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Photometric and Electrochemical Enzyme‐Multiplied Assay Techniques Using β‐Galactosidase as Reporter Enzyme

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β‐Galactosidase (β‐gal) is shown to be a versatile new reporter enzyme in both photometric and electrochemical enzyme‐multiplied assay techniques (EMATs). The well‐known β‐gal substrate analog, o‐nitrophenyl β‐d‐galactopyranoside, yields the visibly colored, o‐nitrophenol product upon hydrolysis, whereas the substrate, p‐aminophenyl β‐d‐galactopyranoside, gives rise to an electrooxidizable product, p‐aminophenol. These β‐gal substrates made possible the demonstration of both photometric and electrochemical signal transduction schemes for β‐gal‐based EMAT detection of estradiol (as the estradiol‐bovine serum albumin (E‐BSA) conjugate). The EMAT system is composed of the reporter enzyme, β‐gal, with covalently attached estradiol, and estrogen antibody, which inhibits enzyme activity of the β‐gal‐estradiol conjugate up to ∼75%. Reporter enzyme inhibition is relieved significantly by addition of ≤2 ng/mL of estradiol (as E‐BSA), which competes for binding with the antibody. Thus, the presence of analyte (E‐BSA) is reported by the enzyme (β‐gal), which amplifies the ligand‐protein dissociation event by turning over its substrate repeatedly. The electrochemical version of EMAT, based on amperometric detection of p‐aminophenol, is responsive to added estradiol within minutes. These results show that β‐gal may serve as a useful alternative to glucose‐6‐phosphate dehydrogenase, which currently is used as reporter enzyme in commercially available EMAT systems.
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Document Type: Research Article

Affiliations: 1: Biomedical Engineering Interdepartmental Graduate Program, University of California, Los Angeles, Los Angeles, California 90095 2: Chemical and Biomolecular Engineering Department, University of California, Los Angeles, Box 951592, Los Angeles, California 90095–1592

Publication date: January 1, 2006

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