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

Anal Chem. 2020 Jun 2;92(11):7683-7689. doi: 10.1021/acs.analchem.0c00573. Epub 2020 May 13.

Abstract

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 Förster 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Batrachoidiformes / metabolism
  • Calcium / analysis*
  • Calcium / metabolism
  • Fluorescence Resonance Energy Transfer*
  • Photochemical Processes
  • Proteolysis
  • Troponin C / chemistry
  • Troponin C / metabolism*

Substances

  • Troponin C
  • Calcium