A new peroxidase from garlic (Allium sativum) bulb: its use in H2O2 biosensing.

Peroxidase POX(1) isoenzyme was purified from garlic (Allium sativum L.) bulb by ammonium sulfate precipitation, gel filtration and anion-exchange chromatography. Native-PAGE profile showed two isoforms, designated POX(1A) and POX(1B). POX(1B) seems to be more attractive for biosensor design since its K(m) (app) for H(2)O(2) is lower than that of POX(1A). In addition to its storage and operational stability, POX(1B) was found to be highly heat-stable, since almost 70% of its activity was conserved at 60 degrees C, whereas full activity was retained at 50 and 40 degrees C for 40 min. The optimal pH was approx. 5 and the optimal temperature was 30 degrees C. Next, gelatin was used as a matrix for enzyme immobilization on a gold electrode surface and electrochemical measurements were performed by using cyclic voltammetry. POX(1B)-based electrodes show great potential for application in H(2)O(2) monitoring of biological samples.

Immobilization of rhodopsin on a self-assembled multilayer and its specific detection by electrochemical impedance spectroscopy.

Rhodopsin, the G protein-coupled receptor (GPCR) which mediates the sense of vision, was prepared from calf eyes and used as receptor enriched membrane fraction. In this study it was immobilized onto gold electrode by two different techniques: Langmuir-Blodgett (LB) and a strategy based on a self-assembled multilayer. We demonstrated that Langmuir and LB films of rhodopsin are not stable. Thus, in this study a new protein multilayer was prepared on gold electrode by building up layer-by-layer a self-assembled multilayer. It is composed of a mixed self-assembled monolayer formed by MHDA and biotinyl-PE, followed by a biotin-avidin system which allows binding of biotinylated antibody specific to rhodopsin. The immobilization of rhodopsin in membrane fraction, by the specific antibody bound previously on self-assembled multilayer, was monitored with electrochemical impedance spectroscopy (EIS). In addition, the specificity and sensitivity of this self-assembled multilayer system to the presence of rhodopsin were investigated. No effect was observed when the system was in contact with olfactory receptor I7 in membrane fraction used for control measurements. All these results demonstrate that rhodopsin can be immobilized efficiently, specifically, quantitatively and stably on gold electrode through the self-assembled multilayer.

Immobilization of native membrane-bound rhodopsin on biosensor surfaces.

In this paper, we evaluated the grafting of G-protein-coupled receptors (GPCRs) onto functionalized surfaces, which is a primary requirement to elaborate receptor-based biosensors, or to develop novel GPCR assays. Bovine rhodopsin, a prototypical GPCR, was used in the form of receptor-enriched membrane fraction. Quantitative immobilization of the membrane-bound rhodopsin either non-specifically on a carboxylated dextran surface grafted with long alkyl groups, or specifically on a surface coated with anti-rhodopsin antibody was demonstrated by surface plasmon resonance. In addition, a new substrate based on mixed self-assembled multilayer that anchors specific anti-receptor antibodies was developed. Electrochemical impedance spectroscopy performed upon deposition of membrane-bound rhodopsin of increasing concentration exhibited a significant change, until a saturation level was reached, indicating optimum receptor immobilization on the substrate. The structures obtained with this new immobilization procedure of the rhodopsin in its native membrane environment are stable, with a controlled density of specific anchoring sites. Therefore, such receptor immobilization method is attractive for a range of applications, especially in the field of GPCR biosensors.