Electrochemical Immunoassay Based On Horseradish Peroxidase/Au Nanoparticles Labeling

Electrochemical immunoassay has attracted much attention in the biomedical analysis area due to its advantages such as good selectivity,high sensitivity,low detection limit,low cost and easy operation of the instrument.In this thesis,on the basis of a brief review on the methods for electrochemistry-related immunoassay and for related signal output/amplification,with human immunoglobulin G（h Ig G）as the detection object and horseradish peroxidase（HRP）/gold nanoparticles（AuNPs）as the signal markers,the immunosensing performance was studied by potentiometry or voltammetry（including potentiostatic amperometry）.The main research contents are as follows.1.On the basis of the HRP-catalyzed oxidation of 2,2’-azino-bis（3-ethylbenzothiazoline-6-sulfonate）（ABTS）to 2,2’-azino-bis（3-ethylbenzothiazole-6-sulfonate）radical cation(ABTS^·+)in the presence of H₂O₂,the enzymatic activity of HRP was measured by potentiometry and ultraviolet-visible spectrophotometry（UV-vis）,and the two methods gave well agreeable results.Electrochemical immunosensing of h Ig G was studied,by electroreducing pyridine-2-carboxylic acid on glassy carbon electrode（2-Py CA/GCE）,covalently immobilizing the primary antibody on 2-Py CA/GCE after activating the carboxyl groups by carbodiimide（EDC）and N-hydroxysuccinimide（NHS）,and labeling HRP on the secondary antibody（HRP-Ab₂）.Electrochemical methods and quartz crystal microbalance（QCM）were used to characterize related materials and electrodes.In the ABTS-H₂O₂ system,the linear detection range（LDR）of h Ig G from potentiometry was from 0.5 pg m L^-1 to 500 ng m L^-1,and the limit of detection（LOD）was 0.024 pg m L^-1（S/N=3）;and the LDR of h Ig G by amperometry was from 0.5 pg m L^-1 to 500 ng m L^-1,and the LOD was 0.089 pg m L^-1（S/N=3）.2.Electrochemical immunosensing of h Ig G was studied,by similarly immobilizing Ab₁ on the above 2-Py CA/GCE,utilizing AuNPs as the secondary antibody label（AuNPs-Ab₂）,and conducting in-situ microliter drop anodic stripping voltammetry to detect the anodic stripping peak of Au enriched at the cathode.Electrochemical methods,UV-vis,scanning electron microscope（SEM）and energy dispersive spectroscopy（EDS）were used to characterize the related materials and electrodes.Under optimized conditions,the LDR of h Ig G was from 5 pg m L^-1 to 500 ng m L^-1,and the LOD was 3.7 pg m L^-1（S/N=3）.3.Electrochemical immunosensing of h Ig G was studied,by similarly immobilizing Ab₁ on the above 2-Py CA/GCE,utilizing AuNPs and HRP as the secondary antibody label（HRP-AuNPs-Ab₂）,and utilizing potentiometry to detect the open-circuit potential and amperometry to detect the reduction current of the enzymatically generated p-benzoquinone（BQ）in a hydroquinone（HQ）-H₂O₂ system.Electrochemical methods,UV-vis,QCM,SEM and EDS were used to characterize related materials and electrodes.Under optimized conditions,the LDR of h Ig G from potentiometry was from 0.5 pg m L^-1 to 500 ng m L^-1,with a LOD of 0.032 pg m L^-1（S/N=3）;and the LDR of h Ig G from amperometry was from 0.500 pg m L^-1 to 500 ng m L^-1,with a LOD of 0.11pg m L^-1（S/N=3）.