Antibody–aptamer functionalized fibre‐optic biosensor for specific detection of Listeria monocytogenes from food

Aim: To develop antibody–aptamer functionalized fibre‐optic biosensor for specific detection of Listeria monocytogenes from food products. Methods and Results: Aptamer, a single‐stranded oligonucleotide ligand that displays affinity for the target molecule, was used in the assay to provide sensor specificity. Aptamer‐A8, specific for internalin A, an invasin protein of L. monocytogenes, was used in the fibre‐optic sensor together with antibody in a sandwich format for detection of L. monocytogenes from food. Biotinylated polyclonal anti‐Listeria antibody, P66, was immobilized on streptavidin‐coated optical waveguide surface for capturing bacteria, and Alexa Fluor 647‐conjugated A8 was used as a reporter. The biosensor was able to selectively detect pathogenic Listeria in pure culture and in mixture with other bacteria at a concentration of approx. 10³ CFU ml^?1. This sensor also successfully detected L. monocytogenes cells from artificially contaminated (initial inoculation of 10² CFU 25 g^?1) ready‐to‐eat meat products such as sliced beef, chicken and turkey after 18 h of enrichment. Conclusion: Based on the data presented in this study, the antibody–aptamer functionalized fibre‐optic biosensor could be used as a detection tool for sensitive and specific detection of L. monocytogenes from foods. Significance and Impact of the Study: The study demonstrates feasibility and novel application of aptamer on fibre‐optic biosensor platform for the sensitive detection of L. monocytogenes from food products.     

A fluorescent coagulation assay for thrombin using a fibre optic evanescent wave sensor

A fibre optic evanescent wave sensor is used for the rapid detection of thrombin. Coagulation of solution phase fluorescently labelled fibrinogen to unlabelled fibrinogen bound to the surface of the fibre optic is observed in real time by the evanescent wave sensor. Thrombin concentrations down to 0.01 NIHml(-1) are detectable within 5 min. The potential application of this technique for rapid amplified immunosensing is discussed.     

Development of an evanescent-field fibre optic sensor for Escherichia coli O157:H7.

An intensity-modulated fibre optic sensor was developed for Escherichia coli O157:H7. The interaction between the whole natural bacteria and the guided lightwave was carried out by means of evanescent-field coupling. A correlation between optical response and the current number of bacteria was achieved. The device sensitivity had been calibrated for initial number of bacteria (N(0)) from 10-800. The sensor sensitivity was 0.016 (+/-0.001) dB/h/N(0). The sensing mechanism starts together with the log phase leading the present sensor response to be five to ten times faster than conventional bacteriological techniques.     

A Novel System for Spectral Analysis of Solar Radiation within a Mixed Beech-Spruce Stand

Abstract: A multi-sensor system is described, based on a 1024 channel diode array spectrometer, to measure spectral radiant flux density in the range of 380 nm to 850 nm, with a resolution of 0.8 nm in minimal 16 milliseconds integration time per sensor (noon, clear sky conditions). 264 space-integrating 4π sensors deployed in the canopies and 2 m above stand floor are sequentially connected to the spectrometer by means of 30-m long fibre optics. During low-level conditions (dawn, overcast sky) the system automatically lengthens the integration time of the spectrometer. About 3 sec per sensor, i.e., 13 min for the total of 264 sensors (worst case) are needed to collect spectral energy data, store them on hard disk and move the channel multiplexer to the next fibre optic position. The detection limit of quartz fibre sensors is 0.2 W/m²; precision and absolute error of radiant flux density are smaller than 3 % and 10 %, respectively.
The system, operating since 1999, is derived from a 20-sensor pilot system developed for PAR measurements (PMMA fibre sensor, 400nm to 700 nm).
Data achieved with the system serve to determine vertical profiles of wavelength dependent radiation extinction, with special respect to R/FR ratios and to develop a model of spectral radiation distribution in a mature forest stand, prerequisites for the computation of carbon gain of the stand and the evaluation of stand growth models.     

A composite fibre optic catheter for monitoring peristaltic transit of an intra‐luminal bead

A fibre optic motion sensor has been developed for monitoring the proximity and direction of motion of a ferrous bead travelling axial to the sensor. By integrating an array of these sensors into our previously developed fibre optic manometry catheters we demonstrate simultaneous detection of peristaltic muscular activity and the associated motion of ferrous beads through a colonic lumen. This allows the motion of solid content to be temporally and spatially related to pressure variations generated by peristaltic contractions without resorting to videoflouroscopy to track the motion of a radio opaque bolus. The composite catheter has been tested in an in‐vitro animal preparation consisting of excised sections of rabbit colon.

Cut‐away image of the fibre optic motion sensor showing the location of the fibre Bragg gratings and the rare earth magnet.     

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation^1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct^2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting^5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical ''gold standard'' of ICP measurement in humans⁸. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%⁹. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters¹⁰ and solid state catheters are the most frequently used^{11,12,13,14,15}. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies¹³ that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.     

Morphology and distribution of Müller cells in the retina of the toad Bufo marinus

We have previously shown that an antibody against neuron-specific enolase (NSE) selectively labels Müller cells (MCs) in the anuran retina (Wilhelm et al. 1992). In the present study the light- and electron-microscopic morphology of MCs and their distribution were described in the retina of the toad, Bufo marinus, using the above antibody. The somata of MCs were located in the proximal part of the inner nuclear layer and were interconnected with each other by their processes. The MCs were uniformly distributed across the retina with an average density of 1500 cells/mm². Processes of MCs encircled the somata of photoreceptor cells isolating them from each other by glial sheath, except for those of the double cones. Some of the photoreceptor pedicles remained free of glial sheath. Electron-microscopic observations confirmed that MC processes provide an extensive scaffolding across the neural retina. At the outer border of the ganglion cell layer these processes formed a non-continuous sheath. The MC processes traversed through the ganglion cell layer and spread beneath it between the neuronal somata and the underlying optic axons. These processes formed a continuous inner limiting membrane separating the optic fibre layer from the vitreous tissue. Neither astrocytic nor oligodendrocytic elements were found in the optic fibre layer. The significance of the uniform MC distribution and the functional implications of the observed pattern of MC scaffolding are discussed.     

Dual wavelength optical fibre sensor for ph measurement

A rapid response, inexpensive, fibre optic pH sensor which uses a two-wavelength time division multiplexed system to measure the change in absorption of an indicator dye and provide a reference channel for other losses in the light path has been constructed and described, for use in solution, with solid state LED sources and detectors being employed.     

Antibody immobilisation on fibre optic TIRF sensors

A study of antibody immobilisation techniques on quartz and fibre optic surfaces for immunosensors has been carried out. Methods of covalent antibody immobilisation which have not previously been applied to optical fibres were investigated, and compared with classical methods found in the literature. Preliminary experiments on covalent immobilisation methods on planar quartz surfaces were conducted to enable us to choose the most suitable protein immobilisation technique for sensor applications. The immobilisation studies were directed in particular towards obtaining a high density of binding sites for the analyte of interest. Two of the most promising methods, antibody immobilisation on surfaces coated with dextran based hydrogel and F(ab')-SH fragments bound to silanised glass, which resulted in surface densities of active sites of above 0.45 pmol/cm2, were selected for further experiments on a fibre optic total internal reflection fluorescence immunosensor and gave satisfactory responses to changes in analyte concentrations of the order of 10(-8) M. The efficiency of polar organic solvents, such as dimethylsulfoxide, in dissociating the antigen-antibody complex and hence to regenerate the immunosensor surface was also evaluated.     

Intramuscular pressures during exercise: an evaluation of a fiber optic transducer-tipped catheter system

The efficacy of a modified fibre optic transducer-tipped catheter system for measuring intramuscular pressures during exercise was determined. A microcapillary infusion technique using a catheter was employed as the standard of comparison due to its established dynamic properties. Pressures were measured in the tibialis anterior muscle of six healthy adults at rest before exercise, during isometric and concentric exercise, and at rest after exercise. The fibre optic system measured contraction pressures equal to the microcapillary infusion technique during all phases of the exercise protocols but recorded a lower relaxation pressure during isometric exercise and a lower rest pressure following 20 min of concentric exercise. Negative relaxation pressures were recorded by the fibre optic system for two subjects during continuous concentric exercise. It is hypothesized that a piston effect, due to the sliding of muscle fibres at the catheter tip following a contraction, rendered falsely low pressures during relaxation and that this artefact was reflected in the subsequent rest pressure following exercise. The larger volume (157 mm3) and area (3.49 mm2) of the fibre optic catheter in the muscle made it more prone to this effect than the conventional catheter (39 mm3 and 0.87 mm2, respectively). The fibre optic system may be preferred when recording the muscle contraction pressures during complex limb movements but should not be used when assessing the relaxation pressures or the pressure at rest following exercise.     

Fine structure of the first optic ganglion (lamina) of the cockroach, Periplaneta americana

The structural organization of the first optic ganglion (lamina) of the cockroach (Periplaneta americana) was investigated by the use of light and electron microscopy. Each compound eye of the cockroach is composed of up to 2000 visual units (ommatidia) of the fused rhabdom type. The ommatidia themselves consist of eight receptor cells which terminate as axons in either the first or second optic ganglion. Three different short visual fibre types end in two separate strata in the lamina, and one long fibre type ends in the second optic ganglion. Monopolar second-order neurons with wide field branching patterns in the middle stratum of the first synaptic region have postsynaptic contacts with short visual fibres. Horizontal fibre elements with branching patterns at different levels of the lamina apparently form three horizontal plexuses with presynaptic and/or postsynaptic connections to first- and secondorder neurons. The lack of well-organized fibre cartridges containing a constant number of first and second order neurons in each fascicle and the presence of only unistratified wide field monopolar cells could represent, as compared to other insect orders, a primitive stage in the development of the first optic ganglion.     

Simultaneous Recording of Input and Output of Lateral Geniculate Neurones

TO understand the way in which the cat dorsal lateral geniculate nucleus (LGN) processes visual information it would be useful to know the number and type of retinal inputs to individual LGN neurones. Using electrical stimulation of the optic nerve Bishop et al.¹concluded that an impulse in a single optic nerve fibre is sufficient to excite a single LGN neurone. From the appearance of excitatory postsynaptic potentials (EPSPs) recorded essentially intracellularly, Creutzfeldt suggested that LGN neurones are driven by perhaps one² or a few³ retinal ganglion cells. Hubel and Wiesel⁴ proposed models of convergence of several retinal inputs on single LGN neurones based on analyses of receptive fields. Guillery⁵ produced anatomical evidence that some types of LGN neurones receive inputs from several different retinal fibres. Now we report direct observations which were made by recording simultaneously from single LGN neurones and from individual retinal ganglion cells which provided excitatory input to them. We shall not consider inhibitory influences, which are currently under study.     

The astrocytes in the retina and optic nerve head of mammals: A special glia for the ganglion cell axons

Summary The neuroglia in the retina and the intraocular portion of the optic nerve of the monkey and cat has been examined by light and electron microscopy. In the retina two types of macroglial cells can be distinguished: 1) Müller cells, and 2) astrocytes. The bipolar radial glial cells of Müller penetrate the entire thickness of the retina and their basal processes align in the nerve fibre layer to form septa that fasciculate the axons of the ganglion cells. In contrast to the Müller cells, the retinal astrocytes are not homogeneously distributed throughout the retina; their number correlates with the thickness of the nerve fibre layer. The processes of the astrocytes are confined to the ganglion cell layer and to the nerve fibre layer. In the latter, the astrocytic processes run parallel to and between the axons of a given nerve fibre bundle. According to cytological criteria, the retinal astrocytes are protoplasmic. In the intraocular portion of the optic nerve, however, the astrocytes are fibrous and their processes run perpendicular to the axon bundles of the prelaminar portion of the optic nerve. Thus, because of their intimate morphological relationship to axons of the nerve fibre layer and the intraocular portion of the optic nerve, the astrocytes in the eye of the monkey and the cat may be considered as a special glia for the axons of ganglion cells.     

The laminar organization of optic nerve fibres in the tectum of goldfish.

Potentials in the tectum of large (12--20 cm) goldfish, evoked by stimulation of the optic nerve, were recorded extracellularly with double-barrelled electrodes (d.c., saline and a.c., Woods metal--Pt). Four fibre groups (E, M1, M2, M3) were recorded at latencies of approximately 2, 3, 5 and 8 ms after stimulation (conduction velocities of approximately 7, 5, 3 and 2 m/s). The same four groups were recorded from the optic nerve when the tectum was stimulated. The fastest fibre groups (E) did not give rise to a postsynaptic wave. Fibre groups M1, M2 and M3 gave rise to postsynaptic potentials which, following computation of their second spatial derivatives with depth, were found to have current sinks at depths of approximately 100-150 micrometers, 150--200 micrometers and 250--350 micrometers respectively. Thus the fastest conducting retinotectal fibres make their synapses most superficially, the opposite of the arrangement in the frog tectum. These postsynaptic waves fatigued at repetitive stimulus rates of 20--50 per second, and in twin pulses at interstimulus intervals of 10--15 ms; and they were reversibly blocked by topical application of pentobarbitol. The fibre potentials, however, were virtually undecremented under these conditions. To compare these electrophysiological findings with the anatomy, the cobalt procedure was used to visualize the profiles of the optic fibres in the various tectal laminae. A thick dense projection filled the superficial grey and white (s.g.w.) layer, and there was a thin satellite band just superficial to it. In addition, there were two deeper bands of sparse innervation, in the middle of the central grey zone (c.g.) and in the deep white (d.w.) layer. These bands were associated with the field potential sinks through lesions made with recording electrodes. The two deep bands correspond to the M3 fibre group. The dense s.g.w. innervation contains both the M1 and M2 fibre groups, the M1 just superficial to the M2. The fastest fibre group, E, which had no postsynaptic wave associated with it, persisted at least six weeks after retinal removal, and probably represents efferent cells with fibres projecting back through the optic nerve to the retina. Filled cell profiles could not be positively identified with the cobalt technique, but could be seen with the HRP technique, when the optic afferents were first allowed to degenerate. The filled cells were the pyramidals of the s.g.w. layer.     

Development of LSPR-Based Optical Fiber Dopamine Sensor Using L-Tyrosine-Capped Silver Nanoparticles and Its Nonlinear Optical Properties

Facile synthesis of L-tyrosine-capped silver nanoparticles (Tyr-AgNPs) was carried out, and its linear and nonlinear optical properties were investigated. Further, the sensing properties of Tyr-AgNPs toward dopamine were explored. Tyr-AgNPs exhibit a decrease in fluorescence intensity while a linear increase in absorption spectrum against increase in dopamine (DA) concentration (0–50 μM) at room temperature. Tyr-AgNPs are used as the sensing material for the fabrication of fiber optic dopamine sensor. Sensitivity, selectivity, and limit of detection of the sensor are evaluated. This proposed fiber optic sensor may offer sensitive and low-cost strategy for DA detection.

     

Wortmannin Blocks Goldfish Retinal Phosphatidylinositol 3-Kinase and Neurite Outgrowth

The goldfish retina has been used extensively for the study of nerve regeneration. A role for phosphatidylinositol 3-kinase (PI3K) in neurite outgrowth from goldfish retinal explants has been examined by means of wortmannin (WT), a selective inhibitor of the enzyme. The presence of PI3K in retinal extracts was determined by means of immunoprecipitation as well as by an in vitro assay system for catalytic activity. The relative amount of the p85 subunit of PI3K detected by western blot in the retina following optic nerve crush was unchanged. WT inhibited goldfish brain PI3K activity at concentrations as low as 10^–9 M, approximating that reported for inhibition of mammalian PI3K's. Daily addition of 10^–8 M WT to retinal explants, activated by prior crush of the optic nerve, significantly inhibited neurite outgrowth during a 7 day in vitro culture period, while a single addition of WT to freshly explanted retina had no effect on neurite outgrowth. These results suggest that a PI3K-mediated process may be critical for nerve regrowth.     

Axonal transport of RNA during regeneration of the optic nerves of goldfish

The distribution of radioactive RNA and RNA precursors in the goldfish optic tecta following intraocular injection of ³H-uridine has been studied during various stages of optic nerve regeneration. ³H-uridine was injected into the posterior chamber of the right eye 17, 30, or 60 days after both optic nerves were crushed. Fish were sacrificed at time intervals ranging from 0.5 to 21 days after injection. One day prior to sacrificing, ¹⁴C-proline was also injected into the right eye as a marker of fast axonal protein transport. Seventeen to 23 days after crushing, the approximate time of nerve reconnection, the amount of radioactive RNA appearing in the left optic tectum was increased by more than ten times control values. Approximately 30 days after crushing the nerve, when the reconnected nerve is maturing, RNA values were still elevated, but significantly decreased from the earlier stage. By 60 days after crushing the optic nerve, the amounts of RNA in the left tectum was close to normal. Evidence suggesting that, at least, some of the radioactive RNA in the tectum originated from RNA transported along optic axons rather than from RNA synthesized locally in the tectum was provided by autoradiographic experiments. Autoradiograms of paraffin sections taken from the goldfish optic tecta after the intraocular injection of ³H-uridine showed a distribution of grains in a linear pattern, suggesting a distribution over the incoming fibers during the reconnection stage of regeneration. Electron microscopic autoradiography of glutaraldehyde fixed epoxy sections confirmed that a significant number of grains (shown to be ³H-RNA) were, in fact, over regenerating optic axons. Intracranial injection of ³H-uridine, during the same stage of regeneration, on the other hand, resulted in a distribution of grains, specifically over cell perikarya. These experiments suggest that during the reconnection phase of nerve regeneration, large amounts of RNA may be carried within regenerating optic axons as they enter the optic tectum.     

Immunocytochemical demonstration ofγ-amino butyric acid and glutamic acid decarboxylase in R7 photoreceptors and C2 centrifugal fibres in the blowfly visual system

Summary Neurons within the compound eye of the flyCalliphora erythrocephala, suspected of containing gamma-aminobutyric acid were revealed immunocytochemically, using antibodies directed against gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD). The GABA content within putative GABAergic neurons was increased by high affinity uptake of GABA and selective blocking of GABA metabolism with Gabaculine. Only neuronal populations which were labelled with the GABA as well as the GAD antibodies were presumed to be GABAergic. The first optic neuropil (lamina) exhibited two distinct GA-BAergic fibre populations amongst a larger population comprised of fourteen cell classes. One fibre population was formed by the axons of the photopic photoreceptors R7 which pass through the lamina and terminate in the second optic neuropil (the medulla). The identity of R7 was established from longitudinal and transverse sections of the retina where R7 can be unequivocally distinguished from the six scotopic photoreceptors R1-6 and the other photopic receptor, R8.The other fibre population matched the profiles in the lamina of terminals of efferent C2 neurons. These neurons project distally from beneath the medulla out to the lamina ganglionaris where each retinotopic unit (cartridge) contains a characteristic hook-like terminal arbor distally. We propose from these data that the photoreceptors R7 and the efferent C2 neurons use GABA as a neurotransmitter.     

Protein phosphorylation: Localization in regenerating optic axons

A number of axonal proteins display changes in phosphorylation during goldfish optic nerve regeneration (Larrivee and Grafstein, 1989). (1) To determine whether the phosphorylation of these proteins was closely linked to their synthesis in the retinal ganglion cell body, cycloheximide was injected intraocularly into goldfish whose optic nerves had been regenerating for 3 weeks. Cycloheximide reduced the incorporation of [³H]proline and³²P orthophosphate into total nerve protein by 84% and 46%, respectively. Of the 20 individual proteins examined, 17 contained less than 15% of the [³H]proline label measured in corresponding controls, whereas 18 proteins contained 50% or more of the³²P label, suggesting that phosphorylation was largely independent of synthesis. (2) To deterine whether the proteins were phosphorylated in the ganglion cell axons, axonal transport of proteins was blocked by intraocular injection of vincristine. Vincristine reduced [³H]proline labeling of total protein by 88% and³²P labeling by 49%. Among the individual proteins [³H]proline labeling was reduced by 90% or more in 18 cases but³²P labeling was reduced only by 50% or less. (3) When³²P was injected into the cranial cavity near the ends of the optic axons, all of the phosphoproteins were labeled more intensely in the optic tract than in the optic nerve. These results suggest that most of the major phosphoproteins that undergo changes in phosphorylation in the course of regeneration are phosphorylated in the optic axons.Abbreviations SDS sodium lauryl sulfate - GAP growth associated protein - TCA trichloracetic acid - kD kilodalton     

Crack Detection in Fibre Reinforced Plastic Structures Using Embedded Fibre Bragg Grating Sensors: Theory,Model Development and Experimental Validation

In a fibre-reinforced polymer (FRP) structure designed using the emerging damage tolerance and structural health monitoring philosophy, sensors and models that describe crack propagation will enable a structure to operate despite the presence of damage by fully exploiting the material’s mechanical properties. When applying this concept to different structures, sensor systems and damage types, a combination of damage mechanics, monitoring technology, and modelling is required. The primary objective of this article is to demonstrate such a combination. This article is divided in three main topics: the damage mechanism (delamination of FRP), the structural health monitoring technology (fibre Bragg gratings to detect delamination), and the finite element method model of the structure that incorporates these concepts into a final and integrated damage-monitoring concept. A novel method for assessing a crack growth/damage event in fibre-reinforced polymer or structural adhesive-bonded structures using embedded fibre Bragg grating (FBG) sensors is presented by combining conventional measured parameters, such as wavelength shift, with parameters associated with measurement errors, typically ignored by the end-user. Conjointly, a novel model for sensor output prediction (virtual sensor) was developed using this FBG sensor crack monitoring concept and implemented in a finite element method code. The monitoring method was demonstrated and validated using glass fibre double cantilever beam specimens instrumented with an array of FBG sensors embedded in the material and tested using an experimental fracture procedure. The digital image correlation technique was used to validate the model prediction by correlating the specific sensor response caused by the crack with the developed model.