title: 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 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.
authors.affiliation: Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei 10608, Taiwan, Republic of China. ws75624@ntut.edu.tw.
authors.forename: Kesavan
authors.initials: K
authors.lastname: Manibalan
authors.forename: Veerappan
authors.initials: V
authors.lastname: Mani
authors.forename: Chih-Hung
authors.initials: CH
authors.lastname: Huang
authors.forename: Sheng-Tung
authors.initials: ST
authors.lastname: Huang
authors.forename: Pu-Chieh
authors.initials: PC
authors.lastname: Chang
mesh_terms: D000840:Anisoles / Q000737:chemistry; D001236:Aspergillus oryzae / Q000201:enzymology; D055664:Electrochemical Techniques; D004566:Electrodes; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D015870:Gene Expression; D006108:Graphite / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D010087:Oxides / Q000737:chemistry; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D000840:Anisoles; D004800:Enzymes, Immobilized; D010087:Oxides; C009760:mequinol; D006108:Graphite; D001616:beta-Galactosidase