Frequency-domain fluorescence lifetime optrode system design and instrumentation without a concurrent reference light-emitting diode

Abstract

We report the design, development, and implementation of an improved instrumentation approach for frequency-domain fluorescence lifetime (FDFL) optrodic sensing without a concurrent reference LED. FDFL traditionally uses a reference LED, at approximately the same wavelength as the sensor fluorophore emission, to measure phase shifts associated with changes in the fluorescence lifetime of fluorophore. For this work we used an oxygen optrode to design, develop, and test the reference-LED-free FDFL approach. Electronics and optics were optimized, and key system parameters, such as inherent system phase shifts, were determined to insure best performance. In our tests with the oxygen optrode, we observed that several key performance characteristics were improved by the implementation of the reference-LED-free instrumentation platform. This system can potentially be adapted to other analyte-selective fluorophores, which will enable scientists and researchers to expand the application of optrodic sensors as basic research tools in biology, medicine, and agriculture.

Light-directed functionalization methods for high-resolution optical fiber based biosensors

“…Microtip growth was performed using a three component mixture containing, (1) a free radical polymerizable monomer base, (2) a UV responsive free radical photoinitiator, and (3) oxygen sensitive dye platinum tetratkis (1,5,10,15,20 pentafluorophenyl) porphyrin (PtTFPP) from Frontier Scientific Inc.…”

“…Differential fluorescence intensity, differential fluorescence lifetime and response times were calculated in each instance [5,10,11]. Optimized microtip sensors were tested against known oxygen concentrations in gas phase by inserting the sensors into a custom-built chamber that was flooded with appropriate mixes of oxygen and nitrogen.…”

“…Physical adsorption, covalent attachment using custom linkers, or entrapment in protective membranes via dip coating, drop coating, direct writing [2,4,5], or are typically used to functionalize the distal end of the fiber. However, these methods are not spatially directed and hence not compatible with miniaturization.…”

“…Previous work in the Physiological Sensing Facility has shown that the use of frequency domain fluorescence lifetime based optrode schemes allows enhanced oxygen detection in both direct and self-referencing mode [2,5]. In these schemes, a protocol based on dip-coating was used to immobilize the oxygen sensitive dye to the end of laser pulled multimode optical fibers and tip size was limited by the size of polymer bead formed upon drying.…”

Non-invasive measurement of real-time oxygen flux in plant systems with a self-referencing optrode

“…However, the electrochemical microelectrode approach still suffered from experimental artifacts when high sensitivity and accuracy of measurement are required because the electrode 3 sensitivity to electromagnetic noise, fouling, and calibration drift. Therefore, an optical sensor, the socalled optrode (optical electrode), was developed to minimize these disadvantages 9,10,11,12 .…”