Long-Wavelength Fluorescence Resonance Energy Transfer (FRET)-Based Biosensor For Glucose Sensing: Development and Interference Testing
Rapid, accurate, and non- or minimally-invasive sensors for glucose measurement have the potential to enhance diabetes control. Recent studies have indicated that implantable optical, affinity biosensors based on Förster Resonance Energy Transfer (FRET) can provide high sensitivity in quantifying glucose concentrations, in continuous and non-destructive fashion. However, there are gaps in the research related to best practices for characterizing the device and factors that alter its performance. First, a standard set of in vitro approaches for evaluating FRET glucose biosensor response has not been established. Second, the potential for chemical interference due to sugars and medications is not well established and standardized methods for quantifying such effects have not been developed. Third, information on the influence of tissue optics on signal detection in these devices is lacking. The general goals of this research project were two-fold: to elucidate the performance and working mechanism of FRET glucose biosensors and identify best practices for assessing performance. Towards these goals, a battery of performance test methods was developed, including spectral response, linearity, sensitivity, limit of detection, kinetic response, reversibility, stability, precision, and accuracy, including error grid analysis. A FRET glucose biosensor was then fabricated and the test methods implemented to fully characterize its response in vitro. Biochemical and optical interference were assessed through a bench-top fluorescence spectroscopy system and phantom measurements, respectively. The biosensor demonstrated a glucose response change of 45%, bias of less than 11%, and a limit of detection of 25 mg/dL. Mannose, maltose, fructose, lactose, and sucrose showed positive results for interference, with concentration estimates over-predicted by up to 64% due to 50 mg/dL concentrations of other sugars. The phantom measurements suggested that non-specific interactions between light and tissue-like samples tended to affect the overall detected signal, with minimal variations in spectral distribution. This was likely due to the choice of fluorophores with long-visible-wavelength emission peaks, where absorption due to hemoglobin is relatively small. The overall results provide evidence of the strengths and weaknesses of the performance of FRET glucose biosensor as well as insight into best practices for thorough objective, quantitative test methods for response evaluation of optical glucose biosensors and factors that influence performance.
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