Chalcogenide Glass Optical Waveguides for Infrared Biosensing

Due to the remarkable properties of chalcogenide (Chg) glasses, Chg optical waveguides should play a significant role in the development of optical biosensors. This paper describes the fabrication and properties of chalcogenide fibres and planar waveguides. Using optical fibre transparent in the mid-infrared spectral range we have developed a biosensor that can collect information on whole metabolism alterations, rapidly and in situ. Thanks to this sensor it is possible to collect infrared spectra by remote spectroscopy, by simple contact with the sample. In this way, we tried to determine spectral modifications due, on the one hand, to cerebral metabolism alterations caused by a transient focal ischemia in the rat brain and, in the other hand, starvation in the mouse liver. We also applied a microdialysis method, a well known technique for in vivo brain metabolism studies, as reference. In the field of integrated microsensors, reactive ion etching was used to pattern rib waveguides between 2 and 300 μm wide. This technique was used to fabricate Y optical junctions for optical interconnections on chalcogenide amorphous films, which can potentially increase the sensitivity and stability of an optical micro-sensor. The first tests were also carried out to functionalise the Chg planar waveguides with the aim of using them as (bio)sensors

Spectroscopie infrarouge déportée : mise au point d'un biocapteur pour l'imagerie métabolique et la sécurité microbiologique

Pr. P. VIGNY, CNRS Orléans, Présidentdu jury. Pr. M. MANFAIT, Université de Reims Champagne-Ardenne, Rapporteur. Dr. A. DESBOIS, Directeur de recherche, CEA /Saclay, Rapporteur. Dr. J.L. ADAM,Directeur de recherche, Université de Rennes 1. Pr. O. SIRE, Université de Bretagne-Sud. Dr. B. BUREAU, Université de Rennes 1.An optical chalcogenide fibre biosensor operating in the Mid-Infrared (MIR) has been developed. It enables to collect infra-Red (IR) spectra by remote spectroscopy thanks to the fibre. The principle of measurement is based on the general concept of Fibre Evanescent Wave spectroscopy. To improve the detection of such a sensor, a tapered fibre is necessary. The transportation section diameter is 450 µm, and 100 µm in the sensing zone, it is this part of the fibre which will be brought into contact with the sample to analyse. Metabolic states of a biological tissue and fluid have been characterised. Samples, mouse livers and serums, are put into contact with the fibre and MIR spectra are collected. The aim is to identify two different metabolic states (pathogenic one and normal) of a same sample. Spectral differences have been identified and assigned to metabolic alterations of glucids, lipids and/or proteins. This differences consist in an increase of the absorption bands intensity and/or shift of characteristic bands. In any case, all spectral results are in agreement with histologic staining and dosages of the sample. The second topics concerns the non-invasive and in situ monitoring of the dynamics of bacterial biofilm. The model used is an uropathogenic micro-organism, called Proteus mirabilis, which exhibits a strong swarming abilities. This bacteria has developed a complex multi-cellular behaviour correlated in space and in time, which permits itself to colonise new surfaces. The change from a vegetative to a differentiated state (pathogenic one) is accompanied by important biochemical modifications in the bacteria membrane. MIR spectra, collected during the P. mirabilis. biofilm development, allow to detect in real time a surface contamination thanks to the fibre, but also biochemical modifications of the bacteria membrane constituents. All spectra have been analysed by Principal Component Analysis (PCA) method to classify spectra by metabolic state but also to determine the spatial cartography of P. mirabilis phenotype.Un biocapteur à base de fibre optique en verre de chalcogénures, transparente dans un large domaine du moyen infrarouge (MIR), a été développé. Il permet l'enregistrement de spectres MIR par spectroscopie déportée. Le principe de la mesure est basé sur le concept de la spectroscopie par ondes évanescentes. Pour améliorer la sensibilité, les fibres utilisées sont effilées sur quelques centimètres et ont un profil en diamètre de 450-100-450 µm. La zone effilée sera mise en contact avec l'échantillon à analyser. Différents états métaboliques (physiologique ou pathologique) dans le foie et le sérum ont été analysés. Les coupes de foies et les sérums ont été mis directement en contact avec la partie senseur de la fibre, et des spectres MIR ont ainsi été collectés. L'objectif de ces études est d'identifier deux états métaboliques différents (sain et pathologique) d'un même échantillon. Des modifications spectrales peuvent être mises en évidence et reliées à une ou des altération(s) du métabolisme des lipides, glucides et/ou protides. Ces modifications peuvent être des différences d'intensité et/ou des décalages en nombre d'onde de bandes d'absorption. Tous les résultats sont corrélés avec les dosages sanguins et les colorations histologiques des échantillons analysés. Le développement d'un biofilm bactérien a été suivi in situ et en temps réel. Proteus mirabilis est un micro-organisme pathogène, opportuniste des voies urinaires, qui a développé un comportement multi-cellulaires complexe, corrélé dans l'espace et le temps, ce qui lui permet de coloniser de nouvelles surfaces. Durant le processus de différenciation, le changement de l'état végétatif à celui migrant (pathogène) est accompagné par des modifications des constituants membranaires. Les spectres MIR, enregistrés pendant le développement d'un biofilm à P. mirabilis, permettent de détecter en temps réel une contamination de surface, mais aussi les modifications biochimiques des constituants de la membrane bactérienne. Tous les spectres ont été traités par Analyse en Composantes Principales (ACP), d'une part pour une reconnaissance non-supervisée d'une pathologie, et d'autre part, déterminer la distribution spatiale des phénotypes de P. mirabilis au sein du biofil

Modulation of the flavin-protein interactions in NADH peroxidase and mercuric ion reductase: a resonance Raman study

NADH peroxidase (Npx) and mercuric ion reductase (MerA) are flavoproteins belonging to the pyridine nucleotide:disulfide oxidoreductases (PNDO) and catalyzing the reduction of toxic substrates, i.e., hydrogen peroxide and mercuric ion, respectively. To determine the role of the flavin adenine dinucleotide (FAD) in the detoxification mechanism, the resonance Raman (RR) spectra of these enzymes under various redox and ligation states have been investigated using blue and/or near-UV excitation(s). These data were compared to those previously obtained for glutathione reductase (GR), another enzyme of the PNDO family, but catalyzing the reduction of oxidized glutathione. Spectral differences have been detected for the marker bands of the isoalloxazine ring of Npx, MerA, and GR. They provide evidence for different catalytic mechanisms in these flavoproteins. The RR modes of the oxidized and two-electron reduced (EH 2 ) forms of Npx are related to very tight flavin-protein interactions maintaining a nearly planar conformation of the isoalloxazine tricycle, a low level of H-bonding at the N 1 /N 5 and O 2 /O 4 sites, and a strong H-bond at N 3 H. They also indicate minimal changes in FAD structure and environment upon either NAD(H) binding or reduction of the sulfinic redox center. All these spectroscopic data support an enzyme functioning centered on the Cys-SO − /Cys-S − redox moiety and a neighbouring His residue. On the contrary, the RR data on various functional forms of MerA are indicative of a modulation of both ring II distortion and H-bonding states of the N 5 site and ring III. The Cd(II) binding to the EH 2 -NADP(H) complexes, biomimetic intermediates in the reaction of Hg(II) reduction, provokes important spectral changes. They are interpreted in terms of flattening of the isoalloxazine ring and large decreases in H-bonding at the N 5 site and ring III. The large flexibility of the FAD structure and environment in MerA is in agreement with proposed mechanisms involving C 4a (flavin) adducts

Infrared fiber sensors for applications in chemistry and biology

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Mapping bacterial surface population physiology in real-time: infrared spectroscopy of Proteus mirabilis swarm colonies.

International audienceWe mapped the space-time distribution of stationary and swarmer cells within a growing Proteus mirabilis colony by infrared (IR) microspectroscopy. Colony mapping was performed at different positions between the inoculum and the periphery with a discrete microscope-mounted IR sensor, while continuous monitoring at a fixed location over time used an optical fiber based IR-attenuated total reflection (ATR) sensor, or "optrode." Phenotypes within a single P. mirabilis population relied on identification of functional determinants (producing unique spectral signals) that reflect differences in macromolecular composition associated with cell differentiation. Inner swarm colony domains are spectrally homogeneous, having patterns similar to those produced by the inoculum. Outer domains composed of active swarmer cells exhibit spectra distinguishable at multiple wavelengths dominated by polysaccharides. Our real-time observations agree with and extend earlier reports indicating that motile swarmer cells are restricted to a narrow (approximately 3 mm) annulus at the colony edge. This study thus validates the use of an IR optrode for real-time and noninvasive monitoring of biofilms and other bacterial surface populations

Infrared glass fibers for in-situ sensing, chemical and biochemical reactions

International audienceInfrared optical fibres based on chalcogenide glasses have been designed for evanescent wave spectroscopy. The sensitivity of the optical sensor is improved in tapering the sensing zone by chemical etching and the working optical domain of the system has been tested on a chloroform sample. This original remote sensor, based on the analysis of infrared signatures, has been applied to follow the fermentation process in cider fabrication as well as to detect and monitor a bacterial biofilm