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.

Graphene based biosensors—Accelerating medical diagnostics to new-dimensions

“…In past few years, many electrochemical graphene-based device schemes, have been proposed for use in vivo for monitoring the dopamine, H 2 O 2 , L-Dopa (an intermediate precursor of the neurotransmitter dopamine), glucose, L-lactate, b-galactosidase-gene expressions. [137][138][139][140][141] An implantable device to study the brain dynamics using rGO wrapped gold-oxide composite (rGO/Au 2 O 3 ) multichannel neural probe with multiple real-time monitoring of neural-chemical and electrical signals by nonenzymatic neural-chemical interface was developed [represented in Fig. 8(a)].…”

The application of graphene for in vitro and in vivo electrochemical biosensing

“…In the last two decades, some devices have been proposed for use in vivo including electrochemical sensors (Caffrey et al, 2015;Grant et al, 2001;Gyetvai et al, 2009;Harreither et al, 2013;Hurst and Clark, 2003;Jacobs et al, 2011;Liu et al, 2012aLiu et al, , 2015Ragones et al, 2015;Shao et al, 2013;Swamy and Venton, 2007;Wang et al, 2001;Zhang et al, 2007); and biosensors (Abel and von Woedtke, 2002;Chai et al, 2013;Deng et al, 2008;Edagawa et al, 2014;Lin et al, 2013;Lowry and Fillenz, 2001;Lu et al, 2013;Pohanka et al, 2009;Ren et al, 2013;Ricci et al, 2007;Santos et al, 2015a;Tian et al, 2005;Yu et al, 2011aYu et al, , 2011bZhang et al, 2004;Zhu et al, 2009). In this context, recently, graphene has also been used in electrochemical sensors (Arvand and Ghodsi, 2013;Manibalan et al, 2015;Zhu et al, 2011) and biosensors (Gu et al, 2014(Gu et al, , 2012 for in vivo applications. The purpose of this section is to present a brief revision of the last 15 years about electrochemical biosensors based on graphene for in vivo applications.…”

Electrochemical Molecular Switch for the Selective Profiling of Cysteine in Live Cells and Whole Blood and for the Quantification of Aminoacylase-1

“…Our research group is interested in designing and making use of latent electrochemical redox probes as effective transducers for the selective detection of non-redox-active biomarkers. , These molecular switches consist of a recognition unit attached via a linker to a latent redox-active reporter. A selective analyte–probe interaction triggers the user-designed chemical or biochemical transformation to unmask the signature electrochemical signal of the reporter . In the current work, ferrocene carbamate phenyl acrylate (FCPA) was designed as the latent electrochemical probe.…”

“…A selective analyte−probe interaction triggers the user-designed chemical or biochemical transformation to unmask the signature electrochemical signal of the reporter. 19 In the current work, ferrocene carbamate phenyl acrylate (FCPA) was designed as the latent electrochemical probe. The aminoferrocene (AF) moiety of FCPA was appended to benzyl alcohol through a carbamate functionality, which masked the redox potential of AF (−0.05 V vs Ag/AgCl) at the negative region of its voltammetric spectrum.…”