Développement d’outils analytiques pour la détection de biomolécules directement dans des fluides sanguins

Cette thèse porte sur le développement de biocapteurs basés sur la technique de résonance des plasmons de surface (SPR) pour effectuer des analyses directement dans un fluide sanguin n’ayant subi aucune purification ou dilution. L’ensemble des biocapteurs discutés exploiteront un instrument SPR portable développé dans le groupe du professeur Masson. Le premier volet de la thèse portera sur le processus d’interférence lié à l’adsorption non spécifique du sérum à la surface du capteur. L’analyse des biomolécules adsorbées sera effectuée en combinant la SPR à la spectrométrie de masse. Les informations obtenues seront exploitées pour la construction de biocapteurs adaptés à l’analyse en milieu sanguin. Un premier biocapteur développé ciblera la protéine antigène prostatique spécifique (APS) contenue dans le sérum servant de biomarqueur pour dépister le cancer de la prostate. Pour détecter les faibles concentrations de cette protéine directement dans le sérum, un matériel plasmonique microstructuré sera utilisé pour amplifier les signaux obtenus et sera recouvert d’une monocouche peptidique minimisant l’adsorption non spécifique du sérum. L’instrument SPR aura été adapté pour permettre également la détection simultanée de fluorescence. Un test ELISA sera ainsi effectué en parallèle du test SPR. Chacune des techniques fournira un contrôle pour la deuxième, tout en permettant de détecter le biomarqueur au niveau requis pour dépister la maladie. La combinaison des deux méthodes permettra aussi d’élargir la gamme dynamique du test de dépistage. Pour terminer, l’instrument SPR portable sera utilisé dans le cadre de détection de petites biomolécules ayant un potentiel thérapeutique directement dans un échantillon de sang. Des peptides ayant une activité anti-athérosclérotique pourront ainsi être détectés à même un échantillon de sang ni purifié ni dilué, et ce à des concentrations de l’ordre du micromolaire. Une modification de la microfluidique via l’introduction d’une membrane poreuse au cœur de celle-ci sera la clé permettant d’effectuer de telles analyses. La présente thèse met de l’avant de nouvelles stratégies et des modifications instrumentales permettant d’analyser des protéines et des petites molécules directement dans un échantillon non purifié de sérum ou de sang. Les modifications apportées au système fluidique, à l’instrument SPR et au niveau du biocapteur employé permettront d’effectuer des biodétections dans des matrices aussi complexes que les fluides sanguins. Les présents travaux mettent en lumière la capacité d’un instrument SPR/fluorescence portable à faire en 12 minutes la biodétection d’un marqueur du cancer de la prostate directement dans un échantillon de sérum. Finalement, on rapporte ici un des premiers articles où un biocapteur SPR est utilisé à même un échantillon de sang non-purifié pour faire des biodétections.

Synthesis, Characterization and Applications of Hybrid Nanocomposites of TiO2 With Conducting Polymers

A nanocomposite is a multiphase solid material where one of the phases has one, two or three dimensions of less than 100 nanometers (nm), or structures having nano-scale repeat distances between the different phases that make up the material. In the broadest sense this definition can include porous media, colloids, gels and copolymers, but is more usually taken to mean the solid combination of a bulk matrix and nano-dimensional phase(s) differing in properties due to dissimilarities in structure and chemistry. The mechanical, electrical, thermal, optical, electrochemical, catalytic properties of the nanocomposite will differ markedly from that of the component materials. Size limits for these effects have been proposed, <5 nm for catalytic activity, <20 nm for making a hard magnetic material soft, <50 nm for refractive index changes, and <100 nm for achieving superparamagnetism, mechanical strengthening or restricting matrix dislocation movement. Conducting polymers have attracted much attention due to high electrical conductivity, ease of preparation, good environmental stability and wide variety of applications in light-emitting, biosensor chemical sensor, separation membrane and electronic devices. The most widely studied conducting polymers are polypyrrole, polyaniline, polythiophene etc. Conducting polymers provide tremendous scope for tuning of their electrical conductivity from semiconducting to metallic region by way of doping and are organic electro chromic materials with chemically active surface. But they are chemically very sensitive and have poor mechanical properties and thus possessing a processibility problem. Nanomaterial shows the presence of more sites for surface reactivity, they possess good mechanical properties and good dispersant too. Thus nanocomposites formed by combining conducting polymers and inorganic oxide nanoparticles possess the good properties of both the constituents and thus enhanced their utility. The properties of such type of nanocomposite are strongly depending on concentration of nanomaterials to be added. Conducting polymer composites is some suitable composition of a conducting polymer with one or more inorganic nanoparticles so that their desirable properties are combined successfully. The composites of core shell metal oxide particles-conducting polymer combine the electrical properties of the polymer shell and the magnetic, optical, electrical or catalytic characteristics of the metal oxide core, which could greatly widen their applicability in the fields of catalysis, electronics and optics. Moreover nanocomposite material composed of conducting polymers & oxides have open more field of application such as drug delivery, conductive paints, rechargeable batteries, toners in photocopying, smart windows, etc.The present work is mainly focussed on the synthesis, characterization and various application studies of conducting polymer modified TiO2 nanocomposites. The conclusions of the present work are outlined below, Mesoporous TiO2 was prepared by the cationic surfactant P123 assisted hydrothermal synthesis route and conducting polymer modified TiO2 nanocomposites were also prepared via the same technique. All the prepared systems show XRD pattern corresponding to anatase phase of TiO2, which means that there is no phase change occurring even after conducting polymer modification. Raman spectroscopy gives supporting evidence for the XRD results. It also confirms the incorporation of the polymer. The mesoporous nature and surface area of the prepared samples were analysed by N2 adsorption desorption studies and the mesoporous ordering can be confirmed by low angle XRD measurementThe morphology of the prepared samples was obtained from both SEM & TEM. The elemental analysis of the samples was performed by EDX analysisThe hybrid composite formation is confirmed by FT-IR spectroscopy and X-ray photoelectron spectroscopyAll the prepared samples have been used for the photocatalytic degradation of dyes, antibiotic, endocrine disruptors and some other organic pollutants. Photocatalytic antibacterial activity studies were also performed using the prepared systemsAll the prepared samples have been used for the photocatalytic degradation of dyes, antibiotic, endocrine disruptors and some other organic pollutants. Photocatalytic antibacterial activity studies were also performed using the prepared systems Polyaniline modified TiO2 nanocomposite systems were found to have good antibacterial activity. Thermal diffusivity studies of the polyaniline modified systems were carried out using thermal lens technique. It is observed that as the amount of polyaniline in the composite increases the thermal diffusivity also increases. The prepared systems can be used as an excellent coolant in various industrial purposes. Nonlinear optical properties (3rd order nonlinearity) of the polyaniline modified systems were studied using Z scan technique. The prepared materials can be used for optical limiting Applications. Lasing studies of polyaniline modified TiO2 systems were carried out and the studies reveal that TiO2 - Polyaniline composite is a potential dye laser gain medium.

Optical Antennas for Biosensing Applications

The aim of the thesis is to theoretically investigate optical/plasmonic antennas for biosensing applications. The full 3-D numerical electromagnetic simulations have been performed by using finite integration technique (FIT). The electromagnetic properties of surface plasmon polaritons (SPPs) and the localized surface plasmons (LSPs) based devices are studied for sensing purpose. The surface plasmon resonance (SPR) biosensors offer high refractive index sensitivity at a fixed wavelength but are not enough for the detection of low concentrations of molecules. It has been demonstrated that the sensitivity of SPR sensors can be increased by employing the transverse magneto-optic Kerr effect (TMOKE) in combination with SPPs. The sensor based on the phenomena of TMOKE and SPPs are known as magneto-optic SPR (MOSPR) sensors. The optimized MOSPR sensor is analyzed which provides 8 times higher sensitivity than the SPR sensor, which will be able to detect lower concentration of molecules. But, the range of the refractive index detection is limited, due to the rapid decay of the amplitude of the MOSPR-signal with the increase of the refractive indices. Whereas, LSPs based sensors can detect lower concentrations of molecules, but their sensitivity is small at a fixed wavelength. Therefore, another device configuration known as perfect plasmonic absorber (PPA) is investigated which is based on the phenomena of metal-insulator-metal (MIM) waveguide. The PPA consists of a periodic array of gold nanoparticles and a thick gold film separated by a dielectric spacer. The electromagnetic modes of the PPA system are analyzed for sensing purpose. The second order mode of the PPA at a fixed wavelength has been proposed for the first time for biosensing applications. The PPA based sensor combines the properties of the LSPR sensor and the SPR sensor, for example, it illustrates increment in sensitivity of the LSPR sensor comparable to the SPR and can detect lower concentration of molecules due to the presence of nanoparticles.

Magneto-optische Oberflächenplasmonresonanzeffekte in austauschverschobenen Dünnschichtsystemen

Die Wechselwirkungen zwischen Biomolekülen spielen eine zentrale Rolle in der biochemischen und pharmazeutischen Forschung. In der biomolekularen Interaktionsanalyse sind dabei Biosensoren auf Basis des Oberflächenplasmonresonanzeffekts (SPR-Effekt) weitverbreitet. Seit Einführung der ersten kommerziellen SPR-Biosensoren Anfang der 1990er Jahre wurden verschiedenste Messanordnungen sowie Materialsysteme mit dem Ziel einer möglichst hohen Empfindlichkeit getestet. Eine Möglichkeit zur Steigerung der Empfindlichkeit klassischer SPR-Systeme bieten sogenannte magneto-optische SPR-Biosensoren (MOSPR-Biosensoren). Grundlage der Empfindlichkeitssteigerung ist die gleichzeitige Messung des SPR-Effekts und des transversalen magneto-optischen KERR-Effekts (tMOKE). Bisherige Untersuchungen haben sich meist auf den Einfluss der Magnetisierung freier ferromagnetischer Schichten beschränkt. Im Rahmen dieser Arbeit wurden erstmals austauschverschobene Dünnschichtsysteme (EB-Systeme), eine Kombination aus Ferromagnet und Antiferromagnet, hinsichtlich ihrer Eignung für SPR- und MOSPR-basierte biosensorische Anwendungen untersucht. Aufgrund der remanenten Magnetisierung der ferromagnetischen Schicht und ihrer magnetischen Strukturierbarkeit sind EB-Systeme eine hochinteressante Plattform zur Realisierung neuer Biosensorkonzepte. Zur Reduzierung der stark dämpfendenden Wirkung magnetischer Materialien wurde das hier betrachtete IrMn/Co EB-System zwischen zwei Goldschichten eingebettet. Eine Gegenüberstellung optimierter Au/ IrMn/Co/Au-Systeme mit einem reinen Au-System, wie es typischerweise in kommerziellen SPR-basierten Biosensoren eingesetzt wird, demonstriert, dass mit den entwickelten EB-Systemen vergleichbare Empfindlichkeiten in SPR-Sensor-Anwendungen erreicht werden können. Die magneto-optische Aktivität der untersuchten Dünnschichtsysteme liegt im Bereich der Literaturwerte für Au/Co/Au-Systeme, mit denen erhöhte Empfindlichkeiten gegenüber Standard-SPR-Biosensoren realisiert wurden. Auf Grundlage magnetisch strukturierter Au/IrMn/Co/Au-Systeme wurden neue Biosensorkonzepte entwickelt und getestet. Erste Experimente belegen, dass mit diesen Schichtsystemen eine gleichzeitige Detektion der magnetisierungsabhängigen Reflektivitäten in ortsauflösenden MOSPR-Messungen möglich ist. Eine solche Messanordnung profitiert von der erhöhten Empfindlichkeit MOSPR-basierter Biosensoren, hohen Messgeschwindigkeiten und einem verbesserten Signal-Rausch-Verhältnis. Weiterhin wurde der domänenwandassistierte Transport (DOWMAT) superparamagnetischer Partikel über der Oberfläche eines exemplarischen EB-Systems, zur Sensorintegration von Misch-, Reinigungs- und Aufkonzentrationsfunktionen erfolgreich getestet. Die Ergebnisse demonstrieren, dass ein Transport von Partikelreihen mit hohen Geschwindigkeiten bei moderaten externen Magnetfeldern über den entwickelten Schichtsystemen möglich ist. Die Agglomeration der Partikel wird dabei intrinsisch vermieden. Diese Beobachtungen verdeutlichen die Vorzüge des DOWMAT-Mechanismus für biosensorische Anwendungen. Die präsentierten Untersuchungen bilden die Grundlage auf dem Weg zur Umsetzung neuer vielversprechender Biosensorkonzepte, die eine Schlüsselfunktion in der medizinischen point-of-care-Diagnostik bei der Detektion kleinster Konzentrationen krankheitsrelevanter Biomarker einnehmen können.

Synthesis, Optical properties and Applications of Water Soluble Conjugated PPPs for Biosensors

In recent years, application of fluorescent conjugated polymers to sense chemical and biological analytes has received much attention owing to its technological significance. Water soluble conjugated polymers are interesting towards the developing sensors for biomolecules. In this present contribution, we describe the syntheses and characterization of a series of water soluble conjugated polymers with sulfonic acid groups in the side chain. Such anionic conjugated polymers are designed to interact with biomolecules such as cytochrome-C. All polymers are water soluble and showed strong blue emission. Significant quenching of the fluorescence from our functionalized PPP was observed upon addition of viologen derivatives or cytochrome -C.

Actividade antioxidante do extracto de Matricaria chamomilla L. e eficácia clínica de formulações cosméticas contendo este extracto e seus compostos isolados

A aplicação tópica de antioxidantes provou ser eficaz em proteger a pele contra danos oxidativos. O extracto de Matricaria chamomilla L. tem sido amplamente utilizado em formulações cosméticas. Assim, o objectivo deste estudo foi avaliar o potencial antioxidante do extracto de camomila, bem como a eficácia clínica na hidratação e nas propriedades mecânicas da pele. A actividade antioxidante do extracto de camomila foi avaliada por quimioluminescência (IC50 = 0,14 ug / mL). As formulações foram suplementadas com a α -bisabolol de fração mássica 0,5 cg/g ou com extracto glicólico de Matricaria chamomilla L. de fração mássica 5,0 cg/g ou com 0,01 cg/g de apigenina e aplicados na parte interna do antebraço e na cara de 25 mulheres. A fisiologia da pele foi avaliada antes e após 2 horas (aplicação única) e 2 e 4 semanas de aplicação diária. Depois de uma única aplicação, todas as formulações aumentaram a água do estrato córneo, mas apenas as formulações com α -bisabolol e extracto de camomila reduziram a PTEA . A formulação com o extracto de camomila mostrou o resultado mais marcante na redução PTEA (27%). No entanto, após 2 - e 4- semanas de aplicação, apenas a formulação contendo o extracto aumentou o a água no estrato córneo. O extracto de camomila, foi eficaz na neutralização de radicais livres e, portanto, apresenta potencial em formulações cosméticas com este propósito.

Biological and chemical assessment of zinc ageing in field soils

As zinc (Zn) is both an essential trace element and potential toxicant, the effects of Zn fixation in soil are of practical significance. Soil samples from four field sites amended with ZnSO4 were used to investigate ageing of soluble Zn under field conditions over a 2-year period. Lability of Zn measured using 65Zn radioisotope dilution showed a significant decrease over time and hence evidence of Zn fixation in three of the four soils. However, 0.01 M CaCl2 extractions and toxicity measurements using a genetically modified lux-marked bacterial biosensor did not indicate a decrease in soluble/bioavailable Zn over time. This was attributed to the strong regulatory effect of abiotic properties such as pH on these latter measurements. These results also showed that Zn ageing occurred immediately after Zn spiking, emphasising the need to incubate freshly spiked soils before ecotoxicity assessments. Ageing effects were detected in Zn-amended field soils using 65Zn isotopic dilution as a measure of lability, but not with either CaCl2 extractions or a lux-marked bacterial biosensor.

Perception and modification of plant flavonoid signals by rhizosphere microorganisms

Flavonoids are a diverse class of polyphenolic compounds that are produced as a result of plant secondary metabolism. They are known to play a multifunctional role in rhizospheric plant-microbe and plant-plant communication. Most familiar is their function as a signal in initiation of the legume-rhizobia symbiosis, but, flavonoids may also be signals in the establishment of arbuscular mycorrhizal symbiosis and are known agents in plant defence and in allelopathic interactions. Flavonoid perception by, and impact on, their microbial targets (e.g. rhizobia, plant pathogens) is relatively well characterized. However, potential impacts on 'non-target' rhizosphere inhabitants ('non-target' is used to distinguish those microorganisms not conventionally known as targets) have not been thoroughly investigated. Thus, this review first summarizes the conventional roles of flavonoids as nod gene inducers, phytoalexins and allelochemicals before exploring questions concerning 'non-target' impacts. We hypothesize that flavonoids act to shape rhizosphere microbial community structure because they represent a potential source of carbon and toxicity and that they impact on rhizosphere function, for example, by accelerating the biodegradation of xenobiotics. We also examine the reverse question, 'how do rhizosphere microbial communities impact on flavonoid signals?' The presence of microorganisms undoubtedly influences the quality and quantity of flavonoids present in the rhizosphere, both through modification of root exudation patterns and microbial catabolism of exudates. Microbial alteration and attenuation of flavonoid signals may have ecological consequences for below-ground plant-microbe and plant-plant interaction. We have a lack of knowledge concerning the composition, concentration and bioavailability of flavonoids actually experienced by microbes in an intact rhizosphere, but this may be addressed through advances in microspectroscopic and biosensor techniques. Through the use of plant mutants defective in flavonoid biosynthesis, we may also start to address the question of the significance of flavonoids in shaping rhizosphere community structure and function.

Hydrolysis of N2O5 on sub-micron mineral salt aerosols

The kinetics of reactive uptake of gaseous N2O5 on sub-micron aerosol particles composed of aqueous ammonium sulfate, ammonium hydrogensulfate and sodium nitrate has been investigated. Uptake was measured in a laminar flow reactor, coupled with a differential mobility analyser (DMA) to obtain the aerosol size distribution, with N2O5 detection using NO chemiluminescence. FTIR spectroscopy was used to obtain information about the composition and water content of the aerosol particles under the conditions used in the kinetic measurements. The aerosols were generated by the nebulisation of aqueous salt solutions. The uptake coefficient on the sulfate salts was in the range [gamma]=0.0015 to 0.033 depending on temperature, humidity and phase of the aerosol. On sodium nitrate aerosols the values were much lower, [gamma]<0.001, confirming the inhibition of N2O5 hydrolysis by nitrate ions. At high humidity (>50% r.h.) the uptake coefficient on liquid sulfate aerosols is independent of water content, but at lower humidity, especially below the efflorescence point, the reactivity of the aerosol declines, correlating with the lower water content. The lower uptake rate on solid aerosols may be due to limitations imposed by the liquid volume in the particles. Uptake on sulfate aerosols showed a negative temperature dependence at T>290 K but no significant temperature dependence at lower temperatures. The results are generally consistent with previous models of N2O5 hydrolysis where the reactive intermediate is NO2+ produced by autoionisation of nitrogen pentoxide in the condensed phase.

Modified rare earth semiconductor oxide as a new nucleotide probe

Recent rapid developments in biological analysis, medical diagnosis, pharmaceutical industry, and environmental control fuel the urgent need for recognition of particular DNA sequences from samples. Currently, DNA detection techniques use radiochemical, enzymatic, fluorescent, or electrochemiluminescent methods; however, these techniques require costly labeled DNA and highly skilled and cumbersome procedure, which prohibit any in-situ monitoring. Here, we report that hybridization of surface-immobilized single-stranded oligonucleotide on praseodymium oxide (evaluated as a biosensor surface for the first time) with complimentary strands in solution provokes a significant shift of electrical impedance curve. This shift is attributed to a change in electrical characteristics through modification of surface charge of the underlying modified praseodymium oxide upon hybridization with the complementary oligonucelotide strand. On the other hand, using a noncomplementary single strand in solution does not create an equivalent change in the impedance value. This result clearly suggests that a new and simple electrochemical technique based on the change in electrical properties of the modified praseodymium oxide semiconductor surface upon recognition and transduction of a biological event without using labeled species is revealed.

A novel antiserum specific to apolipoprotein B-48: application in the investigation of postprandial lipidaemia in humans

1. Apolipoprotein B-48, the transport protein for chylomicrons, is identical with apolipoprotein B-100 for the first 48% of its sequence. No antiserum has yet been reported that can recognize apolipoprotein B-48, but not apolipoprotein B-100. 2. In the present study an antiserum was raised to the C-terminal sequence of apolipoprotein B-48, using specific chemical reactions to ensure that the charged carboxyl group of the C-terminal isoleucine residue was free. In a Western blot the antiserum was shown to bind to a protein band having the characteristics of apolipoprotein B-48, but not to apolipoprotein B-100. 3. In the early evening 11 subjects were given a test meal which contained 40 g of mixed oil and retinyl palmitate. Blood samples were collected over 9 h. Chylomicron-enriched fractions were prepared and analysed for triacylglycerol, retinyl palmitate and apolipoprotein B-48, the latter after separation using SDS/PAGE and visualization by chemiluminescence on a Western blot. Both triacylglycerol and apolipoprotein B-48 showed an early peak at 1 h, which was not seen with retinyl palmitate. All three substances gave a broader peak between 5 and 6 h postprandially. Retinyl palmitate concentrations declined rapidly during the late (6-9 h) postprandial period, but apolipoprotein B-48 concentrations remained elevated. 4. This study has shown that an antiserum has been produced which is specific for apolipoprotein B-48. This has enabled measurement of postprandial concentrations of the protein that revealed features of chylomicron metabolism which have not been reported previously.

Coupling thin layer electrochemistry with epifluorescence microscopy: an expedient way of investigating electrofluorochromism of organic dyes

The first example of thin layer electrochemistry coupled to epifluorescence microscopy in the total internal reflectance mode is described and applied to the investigation of electrochemically modulated fluorescence of an organic dye (chloromethoxytetrazine) in solution. This technique allows to generate full redox switch of fluorescence when converting reversibly the dye into its anion radical, as well as to record the spectral features of the electrogenerated species. Recording simultaneously fluorescence intensity and lifetime along with coulombic charge as a function of the electrode potential will lead to a deep insight into the redox quenching mechanism.

In-situ comparison of the NOy instruments flown in MOZAIC and SPURT

Two aircraft instruments for the measurement of total odd nitrogen (NOy) were compared side by side aboard a Learjet A35 in April 2003 during a campaign of the AFO2000 project SPURT (Spurengastransport in der Tropopausenregion). The instruments albeit employing the same measurement principle (gold converter and chemiluminescence) had different inlet configurations. The ECO-Physics instrument operated by ETH-Zürich in SPURT had the gold converter mounted outside the aircraft, whereas the instrument operated by FZ-Jülich in the European project MOZAIC III (Measurements of ozone, water vapour, carbon monoxide and nitrogen oxides aboard Airbus A340 in-service aircraft) employed a Rosemount probe with 80 cm of FEP-tubing connecting the inlet to the gold converter. The NOy concentrations during the flight ranged between 0.3 and 3 ppb. The two data sets were compared in a blind fashion and each team followed its normal operating procedures. On average, the measurements agreed within 7%, i.e. within the combined uncertainty of the two instruments. This puts an upper limit on potential losses of HNO3 in the Rosemount inlet of the MOZAIC instrument. Larger transient deviations were observed during periods after calibrations and when the aircraft entered the stratosphere. The time lag of the MOZAIC instrument observed in these instances is in accordance with the time constant of the MOZAIC inlet line determined in the laboratory for HNO3.

Investigations into plant biochemical wound-response pathways involved in the production of aphid-induced plant volatiles

Feeding damage to plants by insect herbivores induces the production of plant volatiles, which are attractive to the herbivores natural enemies. Little is understood about the plant biochemical pathways involved in aphid-induced plant volatile production. The aphid parasitoid Diaeretiella rapae can detect and respond to aphid-induced volatiles produced by Arabidopsis thaliana. When given experience of those volatiles, it can learn those cues and can therefore be used as a novel biosensor to detect them. The pathways involved in aphid-induced volatile production were investigated by comparing the responses of D. rapae to volatiles from a number of different transgenic mutants of A. thaliana, mutated in their octadecanoid, ethylene or salicylic acid wound-response pathways and also from wild-type plants. Plants were either undamaged or infested by the peach-potato aphid, Myzus persicae. It is demonstrated that the octadecanoid pathway and specifically the COI1 gene are required for aphid-induced volatile production. The presence of salicylic acid is also involved in volatile production. Using this model system, in combination with A. thaliana plants with single point gene mutations, has potential for the precise dissection of biochemical pathways involved in the production of aphid-induced volatiles