Methodology/Principal Findings: FCS has the advantage of allowing us to determine the number of fluorescent molecules.
FCS measures the fluctuations in fluorescence intensity caused by fluorescent probe movement in a small light cavity with a
defined volume generated by confocal illumination. We thus developed a highly sensitive determination system of H2O2 by
FCS, where horseradish peroxidase (HRP) catalyzes the formation of a covalent bond between fluorescent molecules and
proteins in the presence of H2O2. Our developed system gave a linear calibration curve for H2O2 in the range of 28 to
300 nM with the detection limit of 8 nM. In addition, by coupling with glucose oxidase (GOD)-catalyzed reaction, the
method allows to measure glucose in the range of 80 nM to 1.5 mM with detection limit of 24 nM. The method was
applicable to the assay of glucose in blood plasma. The mean concentration of glucose in normal human blood plasma was
determined to be 4.9 mM.