Electrochemical Immunoassay Technology For Aflatoxin B1Based On Quantum Dots As Labels

Aflatoxins are potent carcinogens, mutagens, teratogens. The excessive aflatoxin in food notonly seriously harms the health of the masses, but also reduces the international competitiveness ofChina’s agricultural products. Since the domestic and international standards are not uniform, theproducts meet our current quality of standards were failure in foreign countries. So, Westerncountries implemente technical trade barriers to the export of China’s agriculture, which has causedtremendous losses.Due to extreme toxicity and wide spread occurrence in staple food and feed, AFB1isregulated in many countries, which puts forward the requirement of high sensitivity ofdetermination methods for AFB1. Currently, the methods for AFB1detection include: thin-layerchromatography (TLC), high performance liquid chromatography (HPLC), immunoassay assayand so on. Because of rapidity, simplicity and accuracy of method, the enzyme-linkedimmunosorbent assay (ELISA) has been widely applied in food safety determination. Bu There area series of problems in the practical application, such as the enzyme activity in the applicationprocess is vulnerable to reaction conditions, short-lived chromogenic reagent needcryopreservation. These problems limit the application of the method.Quantum dots (QDs) is a new nanomaterials with optical characteristics and electrochemicalproperties. QDs can be bound to the antibody as a lable, and then we can use the electrochemicaltechnique to detect the metal content of QDs, thereby detect the concentrations of analyte. Thistechnique can ameliorate these above issues, and effectively improve the sensitivity of ELISA. Themain research contents and conclusions are as follows:1. The synthesis of MAb-PbSThe synthesis of PbS QDs used mercapto acetic acid as capping agent, Pb(NO3)2and Na2S asraw materials. In the condition of ambient temperature and pressure, the amount of mercapto aceticacid was11μL, pH9.5, synthetised uniform size, good water-soluble PbS QDs. The resulting QDswere then characterized by SEM, XRD. Results showed that PbS QDs dispersed with an averageparticle size of about3-5nm, ware face-centered cubic crystal. Then conjugated PbS QDs with anti-aflatoxin B1antibody to synthesise MAb-PbS used NDC, NHS as crosslinking agent. Thisconjugates and the bioactivity of MAb-PbS were proved by fluorescence spectra and traditionalindirect enzyme linked immunosorbent assay. The successful synthesis of MAb-PbS laid thefoundation for the establishment of new electrochemical immunoassay methods for AFB1.2. Optimazition of the test conditions of simultaneity plating Hg anodic stripping voltammetryOptimazed of the test conditions with base solution, pH, mercury concentration, depositionpotential, deposition time, stirring speed, square wave frequency, potential incremental, squarewave amplitude as Influential factors. The optimized conditions were: HAc-NaAc buffer solution;pH4.5; mercury concentration1mg/mL; deposition potential-1.0V; deposition time240s; stirringspeed400r/min; square wave frequency30Hz; potential incremental4mV/s; square waveamplitude25mV. Under these condition, there was a good correlation between the peak current iand Pb concentration C: I（μA）=1.136+0.2175C（μg/L）, correlation coefficent was0.9963. Thelinear range was1~80μg/L, the detection limit was0.3μg/L.3. Established a new electrochemical immunoassay technology for AFB1based on MAb-PbSas LabelsWith the prepared MAb-PbS conjugates, a new electrochemical immunoassay technology forAFB1in peanuts was developed. The results indicate current signal was highest wih incubationtime60min, dilution multiple of MAb-PbS5times. Under these conditions, results showed ahemi-inhibitory concentration (IC50) of0.814ng/mL, a limit of detection (LOD) of0.046ng/mL, adetection linear range of0.1-30ng/mL. Moreover, recoveries ranging from85.3%to117.3%wereobtained by recovery experiment of blank peanut samples, the cross-reactivities of AFB1, AFB2,AFG1, AFG2about Anti-Aflatoxin B1were100%,11%,2.6%,5.7%,3.5%. The results comparedwith HPLC results showed good consistency. In conclusion, the electrochemical immunoassaytechnology for AFB1based on MAb-PbS as Labels had high sensitivity, low detection limits andgood specificity.This method was accurate, reliable, and could be used as a assay for AFB1. TheIC50and detection limit of fluorescence immunoassay technology were5.003μg/L and0.212μg/L.Compared with the fluorescence immunoassay technology, the electrochemical immunoassaytechnology showed higher sensitivity, lower detection limit.4. Established an electrochemical immunoassay technology for AFB1based on MAb-(PbS)2as LabelsAccording layer self-assembly technique, successfully synthesized MAb-(PbS)2as a signalprobe used streptavidin-biotin system. Then established an electrochemical immunoassaytechnology for AFB1based on MAb-(PbS)2as Labels. Experimental results show that the linearrange of this method was0.04-15ng/mL, IC50was0.266μg/L, detection limit was0.018μg/L. This method had a more significant current signal, higher sensitivity and lower detection limit,compared with fluorescence detection and electrochemical detection based on MAb-PbS.