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BEAD-BASED IMMUNOASSAYS WITH ELECTROCHEMICAL DETECTION

by RONKAINEN-MATSUNO, NIINA JOHANNA

Abstract (Summary)
Immunoassays with electrochemical detection have gained popularity due to their versatility, low detection limits, relatively easy procedures, and simple instrumentation. Immunoassays have a wide range of applications in clinical chemistry and biodefense as well as agricultural, environmental, and processed food areas. A variety of sensitive amperometric electrochemical detection methods have previously been used with immunoassays. A microelectrode was used here as a detector for bead-based assays in very small sample volumes (down to 10 ┬ÁL) with low detection limits, fast response times, and fairly good reproducibility. A microelectrode with currents in the pA range and very low charging currents showed great promise as a detector for small volume immunoassays in a biosensor with a microfluidic component. The microelectrode helped overcome the problem of rapid depletion of the electroactive species during detection in small volumes where the macroelectrode would consume the electroactive species faster than the enzymes could produce them. The rapid depletion was seen with macroelectrodes such as the rotating disk electrode. Ovalbumin protein, a simulant for biotoxins, used as an antigen in immunoassays was characterized using ion exchange chromatography, thermal denaturation, and circular dichroism. Ovalbumin varied in the degree of phosphorylation of the two serine residues between preparations. A microvolume sandwich immunoassay for ovalbumin with enzyme labels was developed on paramagnetic beads that were easy to manipulate, presented short diffusional distances, and hence faster assay times. A rotating disk electrode at a fixed potential was used as a detector. The viability of electrochemical immunoassay as a sensitive technique in a water sample matrix was investigated. Water samples from around the country were characterized with respect to pH and metal content. Sandwich immunoassays for ovalbumin were done in finished water from Cincinnati, OH and from Potomac, MD with minor pretreatment with a chelating agent and a buffering agent. The assay was also done successfully in unfiltered pond water from Cedarville, OH.
Bibliographical Information:

Advisor:

School:University of Cincinnati

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:bead based immunoassay in water electrochemical detection ovalbumin microelectrode nonspecific binding

ISBN:

Date of Publication:01/01/2003

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