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

作者：Manibalan Kesavan; Mani Veerappan; Huang Chih Hung; Huang Sheng Tung^*; Chang Pu Chieh

来源：Analyst, 2015, 140(17): 6040-6046.

DOI：10.1039/c5an01036e

A 4-Methoxyphenyl-beta-galactopyranoside (4-MPGal) substrate incorporating 4-methoxy phenol (4-MP) as an electrochemical reporter is described for the monitoring of beta-Galactosidase (beta-Gal) gene expressions. beta-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 beta-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 mu M min(-1) mg of beta-Gal(-1) (E. coli), which is consistent with the previous reports. To detect beta-Gal derived from E. coli, cyclic voltammetry (CV) provides linear ranges of 12-1200 ng mL(-1) and 1.2-12 mu g 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 beta-Gal derived from A. oryzae, CV provides linear ranges of 0.1-100 ng mL(-1) and 0.1-1 mu g 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 beta-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.

• 出版日期2015