La principale originalité de ce travail est la mise en œuvre de deux voies d'immobilisation du biorécepteur sur différents matériaux d'électrodes. Dans un premier temps, nous avons démontré que le polyneutral red (PNR) représente une bonne matrice de rétention pour les enzymes. De plus, de part ses propriétés de médiation vis-à-vis des enzymes et principalement de leur cofacteur (NAD/FAD), ce polymère permet une connexion intime entre le site actif de l'enzyme et l'électrode. L'ensemble de ces caractéristiques nous a permis de mettre en œuvre une bioélectrode applicable en tant qu'anode d'un biocapteur à glucose et d'une cellule de biopile à combustible. Dans un second temps, la glocose oxydase a été immobilisée de façon covalente sur une électrode. L'électro-oxydation de l'éthylène a été menée sur les électrodes de carbone vitreux pour obtenir des fonctions amines. La voie proposée est simple, rapide et efficace. Puis, la glucose oxydase a été greffée avec succès par la méthode EDC / NHS sur les fonctions amines après optimisation des conditions de pH. Ces bioélectrodes ont ensuite été testées en tant que biocapteur à glucose montrant une bonne sensibilité de glucose avec une bonne stabilité sur une période de 4 semaines ce qui prouve l'efficacité de la méthode de greffage pour des applications de détection et dosage. / The main originality of this work is the development of two-way to immobilize a bioreceptor on different electrode materials. Initially, we demonstrated that the polyneutral red (PNR) is a good matrix for retaining enzymes. In addition, its properties of mediation towards enzymes and mainly their cofactor (NAD / FAD), this polymer provides an intimate connection between the active site of the enzyme and the electrode. All these features allowed us to develop an bioelectrodes as the anode of a biosensor for glucose and a fuel cell biopile. In a second step, the glocose oxidase was covalently immobilized on an electrode. The electro-oxidation of ethylene diamine was carried out on glassy carbon electrodes to obtain amine functions. This proposed way is simple, fast and efficient. Then, glucose oxidase was successfully grafted by the method EDC / NHS on amine functions after the optimization of pH conditions. These bioelectrodes were then tested as glucose biosensor and showed good sensitivity with good stability over a period of 4 weeks which proves the effectiveness of the grafting method for detection and assay applications.

La métalloprotéinase matricielle (MMP)-8 ou collagénase-2 est capable de rompre les molécules natives, triples hélices, du collagène interstitiel, initiant ainsi le remodelage cellulaire lors du déplacement dentaire induit par une force orthodontique. C'est un bio-marqueur incontournable du remaniement tissulaire parodontal. L'augmentation de l'expression et de l'activation de MMP-8 dans le fluide gingival reflète l'activité du remodelage parodontal induit par les forces orthodontiques. En moyenne, la concentration de MMP-8 prélevée dans le fluide gingival des patients orthodontiques est 12 fois plus élevée (56 ± 50 µg/l contre 4,6 ± 4 µg/l) que chez les patients non orthodontiques. Le suivi des fluctuations de MMP-8 durant le déplacement orthodontique nécessite la mise au point d'un biocapteur. L'objectif de ce travail est d'utiliser une structure photonique à base de silicium poreux pour la conception d'un biocapteur optique de la MMP-8. Nous avons déterminé le choix du substrat de silicium poreux (PSi) le plus adapté à notre application avec une surface spécifique élevée et pores suffisamment ouverts pour l'infiltration des biomolécules qui sont des anticorps anti-MMP-8. Ensuite nous avons mis en place un procédé de fonctionnalisation chimique et biologique de la surface interne de ces échantillons. / The matrix métalloprotéinase (MMP) 8 or collagenase 2 is able to cleave native molecules, triple helixes, of the interstitial collagen, so introducing the cellular reshaping during orthodontic tooth movement. It is a major biomarker of the periodontal tissular remodeling. The increase of the expression and the activation of MMP-8 in the gingival fluid reflects the activity of the periodontal remodeling. On average, the concentration of MMP-8 taken in the gingival fluid of the orthodontic patients is 12 times as raised(brought up) (56 ± 50 µg / l against 4,6 ± 4 µg / l) that at the not orthodontic patients. Followed it by fluctuations in MMP-8 during the orthodontic movement require the development of a biosensor. The objective of this work is to use a photonique structure with porous silicon for the conception of an optical biosensor of the MMP-8. We determined the choice of the substratum of porous silicon (PSi) the most adapted to our application with a high specific surface and pores opened enough for the infiltration of the biomolecules which are antibodies anti-MMP-8. Then we set up a process of chemical and biological fonctionnalisation of the internal surface of these samples.

Este trabalho teve como objetivo estudar e caracterizar filmes de nanocompósitos de polipirrol dopado com índigo carmim e dodecilsulfato combinados com nanopartículas de ouro para aplicação como biossensores eletroquímicos de pesticida. Primeiramente sintetizou-se três filmes a 10 e 25 ºC por voltametria cíclica: polipirrol dopado com índigo carmim (PPi-IC), polipirrol dopado com índigo carmim contendo nanopartículas de ouro (PPi-IC-NPAu) e polipirrol dopado com índigo carmim e dodecilsulfato de sódio, contendo nanopartículas de ouro (PPi-IC-DS-NPAu). Nesses filmes foram realizadas caracterizações estruturais, morfológicas, ópticas e eletroquímicas, utilizando espectroscopia no infravermelho, espectroscopia Raman, microscopia eletrônica de varredura, espectroscopia UV-VIS-NIR e voltametria cíclica. Os filmes sintetizados a 10 ºC foram modificados com a enzima acetilcolinesterase (AChE), sendo posteriormente caracterizados por microscopia eletrônica de varredura, espectroscopia Raman e voltametria cíclica. Os resultados obtidos mostraram que a interação da enzima com os filmes de polipirrol é dependente de sua composição. A presença de DS não favoreceu a sua imobilização, sendo assim, os filmes de PPi-IC e PPi-IC-NPAu foram os materiais mais adequados para a imobilização física da AChE, sendo capazes de detectar o pesticida metil paration (MP) através da porcentagem de pesticida inibido pela ação da AChE em relação ao cloreto de acetiltiocolina (ATCl). Essa detecção foi realizada por voltametria cíclica, onde a resposta do biossensor é medida a partir da diminuição da corrente produzida. Isso ocorre porque o MP se encontra em contato com a AChE e inibe a ação da mesma, impedindo a hidrólise da ATCl, o que faz diminuir a corrente, pois não há produção de tiocolina, que é produto da hidrólise e uma espécie eletroativa. Os filmes apresentaram sensibilidades (% de inibição por mmol L-1 de pesticida) de 1,9×106 (R2 0,976) e 2,7×106 (R2 0,964) e limites de detecção de 4,15 e 14,8 ng L-1, para PPi-IC-AChE e PPi-IC-NPAu-AChE respectivamente. / The goal of this work was to study and characterize nanocomposite films of gold nanoparticles with indigo carmine and dodecylsulfate doped polypyrrole for application as electrochemical biosensor for pesticide. Initially, three films were synthesized by cyclic voltammetry at 10 and 25 ºC: polypyrrole doped with indigo carmine (PPy-IC), polypyrrole doped with indigo carmine containing gold nanoparticles (PPy-IC-AuNP) and polypyrrole doped with indigo carmine and sodium dodecylsulfate containing gold nanoparticles (PPy-IC-DS-AuNP). These films were studied by structural, morphological, optical and electrochemical characterization, using infrared spectroscopy, Raman spectroscopy, scanning electron microscopy, UV-VIS-NIR spectroscopy and cyclic voltammetry. The films synthesized at 10 ºC were modified with acetylcholinesterase (AChE) enzyme; followed by characterization with scanning electron microscopy, Raman spectroscopy and cyclic voltammetry The results showed a preferential immobilization of the enzymes on films without DS, therefore, PPy-IC and PPy-IC-AuNP were the most suitable materials for the physical immobilization of AChE and for detection of methyl parathion (MP) pesticide through the percentage of pesticide inhibited by the action of AChE on acetylthiocholine chloride (ATCl). This detection was carried out by cyclic voltammetry, where the biosensor response is measured as a decrease of the produced current. The decrease in current occurs because MP reaches the AChE, inhibiting the action of the enzyme, avoiding the hydrolysis of ATCl, leading to the decrease of current as no thiocholine (an electroactive specie, product of the hydrolysis) is produced. The films presented sensitivities (inhibition percentage per mmol L-1) of 1.9×106 (R2 0.976) and 2.7×106 (R2 0.964) and detection limits of 4.15 and 14.8 ng L-1 for PPy-IC-AChE and PPy-IC-AuNP-AChE respectively.

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.

This dissertation evaluates how gold nanoparticle structure and local environment influence resulting sensor function when using these nanomaterials for complex sample analysis. Molecular imprinted polymers (MIPs), a class of plastic antibodies, are engineered and incorporated into these nanosensors thereby facilitating the quantitative detection of a variety of small molecules when Raman spectroscopy and surface enhanced Raman scattering (SERS) are used for detection. First, homogeneous seeded growth gold nanosphere synthesis is evaluated as a function of ionic double layer composition and thickness. Systematically increasing the citrate concentration during synthesis improves nanomaterial shape homogeneity; however, further elevations of citrate concentration increase the number of internal and/or external atomic defects in the nanomaterials which leads to decreasing solution-phase stability. Next, spherical gold nanoparticles are modified with self-assembled monolayer (SAM), modeled using interfacial energy calculations, and experimental characterized using transmission electron microscopy, NMR, extinction spectroscopy, zeta potential, X-ray photoelectron spectroscopy, and flocculation studies to assess the morphology, surface chemistry, optical properties, surface charge, SAM packing density, and nanoparticle stability, respectively. The number of molecules on the nanostructures increases with increasing ionic strength (by decreasing the electrostatic interfacial energy between assembled molecules) which subsequently promotes nanoparticle stability. Third, plastic antibodies that recognize three drugs commonly used to treat migraines are engineered. These methacrylate-based MIPs are synthesized, extracted, characterized, and used to quantitatively and directly detect over-the-counter drugs in complex samples using Raman microscopy. These results along with numerical approximation methods to estimate drug binding site densities and dissociation constants with the MIPs serve as a foundation for understanding how modest recognition selectivity of MIPs coupled with shifts in the vibrational energy modes from the drugs upon hydrogen binding to the polymer backbone promote sensitive and selective drug detection in complex samples. Finally, nanomaterial incorporation into MIPs for applications in SERS-based biosensors is evaluated. Importantly, gold nanorod concentration increases the detectability of the same drugs using MIPs as pre-concentration and recognition elements. This combination of materials, theory, and applications forms a solid foundation which should aid in the design and development of MIP nanobiosensors for specific and sensitive detection of small molecules in complex matrices.

One of the key requirements in the design of wireless sensor nodes and miniature biomedical devices is energy efficiency. For a sensor node, which is a sensor and readout circuit, to survive on limited energy sources such as a battery or harvested energy, its energy consumption should be minimized. Capacitive sensors are candidates for use in energy-constrained applications, as they do not consume static power and can be used in a wide range of applications to measure different physical, chemical or biological quantities. However, the energy consumption is dominated by the capacitive interface circuit, i.e. the capacitance-to-digital converter (CDC). Several energy-efficient CDC architectures are introduced in this dissertation to meet the demand for high resolution and energy efficiency in smart capacitive sensors. First, we propose an energy-efficient CDC based on a differential successive-approximation data converter. The proposed differential CDC employs an energy-efficient operational transconductance amplifier (OTA) based on an inverter. A wide capacitance range with fine absolute resolution is implemented in the proposed coarse-fine DAC architecture which saves 89% of silicon area. The proposed CDC achieves an energy efficiency figure-of-merit (FOM) of 45.8fJ/step, which is the best reported energy efficiency to date. Second, we propose an energy efficient CDC for high-precision capacitive resolution by using oversampling and noise shaping. The proposed CDC achieves 150 aF absolute resolution and an energy efficiency FOM of 187fJ/conversion-step which outperforms state of the art high-precision differential CDCs. In the third and last part, we propose an in-vitro cancer diagnostic biosensor-CMOS platform for low-power, rapid detection, and low cost. The introduced platform is the first to demonstrate the ability to screen and quantify the spermidine/spermine N1 acetyltransferase (SSAT) enzyme which reveals the presence of early-stage cancer, on the surface of a capacitive biosensor. This platform, which is a biosensor combined with a highly energy-efficient digital CDC, is implemented and fabricated in a CMOS technology and can sense and convert the capacitance value from the biosensor to a digital word in an energy efficient way. The platform achieves an ultra-low power consumption: four orders of magnitude less than the state-of-the-art biosensor-CMOS platforms.

No description available.

Detection limit of any method is affected by device (electronics, noise), evaluation, software (timing inaccuracy, rounding mistakes, errors determination of parameters using measured values by numerical methods) and by a chemical reaction (diluting errors, the reaction mixture design, chemical interferents, temperature, the accuracy of pipetting). In this work was proposed a metod for determination of detection limit for measuring of inhibitors of acetylcholine esterase by biosensor analysator of toxicity (BTA). The analysator was developed by BVT Technologies a.s. in 2004. To determine the exact detection limit a calibration of output signal was done. Presently the analysator is able to detect only presence of toxins, not their concentration. Not least are mentioned all founded errors of software, which are continuously eliminated.

This work deals with the preparation of gold nanostructures for future usage in electrochemical sensors and biosensors, methods for their characterization and production. The emphasis is focused on the template-based electrodeposition method of gold and on study of the effect of manufacturing conditions on physical properties, mainly electrical and topological of nanostructures. Thesis is focused also on overall function and sensitivity of the gold nanostructured electrode.

The master’s thesis deals with two applications of gold discoidal nanostructures exhibiting plasmon resonance for biodetection. The first approach considers the detection of changes in the phase on plasmonic antennas using coherence-controlled holography microscope. It was found that the steepness of the phase is increasing with the illumination wavelength when plasmon resonance is excited in larger antennas. The sensitivity of the phase to refractive-index changes of the surrounding media was observed when the largest response was given by antennas in resonance with wavelength of illumination. Next part deals with plasmon resonance detection by means of optical spectroscopy combined with voltametry which characterizes the electrochemical activity. Changes in resonance wavelength induced by the presence of SSC buffer were observed, although this influence seems to diminish in time. Conducted experiments have also shown that oxygen-plasma cleaning is not suitable for sample surface cleaning because of oxidation of metals including gold as well.