Recent advances in miniaturization and analyte-sensitive fluorescent indicators make optical fiber biosensors promising alternatives to microelectrodes. Optical sensing offers several advantages over electrochemical methods including increased stability and better spatial control to monitor physiological processes at cellular resolutions. The distal end of an optical fiber can be functionalized with different fluorophore/polymer combinations through mechanical, dip-coating or photopolymerization techniques. Unlike mechanical and dip-coating schemes, photopolymerization can spatially confine the sensing layer in the vicinity of light in a more reproducible and controllable manner. The objective of this study was to fabricate microscale fluorescence lifetime based optrodes using UV-induced photopolymerization. Six commercially available acrylate based monomers were investigated for stable entrapment of the oxygen sensitive porphyrin dye (PtTFPP) dye via photopolymerization at the end of optical fibers. Of these, the acrylate-functionalized alkoxysilane monomer, 3-methacryloxypropyl-trimethoxysilane (tradename Dynasylan MEMO) showed maximal response to changes in oxygen concentration. Dye-doped polymer microtips were grown at the ends 50 μm optical fibers and sensitivity and response time were optimized by varying both the concentration of doped dye and the excitation power used for polymerization. The resulting sensors showed linear response within the physiologically relevant range of oxygen concentrations and fast response times. While applied here to oxygen sensing, the photopolymer formulation and process parameters described are compatible with a wide range of available organic dyes and can be used to pattern arrays of spots, needles or more complex shapes at high spatial resolution.
Optically transduced sensors (optrodes, or optodes) offer significant advantages over polarographic techniques for
measuring oxygen. In biology and medicine, how we make measurements is very important, and this is especially true in
terms of physiological exchange. Cellular and tissue oxygenation is a function of background concentration and
respiratory demand, and in pure physical terms this is best expressed in terms of molecular flux based on Fick's law.
Measuring dynamic flux from biological systems requires sensing technology that can measure activity in multiple
dimensions. Here we report the development of a self-referencing oxygen optrode (SRO) for reliably making noninvasive
measurements of oxygen flux from a variety of biological systems. The self-referencing microsensor technique
was adapted to operate optrodic oxygen sensors through the integration of optical sensing instrumentation with software-controlled
data acquisition and micro-stepping motion control. This allows the sensor to scan biologically active
gradients of oxygen flux directly, as it relates to cellular and tissue respiratory activity. The technique was validated first
using artificially generated oxygen gradients, which are theoretically modelled and compare with measured signals.
Subsequently, the SRO was applied in basic research applications to non-invasively measure molecular oxygen flux
from a variety of animal and plant systems.
A mammalian cell-based optical biosensor was built to detect pathogenic <i>Listeria</i> and <i>Bacillus</i> species. This sensor measures the ability of the pathogens to infect and induce cytotoxicity on hybrid lymphocyte cell line (Ped-2E9) resulting in the release of alkaline phosphatase (ALP) that can be detected optically using a portable spectrophotometer. The Ped-2E9 cells were encapsulated in collagen gel matrices and grown in 48-well plates or in specially designed filtration tube units. Toxin preparations or bacterial cells were introduced and ALP release was assayed after 3-5 h. Pathogenic <i>L. monocytogenes </i> strains or the listeriolysin toxins preparation showed cytotoxicity ranging from 55% - 92%. Toxin preparations (~20 μg/ml) from <i>B. cereus </i>strains showed 24 - 98% cytotoxicity. In contrast, a non-pathogenic <i>L. innocua </i>(F4247) and a <i>B. substilis </i>induced only 2% and 8% cytotoxicity, respectively. This cell-based detection device demonstrates its ability to detect the presence of pathogenic <i>Listeria</i> and <i>Bacillus</i> species and can potentially be used onsite for food safety or in biosecurity application.
Biomolecules encapsulated in porous silicate glass using the sol-gel process form optically transparent materials capable of biorecognition. We are working to design biosensors from these materials for the detection of glutamate, the major excitatory neurotransmitter in the central nervous system. Previously we demonstrated the ability of glutamate dehydrogenase (GDH)-doped sol-gel bulk materials to measure glutamate at varying concentrations. Here we show that GDH can be encapsulated in a thin film while retaining its enzymatic activity. The films are likely to be reaction limited rather than diffusion limited, as the reaction rate at saturating glutamate concentrations varies linearly with enzyme loading. At a given enzyme loading, the film reaction rate increases with increasing glutamate concentration, demonstrating its potential as a glutamate sensor material. In addition we have shown that the enzyme-doped sol-gel glass can be deposited onto the tip of an optical fiber. The fiber is active and responds to the presence of glutamate.
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Smart Biomedical and Physiological Sensor Technology V