DIRECT ELECTRON-TRANSFER BIOELECTRONIC INTERFACES - APPLICATION TO CLINICAL ANALYSIS

Bioelectronic interfaces based on direct electron transfer to proteins and enzymes immobilised at functional electrode surfaces are currentl y under development and the potential of two such systems for applicat ion to clinical measurement will be outlined. The first is the detecti on of free radical production via direct electrochemistry of cytochrom e c immobilised covalently at modified gold electrodes. The redox prot ein cytochrome c has been immobilised covalently to gold electrodes su rface-modified with N-acetyl cysteine via carbodiimide condensation. T he electrodes thus produced were used to measure directly the enzymati c and cellular production of the superoxide anion radical (O-2(-)) The superoxide radical reduced the immobilised cytochrome c which was imm ediately re-oxidised by the surface-modified gold electrode poised at a potential of +25 mV (vs Ag/AgCl). The electron transfer rate constan t (k(et)) of this process was 3.4 +/- 1.2 s(-1). The rate of current g eneration was directly proportional to the rate of O-2(-) production. The essentially reagentless system produced was designed to be applied ultimately to continuous monitoring of free radical activity in vivo since there is evidence that oxygen-derived free radical species act a s mediators which cause and perpetuate inflammation in disease states, including rheumatoid arthritis and neurodegenerative disorders. The s econd systems are pseudo-homogeneous immunoassays based on direct elec tron transfer to horseradish peroxidase. Horseradish peroxidase enzyme electrodes based on activated carbon (HRP-ACE) have been constructed by simple passive adsorption. Direct electron transfer between the ele ctrodes and HRP resulted in the electroenzymatic reduction of hydrogen peroxide at potentials lower than +480 mV (vs Ag/ AgCl) and a current sensitivity for peroxide of 637 nA mu M(-1) cm(-2) at +50 mV (vs Ag/A gCl). A linear response to hydrogen peroxide measurement was obtained over the range 0.2-150 mu M. Kinetic analysis of the HRP-ACE system ga ve a heterogeneous rate constant (k'(ME)) of 5.3 x 10(-3) cm s(-1) and an enzyme turnover number (k'(cat,E)) of 2.8 x 10(-2) cm s(-1). The H RP-ACE electrode showed excellent storage stability in phosphate buffe red saline (pH 7.4) at 4 degrees C, with a calculated half-life of 235 days. The potential for electrodes, based on the HRP/ACE interaction, to be applied to clinical measurement in model no wash competitive an d non-competitive ('pseudo-homogeneous') immunoassays is described.