Fast Detection of Beta Galactosidase and Enzyme Kinetics with 4-Aminophenyl-β-D-Galactopyranoside as Substrate

Abstract

Beta-galactosidase (β-Gal) is an important enzyme utilized to determine cell senescence and understand disease pathology, among being utilized for several biosensing applications. This key biomarker can be measured indirectly using electrochemical methods by detecting the electroactive p-aminophenol (PAP) produced from the enzymatic conversion of a substrate. Here, we present a quick electrochemical method based on cyclic voltammograms (CVs) to quantify the amount of β-Gal in aqueous solution in under 5 minutes. Using timed CVs, the enzyme kinetics of β-Gal versus 4-aminophenyl-β-D-galactopyranoside (PAPG) was estimated. The detection limit for β-Gal was 40 ng ml⁻¹ using the timed quick CV method. K_M was found to be 18.7 ± 0.7 µM, and V_max 10.1 ± 0.6 µmol min⁻¹ mg⁻¹, for β-Gal with PAPG as a substrate. By adding divalent salts Mg²⁺ and Ca²⁺, K_M was lowered to nearly half, and V_max increased by 20%. The electrochemical protocol utilized neutral pH and ambient room temperature benchtop conditions, making it amenable to scanning electrochemical microscopy and lab-on-chip detection platforms. In addition to cell senescence, this quick and simple electrochemical method can be used in broad biosensing applications, including the detection of water and foodborne pathogens and the identification of toxic contaminants.

Keywords:

• Cell senescence
• cyclic voltammetry
• enzyme

Additional information

Funding

This project was funded by the National Institute of Health (NIH 1-R21-EB023595-01).