The early stage development studies of novel implantable continuous metabolite sensor systems for glucose, lactate and fatty acids are discussed. These sensors utilize non-enzymatic "reagentless" sensor systems based on NIR fluorophore-labeled binding proteins. For in vivo applications, NIR fluorescence based systems (beyond 600 nm) have the added benefit of reduced interference from background scattering, tissue and serum absorption and cell auto-fluorescence. The long wavelength emission facilitates implanted sensor disks to transmit fluorescence to an external reader through wireless connections and the resulting fluorescence signals can be correlated to metabolite concentrations. We have developed a prototype optical system that uses a bifurcated optical fiber to transmit excitation and read emission at the surface of the skin. With this system, fluorescence signals were read over time through animal skin. The changes in glucose concentration were studied using immobilized sensor proteins and were compared to non-immobilized sensors in solution. For sensors in solution, no response delay was observed. For immobilized systems, the fluorescence response showed a delay corresponding to the diffusion time for the metabolite to equilibrate within the sensor.
The design and synthesis of an environmentally sensitive long wavelength fluorescing squaraine dye for conjugation to proteins is dsecribed. Environmentally sensitive dyes are valuable for probing environmental changes that occur when labeled proteins bind their corresponding ligands and can be used to construct flyorescent sensors. Long wavelength (>650 nm) dyes would enable through-skin wireless sensing with minimum interference from the background. While several environmentally sensitive dyes are known in the visible spectrum, only a few are available in the long wavelength region, and none of them are available with reactive groups suitable for protein conjugation. Several derivatives of squarain dyes are known to be environmentally sensitive and fluorescent in the long wavelength region, but none of them are available with linkers for protein conjugation. In order to achieve this goal, we developed a synthetic scheme to introduce a reactive linker onto an anilinic squaraine that is highly sensitive to its environment. The synthesis involves the preparation of the dye with an iodoacetyl ester linker that readily reacts with a thiol on a cysteine residue of the binding protein. The squaraine dye was conjugated to known binding proteins that were evaluated as optical sensors. Ultimately, we expect these systems to measure analytes in the body and transmit information through the skin to an external monitor.