Organic phase enzyme electrodes: kinetics and analytical applications

Abstract

The suitability of organic media for enzymatic reactions has led to tremendous advancement in biosynthesis and, more recently, biosensing. Organic phase enzyme-based biosensors (OPEE), i.e. biosensors that operate in organic media, combine the high selectivity and specificity of enzymes with the ability to control their reactivity by altering some physiochemical properties of the reaction media, such as solvent polarity and degree of hydration. Classical enzymology predicts that hydrophobic organic solvents, rather than hydrophilic solvents, are suitable for enzyme activity, on account of the latter's ability to strip the enzyme of essential water of hydration necessary for the flexibility and polarity of the active site micro-environment. We have demonstrated with solvents, such as acetonitrile, acetone, butan-2-ol, chloroform, hexane and tetrahydrofuran, that organic-phase amperometric biosensors can be developed to exhibit excellent reactivities in both hydrophilic and hydrophobic organic solvents by controlling the degree of hydration of the biosensing environment. Amperometric biosensors containing tyrosinase, glucose oxidase (GOx), horseradish peroxidase (HRP), and more recently chemically modified HRP, have been constructed and successfully applied to assays in organic solvents. Several enzyme immobilization matrices have been used in our laboratory for the fabrication of sensors. Poly(estersulphonic acid)-entrapped tyrosinase and HRP electrodes have been found to be very effective biosensors in a variety of organic solvents for determining phenols, organic peroxides and pesticide compounds. The Os-polymers, [Os(bpy) 2(PVP) nCl]Cl [bpy = 2,2′-bipyridyl, PVP = poly(4-vinylpyridine); n = 10, 20 or 25] and [Os(byp) 2(PVI) 10Cl]Cl [PVI = poly(4-vinylimidazole)] have been used with bifunctional cross-linking agents, such as glutaraldehyde or polyethylene glycol, to form reagentless biosensors with GOx, tyrosinase and HRP (native and modified forms of HRP). These biosensors, based on the electrostatic complexation of the enzyme and the positively charged Os-polymer, have been found to be very stable in organic phase. GOx sensors that employ soluble mediators, such as, ferrocenemonocarboxylic acid, exhibited better catalytic efficiency, k cat ′ K M ′ in the organic phase than in the aqueous phase. In our studies we have demonstrated that the reactivities of amperometric organic phase biosensors follow the Michaelis-Menten kinetic paradigm. However, kinetic analyses have shown that the values of the sensor biocatalytic parameters, including I max, k cat′ and K M′ depend on solvent media. Under certain conditions, k cat ′ K M ′ is the second order rate constant through ΔG =/ −RT ln[ k cat ′ K M ′ )hlk B T], where T, h, and k B are the absolute temperature, Planck's constant and Boltzmann's constant, respectively. Thus, k cat ′ K M ′ is a measure of the activation of the biosensor redox-catalytic reaction in different solvents. This parameter has been evaluated at different temperatures in both aqueous and organic media. The ΔG =/ values (the difference in Gibbs energy between transition and ground state of sensor reaction for a given solvent and substrate) in organic and aqueous media have been used to evaluate the role of the solvent on the stabilization of the transition state on the electrocatalytic reaction. Organic phase HRP biosensors have also been applied in our laboratory for the determination of thiourea, ethylenethiourea and other thio-compounds (which are the parent compounds for some pesticides). The thio-compounds act as inhibitors. Their determination is based on the change in the electrocatalytic current of peroxide reduction by the biosensor, which accompanies the addition of the organic sulphides. Tyrosinase electrodes have been applied as detectors in reversed-phase HPLC for the detection of the eight phenols that are normally found in cigarette filters. Native HRP and bis-succinimide-modified-HRP electrodes have also been applied in the amperometric determination of organic peroxides, the aim being to produce a more sensitive, reproducible and stable organic phase HRP electrode.