Date of Award
Doctor of Philosophy (PhD)
Diabetics are often required to self-monitor blood glucose levels to effectively deliver prescribed therapies. However, the pain and bother associated with traditional finger-prick measurements often result in decreased patient compliance and therefore poor disease management, which could result in the early-onset of complications. Enzymatic "smart tattoos"—implantable luminescent particles that may be transdermally interrogated with light—are being pursued as minimally-invasive diabetic monitoring devices, with hopes of increasing diabetic compliance by reducing excessive pain and bother associated with finger-prick measurements. These devices typically comprise an oxygen-quenched luminescent dye and glucose oxidase (GOx), an enzyme that catalyzes the oxidation of β-D-glucose. Under glucose-limited reaction conditions, local glucose concentrations can be extracted from oxygen-dependent emission spectra or luminescence lifetimes.
Previously, enzymatic smart tattoos comprising enzyme-doped alginate hydrogel microsphere sensors and ruthenium complexes as oxygen indicators were reported. In this dissertation, however, the integration of a more sensitive metalloporphyrin oxygen indicator, Pt(II) Octaethylporphine (PtOEP), and the reference probe, Rhodamine B Isothiocyanate (RITC), into enzyme-doped alginate-modified silica ("algilica") particles is presented. A particularly important feature of these sensors is the shift from traditional ruthenium-based oxygen indicators to metalloporphyrin complexes, due in part to higher excitation wavelengths which reduce the effects of tissue scatter and absorption, increased photostability, and higher oxygen sensitivity. Using the novel algilica matrix and diffusion-limiting nanofilms, glucose sensitivities of two orders of magnitude greater than ruthenium-based enzymatic smart tattoos were achieved with porphyrin oxygen indicators. Of central importance was the demonstration that surface adsorbed polyelectrolyte nanofilms allowed glucose sensitivity and range to be controlled by modulating substrate flux into the sensor, resulting in sensitivities (change in intensity ratio) of 1–5 Wing dL-1 and upper range limits of 90–250 mg/dL. Remarkably, it was shown that nanofilms only 12 nanometers thick could significantly affect response behavior, confirming theoretical predictions based on models of reaction-diffusion kinetics.
To approach clinical utility, implantable smart tattoos must maintain appropriate function for at least 6 months. Therefore, to examine the effects of long-term operation on sensor function, a mathematical model was developed and the output validated with experimental results. Both theoretical and experimental results demonstrated limited device lifetime (∼ 90% loss of sensitivity over 24 hours) due to enzyme inactivation resulting from hydrogen peroxide, a byproduct of glucose oxidation. To improve longterm stability, a first-generation bi-enzymatic smart tattoo prototype was constructed via the co-incorporation of catalase, an enzyme that consumes hydrogen peroxide, which enhanced response stability two fold over time. Furthermore, to design clinically viable implantation schemes, it is important to understand how individual sensors within a population contribute to overall response properties. Thus, an imaging technique was developed to perform real-time ratiometric imaging of individual sensor function. The results indicated significant differences in sensor behavior depending on location within the sensor population and/or physical parameters, as expected. These findings demonstrate the feasibility of engineering highly sensitive enzymatic-based glucose sensors and lay the groundwork for developments of additional enzymatic analyte sensors.
Stein, Erich Wayne, "" (2006). Dissertation. 544.