Novel bienzyme lactate-sensing electrode was fabricated by physical inclusion of lactate oxidase(LOx), horseradish peroxidase (HRP), and methylene blue (MB) into the polyelectrolytes-complex layer consisting of double-stranded DNA and poly(allylamine) (PAA) prepared on a glassy carbon (GC) electrode. A hydrogen peroxide (H2O2) produced during the LOx reaction at outer LOx/PAA layer was cathodically detected by MB-mediated bioelectrocatalytic reduction by HRP at inner DNA/MB/HRP/PAA layer. Steady-state cathodic current responses to L-lactate obtained at the applied potential of −0.25 V (Ag/AgCl) increased linearly up to 150 M, and the response time (100% response) was less than 15 sec. The sensitivity (slope of the linear calibration region) was 1.51 A/mM, and lower detection limit was found to be 10 M (S/N = 3). Relative standard deviation (RSD) of seven successive measurements of 20 M L-lactate was 2.4%. The steady-state response for 100 M L-lactate was scarcely influenced by subsequent addition of 100 M L-ascorbic acid (AA). The layered structure and/or ion network structure of DNA/PAA membrane may prohibit the reaction of AA to oxidized MB and HRP leading to electrocatalytic interference. This lactate biosensor retained ca. 45% of original activity (sensitivity) after one week storage in phosphate buffer (0.1 M, pH 7.0) at 4 °C.
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