Construction And Application Of Novel Electrochemical Bio-sensors Based On Carbon Nanocomposites

T-2 toxin is a kind of mycotoxin produced by Fusarium in which T-2 is the most toxic toxin primarily being in wheat,barley,oat,corn and other grains and is heavily harmful to human and animal health.Beta-lactam antibiotics are one of the most widely used antibiotics in the prevention and treatment of animal bacterial infectious diseases.However,drug abuse causes antibiotic residue problems in animal-derived food,which has brought great impact and harm to human health and ecological environment.The commonly used rapid screening methods for toxic and harmful substances include enzyme-linked immunosorbent assay（ELISA）,liquid chromatography-tandem mass spectrometry（LC-MS）and so on.The electrochemical biosensor has the advantages of simplicity,high sensitivity and low-cost gradually playing an increasingly important role in drugs and environmental analysis.In our research,we hope to amplify the detection signal and improve the poor reproducibility by quantum size effect,surface effect and other excellent physical and chemical properties of nano-materials The research work is as follows:Method 1:An electrochemical immunosensor based on gold nanoparticles/single-walled carbon nanotubes/chitosan composites（AuNPs/SWNTs/CS）was developed.It can be used to detect T-2 toxin in cereal,feed and animal tissue samples with high efficiency and good sensitivity.1.The hapten compound T-2-HS of T-2 was synthesized by using T-2 and succinic anhydride（HS）as raw materials and identified by mass spectrometry as the expected product.Then conjugated the expected hapten T-2-HS to OVA by EDC method.The synthesized antigen was identified by UV full-wavelength scanning indicating that the reaction was successful.2.Gold nanoparticles and carboxylated single-walled carbon nanotubes were modified onto polished bare electrodes by electrodeposition and physical deposition respectively.The modified electrodes were characterized by scanning electron microscopy（SEM）and electrochemical methods（cyclic voltammetry,CV;electrochemical impedance spectroscopy,EIS）.The results showed that we successfully modified the electrodes by observing electron microscopy and current response values.3.The T-2-OVA was covalently bonded with the functionalized AuNPs/SWNTs/CS material to modify the surface of the glassy carbon electrode（GCE）.The modified electrodes were characterized by transmission electron microscopy（TEM）and electrochemical methods,cyclic voltammetry（CV）and electrochemical impedance spectroscopy（EIS）.The electrochemical immunosensor for the detection of T-2 toxin was successfully developed by observing the results of electron microscopy and current response values.4.This method was based on the competitive binding between free T-2 toxin and different concentrations of T-2-OVA to T-2 monoclonal antibody.Finally,the second antibody of sheep anti-rat IgG labeled by alkaline phosphatase（ALP-anti-antibody）was added to the electrode surface,and the electrochemical signal was generated byα-naphthol phosphate（α-NP）in the substrate of ALP catalytic reaction.5.The linear range of the detection of T-2 toxin by the immunosensor was 0.01-100μg/L where the peak current signal of DPV was linearly related to the logarithm of T-2concentration.The regression equation was y=-54.839x 137.57,the index of correlation R²=0.9986,and the detection limit was 0.14μg/L much lower than the minimum detection limit of 0.5-100μg/L.In addition,the sensor was successfully applied to detect T-2 toxin spiked in feed and tissue samples,and the recovery rate was good（92.34%-108.65%）.Furthermore,the electrochemical immunoassay had a good correlation with liquid chromatography tandem mass spectrometry（LC-MS/MS）,and the index of correlation was 0.9975,which indicated that the electrochemical immunosensor effective and reliable.A highly sensitive,specific and reproducible immunosensor was successfully established,which provides a new technique for the field analysis of T-2toxin.Method 2:A novel electrochemical receptor sensor was established for the rapid detection ofβ-lactam antibiotics with the combination of receptor analysis and electrochemical detection,so that the receptor analysis method was fast based on the sensor technology.The development of sensitivity,trace,accuracy and on-line analysis.The following works were carried out:1.The receptor protein was purified according to the research progress of biological elements in this experiment,and graphene/thionine composite and enzyme-labeled drug（HRP-AMP）were synthesized and identified.2.The electrochemical receptor sensor was constructed and characterized by CV and EIS.The modified electrode was successfully constructed by differential voltammetry.3.Finally,through the direct immune reaction principle and the catalytic reaction of horseradish peroxidase to H₂O₂,thionine was used as the effective electron transfer medium for the detection of antibiotics.4.The regression equation was y=-66.745x 117.06,and the index of correlation was 0.9946.The detection of limit was 0.16μg/L much lower than the minimum detection limit of 5μg/L stipulated by national legislation.In addition,the LOD and LOQ was 14.88μg/L,2.46μg/L,17.16μg/L,0.06μg/L,0.21μg/L and 36.09μg/L,5.40μg/L,41.45μg/L,0.13μg/L,0.42μg/L respectively for the milk samples spiked with Cefalexin,Cefquinoxime,Cefotafur,Penicillin G and Ampicillin.The recovery rate was between 84.89%and 102.44%and the coefficient of variation was less than 11.34%.Furthermore,the electrochemical receptor analysis method had a good correlation with liquid chromatography tandem mass spectrometry（LC-MS/MS）,and the index of correlation was 0.9995,which indicated that the electrochemical receptor-sensor was effective and reliable.A new method with excellent properties for rapid screening and field analysis ofβ-lactam antibiotics was successfully constructed.