The development of highly specific and sensitive implantable sensors for (i) chronic in vivo monitoring of analyte and drug levels in the intensive care unit, (ii) determining glucose levels within the diabetic patient population, or (iii) detecting toxins and harmful agents on the battlefield has been an intensive research area for several years. While there are several other indications which call for chronic monitoring via an implantable sensors, it is the total economic impact of Diabetes in the United States of greater than $20 Billion annually that makes glucose the analyte of interest in studies of implantable sensor design. Owing to the exquisite sensitivity and specificity of fluorescence spectroscopy, implantable sensors based upon fluorescence decay kinetics have evolved using protein/fluorophore chemistry combinations that are highly specific to glucose. Yet to date, there has been little progress in the development of a quantitative fluorescence sensor involving (i) fluorophores excitable in the near-infrared wavelength regime for maximal penetration into tissue and (ii) measurement approaches that would be unaffected by normal physiological changes in tissue optical properties. Furthermore, the fluorescence decay kinetics of a conjugated fluorophore tethered on the backbone of a biocompatible polymer are not well understood, especially in the case where fluorescence resonance energy transfer (FRET) in which the distance between separately tethered donor and acceptor (D-A) fluorophores are specifically influenced by the presence of an agent or analyte of interest. In this research application, we propose to expand frequency-domain photon migration (FDPM) techniques developed in our laboratory for fluorescence lifetime sensing in the presence of tissue-like scattering specific for the detection of glucose using an immobilized FRET D-A pair conjugated on a tethered glucose-binding protein and a competitive binder.
Our specific aims are to (1) Demonstrate sensitivity to glucose (0-30 mM) of the freely diffusing Cy5(tm) concavalinA donor with a sugar-laden dextrans conjugated to the acceptor Malachite Green within tissue-like scattering and non-scattering buffers using time-domain, FDPM, and conventional phase modulation spectroscopy; (2) Immobilize the conjugated D-A and evaluate the FRET characteristics of D-A donor pair distribution, p(r), within precursor and crosslinked polyethylene glycol (PEG); (3) Develop optimization routines to evaluate p(r), as a function of glucose concentration from spectroscopic measurements; (4) Demonstrate quantitative glucose sensing in the PEG system in response to physiologic concentrations of glucose with and without scattering; and (5) Perform feasibility trials within acute and chronic preparations of the normal and BB/Wor diabetic rat models.
| Liang, Feng; Pan, Tianshu; Sevick-Muraca, Eva M (2005) Measurements of FRET in a glucose-sensitive affinity system with frequency-domain lifetime spectroscopy. Photochem Photobiol 81:1386-94 |
