Amplifying fluorescent polymers developed in the Swager research group have shown a remarkable ability to act as sensitive and specific sensors for various analytes.  This proposal seeks to harness this sensing ability of amplifying fluorescent polymers for the development of new assays for protease detection.  To do so, the synthesis of an amplifying mixed co-polymer with some percentage of macrocyclic units is proposed.  These macrocycles will encapsulate squaraine dyes in close proximity to the polymer backbone, and will contain oligopeptide linkers that can be cleaved by a target protease.  In the absence of the target protease, the dye will be held in the macrocycle and efficient Dexter-type energy transfer will occur from the polymer backbone to the squaraine dye.  Excitation of the polymer will result in selective amplified emission from the squaraine dye.  Protease-mediated cleavage of the macrocycle will release the squaraine dye, leading to a quenching of the Dexter energy transfer between the polymer and the dye.  The use of Dexter-type energy transfer will increase the sensitivity of the system, as the emission of the squaraine dye will occur on a completely dark background, without residual polymer fluorescence.  The efficiency of energy transfer will be monitored using fluorescence spectroscopy to detect the emission of the squaraine dye, which occurs in the near-infrared spectral region.  Fluorescence spectroscopy will further be used to measure the rate at which the target protease cleaves the macrocycle, as this will result in a quenching of the Dexter energy transfer between the polymer and a dye and a silencing of the dye's emission.  PUBLIC HEALTH RELEVANCE This proposal describes the synthesis of a new class of amplifying fluorescent polymers that can be utilized for ultrasensitive protease detection.  Protease activity has been implicated in a variety of diseases, including cancer;the detection of proteases in vivo is therefore critical both for the diagnosis of multiple diseases and for the development of new disease treatments.  The proposed macrocyclic polymer can easily be adapted to detect virtually any target protease, as well as other biologically relevant targets.