Electrochemical Biosensor For The Rapid Detection Of Aflatoxin B1

Food safety has drawn great attention from the society.Aflatoxin B1（AFB₁）,which is hepatotoxic and mutagenic,poses a great threaten to the food safety and economic development of the world.Thus,searching for reliable methods for AFB₁detection has become important to ensure food safety.Many methods have been developed for the detection of AFB₁,such as thin layer chromatography,high performance liquid chromatography and enzyme-linked immunosorbent assay.Unfortunately,most of the reported methods still have some limitations in processing,such as low accuracy,complex operation and high cost.In order to develop an AFB₁detection system of simple operation,low detection limit,and stable performance,this project used two different structures of carbon materials including graphene（GN）and carbon felt（CF）to build electrochemical biosensors.The structures of the resulting electrochemical sensors were characterized by field emission scanning electron microscopy,transmission electron microscopy and X-ray diffraction.The electrochemical tests indicated that the developed electrochemical biosensors have the advantages of low detection limit（LOD）,high stability and strong specificity.Both of the two electrodes have wide detection range and good reproducibility,and are feasible in the actual sample detection.The main content of this thesis was summarized as follows:（1）Electrochemical biosensors based on layer-by-layer（LbL）self-assembly technology.Modified-doped GN and carboxylated polystyrene nanospheres（PS-COOH）were alternatively assembled on the glassy carbon electrode via LbL self-assembly technique.Combined with the advantages of high specific surface area of GN and good biocompatibility of PS-COOH,the electrochemical sensors possesses excellent performance owing to the synergistic effects arisen from the sandwich-like structure.The activated carboxyl group was bonded with an aptamer（Apt）.The free succinimide group was passivated with ethanolamine.The non-specific active sites were blocked with bovine serum albumin（BSA）.After incubation with AFB₁,the sensor was evaluated in the electrolyte of K₃[Fe（CN）₆].The main experimental parameters were optimized as follows:PDDA concentration of 0.3 mg mL^-1,electrostatic adsorption for 10 min,self-assembly of 4 layers,2μM Apt incubation for 1 h.The performance of the resulting sensor was optimized as follows:the detection concentration ranged of AFB₁are from 0.001 to 0.10 ng mL^-1with a LOD of 0.002 ng mL^-1.After 30 days of storage at 4℃,the recovery rate decreased less than 10%.The recoveries were 94.5～103.3%in edible oil and soy sauce.The sensors are not affected by ochratoxin（OTA）and other interfering substances.（2）Electrochemical biosensors based on horseradish peroxidase（HRP）as a signal amplification probe and self-supporting electrode.SSE was used as the base material of the sensor,which has a stable three-dimensional structure of porous space and abundant active sites.It can not only reduces the LOD of the sensor,but also improves stability of the sensors.PS-COOH was used as a carrier to fix HRP which is the signal amplification probe.HRP can amplify electrochemical signals to improve the sensitivity of the sensors.SSE consisting of carbon nanotube arrays assembled on CF support was fabricated via a typical chemical vapor deposition process in which Ni-Cu oxalates were used as a catalyst percussor and methane was used as a carbon source Au was introduced via a replacement reaction with NiCu particles located on the tip of CNTs and subsequently bonded with Apt.After passivated by BSA,the sensor was incubated with AFB₁.Owing to the competitive relationship of PS-COOH and AFB₁,the excess Apt can bind to PS-COOH.PS-COOH is used as a carrier of HRP which was utilized as a signal amplification probe.The free carboxyl group of PS-COOH is capable of binding HRP,thereby immobilizing HRP on the electrode.HRP catalyzes the oxidation of H₂O₂in the electrolyte,which significantly amplifies the electrochemical signal of the electrode.The experimental parameters were optimized as follows:Apt concentration of 1μM,PS-COOH concentration of 0.3 mg mL^-1,HRP concentration of 2 mg mL^-1,and H₂O₂concentration of 10 m M.The optimized electrochemical sensor possessed LODs of 0.016 pg mL^-1and 0.3 ng mL^-1in the ranges of 0.1～10 pg mL^-1and 1～10 ng mL^-1,respectively.After four-week sealed storage at 4℃,the recovery rate was decreased only by 10.4%.It was not affected by OTA and other interfering substances.The recovery rates of spiked AFB₁in wine and soy sauce were in the range of87.53～106.71%.