Detection of H2O2 by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a technique in which measurement of fluorescence intensity fluctuations is used to clarify dynamic molecular interactions within a very small space in a solution containing a small number of fluorescent molecules. The FCS-based analysis gives the average number and average diffusion time of the fluorescent molecules during their passage through a very small space. One advantage of FCS is that physical separation between free and bound fluorescent probes is not required because the properties of fluorescence fluctuations are accounted for. Therefore, when fluorescent probes are bound with proteins by peroxidase and hydrogen peroxide (H2O2), FCS enables us to detect H2O2 with high sensitivity. In addition, because H2O2 is generated by oxidase-catalyzed reactions, a highly sensitive method for detecting H2O2 is applicable to the measurement of low levels of various oxidases and their substrates, such as glucose. We here describe the protocol of a de novo, highly sensitive method for the measurement of H2O2 and glucose using FCS.