Figure 1.
Scheme of the enzymatic reaction.
This scheme shows binding between tyramide labeled with tetramethyl rhodamine (TMR) and proteins by peroxidase and H2O2. Because the H2O2 concentrations correspond to those of the proteins labeled with tyramide-TMR, we can determine the concentrations of H2O2 by FCS. Further, because H2O2 is produced by the reaction between glucose and glucose oxidase, we can determine the concentrations of H2O2 and thus deduce the concentrations of glucose.
Figure 2.
Determination of H2O2 concentration.
A. FCS curve proves that our FCS measurements are successful for the detection of 0.2 µM H2O2. B. Calibration curve by an FCS method. F2 indicates the existence ratio for tyramide-TMR-BSA. The SD values are too small to set the error bars in this figure. For example, when the H2O2 concentration was 0.008 µM, the average value of F2 was 1.2% and the SD value was 1.0%. When the H2O2 concentration was 0.2 µM, the average value of F2 was 29.1% and the SD value was 2.8%. C. Calibration curve by the Amplex™ Red method. The measurements by FCS and Amplex™ Red were performed 3 times each.
Figure 3.
Determination of glucose concentration.
A. Calibration curve by an FCS method. F2 indicates the existence ratio for tyramide-TMR-BSA. The SD values are too small to set the error bars in this figure. For example, when the glucose concentration was 1.0 µM, the average value of F2 was 41.8% and the SD value was 1.3%. When the glucose concentration was 1.5 µM, the average value of F2 was 61.4% and the SD value was 2.4%. B. Calibration curve by the Amplex™ Red method. The measurements by FCS and Amplex™ Red were performed 3 times each.
Figure 4.
Determination of glucose concentration in human blood plasma.
The measurements by FCS were performed 3 times. The F2 values obtained here were calibrated by Figure 3A, and then the glucose concentrations were calculated by taking account of dilution.