This paper describes a Raman study of enzyme reactions that use potassium ferricyanide as a reaction mediator. The enzyme reaction systems investigated consisted of glucose oxidase (GOD), glucose, and potassium ferricyanide, and of lactate oxidase (LOD), lactate, and potassium ferricyanide. Raman spectra were measured for the above enzyme systems every 2 s to monitor the progress of the enzyme reactions at real time. The observed Raman spectra showed only three peaks at 2186, 2095, and 2022 cm-1 due to C N stretching modes of potassium ferricyanide (2186 cm-1) and potassium ferrocyanide (2095 and 2022 cm-1); with the progress of the enzyme reactions, the band at 2186 cm-1 decreases while those at 2095 and 2022 cm-1 increase. From the differentiation of the Raman intensity at 2022 cm-1, we could calculate the velocity (V') of the intensity change that directly reflects the rate (V) of the enzyme reactions. By plotting the velocity thus obtained (V') vs. the concentration of the enzyme substrate ([S]), we were able to develop a calibration curve to predict the concentration of the enzyme substrate. For GOD, the correlation coefficient (R) and the detection limit of this calibration curve were 0.99 and 20 mg/dL, respectively. This detection limit was better than that obtained from conventional glucose sensors. For LOD, we were able to determine Michaelis constant (Km) from the maximum velocity (Vmax). The method proposed here is applicable to various kinds of enzymes that use potassium ferricyanide as a reaction mediator.
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