A novel probe for multiplexed detection of intracellular proteases based on fret and fluorescence polarization

Currentprobes for proteases detection are susceptible tophotobleachingandinefficientin cell penetration. Also it is not applicable for multiplexed detection. Anovel probe based on quantum dot (QD), nanogold, substrate peptide and EGF is studied as the sensor for intracellular proteases detection. Due to the less fluorescence overlapping and the excellent propertiesagainst photobleaching of QDs, the strong photo quenching capacities of nanogolds, and the high transfer efficiency of EGF, this probe can be employedforlong time multiplexed detection, which was difficult for conventional probes. A comprehensive analysis on how to effectively design a fluorescent probe based on substrate peptide containing cleavage sites was first presented. Furthermore, the detection was achieved based on fluorescence polarization. The sensitivity was improved based on the concentration change of the fluorescent molecules, which has not been effectively utilized before.