Development of electrochemical histamine biosensor for histamine using genetically engineered histamine dehydrogenase from Rhizobium SP with gold-binding peptide tag

Abstract

Histamine is an essential biogenic amine that acts as a neurotransmitter and has been found to play a key role in many neurological and psychiatric diseases. Thus, there exists a considerable demand for determining histamine for point-of-care analysis aimed to disease diagnosis and prevention. To this end, we have evaluated a high-turnover redox couple involving the enzyme histamine dehydrogenase from Rhizobium sp. 4-9 (HDR) which contains a covalently attached flavin mononucleotide and an iron-sulfur center that shuttles electrons between the reduced flavin and external electron acceptors. There is a significant interest in the immobilization of this enzyme to improve catalytic stability on conductive surfaces towards biosensing applications. Here we engineered HDR construct with a functional cyclic gold-binding peptide (cAuBP) tag, at the C and N terminus, to immobilize the enzyme onto gold surfaces and evaluate the catalytic activity of fusion proteins. The electron transfer efficiency of redox enzymes can be difficult in the absence of mediator, thus 1,1-ferrocenedimethanol was chosen to perform all the experiments, on its oxidized form. The enzyme exhibits high substrate selectivity without substrate inhibition even at high turnover rates (apparent Kcat>127s-1) observed using soluble ferricenium substrates, unlike the closely related histamine dehydrogenase from Nocardioides simplex (nsHDR). Cyclic voltammetry (CV) and Amperometry experiments were carried out using a three electrodes system with a conventional cell equipped with an Ag/ AgCl electrode as the reference electrode, a platinum electrode as the counter electrode, and gold disc electrode as the working electrode. The possibility of Direct Electron Transfer (DET) between the enzyme and electrode was evaluated and mediated by the ferrocene derivative as well. Cyclic voltammograms and Amperometry of oxidized ferrocene and HDR from Rhizobium sp. 4-9 were recorded in the presence and absence of histamine, in Phosphate Buffer pH 7.4. High rates of electron transfer between histamine and a gold electrode were observed when the oxidized ferrocene is in solution. Atomic force microscopy, Localized Surface Plasmon Resonance (LSPR) and Surface Plasmon Resonance (SPR) were performed to evaluate the binding affinity to gold surfaces. The specificity, kinetic properties, lack of substrate inhibition and electrochemical studies indicate the suitability of the HDR for use in biosensing applications.