Development of multianalyte electrochemical immunoassays.

摘要

This dissertation work summarizes preliminary studies for the development of multianalyte electrochemical immunoassays based on microfabricated planar amperometric microcells (MPAMs) consisting of gold microelectrode arrays (MEAs) for the simultaneous detection of three cardiac markers associated with diagnosis of acute myocardial infarction, creatine kinase-MB, cardiac troponin I, and cardiac troponin T.;Electrochemical surface areas of gold MEAs were determined before and after surface modifications. Surface modifications of MEAs include air plasma cleaning, protein/enzyme immobilization, antigen/antibody binding, and thiol SAM adsorption/electrochemical desorption.;This work describes enzyme immobilization, which is the model experiment for antibody immobilization. Glucose oxidase, horseradish peroxidase, alkaline phosphatase, and beta-galactocidase were immobilized on the surfaces of gold MEAs in MPAMs. Scanning electrochemical microscopy was used to study the layer-by-layer construction of enzyme immobilization with a glass-sealed Pt disc microelectrode probe above the MEAs with immobilized enzymes. The activities of immobilized horseradish peroxidase, alkaline phosphatase, and beta-galactocidase were also measured using gold MEAs with chronoamperometry.;One electrochemical protein patterning method was proposed in this dissertation to site-specifically immobilize multiple enzymes/proteins on three individually addressable reactivating MEA surfaces by long-chain thiol SAM adsorption and its electrochemical desorption.;Alkaline phosphatase and beta-galactocidase were site-specifically immobilized onto the surfaces of two individual MEAs in an MPAM with three MEAs. The two MEAs with alkaline phosphatase and beta-galactocidase were characterized separately and simultaneously.;A simplified electrochemical immunoassay was developed in this work by coupling anti-bovine serum albumin-alkaline phosphatase to bovine serum albumin immobilized on the surface of gold MEAs. The MEAs were characterized with chronoamperometry when coupled anti-bovine serum albumin-alkaline phosphatase was of different concentrations by measuring the activity of conjugated alkaline phosphatase.