Fluorescence Lifetime Imaging of Quantum Dot Labeled DNA Microarrays

Quantum dot (QD) labeling combined with fluorescence lifetime imaging microscopy is proposed as a powerful transduction technique for the detection of DNA hybridization events. Fluorescence lifetime analysis of DNA microarray spots of hybridized QD labeled target indicated a characteristic lifetime value of 18.8 ns, compared to 13.3 ns obtained for spots of free QD solution, revealing that QD labels are sensitive to the spot microenvironment. Additionally, time gated detection was shown to improve the microarray image contrast ratio by 1.8, achieving femtomolar target sensitivity. Finally, lifetime multiplexing based on Qdot525 and Alexa430 was demonstrated using a single excitation-detection readout channel

Fast DNA and protein microarray tests for the diagnosis of hepatitis C virus infection on a single platform

Hepatitis C virus (HCV) is a major cause of chronic liver disease and liver cancer, and remains a large health care burden to the world. In this study we developed a DNA microarray test to detect HCV RNA and a protein microarray to detect human anti-HCV antibodies on a single platform. A main focus of this study was to evaluate possibilities to reduce the assay time, as a short time-to-result (TTR) is a prerequisite for a point-of-care test. Significantly reducing hybridisation and washing times did not impair the assay performance. This was confirmed first using artificial targets and subsequently using clinical samples from an HCV seroconversion panel derived from a HCV-infected patient. We were able to reduce the time required for the detection of human anti-HCV antibodies to only 14 min, achieving nanomolar sensitivity. The protein microarray exhibited an analytical sensitivity comparable to that of commercial systems. Similar results were obtained with the DNA microarray using a universal probe which covered all different HCV genotypes. It was possible to reduce the assay time after PCR from 150 min to 16 min without any loss of sensitivity. Taken together, these results constitute a significant step forward in the design of rapid, microarray-based diagnostics for human infectious disease, and show that the protein microarray is currently the most favourable candidate to fill this role

Peptide-tags for enhanced DNA microarray performance

DNA microarrays are powerful tools for gene expression analysis and genotyping studies in research and diagnostic applications. A high sensitivity and short time-to-result are prerequisites for their practical application in the clinic. The hybridization efficiency of DNA microarrays depends on the probe density and the probe orientation and thus their accessibility for target molecules. In order to find an optimal probe immobilization procedure a set of different oligonucleotide modifications was tested on epoxy silane functionalized glass slides. It was found that histidine-tagged oligonucleotides resulted in the highest amount of bound probe and by far the best hybridization efficiencies. The detection limit obtained with histidine-tagged probes was up to two orders of magnitude lower compared to commonly used probe modifications. In order to further investigate the binding mechanism of histidine-tags towards functionalized glass substrates a set of different peptide-tags with and without free terminal amino-groups and with different amino acid compositions was tested. The results indicate an impact of the terminal amino group on the covalent surface binding and of aromatic amino acid residues on the enhanced hybridisation efficiency

Etude et réalisation de biocapteurs impédancemétriques en utilisant différentes approches d'immobilisation

Le but de cette thèse est d'élaborer des biocapteurs impédancemétriques utilisant différents types de matériaux. Nous avons étudié, l'insertion d'anticorps de type IgG dans des monocouches de molécules amphiphiles ODA (octadécylamine) à l'interface air-eau. La couche mixte anticorps/ODA est transférée ensuite sur la surface de l'électrode par la technique de Langmuir-Blodgett. La spectroscopie d'impédance a été utilisée pour suivre l'interaction entre l'anticorps et l'antigène spécifique. Les résultats montrent que cette méthode présente une sensibilité élevée et d'excellentes performances. Nous avons également greffé des oligonucléotides (ODNs) portant un groupe amine sur un film de polypyrrole préalablement fonctionnalisé par un groupement ester activé. La spectroscopie d'impédance a révélé le greffage des ODNs sur le film de polypyrrole ainsi que la réaction d'hybridation avec les ODNs complémentaires. Nous avons montré l'origine des variations des propriétés électriques du film de polypyrrole après une telle interaction. Nous avons utilisé des monocouches auto assemblées (SAMs) pour greffer des molécules en utilisant deux configurations deux configurations de monocouches sur lesquelles nous avons greffé de la biotine hydrazide afin d'étudier l'interaction entre la biotine et l'avidine. Nous avons montré par la spectroscopie d'impédance que la structure chimique de la monocouche influence fortement la sensibilité du biocapteur d'affinité.The goal of this thesis is to design impedancemetric biosensors using various types of materials. We studied, the adsorption of antibody onto the monolayers of amphiphile molecules ODA (octadecylamine) at the air-water interface. The mixed monolayer IgG/ODA was transferred onto a modified SAMs electrode by the technique of Langmuir-Blodgett. The impedance spectroscopy was used to follow the interaction between the antibody and the specific antigen. The results show that this method has a high sensitivity and a quite good characteristics. We had also grafted an oligonucléotides (ODNs) carrying an amino group on a polypyrrole film functionalized by an activated ester. The impedance spectroscopy revealed the grafting of the ODN on film of polypyrrole as well as the reaction of hybridization with the complementary ODNs. We showed the origin of the variations of the electric properties of polypyrrole film after such a reaction. We have also used the self assembled monolayer (SAMs) to graft moleculesusing two SAMs configurations with grafted biotine hydrazide in order to study the interaction between the biotin and an avidin. We showed by impedance spectroscopy that the chemical structure of SAMs influence strongly the sensitivity of the affinity biosensors.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Elaboration of odorant biosensors based on Langmuir-Blodgett technique

International audienceThe advent of electronic noses opened a new kind of analytical approach, which offers an easy, quick, and cheap measurement comparing to conventional analytical approaches. It has potential applications on many different fields, such as assessment of various foodstuffs and beverages, environmental contamination monitoring and medical diagnostics etc. In this study, we used odorant binding protein (OBP) as odor-sensing material for developing such kind of artificial odorant biosensor. And biological films of OBP-1F (rat OBP) were deposited by Langmuir-Blodgett (LB) technique on gold electrode previously functionalised with 1-octadecanethiol (ODT) by self-assembled monolayers (SAMs). It is known that successful immobilization of the Langmuir-Blodgett films depends strongly on the characteristics, particularly stability, of the corresponding Langmuir films at the air/water interface. Hence the characteristics of monolayer of amphiphile octadecylamine (ODA) and the mixed monolayer of ODA/OBP were studied. Electrochemical impedance spectroscopy (EIS) was used to monitor the response of the system to a specific odorant molecule, isoamyl acetate

Detection of ESAT-6 by a label free miniature immuno-electrochemical biosensor as a diagnostic tool for tuberculosis

International audienceTuberculosis is a worldwide disease considered as a major health problem with high morbidity and mortality rates. Poor detection of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis remains a major obstacle to the global control of this disease. Here we report the development of a new test based on the detection of the major virulent factor of Mtb, namely the early secreted antigenic target 6-kDa protein or ESAT-6. A label free electrochemical immunosensor using an anti-ESAT-6 monoclonal antibody as a bio-receptor is described herein. Anti-ESAT-6 antibodies were first covalently immobilized on the surface of a gold screen-printed electrode functionalized via a self-assembled thiol monolayer. Interaction between the bio-receptor and ESAT-6 antigen was evaluated by square wave voltammetry method using [Fe(CN)6](3-/4-) as redox probe. The detection limit of ESAT-6 antigen was 7ng/ml. The immunosensor has also been able to detect native ESAT-6 antigen secreted in cell culture filtrates of three pathogenic strains of Mtb (CDC1551, H37RV and H8N8). Overall, this work describes an immune-electrochemical biosensor, based on ESAT-6 antigen detection, as a useful diagnostic tool for tuberculosis

Facile and Controllable Synthesis of Large-Area Monolayer WS₂ Flakes Based on WO₃ Precursor Drop-Casted Substrates by Chemical Vapor Deposition

Monolayer WS2 (Tungsten Disulfide) with a direct-energy gap and excellent photoluminescence quantum yield at room temperature shows potential applications in optoelectronics. However, controllable synthesis of large-area monolayer WS2 is still challenging because of the difficulty in controlling the interrelated growth parameters. Herein, we report a facile and controllable method for synthesis of large-area monolayer WS2 flakes by direct sulfurization of powdered WO3 (Tungsten Trioxide) drop-casted on SiO2/Si substrates in a one-end sealed quartz tube. The samples were thoroughly characterized by an optical microscope, atomic force microscope, transmission electron microscope, fluorescence microscope, photoluminescence spectrometer, and Raman spectrometer. The obtained results indicate that large triangular monolayer WS2 flakes with an edge length up to 250 to 370 μm and homogeneous crystallinity were readily synthesized within 5 min of growth. We demonstrate that the as-grown monolayer WS2 flakes show distinctly size-dependent fluorescence emission, which is mainly attributed to the heterogeneous release of intrinsic tensile strain after growth

Impedance-Probing of Mixed Amphiphile-Antibody Films Transferred onto Silver Electrodes

International audienceThe impedance spectroscopy was employed to follow the different steps of the immunosensor fabrication and the electronic transduction for the detection of the specific rabbit antigen IgG. A mixed amphiphile (octadecylamine)-antibody Langmuir-Blodgett (LB) film was structured at the air-water interface and transferred onto an octadecyl thiol (ODT) modified silver electrode. Impedance spectroscopy allows one to model the different layers with equivalent electrical circuits. A real probing of the ODT self-assembled monolayer (SAM) was done: electrical defects were found in the ODT SAM and they disappeared after deposition of the LBfilm. It can be suggested that these defects were suppressed by inclusion of octadecylamine (ODA) in the ODT layer. The total impedance of the mixed LBfilm increased in the presence of an increasing concentration of specific antigen. The equivalent circuit shows a decrease of the capacitance of the LBbilayer and an increase of its resistance. Such a structured building-up including biomolecules leads to a reproducible immunosensor, allowing detection of the specific antigen in the range from 10 ng ml –1 to 1200 ng ml –1

Synthesis and Characterization of MgO Thin Films Obtained by Spray Technique for Optoelectronic Applications

Magnesium oxide (MgO) thin films with different magnesium concentrations ([Mg2+] = 0.05, 0.1, 0.15 and 0.2 mol·L−1) in a spray solution have been successfully grown using a spray pyrolysis technique. X-ray diffraction (XRD), Maud software, FTIR spectroscopy, a confocal microscope, Wien2k software, spectrophotometry and a Photoluminescence spectrometer were used to investigate the structural, morphological and optical properties. XRD analysis revealed a better crystalline quality of the MgO thin layer synthesized with [Mg2+] = 0.15 mol·L−1, which crystallized into a face-centered cubic structure along the preferred orientation (200) lattice plan. The enhancement of the crystalline quality for the MgO thin film ([Mg2+] = 0.15 mol·L−1) was obtained, which was accompanied by an increment of 94.3 nm of the crystallite size. No secondary phase was detected and the purity phase of the MgO thin film was confirmed using Maud software. From the transmission spectra results, high transparent and antireflective properties of the MgO thin film were observed, with an average transmission value of about 91.48% in the visible range, which can be used as an optical window or buffer layer in solar cell applications. The films also have a high reflectance value in the IR range, which indicates that the highly reflective surface will prevent an increase in surface temperature under solar irradiation, which could be beneficial in solar cell applications. A direct band gap type was estimated using the Tauc relation which is close to the experimental value of 4.0 eV for optimal growth. The MgO material was tested for the degradation of methylene blue (MB), which reached a high photodegradation rate of about 83% after 180 min under sunlight illumination. These experimental trends open a new door for promising the removal of water contaminants for photocatalysis application