A fluorescent biosensor for the detection and imaging of a cancer biomarker

Purpose: Cancer is among the leading cause of death worldwide with approximately 8.2 million deaths. Cancer mortality can be reduced if it can be detected and treated at early stages. Certain biomarkers like hyaluronidase are reported in the literature for the screening and detection of cancer. It is an endoglycosidase that degrades hyaluronic acid (HA) and this enzyme is overexpressed in various cancers. Therefore, it is of great interest to develop a simple, sensitive and fast technique with which one can estimate the activity/level of hyaluronidase. We have developed a fluorescent biosensor for the detection of hyaluronidase activity/level. This probe was developed by labeling hyaluronic acid with a long lifetime fluorophore. Monitoring the cleavage of hyaluronic acid by measuring the changes in the fluorescent properties of the biosensor will be a simple and precise tool reflecting hyaluronidase activity and can be used for the detection of cancer. Methods: This fluorescent biosensor was developed by heavy labeling of hyaluronic acid with an orange/red emitting azadioxatriangulenium (ADOTA) fluorophore. ADOTA in water emits at 560 nm with a long fluorescence lifetime of ~ 20 ns. The hyaluronidase activity was measured as a function of the change in the steady-state fluorescence intensity and fluorescence lifetime of the biosensor. Fluorescence lifetime imaging microscopy (FLIM) was also used to image hyaluronidase activity in cancer cells. Results: 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. Recovery in the fluorescence lifetime was used for fluorescence lifetime-based imaging of cancer. Conclusions: Our results show the ability of this the HA-ADOTA probe to detect activity/level of hyaluronidase in biological samples. Due to long fluorescence lifetime of the dye, it can be also be used to remove the background in cellular images. In future, this novel technology can be used to design a small device which can be used in primary care settings for the screening of cancer.