Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List

Full text of this paper is not available in the ETD Center. Copies may be available for inter-library loan from University of Cincinnati or may be available for purchase from Proquest/UMI

ETD Abstract Container

Abstract Header

DEVELOPMENT OF IMMUNOMAGNETIC BEAD ASSAY WITH ELECTROCHEMICAL DETECTION FOR USE IN A MINIATURIZED SENSOR

Purushothama, Shobha
http://rave.ohiolink.edu/etdc/view?acc_num=ucin984589640

Abstract Details

2001, PhD, University of Cincinnati, Arts and Sciences : Chemistry.
A bead based enzyme immunoassay for the model antigen mouse IgG has been developed with rotating disk electrode (RDE) detection. Alkaline phosphatase was used as the enzyme label to convert substrate 4-aminophenyl phosphate to the electroactive 4-aminophenol (PAP). The detection scheme allows enzyme turnover to be monitored continuously and thus reduces errors associated with obtaining data at fixed times as in flow injection analysis. The determination of PAP by the RDE has a linear range from 5 x 10 -9 to 5 x 10 -5 M. An 800 µl electrochemical cell was used to minimize enzyme product dilution. A linear response, for mouse IgG was obtained in this non-optimized system over the concentration range of 50 to 5000 ng/ml. The third chapter describes an immunoassay with dendrimer-modified 2.8 µm magnetic beads for the artificial microorganism, Bugbead. Bugbead consists of a 0.39 µm diameter polystyrene bead coated with proteins via avidin-biotin binding to represent the epitopes on a real microorganism. Starburst© PAMAM dendrimer (G 0.5, G 2.5, G 5.5) was covalently attached to the beads and capture antibody was attached to dendrimer termini by means of the avidin-biotin binding. The effect of dendrimer on the immunoassay was evaluated by comparison to an assay with commercial streptavidin beads to which the same capture antibody was bound. RDE amperometry in a micro drop was used to continuously monitor the enzyme turnover. The antibody bound/bead increases with the dendrimer generation used. With G 5.5 beads there is an improvement in assay sensitivity over the range of analyte concentrations studied. Beads with G 2.5 had assay sensitivity similar to the streptavidin beads and the beads with G 0.5 had poorer sensitivity. Different surface modifications and ion-pair agents were also studied in an attempt to reduce non-specific signal. The assay for ovalbumin was developed using a streptavidin bead based sandwich immunoassay coupled to electrochemical detection using the RDE in a small volume to minimize product dilution and improve detection limits. The assay developed has a detection limit of 0.1 ng/ml. Assay sensitivity in complex matrices such as food and serum was also evaluated.
William Heineman (Advisor)
immunomagnetic beads; electrochemical detection; dendrimers

Recommended Citations

Citations

  • Purushothama, S. (2001). DEVELOPMENT OF IMMUNOMAGNETIC BEAD ASSAY WITH ELECTROCHEMICAL DETECTION FOR USE IN A MINIATURIZED SENSOR [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin984589640

    APA Style (7th edition)

  • Purushothama, Shobha. DEVELOPMENT OF IMMUNOMAGNETIC BEAD ASSAY WITH ELECTROCHEMICAL DETECTION FOR USE IN A MINIATURIZED SENSOR. 2001. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin984589640.

    MLA Style (8th edition)

  • Purushothama, Shobha. "DEVELOPMENT OF IMMUNOMAGNETIC BEAD ASSAY WITH ELECTROCHEMICAL DETECTION FOR USE IN A MINIATURIZED SENSOR." Doctoral dissertation, University of Cincinnati, 2001. http://rave.ohiolink.edu/etdc/view?acc_num=ucin984589640

    Chicago Manual of Style (17th edition)

ucin984589640
© 2001, all rights reserved.
This open access ETD is published by University of Cincinnati and OhioLINK.