Photostable and Proteolysis-Resistant Forster Resonance Energy Transfer-Based Calcium Biosensor

Molecular sensors from protein engineering offer new methods to sensitively bind to and detect target analytes for a wide range of applications. For example, these sensors can be integrated into probes for implantation, and then yield new and valuable physiological information. Here, a new Forster resonance energy transfer (FRET)based sensor is integrated with an optical fiber to yield a device measuring free Ca2+. This membrane encapsulated optical fiber (MEOF) device is composed of a sensor matrix that fills poly(tetrafluoroethylene) (PTFE) with an engineered troponin C (TnC) protein fused to a pair of FRET fluorophores. The FRET efficiency is modulated upon Ca2+ ion binding. The probe further comprises a second, size-excluding filter membrane that is synthesized by filling the pores of a PTFE matrix with a poly(ethylene glycol) dimethacrylate (PEGDMA) hydrogel; this design ensures protection from circulating proteases and the foreign body response. The two membranes are stacked and placed on a thin, silica optical fiber for optical excitation and detection. Results show the biosensor responds to changes in Ca2+ concentration within minutes with a sensitivity ranging from 0.01 to 10 mM Ca2+, allowing discrimination of hyper- and hypocalcemia. Furthermore, the system reversibly binds Ca2+ to allow continuous monitoring. This work paves the way for the use of engineered structure-switching proteins for continuous optical monitoring in a large number of applications.