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

• Published in: Analyst (London) 2015-09, Vol.140 (17), p.6040-6046
• Main Authors: Manibalan, Kesavan, Mani, Veerappan, Huang, Chih-Hung, Huang, Sheng-Tung, Chang, Pu-Chieh
• Format: Article
• Language: English
• Subjects: Anisoles - chemistry; Aspergillus oryzae; Aspergillus oryzae - enzymology; beta-Galactosidase - genetics; beta-Galactosidase - metabolism; Electrochemical Techniques; Electrodes; Enzymes, Immobilized - chemistry; Enzymes, Immobilized - metabolism; Escherichia coli; Escherichia coli - enzymology; Gene Expression; Graphene; Graphite - chemistry; Hydrolysis; Hydroxyl groups; Kinetics; Oxides - chemistry; Phenol; Platforms; Substrate Specificity; Transducers; Voltammetry
• ISSN: 0003-2654; 1364-5528
• DOI: 10.1039/c5an01036e

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 Km and Vmax 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.