A new electrochemical substrate for rapid and sensitive in vivo monitoring of β-galactosidase gene expressions

Abstract

A 4-Methoxyphenyl-β-galactopyranoside (4-MPGal) substrate incorporating 4-methoxy phenol (4-MP) as an electrochemical reporter is described for the monitoring of β-Galactosidase (β-Gal) gene expressions. β-Gal derived from Escherichia coli (E. coli) and Aspergillus oryzae (A. oryzae) were investigated, while a graphene oxide film modified electrode was employed as the transducer. The electrochemical signal of 4-MPG within 4-MPGal was masked by protecting their hydroxyl group with galactose. The externally added β-Gal triggered the deprotection through specific enzymatic hydrolysis with concomitant release of 4-MP. The apparent K_m and V_max values of 4-MPGal are determined to be 0.21 mM and 0.51 μM min⁻¹ mg of β-Gal⁻¹ (E. coli), which is consistent with the previous reports. To detect β-Gal derived from E. coli, cyclic voltammetry (CV) provides linear ranges of 12–1200 ng mL⁻¹ and 1.2–12 μg mL⁻¹ with a limit of detection (LOD) of 5 ng mL⁻¹, while differential pulse voltammetry (DPV) shows a linear range of 1.2–120 ng mL⁻¹ and LOD of 1 ng mL⁻¹. To detect β-Gal derived from A. oryzae, CV provides linear ranges of 0.1–100 ng mL⁻¹ and 0.1–1 μg mL⁻¹ with a LOD of 0.06 ng mL⁻¹, while DPV shows a linear range of 10 pg mL⁻¹–10 ng mL⁻¹ with a LOD of 8 pg mL⁻¹. Moreover, we set up a platform for the real-time in vivo monitoring of β-Gal gene expressions in E. coli cultivated through microbiological culture. The developed sensing platform using 4-MPGal as a substrate is simple, rapid, sensitive, specific and advantageous over its laborious optical analogues.