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

Abstract

Beta-galactosidase (beta-gal) is shown to be a versatile new reporter enzyme in both photometric and electrochemical enzyme-multiplied assay techniques (EMATs). The well-known beta-gal substrate analog, o-nitrophenyl beta-d-galactopyranoside, yields the visibly colored, o-nitrophenol product upon hydrolysis, whereas the substrate, p-aminophenyl beta-D-galactopyranoside, gives rise to an electrooxidizable product, p-aminophenol. These beta-gal substrates made possible the demonstration of both photometric and electrochemical signal transduction schemes for beta-gal-based EMAT detection of estradiol (as the estradiol-bovine serum albumin (E-BSA) conjugate). The EMAT system is composed of the reporter enzyme, beta-gal, with covalently attached estradiol, and estrogen antibody, which inhibits enzyme activity of the beta-gal-estradiol conjugate up to approximately 75%. Reporter enzyme inhibition is relieved significantly by addition of < or =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 (beta-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 beta-gal may serve as a useful alternative to glucose-6-phosphate dehydrogenase, which currently is used as reporter enzyme in commercially available EMAT systems.

Fabricating cholyglycine–glucose‐6‐phosphate dehydrogenase conjugates for cholyglycine detection

“…Ko [17] has successfully utilized UV wavelength scanning to identify estradiol-β-galactosidase conjugates. Here, UV wavelength scanning was thus used to compare CG-G6PD conjugates with CG and G6PD.…”

Protein G: β‐galactosidase fusion protein for multi‐modal bioanalytical applications

“…[7][8][9][10][11] Additionally, analogous to HRP, β-gal can be used to produce an electrochemical response through the cleavage of 4-Amino-phenyl β-Dgalactopyranoside (PAPG) yielding redox active p-aminophenol (PAP). [12][13][14][15] We constructed a protein G: β-gal fusion protein that can be used for analytical applications involving IgG. Importantly, because β-gal exhibits activity toward a variety of substrates the fusion protein enables multiple modes of detection (i.e., optical, electrochemical).…”

“… 7 , 8 , 9 , 10 , 11 Additionally, analogous to HRP, β‐gal can be used to produce an electrochemical response through the cleavage of 4‐Amino‐phenyl β‐D‐galactopyranoside (PAPG) yielding redox active p‐aminophenol (PAP). 12 , 13 , 14 , 15 …”

Electrochemical Measurement of the β-Galactosidase Reporter from Live Cells: A Comparison to the Miller Assay

“…Such techniques would enable more predictive models of metabolic and cellular networks. This paper is largely motivated by many reports of the biosensor and biochip community, where researchers have used the redox-active molecule p -aminophenyl β- d -galactopyranoside (PAPG) as a substrate for electrochemically measuring β-gal activity both inside and outside of cells. − The focus to-date, has targeted the development of sensitive electrode and biochip systems that provide cost-efficient real-time measurements on the microscale that are easily integrated into more elaborate electronic systems.…”