Electrochemical Glucose and Lactate Sensors Based on “Wired” Thermostable Soybean Peroxidase Operating Continuously and Stably at 37 °C

Abstract

Following a recent report from our laboratory on a thermostable amperometric H₂O₂ sensor based on “wiring” soybean peroxidase, glucose and lactate sensors maintaining stable output under continuous operation at 37 °C for 12 and 8 days, respectively, were built. The vitreous carbon base of the sensor was coated with four polymer layers. The first was made by cross-linking thermostable soybean peroxidase and the redox polymer formed through complexing part of the rings of poly(vinylpyridine) with [Os(bpy)₂Cl]^+/2+ (bpy = bipyridine) and quaternizing part of the rings with bromoethylamine. The second was an insulating and H₂O₂ transport controlling cellulose acetate layer. The third was an immobilized glucose oxidase or lactate oxidase layer. The fourth was a substrate transport controlling cellulose acetate layer In the case of the glucose sensor, the current output was independent of potential between −0.2 and +0.3 V (vs SCE), and the response time (t_10/90)was 2 partial pressure above 15 Torr. The sensor was relatively insensitive to motion and to interferants. Changing the rotation speed of the electrode from 50 to 2500 rpm increased the current by <10%. At a glucose concentration of 4 mM, the addition of 0.1 mM ascorbate decreased the current by <1%. The operational stability was glucose oxidase loading dependent. Though the current decreased by 85% after 100 h of operation at 37 °C when the 3-mm-diameter electrode was loaded with only 1.3 μg of glucose oxidase, it decreased by <1% after such operation when loaded with 52 μg of the enzyme. Similar results were obtained for the lactate sensor, with the exception of a more noticeable oxygen concentration dependence of the lactate response at low oxygen concentrations.