Nous avons développé un nano-fil conducteur, constitué uniquement de protéines et bio-inspiré des nano-fils bactériens conducteurs. Pour cela, une protéine chimère a été créée par l'association d'une protéine prion capable de s'auto-assembler en fibre et d'une métalloprotéine, une rubrédoxine, capable d'effectuer des transferts d'électrons. Comme montré par des techniques de microscopies et de spectroscopies (absorbance UV-visible et RPE), la protéine chimère est capable de former des fibres à la surface desquelles on retrouve les rubrédoxines. Les propriétés électroniques des nano-fils ont été caractérisées par des mesures courant-tension sur des échantillons secs et par électrochimie. Les mesures courant-tension ont montré que la conduction se faisait par plusieurs mécanismes. Les acides aminés aromatiques présents au centre du domaine prion semblent impliqués dans un des mécanismes de conduction. Les mesures électrochimiques ont quant à elles montré une conduction par sauts entre rubrédoxines. De plus, nous avons utilisé les nano-fils comme interface entre une enzyme, la laccase, et une électrode. Un courant électrocatalytique dû à la réduction de l'oxygène a été obtenu prouvant ainsi la capacité de nos nano-fils à agir comme médiateurs d'électrons. Les nano-fils conducteurs faits de protéines sont une structure intéressante pour comprendre le transport de charges dans les systèmes biologiques et sont également très prometteurs pour le développement de la bioélectronique et plus particulièrement de biocapteurs et de biopiles enzymatiques / The discovery of bacterial nanowires able to transport electrons on long range within biofilms and transfer them to electrodes is very promising for the development of bioelectronics and bio-electrochemical interfaces. However, their assembling process, their molecular composition and the electron transport mechanism are not fully understood yet. We took inspiration from bacterial nanowires to design conductive protein nanowires. We fused the sequence of a rubredoxin, an electron transfer iron-sulfur protein, to the sequence of HET-s(218-289), a prion domain that forms amyloid fibril by self-assembling under well-defined conditions. The resulting chimeric protein forms amyloid fibrils and displays redox proteins organized on the surface as shown by microscopy techniques and UV-Vis and EPR spectroscopy. Electron transfer mechanisms were studied in “dry state” current-voltage (I-V ) measurements and as hydrated film by electrochemistry. Dry state measurements allowed to evidence several conduction pathways with a possible role of aromatic residues in the conduction. Electrochemistry revealed electron transport by hopping between adjacent redox centers. This property allowed the use of our protein as mediator between a multicopper enzyme (laccase) and an electrode for electrocatalytic reduction of oxygen. These protein nanowires are interesting structures for the study of charge transport mechanisms in biological systems but are also very promising for the design of biosensors and enzymatic biofuel cells.

SiOx nanowires grown through the VLS mechanism were electrophoretically deposited on top of Au electrodes. GOx was immobilized using APTES and the EDC-NHS chemistry. Cyclic Voltammetry was used as the method to characterize the electrodes through their processing steps, and CV was also used to detect glucose in a PBS based solution. Ferro-Ferri Cyanide couple was used as the mediator.

Silicon carbide (SiC) has been around for more than 100 years as an industrial material and has found wide and varied applications because of its unique electrical and thermal properties. In recent years there has been increased attention on SiC as a viable material for biomedical applications. Among these applications are those where SiC is used as a substrate material for biosensors and biotransducers, taking advantage of its surface chemical, tribological and electrical properties. In this work we have used the proven bio- and hema-compatibility of SiC to develop a viable biorecognition interface using SiC as the substrate material for myocardial infarction detection. The approach followed included the development of an electrochemical-based sensor in which 3C-SiC is used as the active electrode and where flat band potential energy changes are monitored after successive modification of the SiC with aminopropyltriethoxysilane, anti-myoglobin and myoglobin incubation. We have studied the quality of self assembled monolayers obtained by surface modification of SiC using organosilanes such as aminopropyltriethoxysilane and octadecene, which is the starting point for the immobilization of cells or proteins on a substrate. We employed this technique on 6H-SiC where we were able to control the proliferation of H4 human neuroglioma and PC12 rat pheochromocytoma cells in vitro. Finally, aminopropyltriethoxysilane (APTES) was successfully used to immobilize anti-myoglobin on the 3C-SiC electrodes as demonstrated by fluorescence microscopy results. The electrical characterization of the surfaces was performed via impedance spectroscopy and by measuring changes in flat band potential using the Mott-Schottky plot technique. Changes in flat band and impedance of the SiC/antibody/protein interface would allow us to detect changes in the space charge region of the semiconductor. However, we believe that because of the presence of surface states and different crystal defects on the 3C-SiC we did not observed repeatable results that allowed us to identify the presence of myoglobin in solution. In addition, certain modifications need to be performed to the electrochemical cell in order to confirm the presence of the myoglobin immobilized on the functionalized SiC surfaces.

Food poisoning is mainly caused by pathogenic microorganisms and is now a severe problem worldwide. Therefore, rapid and sensitive methods are required to detect foodborne pathogens. A locally isolated bacterium, Bacillus licheniformis B38 was selected for this study. The spores of B. licheniformis B38 were induced by Schaeffer’s sporulation medium containing KCl, MgSO4.7H2O, Ca(NO3)4, MnCl2 and FeSO4. Schaeffer-Fulton endospore staining was used to differentiate spores and vegetative cells, where spores were stained green and vegetative cells were stained red. In order to separate the spores from the cells, a two-phase system was used to obtain pure spore suspension for following experiments. Spore coat proteins were extracted by SDS-8 M urea sample buffer and visualized by two different types of coomassie brilliant blue staining solutions. One of the staining solutions was more suitable for gel elution by diffusion. An ~10 kDa spore coat protein was selected for protein purification. Based on the given results, the protein purification by liquid chromatography was less convincing than using gel elution by diffusion technique. The two hypothetical protein sequences, P06552 and P45693, from the ~10 kDa spore coat protein were identified. In the preliminary study of B. licheniformis B38 spores detection by surface plasmon resonance, several binding parameters were studied. Dot blot was done to verify the reaction between the Bacillus spores polyclonal antibody against the B. licheniformis B38 spore coat protein. The most promising result was the binding of 0.1 mg/mL polyclonal antibody (analyte) to the 0.2 mg/mL spore coat protein at pH 2 (ligand) which showed 5.74 RU. The differences between a dot blot and a SPR detection techniques are described.

This dissertation describes the assessment and use of carbon optically transparent electrodes (C-OTEs) based on pyrolyzed photoresist films (PPFs) as a platform for spectroelectrochemical investigations. C-OTEs are examined for use in UV-Vis spectroelectrochemistry and electrogenerated chemiluminescence and compared to non-transparent glassy carbon (GC) and the conventional transparent electrode indium tin oxide (ITO). Chapter 1 provides a general overview of transparent electrodes, carbon electrodes, and spectroelectrochemistry. Chapter 2 details a UV-Vis spectroelectrochemical investigation of electrogenerated graphitic oxides (EGO) on the surface of the C-OTE in the presence of KCl. X-ray photoelectron spectroscopy and time of flight secondary ion mass spectroscopy are used to determine EGO composition. Several supporting electrolytes are investigated to determine the mechanism of EGO formation. Chapter 3 details experiments to electrochemically access the exciton emission from self-assembled double-walled tubular J-aggregates via electrogenerated chemiluminescence (ECL). Optimization of ECL intensity with respect to the coreactant concentration and the supporting electrolyte pH is performed on opaque glassy carbon electrodes. ECL and fluorescence spectra are compared, and C-OTEs are utilized to determine the source of disagreement between the spectra. Chapter 4 describes the preparation and characterization (i.e. transparency, thickness, sheet resistance, rms roughness, and electroactive surface area) of C-OTEs and explores C-OTEs for general use in ECL under a variety of conditions. Simultaneous cyclic voltammograms and ECL transients are obtained for three thicknesses of PPFs and compared to non-transparent GC and the conventional transparent electrode ITO in both front face and transmission electrode cell geometries. Despite positive potential shifts in oxidation and ECL peaks, attributed to the internal resistance of the PPFs that result from their nanoscale thickness, the PPFs display similar ECL activity to GC, including the low oxidation potential observed for amine coreactants on hydrophobic electrodes. Overall, C-OTEs are promising electrodes for spectroelectrochemical applications because they yield higher ECL than ITO in both oxidative-reductive and reductive-oxidative ECL modes, are more stable in alkaline solutions, display a wide potential window of stability, and have tunable transparency for more efficient detection of light in the transmission cell geometry. Future directions for this research are discussed in Chapter 5, which outlines several approaches to designing and improving spectroelectrochemical sensors. / text

In this thesis, a systematic study has been carried out on the synthesis, characterization and applications of microcrystalline diamond (MCD) and nanocrystalline diamond (NCD) thin films deposited by the chemical vapor deposition (CVD) method. Firstly, an overview of diamond films synthesized from carbon-containing gas plasmas is presented. A parameter study was performed to grow diamond thin films. The transition from micro- to nanocrystallinity of diamond grains was achieved by controlling the Ar/Hydrogen gas ratio. The nanocrystallinity is the result of a new growth mechanism which involves the insertion of carbon dimmer into carbon-carbon and carbon-hydrogen bonds. Secondly, characterization of diamond films has been carried out by different techniques including electron microscopy, near edge X-ray absorption fine structure (NEXAFS), nanoindentation, and Raman spectroscopy. Unique properties of NCD, compared to those of MCD grown by conventional hydrogen rich plasma, have been observed and investigated. Thirdly, various applications of diamond films are discussed: a). Well-adhered MCD coatings have been deposited on WC-Co substrates with proper surface pretreatment. A diffusion barrier Cr/CrN/Cr was deposited on the cemented carbide substrate and the substrate was short peened with 150 micron friable diamond powders to achieve higher nucleation density and stronger adhesion strength; b). A nitrogen doped NCD based biosensor was fabricated for glucose sensing. Carboxyl functional group and conducting polymer (polyaniline) have been utilized respectively to electrochemically functionalize the diamond surface. A linear response to glucose concentration has been obtained from the electrode with good sensitivity and stability; c). A novel approach to synthesize NCD wires has been developed for the first time. The NCD coating was successfully coated on Si nanowires (SiNWs) to form NCD wire with diameter around a few microns. This study opens a whole new area for applications based on diamond wires such as neural transmission electrodes, field emission emitters, and electrochemical electrodes with improved properties

The use of nanotechnology to develop methods for fabrication and characterization of organized hybrid nanostructures that include integrated polymeric, biological and inorganic compounds has increased exponentially during the last decade. Such bio-nano-composite materials could be used in solving current biomedical problems spanning from nanomedicine to tissue engineering and biosensing. In this dissertation, a systematic study has been carried out on the synthesis, characterization, of two interfaces between naturally derived and synthetic nanostructures. Carbon nanotubes and porous silicon represent the synthetic nanostructures that were developed for the purpose of interfacing with the naturally derived bovine type I collagen and respiratory syncytial virus DNA respectively. Firstly, the synthesis of collagen-carbon nanotubes by two different techniques: fibrillogenesis through slow wet fiber drawing (gelation process) and electrospinning has been highlighted. Characterization of the novel nanocomposite was conducted using electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, nanoindentation, and Raman spectroscopy. The collagen-carbon nanotube gelation process was found to have superior nanoscale surface mechanical properties that were more conducive to higher osteoblast specific protein expression such as osteocalcin. Applications of the developed nanofibers are detailed in the fields of orthopaedics and tissue engineering. Secondly, an overview of porous silicon synthesized by hydrofluoric acid is presented. A parametric study was performed to determine the optimal pore size was carried out. The use of porous silicon as a biosensor to detect RSV virus by DNA hybridization was then provided and the importance of the interface chemistry was highlighted.

Studies of biomolecular interactions are of interest for several reasons. Beside basic research, the knowledge gained from such studies is also very valuable in for example drug target identification. Medical care is another area where biomolecules may be used as biomarkers to aid physicians in making correct diagnosis. In addition, the highly specific interactions between antibodies and almost any substance opens up the possibilities to design systems for detection of trace amounts of both biological and non-biological substances within environmental restoration, law enforcement, correctional care, customs service and national security. A biochip, which contains a biologically active material, offers a means of monitoring the molecular interactions in the above applications in a sensitive and specific manner. The biochip is a key component of a biosensor, which also includes components for transforming the interaction events into a human-readable signal. This thesis describes the use of poly(ethylene glycol) (PEG) in biochip design. Two different approaches are presented, the first based on ethylene glycol (EG)-containing alkyl thiol self-assembled monolayers (SAMs) on flat gold and the second on photo-induced graft copolymerisation of PEG-containing methacrylate monomers onto various substrates. The former is a two dimensional system where EG-terminated thiols are mixed with similar thiols presenting tail groups that mimic the explosive substance 2,4,6-trinitrotoluene (TNT). In an immunoassay, the detection limit for TNT was determined to fall in the range 1-10 µg/L. In the second approach, a branched three dimensional biosensor matrix (hydrogel) is proposed. The carboxymethylated (CM) dextran matrix, which is commonly used within the biosensing community, is not always ideal for studies of biointeractions, due to the non-specific binding frequently encountered in work with complex biological solutions and various proteins. To employ PEG, which displays a low non-specific binding of such species, is therefore an interesting option worth investigating. The use of a branched graft polymerised PEG matrix in biosensor applications is novel as compared to previous reports which have focused on linear PEG chains. The latter approach provides, at maximum, one functional group, per surface anchoring point, for immobilisation of sensor elements. Thus, it has the inherited disadvantage that it limits the number of available immobilisation sites. The present PEG matrix contains a large number of functional groups, for immobilisation of sensor elements, per grafting site and offers the potential of improved response upon binding to the analyte as demonstrated in a series of successful sensor experiments. Furthermore, the nature of the process enables easy preparation of matrix patterns and gradients. In a PEG matrix gradient, protein permeability is studied and the capabilities of immobilising proteins are demonstrated. By combining the patterning technique with different monomers in a two-step process, an inert platform, lacking chemical attachment sites, is provided with arrays of spots (with immobilisation capabilities), which are conveniently addressed via microdispensing and used for biosensor purposes. The EG-terminated thiols present another means of generating such inert platforms, a route which is also investigated. To further explore the sensor quality of these spots, the concepts of patterning and gradient formation are combined and studied. / Det är intressant att studera biomolekylära interaktioner av många anledningar. För att kunna bedriva framgångsrik läkemedelsutveckling är det oerhört viktigt att känna till hur olika molekyler samverkar i människokroppen. Inom sjukvården kan biomolekyler användas som biomarkörer, då närvaro av dem eller förändringar av deras koncentrationer är kopplade till sjukdomstillstånd, och därmed hjälper läkaren att ställa rätt diagnos. Dessutom kan de mycket specifika interaktionerna mellan antikroppar och (i princip) valfri substans användas för detektion av spårämnen vid miljösaneringsarbete, gränskontroller, polisarbete, fängelser och arbete med nationell säkerhet. Den här avhandlingen beskriver hur polymeren polyetylenglykol (PEG) kan användas vid design av biochip. Ett biochip är en liten anordning, som kan användas för att detektera specifika molekyler med hjälp av en biologisk interaktion. Traditionellt har PEG använts inom biomaterialsektorn, men återfinns även i hygienartiklar som tvål och tandkräm. Ett annat användningsområde är konservering av bärgade träskepp och i en del litiumjonbatterier ingår PEG som en komponent. Dessutom pågår utveckling av PEG-innehållande skyddsvästar. I det här arbetet används PEG framför allt på grund av sin förmåga att minimera ospecifik inbindning av proteiner, som utgör en stor del av gruppen biomolekyler, till ytor på biochip. Två olika typer av ytbeläggningar, som innehåller den här polymeren, har använts. Den första typen ger mycket tunna (~0.000003 mm), tvådimensionella filmer medan den andra ger en något tjockare (~0.00005 mm), tredimensionell struktur (matris). De tvådimensionella filmerna har använts för att utveckla en sprängämnesdetektor med mycket hög känslighet (detektionsgräns mellan 1-10 ppb). En viktig beståndsdel i detta system är antikroppar riktade mot sprängämnet trinitrotoluen (TNT). Den tredimensionella matrisen är mer generell och kan användas för att studera många olika molekylära interaktioner. Tillverkningsmetoden av matrisen är baserad på belysning med ultraviolett ljus och är därmed lämpad för att skapa mönstrade ytor. Genom att blockera delar av ljusflödet begränsas tillväxten av matrisen till de belysta delarna. På så sätt har bland annat så kallade mikro-arrayer, bestående av mikrometerstora (tusendels millimeter) strukturer i ett regelbundet mönster, tillverkats. Tekniken tillåter även tillverkning av gradienter, där matrisens tjocklek varierar längs med provet, genom att belysa olika delar av provytan olika länge. Genom att undersöka dessa gradienter har information om matrisens genomsläpplighet för proteiner kunnat extraheras. Gradientkonceptet har även kombinerats med mikro-arraytillverkningen och gett möjlighet att studera interaktioner mellan flera olika modellproteiner och deras motsvarande antikroppar i olika tjocka matriser på en och samma yta. Det finns ett stort antal sätt att utnyttja interaktionerna mellan olika molekyler på ett biochip. Ett tilltalande tillvägagångssätt är exempelvis att i en mikro-array binda in olika molekyler som kan fånga kliniskt intressanta biomolekyler, i syfte att skapa en hälsoprofil. Ett sådant biochip skulle ge möjlighet att parallellt detektera eller bestämma koncentrationen av ett stort antal biomolekyler i till exempel en droppe blod. På så sätt kan en diagnos snabbt ställas, kanske till och med utan att patienten behöver uppsöka sjukvården. Den utvecklade PEG-matrisen har god potential att fungera i en sådan applikation.

Cells utilize various signal transduction networks to regulate metabolism. Nevertheless, a quantitative understanding of the relationship between growth factor signaling and metabolic state at the single cell level has been lacking. The signal transduction and metabolic states could vary widely among individual cells. However, such cell-to-cell variation might be masked by the bulk measurements obtained from conventional biochemical methods. To assess the spatiotemporal dynamics of metabolism in individual intact cells, we developed genetically encoded biosensors based on fluorescent proteins. As a key redox cofactor in metabolism, NADH has been implicated in the Warburg effect, the abnormal metabolism of glucose that is a hallmark of cancer cells. To date, however, sensitive and specific detection of NADH in the cytosol of individual live cells has been difficult. We engineered a fluorescent biosensor of NADH by combining a circularly permuted green fluorescent protein variant with a bacterial NADH-binding protein Rex. The optimized biosensor Peredox reports cytosolic \(NADH:NAD^+\) ratios in individual live cells and can be calibrated with exogenous lactate and pyruvate. Notably pH resistant, this biosensor can be used in several cultured and primary cell types and in a high-content imaging format. We then examined the single cell dynamics of glycolysis and energy-sensing signaling pathways using Peredox and other fluorescent biosensors: AMPKAR, a sensor of the AMPK activity; and FOXO3-FP, a fluorescently-tagged protein domain from Forkhead transcription factor FOXO3 to report on the PI3K/Akt pathway activity. With perturbation to growth factor signaling, we observed a transient response in the cytosolic \(NADH:NAD^+\) redox state. In contrast, with partial inhibition of glycolysis by iodoacetate, individual cells varied substantially in their responses, and cytosolic \(NADH:NAD^+\) ratios oscillated between high and low states with a regular, approximately half-hour period, persisting for hours. These glycolytic NADH oscillations appeared to be cell-autonomous and coincided with the activation of the PI3K/Akt pathway but not the AMPK pathway. These results suggest a dynamic coupling between growth factor signaling and metabolic parameters. Overall, this thesis presents novel optical tools to assess metabolic dynamics – and to unravel the elaborate and complex integration of glucose metabolism and signaling pathways at the single cell level.

For the last decade, semiconducting nanowires synthesized by bottom-up methods have opened up new opportunities, stimulated innovative scientific research, and led to applications in materials science, electronics, optics, and biology at the nanoscale. Notably, nanowire building blocks with precise control of size, structure, morphology, and even composition in one, two, and three dimensions can successfully demonstrate high-performance electrical characteristics of field-effect transistors (FETs) and highly sensitive, selective, label-free, real-time biosensors in the fields of nanoelectronics and nano-biosensing, respectively. This thesis has focused on the design, synthesis, assembly, fabrication and electrical characterization of nanowire heterostructures for a proof-of-concept nanoprocessor and morphology-modulated kinked nanowire molecular nanosensor. / Physics