Pentacyanoferrate-bound poly(1-vinylimidazole) (PVI[Fe(CN)5]) was selected as a mediator for amperometric creatinine determination based on the reductive H2O2 detection. Creatinine amidohydrolase (CNH), creatine amidohydrolase (CRH), sarcosine oxidase (SOD), peroxidase (POD), and PVI[Fe(CN)5] were crosslinked with poly(ethylene glycol) diglycidyl ether (PEGDGE) on a glassy carbon (GC) electrode for a creatinine biosensor fabrication. Reduction current was monitored at -0.1V in the presence of creatinine and O2. It is revealed that PVI[Fe(CN)5] is suitable as a mediator for a bioelectrocatalytic reaction of POD, since PVI[Fe(CN)5] neither reacts with reactants nor works as an electron acceptor of SOD. The amounts of PVI[Fe(CN)5], PEGDGE, and enzymes were optimized toward creatinine detection. Nafion as a protecting film successfully prevented the enzyme layer from interferences. The detection limit and linear range in creatinine determination were 12μM and 12-500μM (R(2)=0.993), respectively, and the sensitivity was 11mAcm(-2)M(-1), which is applicable for urine creatinine tests. The results of the creatinine determination for four urine samples measured with this proposed method were compared with Jaffe method, and a good correlation was obtained between the results.

A sensitive d-amino acid oxidase (DAAO)/peroxidase (POD) bienzyme biosensor is constructed, in which pentacyanoferrate-bound poly(1-vinylimidazole) polymer (PVI[Fe(CN)5]) is selected as a mediator. Reductive current of PVI[Fe(CN)5] related to the H2O2 concentration generated in the DAAO reaction was measured at -0.1V vs. Ag|AgCl with DAAO/POD/PVI[Fe(CN)5]-modified electrode. The result revealed that PVI[Fe(CN)5] is suitable as a mediator for this bienzyme system due to its appropriate formal potential and its extremely low reactivity against DAAO. The stability of DAAO was improved by adding free flavin adenine dinucleotide and the electrode composition was optimized for the detection of d-alanine. Nafion and ascorbate oxidase-immobilized films worked successfully to prevent severe interference from uric acid and ascorbic acid. The low detection limits of d-alanine (2μM) and d-serine (2μM) imply its possibility for the determination of extremely low concentration of d-amino acids in physiological fluids. The proposed bienzyme biosensor is proved to be capable of detecting d-amino acids in urine.

H 2 O 2-generating oxidase/peroxidase (POD)-based mediated biosensors are very useful to minimize interference, but require suitable mediators which work well only for POD but not against the oxidase. Pentacyanoferrate-bound poly(1-vinylimidazole) (PVI[Fe(CN) 5 ]), PVI[Os(dcbbpy) 2 Cl] (dcbbpy = 4,4'-dicarboxy-2,2'-bipyridine) and PVI[Os(dmebpy) 2 Cl] (dmebpy = 4,4'-dimethyl-2,2'-bipyridine) have been utilized to investigate the interaction with four kinds of H 2 O 2-generating oxidases: glucose oxidase, sarcosine oxidase, choline oxidase (ChOD) and lactate oxidase. The mediated bioelectrocatalytic activities of the redox polymers for the enzymes have been determined by cyclic voltammetry in the presence of the substrates. The highly negatively charged PVI[Fe(CN) 5 ] shows practically no mediating activity against the four flavoenzymes, but strong one to POD. On the other hand, PVI[Os(dmebpy) 2 Cl] with neutral ligands shows a high activity for the oxidases except ChOD. The mediating activity of PVI[Os(dcbbpy) 2 Cl] with negatively charged ligands is much smaller than that of PVI[Os(dmebpy) 2 Cl]. These results reveal that electrostatic repulsion and steric hindrance are enhanced by using negatively charged polymers to realize minimum activity against the oxidases.

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