Highly selective microbiosensors for in vivo measurement of glucose, lactate and glutamate

An alternative approach to production of amperometric microbiosensors, which combines electrochemical electrometallization and electropolymerisation of phenylene diamine film with covalent binding enzymes, is presented. In this respect, for a sensitive detection of hydrogen peroxide (HP) at +0.4 V versus Ag/AgCl (detection limit of 0.5 μM, s/n = 3), carbon fiber microelectrodes (30 μm in diameter and 500 μm long) were covered with ruthenium. To obtain a highly selective detection of HP, in the presence of different interfering compounds (ascorbic acid, uric acid, etc.), an additive semi-permeable polymer film was formed on the top of the ruthenium layer by electropolymerisation of m-phenylene diamine (m-PD). The enzymatic selective layers were formed by covalent cross-linking the enzymes (glucose oxidase, lactate oxidase or glutamate oxidase) with BSA by glutaraldehyde in the presence of ascorbate oxidase. An additional polymeric layer based on polyurethane and Nafion was deposited on the top of the enzymatic membrane (glucose oxidase, lactate oxidase, or glutamate oxidase) in order to extend the dynamic range of biosensors up to 4 mM for glucose (R = 0.997; Y[nA] = −0.22 + 9.68x[glucose, mM]), 1.75 mM for lactate (R = 0.991; Y[nA] = 0.43 + 15.36x[lactate, mM]) and 0.25 mM for glutamate (R = 0.999; Y[nA] = 0.02 + 29.14x[glutamate, mM]). The developed microbiosensors exhibited also negligible influences from interfering compounds at their physiological concentrations. Microbiosensors remained stable during 10 h in a flow injection system at 36 °C and pH 7.4. The microbiosensors developed are now used in vivo and, as an example, we report here the data obtained with the glucose biosensor.