Shabbir B. Bambot

Sensing oxygen through skin using a red diode laser and fluorescence lifetimes

The most difficult impediments to transcutaneous optical sensing are the absorbance and scatter of light caused by skin and the lack of fluorescent sensing probes which can be excited at wavelengths over 600 nm. Furthermore, current optical sensing techniques rely on absorbance or fluorescence intensity measurements, both of which are sensitive to drifts in lamp intensity, changes in probe concentration and inner filter effects. We demonstrate oxygen sensing through a layer of skin by using red light which readily penetrates skin as diffusely scattered light. The oxygen sensitive osmium-ligand complex used in this study can be excited at 635-680 nm. In addition, we measure fluorescence lifetimes, which are inherently unaffected by factors that limit absorbance and fluorescence intensity measurements. By using phase fluorimetry and long lived fluorophores, we are able to demonstrate the potential for subdermal oxygen sensing with simple and inexpensive instrumentation. This work describes a paradigm for future non-invasive measurements of other analytes.

Potential applications of lifetime-based, phase-modulation fluorimetry in bioprocess and clinical monitoring

The measurement of analyte concentration is a critical part of successful bioreactor and clinical monitoring. Although strategies exist for measuring the majority of relevant analytes, industrial on-line bioreactor control is carried out primarily by measurement and control of pH, pO2 and, in some cases, cell density. This is because the available technology cannot be easily and inexpensively adapted to (a) measure the analyte in an aseptic manner and/or allow for remote sensing, and (b) measure in real time so that on-line control is possible. Similar issues need to be addressed for biosensors for clinical applications. A rapidly emerging technology that has the potential of meeting these challenges is liftime-based phase-modulation fluorimetry, an optical technique that uses the measurement of fluorescence lifetime rather than intensity for determining the concentration of an analyte.

Lifetime-based optical sensing of pH using resonance energy transfer in sol-gel films

Abstract We describe the fabrication and testing of an optical pH sensor based on fluorescence lifetime measurements and sol-gel technology. These sensors are potentially suitable for use with simple laser-diode light sources. The sensor is based on the phenomenon of fluorescence resonance energy transfer (FRET) from a pH-insentive donor to a pH-sensitive acceptor. The pH-dependent increase in the bromothymol blue (BTB) acceptor absorbance results in increased energy transfer, reducing the lifetime of the Texas Red Hydrazide (TRH) donor. The lifetimes are measured by the phase and modulation of the emission, relative to the modulated incident light, and are found to be insentive to the total signal level and fluctuations in light intensity. However, the present sensors are sensitive to salt concentration and/or ionic strength. Importantly, this sol-gel sensor is not fragile: it provides stable readings for days and can be repeatedly autoclaved without loss of sensitivity to pH. The use of FRET as the pH transduction mechanism can be reliably extended to longer wavelenghts, and allows the future use of laser-diode excitation sources. Such simple and robust sensors have numerous potential applications in analytical and clinical chemistry.

Optical Oxygen Sensor Using Fluorescence Lifetime Measurement

The industry standard for oxygen measurement, the modified Clark electrode has now been available for more than three decades (Clark, L. C., 1956), and is still being constantly modified and perfected. It is a membrane covered electrode that encloses a platinum cathode, a silver anode and a KCl or Ag/AgCl electrolyte. Oxygen diffusing from the surrounding medium through the membrane gets reduced at the surface of the cathode. The magnitude of the negative bias on the platinum electrode (-0.8 to -1 V) is maintained such that the electrode operates under conditions of membrane controlled diffusion. Since the diffusive flux is a function of the partial pressure of oxygen in the fluid, it is possible to calibrate the electrode current versus oxygen tension. Clark electrodes are calibrated by equilibrating a sample with nitrogen to read zero and followed with either air or oxygen to read 100%. The readings are usually expressed as a percentage of air saturation.

Lifetime-based optical sensing of pH using resonance energy transfer in sol-gel sensors

We describe the fabrication and testing of an optical pH sensor based on fluorescence lifetime measurements and sol-gel technology. The sensor is based on the phenomenon of fluorescence resonance energy transfer (FRET), from a pH-insensitive donor to a pH-sensitive acceptor. The pH-dependent increase in the bromothymol blue acceptor absorbance results in increased energy transfer, reducing the lifetime of the Texas red hydrazide donor. The lifetimes were measured by the phase and modulation of the emission, relative to the modulated incident light, and were found to be insensitive to the total signal level and fluctuations in light intensity. However, the present sensors are sensitive to salt concentration and/or ionic strength. Importantly, this sol-gel sensor is not fragile, providing stable readings for days and can be repeatedly autoclaved without loss of sensitivity to pH. The use of FRET as the pH transduction mechanism can be reliably extended to longer wavelengths, and allows the future use of laser diode excitation sources.