L^-1.Like the above strategy,the signal on/reference signal response mode can be achieved by labeling Fc to c DNA.The linear range of I_MB/I_Fc to AFB1 was 1-10000 pg m L^-1,and the LOD was 0.33 pg m L^-1.It was found that the signal on/off strategy had a large signal variation and higher sensitivity.3)In order to further improve the sensitivity of detection,a cascade signal amplification strategy based on the combination of DNA walker(DW)and HCR was proposed,and a signal on/off ratiometric electrochemical sensor was constructed.This electrochemical aptasensor used the specific binding of AFB1 and Fc-labeled aptamer to activate the DW which fixed on the electrode surface.Meanwhile,the freed DW strand would hybridizes with hairpin DNA via a toehold-mediated strand exchange and form the ds DNA.Further,above-mentioned ds DNA was used as a substrate,produced a longer ds DNA by HCR to absorb MB and thus generating I_MB.As the concentration of AFB1 increased,I_Fc gradually decreased,while I_MB increased significantly.The ratio of I_MB/I_Fc could acted as a yardstick for detection of AFB1 by taking advantage of dual-amplification strategy.Its linear range was 0.003-3 pg m L^-1,and the LOD was as low as 0.88 fg m L^-1.This research work had achieved a significant improvement in the detection performance of ratiometric electrochemical sensors by introducing a dual-signal amplification strategy,which was expected to provide new forms for the research of high-performance agricultural sensing methods and devices.