Fluorescent Biosensor For Hyaluronidase: Intensity Based Ratiometric Sensing And Time-Gated Detection Using A Long Lifetime Azadioxatriangulenium (Adota) Fluorophore

We have designed a rapid and sensitive, intensity based ratiometric sensing and lifetime based sensing probe for the detection of hyaluronidase activity. Hyaluronidase has been reported to have upregulated in various pathological conditions like cancer. Therefore, developing an assay to estimate the enzyme concentration is of utmost importance and can be used as a diagnostic method for cancer. We have developed a fluorescent probe by using hyaluronic acid as template and heavily labeling it with an orange/red emitting azadioxatriangulenium (ADOTA) fluorophore, which also has a long fluorescence lifetime. The heavily ADOTA labeled hyaluronic acid (HA-ADOTA) has a red shift in the peak emission wavelength (605 nm), a weak fluorescence signal and a short fluorescence lifetime due to efficient self-quenching. In the presence of enzyme hyaluronidase, the brightness and fluorescence lifetime of the sample increases with a shift in the peak emission to its original wavelength at 560 nm. The ratio of the fluorescence intensity of the HA - ADOTA probe at 560 nm and 605 nm can be used as the sensing signal for detecting hyaluronidase. Long fluorescence lifetime and red emission also makes it suitable for time-gated lifetime imaging. The proposed method makes it a rapid and sensitive assay, making it useful to assay hyaluronidase in relevant clinical samples.