ORGANIC-PHASE ENZYME ELECTRODES - KINETICS AND ANALYTICAL APPLICATIONS

The suitability of organic media for enzymatic reactions has led to tr emendous advancement in biosynthesis and, more recently, biosensing. O rganic phase enzyme-based biosensors (OPEE), i.e. biosensors that oper ate 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 polar ity and degree of hydration. Classical enzymology predicts that hydrop hobic 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 a nd 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 biosensor s can be developed to exhibit excellent reactivities in both hydrophil ic and hydrophobic organic solvents by controlling the degree of hydra tion of the biosensing environment. Amperometric biosensors containing tyrosinase, glucose oxidase (GOx), horseradish peroxidase (HRP), and more recently chemically modified HRP, have been constructed and succe ssfully applied to assays in organic solvents. Several enzyme immobili zation matrices have been used in our laboratory for the fabrication o f sensors. Poly(estersulphonic acid)-entrapped tyrosinase and HRP elec trodes have been found to be very effective biosensors in a variety of organic solvents for determining phenols, organic peroxides and pesti cide compounds. The Os-polymers, [Os(bpy)(2)(PVP)(n)Cl]Cl [bpy = 2,2'- bipyridyl, PVP = poly(4-vinylpyridine); n = 10, 20 or 25] and [Os(byp) (2)(PVI)(10)Cl]Cl {PVI = poly(4-vinylimidazole)} have been used with b ifunctional cross-linking agents, such as glutaraldehyde or polyethyle ne glycol, to form reagentless biosensors with GOx, tyrosinase and HRP (native and modified forms of HRP). These biosensors, based on the el ectrostatic complexation of the enzyme and the positively charged Os-p olymer, have been found to be very stable in organic phase. GOx sensor s that employ soluble mediators, such as, ferrocenemonocarboxylic acid , exhibited better catalytic efficiency, k(cat)'/K-M' in the organic p hase than in the aqueous phase. In our studies we have demonstrated th at the reactivities of amperometric organic phase biosensors follow th e Michaelis-Menten kinetic paradigm. However, kinetic analyses have sh own that the values of the sensor biocatalytic parameters, including I -max, k(cat)' and K-M' depend on solvent media. Under certain conditio ns, k(cat)'/K-M' is the second order rate constant for the electrode-e nzyme reaction and is related to the activation Gibbs energy through D elta G(not equal) = -RT In[k(cat)'/K-M')h/k(B)T], where T, h, and k(B) are the absolute temperature, Planck's constant and Boltzmann's const ant, respectively. Thus, k(cat)'/K-M' is a measure of the activation o f the biosensor redox-catalytic reaction, in different solvents. This parameter has been evaluated at different temperatures in both aqueous and organic media. The Delta G(not equal) values (the difference in G ibbs energy between the 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 of the electrocatalytic reaction. Organic phase H RP biosensors have also been applied in our laboratory for the determi nation of thiourea, ethylenethiourea and other thio-compounds (which a re the parent compounds for some pesticides). The thio-compounds act a s inhibitors. Their determination is based on the change in the electr ocatalytic current of peroxide reduction by the biosensor, which accom panies the addition of the organic sulphides. Tyrosinase electrodes ha ve also been applied as detectors in reversed-phase HPLC for the detec tion of the eight phenols that are normally found in cigarette filters . Native HRP and bis-succinimide-modified-HRP electrodes have also bee n applied in the amperometric determination of organic peroxides, the aim being to produce a more sensitive, reproducible and stable organic phase HRP electrode. (C) 1996 Elsevier Science Limited