Search

Despite numerous innovations, measuring bacteria concentrations on a routine basis is still time consuming and ensuring accurate measurements requires careful handling. Furthermore, it often requires sampling small volumes of bacteria suspensions which might be poorly representative of the real bacteria concentration. In this paper, we propose a spectroscopy measurement method based on a description of the absorption/attenuation spectra of ESKAPEE bacteria. Concentrations were measured with accuracies less than 2%. In addition, mixing the mathematical description of the absorption/attenuation spectra of mammalian T-cells and bacteria allows for the simultaneous measurements of both species’ concentrations. This method allows real-time, sampling-free and seeder-free measurement and can be easily integrated into a closed-system environment.

Advanced Therapy Medicinal Products are promising drugs for patients in therapeutic impasses. Their complex fabrication process implies regular quality controls to monitor cell concentration. Among the different methods available, optical techniques offer several advantages. Our study aims to measure cell concentration in real time in a potential closed-loop environment using white light spectroscopy and to test the possibility of simultaneously measuring concentrations of several species. By analyzing the shapes of the absorption spectra, this system allowed the quantification of T-cells with an accuracy of about 3% during 30 h of cultivation monitoring and 26 h of doubling time, coherent with what is expected for normal cell culture. Moreover, our system permitted concentration measurements for two species in reconstructed co-cultures of T-cells and Candida albicans yeasts. This method can now be applied to any single or co-culture, it allows real-time monitoring, and can be easily integrated into a closed system.

We investigated the possibility to grow molecularly porous amino-silane layers on glass-like substrates. The goal of this work is to show that it is possible to substantially increase the sensitivity of a fluorescence sensor by adjusting the functionalization strategy. Two methods are studied, one using APTMS only and another one using both APTMS and APDMS. We show that, using the second method, sensor sensitivity is improved by a factor of about 5. In order to demonstrate this, we applied the technique to the grafting of fluorescein in order to build a fluorescence pH sensor.

This article presents a novel low cost force sensing platform for the mechanical characterisation of human oocytes. This platform is compatible with the specific context of Assisted Reproductive Technology (ART). The oocyte is placed inside a standard Petri dish filled with a culture medium. It is immobilized using a holding pipette. The disposable parts used to mechanically test each oocyte are made of glass. This configuration is very similar to the one used in IntraCytoplasmic Sperm Injection protocol excepted that the injection pipette is replaced by a glass indenter. This platform uses two passive and linear magnetic springs to measure the nanoforce applied to the oocyte. The stabilisation of the unstable magnetic springs is passively achieved using the reaction force generated by the compressed oocyte. Some preliminary results obtained with an experimental platform are presented. The global magnetic stiffness of the indenter, evaluated by simulation and experimentally identified, is about 0.0013 N m−1.

In the field of blood transfusion, there is a need to improve the bedside pre-transfusion ABO compatibility test. In France, this test is mandatory for each red cell concentrates transfusion. It is performed manually and serious transfusion accidents still occur, principally due to human errors. Therefore, an automated ABO compatibility test is required. Works concerning objective interpretation of ABO compatibility test have been reported but the proposed techniques cannot be easily translated to the patient's bedside. We propose a prototype device which demonstrates the easy use of biochip technology to perform this test: it contains a fluidic system, biochips (two to test the patient and two to test the red cell concentrates) and an optical absorbance detection module. When blood is applied to the biochips, red blood cells are trapped onto the surface if antigens and antibodies are complementary (positive chips). If they are not complementary, very little red blood cells are adsorbed (negative chips). Percentages of surface covered with red blood cells in negative biochips are 2% ± 2 (red cell concentrates) and 1% ± 1 (whole blood). This proves that the fluidic configuration leads to an optimum control of fluids flows with little retention of red blood cells in the circuitry. These percentages increase to 96% ± 3 and 82% ± 8 for red cell concentrates and whole blood respectively. This demonstrates a strong and specific immunocapture of red blood cells on positive chips. Furthermore, optical detection proves to be efficient at critical red blood cells concentrations (108 C/mL) and absorbance strongly correlates to the percentage of red blood cells captured by antibodies.

International audience; We present a simple optical micro-system used to measure the transmission spectra of oocytes in order to qualify their maturation stage. Two applications of the device are possible: (i) the evaluation of the maturation stages of oocytes, and (ii) the development of a fertilization indicator. For the first application, CV, MI and MII oocytes were also analysed. Transmission spectra allow the 3 maturation stages to be identified but cannot be used to estimate the maturity of an unknown oocyte. Oocytes are subject to continuous development. This is why spectral separation of the 3 maturation stages cannot be made although they can be visually identified. However, the visual observation remains biologist-dependent. We therefore investigated the temporal maturation evolution of the oocytes in terms of transmission spectra and probability analysis. Results show that oocytes to be fertilized should not only be chosen in the MII stage, but also at the right time during the MII stage. This particular aspect requires further investigation. However, spectral measurements could be used as a technique for monitoring the maturation evolution of the oocytes. Fertilized oocytes exhibiting fertilization abnormalities were also tested. The device proved to be an efficient fertilization indicator.

International audience; We report Gold/Silica biochips for low cost biosensor devices. Firstly, the study of biochemical interactions on silica by means of Surface Plasmon Resonance (SPR) is presented. Secondly, Gold/Silica biochips are employed to reduce the strong quenching that occurs when a fluorophore is close to the gold surface. Furthermore, the control of the Silica-like thickness allows optimizing the distance between the metallic surface and the fluorophore in order to enhance the fluorescent signal. These results represent the first steps towards highly sensitive, specific and low cost biosensors based, for example, on Surface Plasmon Coupled Emission (SPCE) techniques.

International audience; In vitro Fertilisation (IVF) is one of the therapeutic techniques offered to patients suffering from reproduction difficulties. Among the various steps involved in gamete manipulation, especially in intra cytoplasmic sperm injection (ICSI), the determination of the cell to be fertilised is critical. Usually, the choice of the oocyte relies on a visual checking of the maturity of the cell and on its morphological properties. Our current work deals with the fabrication of labs-on-chip (LOC), devoted to the characterisation of the cells before fertilisation. They consist of a platform that includes different analysis tools and a system that allows the accurate displacement between the different analysis bays. The first issue that must be addressed is the capture, the transportation and the trapping of a single oocyte on the LOC surface.We recently proposed a system based on micro-translators used for long distance transportation and micro-fluidics for accurate trapping. In this paper, we demonstrate a full micro-fluidic system which allows both, the long range displacement and the accurate trapping of a single oocyte. This simplifies the LOC because there is no more need for any micro-motor or complex mechanical systems.We also demonstrate the first optical sensor that quantifies the degree of maturity of the cell to be fertilised. This sensor can also be used as a fertilisation indicator.

Compensation techniques for producing well defined corner in KOH-etched (1 0 0) silicon have widely been proposed. However, the case of apertures connected with V-grooves has apparently not yet been published. In this paper, we present a compensation technique used in order to produce well defined square apertures connected with V-grooves. A full description of the KOH etching process, simulation methods as well as experimental confirmations are presented here. The technique is very easy to use and is convenient for any size of apertures and V-grooves. The structure obtained with this technique are of interest in many micro-machining domains, especially in the field of micro-fluidics and micro-biology.

International audience; The work described here concerns the fabrication of cost-effective biosensors that permit to amplify a fluorescence signal without a complex nano-structuration of the surface. The idea is to put to profit the natural pseudo nano-structuring that is observed when depositing metallic layers by various micro-fabrication techniques. This new architecture consists of a glass substrate. A gold film is deposited on the top of it and a silica layer onto the gold. A dye (Cy5) is then absorbed onto the surface and the fluorescence intensity is measured. This intensity depends on the distance between the dye and the metal. It also depends on the properties of the metallic film. The goal of the work is to determine which gold deposition method leads to the highest fluorescence amplification and which silica thickness is required to achieve this amplification.

International audience; The detection of DNA hybridization can be either complicated or require expensive devices. Some methods use a fluorescence signal to investigate hybridization. Here, we present an optical probe based on optical fibers both for illumination and for fluorescence collection. The detection is made with a microspectrophotometer and the signal is then treated with a PC. We also developed the DNA biochips (glass plate with a gold layer functionalized with target DNA) as well as a dedicated microtank which maintains a constant flow of probe DNA over the target one. Finally, the detection of hybridization with this simple and versatile system is presented.

There are various difficulties which reduce the percentage of success in human in vitro fertilization (IVF). One of them concerns the choice of the oocyte to be fertilized. Up to now, the selection of the mature cell to be employed essentially relies on subjective criteria. Most biologists visually estimate the morphological properties of the oocyte. Some of the microsystems we are currently developing in our laboratory propose a solution to the objective determination of some of the oocyte properties, with the ulterior motive of grouping the microsystems in a single Lab-On-Chip (LOC). In this paper, we present these microsystems or techniques (microfluidic platform, microoptics, micromechanics, and image processing) together with the first experimental results that have been achieved. To conclude, we would like to say that the systems we describe are unique and that the results we have obtained up to now cannot be achieved using any other IVF techniques. Also, these studies have been approved by the local ethics committee.

International audience; We present a new combination of a fluorescent film deposited onto a chip and a new kind of lowcost optical fluorescence probe. The probe consists of an optical fiber which guides the excitation beam to the chip and a second one which collects the fluorescence light toward a spectrometer slotted into a PC. The resulting data are then processed to extract the pertinent information. This system is designed for the detection of DNA but, at present, we have achieved the detection of functionalized lipidic vesicles and studied the photobleaching process of such films.

International audience; Compensation techniques for producing well-defined bent V-grooves in KOH etched (1 0 0) silicon have been widely proposed. However, many of the methods presented in the literature suffer from two drawbacks: the etching depth is supposed to be equal to the V-groove depth and the V-groove must be wide enough for the compensation structures to fit in the pattern. A few solutions have been proposed in order to get round these disadvantages, but they are generally complicated to implement. In this paper, we propose a very simple compensation technique to obtain well-defined structures with any arbitrary etching depth and/or V-groove width. The only condition for the compensation is that the V-groove must be long enough.

In vitro fertilising (IVF) probably belongs to the most exciting subjects of research, not only for human assisted reproduction but also for veterinary medicine. Among the various steps of gamete manipulation, the determination of the cell to be fertilised is perhaps the most critical. Up to now, the choice of the oocyte is made using subjective criteria. It is generally the results of a visual estimation of the morphological properties of the cell. In this paper, we describe our first studies concerning the fabrication of a micro-machined lab-on-chip devoted to the analysis of the cells to be fertilised. The overall idea is to end with a system able to capture the cell on the lab-on-chip surface, to transport it to different analysis stages, and to establish an objective catalogue of the oocyte characteristics. The first issue that must be addressed is capturing, transportating and trapping a single cell at different locations on the chip surface. In this article, we present some of the silicon microsystems used for the capture and the transportation of a single oocyte. We also present a lab-on-chip concept, which exploits the microfluidic technology in order to accurately trap the cell at the desired position.

International audience; During intracytoplasmic sperm injection (ICSI) attempts, oocytes reaching metaphase II are microinjected. A morphological examination under a microscope is the usual method for determining oocyte maturity. The level of oocyte maturity is based on the meiotic status (Germinal Vesicle, metaphase I and metaphase II) of the oocytes with respect to their increasing maturity. In this letter, we summarize the studies conducted to analyze cytoplasm maturity using various microsystems and image processing. Optical microsystems are used to measure the transmission spectra and refractive index of the oocytes. We compared the transmission spectra measurements to the transmission electron microscopy results. Karhunen-Loeve transform is also used to evaluate the maturity of the oocytes. To summarize, optical analysis techniques are a minimally invasive technology allowing cytoplasm maturity to be assessed. Oocytes should not only be qualified in terms of GV, MI or MII, but also regarding their temporal evolution over the course of these maturation stages. The ultimate aim of this work is to describe the maturation of the oocytes by a trajectory in a multidimensional space and to determine when would be the best time for successful fertilization.

The aim of this activity is the development of a mono point imaging fiber system which uses hyperspectral multi-excitation auto fluorescence technique for biological tissues and the study of an Optical Coherence Tomography system like another modality. At first, this report presents the optical properties of biological tissues and the relevant fluorophores for cancerous tumors detection. Secondly, the fluorescence imaging system instrumentation and hyperspectral analysis are presented with in vitro results. The third part presents the "scan free" optical coherence tomography system which is able to image without optical displacement. It's characterized and have interesting functionality like depth dependant dispersion compensation. These both techniques are complementary because they get different kind of information. The information of the first one is about biochemical composition of the tissues and the information of the second one is about the stucture.; L'objectif de ce travail de recherche est le développement d'un système fibré d'imagerie point par point d'auto fluorescence multi-excitation, de tissus biologiques en utilisant la technique de fluorescence hyperspectrale et l'étude d'un système de tomographie optique cohérente comme possible modalité supplémentaire. La première partie de ce rapport présente les propriétés optique des tissus biologiques et les fluorophores pertinents pour la détection de tumeurs cancéreuses. La deuxième partie présente l'instrumentation du système d'imagerie de fluorescence et l'analyse hyperspectrale des résultats obtenus in vitro.Il est démontré la pertinence de ce type d'analyse qui permet de déterminer la concentration de certains fluorophores. La troisième partie présente le système de tomographie optique cohérente appelé "scan free" OCT car il permet de réaliser des images sans déplacement d'éléments optiques. Ce système est caractérisé et présente des fonctionnalités intéressantes comme la compensation de la dispersion dépendante de la profondeur. Les divers résultats obtenus montrent que ces deux techniques sont complémentaires car elles apportent des informations de nature différentes. La première technique donne de se informations sur la composition biochimique des tissus, la seconde donne des information sur la structure.

The aim of this activity is the development of a mono point imaging fiber system which uses hyperspectral multi-excitation auto fluorescence technique for biological tissues and the study of an Optical Coherence Tomography system like another modality. At first, this report presents the optical properties of biological tissues and the relevant fluorophores for cancerous tumors detection. Secondly, the fluorescence imaging system instrumentation and hyperspectral analysis are presented with in vitro results. The third part presents the "scan free" optical coherence tomography system which is able to image without optical displacement. It's characterized and have interesting functionality like depth dependant dispersion compensation. These both techniques are complementary because they get different kind of information. The information of the first one is about biochemical composition of the tissues and the information of the second one is about the stucture.; L'objectif de ce travail de recherche est le développement d'un système fibré d'imagerie point par point d'auto fluorescence multi-excitation, de tissus biologiques en utilisant la technique de fluorescence hyperspectrale et l'étude d'un système de tomographie optique cohérente comme possible modalité supplémentaire. La première partie de ce rapport présente les propriétés optique des tissus biologiques et les fluorophores pertinents pour la détection de tumeurs cancéreuses. La deuxième partie présente l'instrumentation du système d'imagerie de fluorescence et l'analyse hyperspectrale des résultats obtenus in vitro.Il est démontré la pertinence de ce type d'analyse qui permet de déterminer la concentration de certains fluorophores. La troisième partie présente le système de tomographie optique cohérente appelé "scan free" OCT car il permet de réaliser des images sans déplacement d'éléments optiques. Ce système est caractérisé et présente des fonctionnalités intéressantes comme la compensation de la dispersion dépendante de la profondeur. Les divers résultats obtenus montrent que ces deux techniques sont complémentaires car elles apportent des informations de nature différentes. La première technique donne de se informations sur la composition biochimique des tissus, la seconde donne des information sur la structure.