Flow-amperometric biosensor for respiratory toxins using myoglobin-adsorbed carbon-felt, based on an inhibitory effect on bioelectrocatalytic reduction of oxygen

Novel, simple and highly sensitive reagentless amperometric flow-biosensor for respiratory toxins (i.e. azide and cyanide) has been developed by using myoglobin (Mb)-adsorbed carbon-felt (CF), based on the inhibitory event of azide and cyanide on the Mb-catalyzed O2 reduction. The Mb-adsorbed CF (Mb–CF) showed a sufficient bioelectrocatalytic activity for O2 reduction in the potential region from 0 to −0.4V vs. Ag/AgCl at pH 5.0, due to the direct electron transfer (DET) between Mb-heme and the CF electrode. The heterogeneous electron transfer rate constant (ks) of the electrochemical redox reaction of Mb-heme–Fe(II)/(III) was estimated to be 15.5s−1. This Mb–CF-catalyzed O2 reduction was reversibly inhibited by azide and cyanide, which bind to sixth coordination position of heme-iron center of Mb. When air-saturated 0.1M phosphate/citrate buffer (pH 5.0) was used as a carrier under the applied potential of −0.2V vs. Ag/AgCl, the steady-state current due to the Mb-catalyzed O2 reduction was reversibly inhibited by the injection (200μl) of azide and cyanide, resulting in peak-shape current responses. The magnitude of the inhibition peak currents linearly increased with increasing concentrations of azide (up to 3μM) and cyanide (up to 5μM), and the detection limit of azide and cyanide were found to be 0.12 and 0.23μM, respectively (S/N=2). The apparent inhibition constant, K′i, of azide and cyanide were estimated to be 5.71 and 8.95μM, respectively.