| Pufferfish is one of the most popular fish in China,which is nutritious and delicious with high economic value.Tetrodotoxin（TTX）,a kind of marine biological toxin in pufferfish,is the most toxic natural alkaloid neurotoxin.TTX is highly toxic,with rapid onset and short poisoning incubation period.Due to its specific toxic mechanism,TTX has promising application in medicine.Its pure extract can be used in clinical practice.TTX residue in aquatic products such as pufferfish has acritical impact on the public food safety.The residue concentration in species of wild pufferfish is varying.Relevant authorities allowed the processing and management of two kinds of farmed pufferfish（Takifugu rubripes and Takifugu obscurus）.The market management and monitoring of TTX residues should be intensified.In the meanwhile,real-time supervision on the breeding,processing,storage and circulation stages of pufferfish is necessary.The current research is mostly focused on the detection of TTX,and the way to achieve rapid,qualitative and quantitative determination is an important factor to promote the development of new detection methods.Based on the merits of electrochemistry and immunological analysis,three types of modified electrochemical immunosensor for the detection of TTX are fabricated in this work.The reaction conditions were optimized and the method performance were evaluated by stability,repeatability and recovery rate.Additionally,the methods have simple method for sample preparation,short detection period,high accuracy,and lower detection limit.This method could meet the requirements for rapid on-site testing.The main purpose of this study is to develop the electrochemical immunosensor for TTX detection,and provide ideas for the electrochemical immunodetection,trace analysis of TTX and other small molecule substances.The main research results are listed as follows:The glassy carbon electrode（GCE,working electrode）,saturated calomel electrode（SCE,reference electrode）and platinum wire electrode（auxiliary electrode）were used to establish a three-electrodes system.The sensor construction and detection process were carried out in this electrochemical system.1.Establishment and evaluation of glutaraldehyde based electrochemical immunosensorThe GCE/glutaraldehyde/BSA-TTX/BSA sensor was constructed on the glassy carbon electrode interface based on the competitive ELISA.In this system,the modified interface competes with free TTX to bind antibody.The optimal concentration of BSA was 2%,and the incubation time was 90 min（37℃）.In addition,the linear range of TTX was 2 ng/mL to 1250 ng/mL（r2>0.990）,and the detection limit（LOD,S/N>3）was 2.0 ng/mL with the recoveries between 81.36%-96.96%,and acceptable stability（RSD=5.27%,n=3）.In following studies,different types of nanomaterials were selected to enhance the electron transfer rate and amplify the electrical signal in the detection process,so as to improve the sensitivity of the method.2.Establishment and evaluation of chitosan-based electrochemical immunosensorThe free amino group in the molecular structure of chitosan carries positive charge,which can form electrostatic adsorption with the anion[Fe（CN）6]3-/4-which enhanced the redox current intensity at the electrode interface and amplified the electrical signal.Firstly,chitosan was modified on the GCE interface,then GCE/CS/AuNPs-BSA-TTX/BSA sensor was constructed step by step.The different concentrations of TTX were detected by competitive immunoassay on the modified surface.The final modified surface with different antibody capture quantities resulted in different response during the characterization of GCE/CS/AuNPs-BSA-TTX/BSA/anti-TTX.After optimization,the optimal concentration of BSA was set at 2%and the incubation time was set at 60 min（37℃）.The linearity was good in the range from 2 ng/mL to 1250 ng/mL（r2>0.955）,with the LOD（S/N>3）at 2.0 ng/mL,the recovery rate between 78.40%and 105.74%,and for recovery experiment at 5.06%.3.Establishment and evaluation of Nafion based electrochemical immunosensorNafion is stable and excellent membrane of cationic permeable,it can permeate the electroactive substance Ru（bpy）32+to enhance the interfacial conduction efficiency of the electrode.After the GCE/Nafion/AuNPs-BSA-TTX/BSA sensor was constructed,the signal response on the modified surface was influenced by the specific binding of antigen and antibody.The optimal BSA concentration was 1%,and the incubating time was 60min（37℃）.There was a negative correlation between TTX concentration and the change in the ACV peak current in the range of 2 ng/mL to 1250 ng/mL（r2>0.990）with the LOD（S/N>3）at 2.0 ng/mL,the recovery rate between 80.33%-115.44%,and the RSD of 4.24%for recovery experiment. |
