Abstract
Numerous optodes, with fluorophores as the chemical sensing element and optical fibres for light delivery and collection, have been fabricated for minimally invasive endoscopic measurements of key physiological parameters such as pH. These flexible miniaturised optodes have typically attempted to maximize signal-to-noise through the application of high concentrations of fluorophores. We show that high-density attachment of carboxyfluorescein onto silica microspheres, the sensing elements, results in fluorescence energy transfer, manifesting as reduced fluorescence intensity and lifetime in addition to spectral changes. We demonstrate that the change in fluorescence intensity of carboxyfluorescein with pH in this “high-density” regime is opposite to that normally observed, with complex variations in fluorescent lifetime across the emission spectra of coupled fluorophores. Improved understanding of such highly loaded sensor beads is important because it leads to large increases in photostability and will aid the development of compact fibre probes, suitable for clinical applications. The time-resolved spectral measurement techniques presented here can be further applied to similar studies of other optodes.
Highlights
The time-resolved spectrometer was based on a 256 × 1 pixels complementary metal–oxide–semiconductor (CMOS) single-photon avalanche diode (SPAD) line sensor which allows fast histogramming of arriving photons with time-correlation single photon counting (TCSPC)
The TCSPC capable CMOS SPAD line sensor detects single photons and generates histograms according to their arrival time for 256 pixels simultaneously, each correlated to a different wavelength
The need for miniaturised optodes introduces a requirement for high fluorophore density due to the small practical volume
Summary
Sensing of physiological parameters such as pH [1,2,3,4,5,6,7,8], oxygen [2,3,8,9,10,11], glucose [12,13,14], and lactate [15] using fluorescence spectroscopy offers a sensitive technique that has the potential for clinical diagnosis [16,17]. The approaches reported include fibre intrinsic interferometers [5], a variety of responsive coatings [2,3,6], and nano or microparticles attached onto the end of the optical fibre [4,8,19,20] In the latter cases, these sensors often require a high loading density to provide sufficient signal and sensitivities, which we show here may lead to interesting quenching dynamics [21,22,23]. We demonstrated a new architecture for fibre-based in situ pH and oxygen ratiometric sensing [8], with the sensors based on amino-modified silica microspheres covalently conjugated to fluorophores placed into pits etched on the ends of multi-core optical fibres Using this approach, we reported pH and oxygen sensing in an ex vivo lung perfusion model with an accuracy of 0.02 pH units and 0.6 mg/L dissolved oxygen sensitivity [8]. Optical fibrefluorophore sensors in which limited space is likely to lead to high fluorophore loading regimes
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