In this study we use AFM-based nanolithography technique to produce nanofeatures of the single strand DNA and LNA probe molecules which are prepared via thiolated nucleic acid self-assembled monolayers (SAMs) on gold substrates. The goal is to observe the topographic changes of the DNA film structures resulting from the formation of rigid double strand DNA when the target and probe DNAs bind together. The so-called hybridization depends strongly on the probe density on the substrate surface. To find the proper probe density for hybridization, we vary the concentration of the probe DNA and search for the optimal conditions for measuring the height changes of the nanofeatures. We also monitor the topographic changes of the DNA nanofeatures in the different target DNA concentrations as a function of time, and the binding isotherms are fitted with the Langmuir adsorption model to derive the equilibrium dissociation constant and maximum hybridization efficiency. In addition, we extend the nanoscale hybridization reaction detection to mismatched DNA:DNA and LNA:DNA hybridization, and observe that topographic change of mismatched hybridization is inconspicuous and rapidly reach equilibrium. The results reveal the apparent difference between the perfect match and mismatch conditions, and validate that this approach can be applied to differentiate the situations for both perfect match and mismatch cases, demonstrating its potentials in the gene chip technology.

Improving the identification ability of surfaced-immobilized nucleic acid probes for small size DNA or RNA targets, utilizing optical or electrochemical methods, has been the goal for the gene chip technology. This study focuses on new probe design for introducing hairpin structural features and locked nucleic acid modification. We use three kinds of probes (DNA-LN, DNA-HP and LNA-HP) to prepare recognition layers via self-assembly processes on a gold substrate, and utilize AFM-based nanolithography technique to produce nanofeatures to observe the stiffness changes of oligonucleotide chains resulting from the formation of rigid double stranded duplexes when target sequence hybridizes to the probe. We also monitor the topographic changes upon exposure to the single mismatched and non-complementary targets as a function of time. The results reveal LNA-HP probes exhibit the highest response to discriminating single-point mutation in the base sequence. In addition, we study the effects of salt concentration, reaction temperature and the small size on the hybridization efficiency.

In order to gain insights into the electronic interactions between metallic gold and self-assembled monolayers composed of π-conjugated thiols, a series of thiol-containing molecules based on a stilbene backbone were synthesized and assembled on gold surface. The resulted monolayers were characterized with a variety of surface-sensitive techniques and the electronic properties of the obtained surfaces were studied with the use of ultraviolet photoelectron spectroscopy. Work-function changes and alignment of the molecular energy levels with respect to the Fermi level of the metal were investigated and important insights regarding the electronic properties of the metal / organic interfaces were obtained. Another aspect of interactions between organic dyes and metallic gold was studied in the context of spectroscopic properties of systems incorporating gold nanoparticles with organic fluorophores covalently attached to the nanoparticle surface. Ultrafast dynamics of the excited-state deactivation of the organic fluorophores attached to the surface of gold nanoparticles were studied with the use of a fs transient absorption technique. It was found that the close proximity of a gold nanoparticle had a profound impact on the excited-state lifetime of the studied organic fluorophore. The influence of the structure of the studied systems on the excited-state deactivation dynamics of the organic fluorophores was described.

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.

Molecular self-assembly has recently attracted significant attention for possible application in organic electronic and optoelectronic devices, such as self-assembled monolayer field-effect transistors (SAMFETs) and functional self-assembled integrated circuits. Self-assembly combines the advantages of low temperature solution processability, regio-selective monolayer adsorption and nano-scale control of film thickness. Much progress has been made in improving device performance using self-assembled monolayers (SAMs). However, most SAMFET devices reported to date showed current modulation only with submicrometre channels, with low device yields and poor reproducibility as a result of limited lateral interconnection of the semiconducting layer.In an attempt to address these issues, this thesis presents an investigation of the synthesis and properties of conjugated SAM molecules for use as the charge transporting layer in SAMFETs. Chapter 1 gives a comprehensive introduction to SAM-based surface systems, organic semiconductors and their use in OFETs and SAMFETs. Chapter 2 discusses attempts to design and synthesise p-type conjugated molecules capable of self-assembly on oxide surfaces based on a phenylene-bithiophene semiconducting core. The optical and electrochemical properties, as well as the thermal behaviour of these molecules are studied in detail. This theme is carried over to Chapter 3, which describes the synthesis, chemical and physical characterisation of two families of n-type SAM molecules. These molecules consist of NTCDI cores with hexyl or cyclohexyl chains as end-capping groups. Incorporation of a selection of materials as the active layer in OFETs or SAMFETs to evaluate the charge transport is demonstrated in Chapter 4. Monolayer films based on p-type monochlorosilane-terminated SAM molecules are made using the solution assembly technique and characterised by contact angle and AFM. OFETs made from DH-PTTP by both thermal evaporation and spin coating show high mobilities comparable to the best values reported in the literature. Top-contact SAMFETs show a hole mobility of 1.1 × 10-3 cm2V-1s-1 in air, consistent with those of solution processed DH-PTTP based OFETs. Finally, an overview of the project and some suggestions for future work are presented in Chapter 5.

Reactive ion scattering spectrometry (RISS) utilizing low energy (tens of eV) polyatomic ions was employed to characterize self-assembled monolayers (SAMs) on gold. The terminal composition of halogenated SAMs, chemisorption motifs of disulfide and diselenide SAMs, and electron transfer properties of molecular wire containing SAMs were interrogated to develop the versatility of RISS as an analytical surface characterization technique.Novel halogen terminated SAMs were examined for their ability to convert translational to vibrational energy of colliding projectile ions. A general increasing energy deposition trend correlated with increasing terminal mass with the exception of the iodine functionality. Increased amounts of surface abstractions and sputtering from C12I suggest competitive ion-surface interactions account for less than predicted energy deposition results. Mixed films of CH2Br and CH3 terminal groups elucidated interfacial surface crowding discerned by energy deposition results.Thiol and disulfide based SAMs were shown by RISS comparisons to be dissimilar in structure. Terminal orientation, however, was the same based on ion-surface reactions, disproving the proposed dimer model of disulfide SAMs. Ion-surface reactions and electron transfer properties of disulfide surfaces suggested greater percentages of c(4x2) superlattice structure than in thiol SAMs. Based on increased hydrogen reactivity, decreased methyl reactivity, and increased energy deposition results, diselenide based SAMs were more disordered than S-Au based SAMs. Electron transfer results monitored through total ion currents (TIC) showed Se-Au contacts are more conductive than S-Au attachments.Molecular wire candidates whose electron transfer capabilities are difficult to characterize by traditional techniques were characterized by RISS after being doped into matrix SAMs. Electron transfer properties were dependent on the isolating SAM matrix, dipole moments of the wires, and the potential applied to the surface. Changes in surface voltage dictated molecular wire geometry and electron transfer. Wires were annealed into preferential geometries by colliding ions, but did not operate as switches.While not related to the advancement of RISS, structural elucidation of the pharmaceutical carvidioliol was investigated by collision-induced dissociation, surface-induced dissociation, sustained off-resonance irradiation, and sustained off-resonance irradiation-resonant excitation and through gas-phase hydrogen/deuterium exchange. This molecule fragmented easily by all methods and demonstrated the chemical specificity of gas-phase hydrogen/deuterium exchange experiments.

Nowadays, material, energy and information technologies are three pillar industries. The materials that have close relation with our life have also been the foundation for the development of energy and information technologies. As the new member of the material family, functional molecular materials have become increasingly important for many applications, for which the design and characterization by the theoretical modeling have played the vital role. In this thesis, three different categories of functional molecular materials, the endohedral fullerenes, the fullerene derivatives and the self-assembled monolayers (SAMs), have been studied by means of first principles methods. The non-metal endohedral fullerene N@C60 is a special endohedral fullerene that is believed to be relevant to the construction of future quantum computer. The energy landscape inside the N@C60 has been carefully explored by density functional theory (DFT) calculations. The most energy favorable potential energysurfaces (PESs) for the N atom to move within the cavity have been identified. The effect of the charging on the PESs has also been examined. It is found that the inclusion of dispersion force is essential in determining the equilibriumstructure of N@C60. Furthermore, the performance of several commonly useddensity functionals with or without dispersion correction has been verified for ten different endohedral fullerenes A@C60 with the atom A being either reactive nonmetal or nobel gases elements. It shows that the inclusion of the dispersion forcedoes provide better description for the binding energy (BE), however, none ofthem could correctly describe the energy landscape inside all the ten endohedral fullerenes exclusively. It thus calls for the further improvement of current density functionals for weak interacting systems. Soft X-ray spectroscopy is a powerful tool for studying the chemical and electronic structures of functional molecular materials. Theoretical calculations have been proven to be extremely useful for providing correct assignments for spectraof large systems. In this thesis, we have performed first principles simulations forthe near-edge X-ray absorption fine structure (NEXAFS) and X-ray photoelectron spectra (XPS) of fullerene derivatives and aminothiolates SAMs. Our calculatedspectra can accurately reproduce experimental results available for all the systemsunder investigations, and identify the species or structures that are responsible for those unexpected spectral features observed in experiments. We have suggested a modified building block (MBB) approach that allows to calculate NEXAFS spectraof a large number of fullerene derivatives with very small computational cost, and resolved the long standing puzzle around the experimental XPS and NEXAFS spectra of SAMs with aminothiolates. / <p>QC 20120523</p>

Implanted combination devices comprising both biological as well as biomaterial components may trigger non-specific inflammatory responses against the biomaterial component as well as specific immune responses against the biological component. This specific immune response may be enhanced by the biomaterial, thereby implying a biomaterial-mediated adjuvant effect, or in contrast may be mitigated by the biomaterial. Since adjuvants function by triggering dendritic cell (DC) maturation, biomaterials may regulate DC responses and hence facilitate DC-orchestrated host responses. This research work has focused on examining DC responses to different model self-assembled monolayer (SAM) biomaterial chemistries, as an in vitro readout of the potential of these biomaterials to trigger DC maturation. The underlying hypothesis was that DCs recognize and respond to biomaterials either indirectly through the adsorbed protein layer, specifically through carbohydrate modifications of these proteins, or through carbohydrates inherent in the biomaterial chemistry, using PRRs to initiate an immune response. Towards this goal, DCs were derived from human peripheral blood mononuclear cells (PBMCs) by culture with DC differentiation cytokines and the culture systems were characterized as being composed of DCs as well as associated T and B lymphocytes. Culture of DCs on different SAM chemistries implied differential DC responses in terms of morphology, maturation marker expression and allostimulatory capacities as well as distinct underlying mechanisms responsible for these responses. Enzyme-linked lectin (ELLA) assays were used to characterize the profiles of carbohydrates associated with serum/plasma proteins adsorbed to different SAM chemistries. Differential profiles of DC carbohydrate ligands of CLRs were present on different chemistries. Furthermore, the profiles of human serum/plasma proteins adsorbed to and eluted from different SAM chemistries were assessed using immunoblot analysis. Finally, to observe the roles of carbohydrates in supporting DC maturation in the presence of a biomaterial, DCs were cultured in the presence of partially de-glycosylated FBS from which DC carbohydrate ligands were selectively removed. This research is significant towards the ultimate development of optimal design criteria for biomaterials for use in diverse tissue-engineering or vaccine development applications for which a wide spectrum of adjuvant effects are required.

Nanoelectrodes are defined as electrodes which have a critical dimension on the order of nanometres. Due to their smaller dimensions they have a reduced iR drop and enhanced mass transport, which results in the rapid establishment of an enhanced steady-state diffusion profile and a greater Faradaic current density, along with a smaller relative double layer capacitance, which together give a significantly increased signal to noise ratio compared to macroelectrodes. This potentially makes nanoelectrodes better sensors and analytical tools than macroelectrodes in terms of their having lower limits of detection and faster detection times. However, due to difficulties with fabrication most nanoelectrode designs are highly irreproducible which has inhibited their characterisation and commercial development. The Mount group has previously reported the design, fabrication and characterisation of a novel nanoelectrode design in conjunction with Engineers from the Scottish Microelectronic Centre (SMC). Microsquare Nanoband Edge Electrode arrays (MNEEs) consist of an array of cavities with nanoscale Pt bands (formed by sandwiching the metal between insulating layers) exposed around their perimeter. MNEEs are fabricated using a photolithographic process so can be reproducibly made in large quantities to high fidelity. The purpose of this work is to develop our understanding of the fundamental electrochemical behaviour of MNEEs for biosensing. First, a quantitative analysis of the cyclic voltammograms (CVs) and Electrochemical Impedance Spectroscopy (EIS) of macroelectrodes, microelectrodes and MNEE are compared and discussed. Second, their fundamental response is compared in terms of their biosensing properties by using a pre-established impedimetric biosensing protocol developed on macroelectrodes. This protocol uses a PNA probe to detect the mecA cassette of methicillin resistant staphylococcus aureus (MRSA). The procedure has been optimised and compared for macroelectrodes, microelectrodes and MNEE so as to compare their performances as biosensors. It was observed that MNEE’s: (a) form thiol films faster than electrodes with larger dimensions, determined by kinetic studies of 6-mercaptohexan-1-ol film formation (b) form films with different packing structures dependant on the electrode bulk to edge ratio (c) can detect the same concentration of target in less time than larger electrodes because of their increased sensitivity. The film packing has also been quantitatively investigated using EIS and it can be seen that films formed n MNEE were better able to incorporate target DNA into their more splayed out structure. Unique to this project has been the establishment of a protocol to form heterogeneous carbazole-alanine hydrogel matrices on nanoelectrodes, whose polymerisation is initiated by a pH swing at the electrode surface induced by the oxidation of hydroquinone. The gels growth pattern follows the diffusion field at the electrode and can be monitored using EIS. This also gives a measure of the permeability of the gel by fitting to the correct equivalent circuit. The gel structure has been imaged using light microscopy, confocal microscopy and scanning electrochemical microscopy (SEM). The results give a visual demonstration that MNEE has enhanced diffusion at the corners of the cavities, which is in agreement with previously published simulations, and give evidence as to the onset of hemispherical diffusion and the conditions at which the diffusion field between neighbouring electrodes begin to overlap, a phenomenon which can be observed visually and correlated to changes in the EIS data. Hydrogels have been grown chronopotentiometrically at different currents and the permittivity (through the diffusion coefficients) has been measured of redox couples through gels grown at different speeds. It was found that the hierarchical structure of the hydrogels can be tuned; potentially opening the door to a new breed of tuneable, biocompatible anti-biofouling matrices on bio-functionalised electrodes. The system was characterised using the same MRSA detection protocol as optimised for the MNEE and the target DNA was found to be able to permeate through the hydrogels and bind to the probe, which resulted in a significant change in impedance.

Orientador: Paulo Roberto Bueno / Coorientador: Flávio Cesar Bedatty Fernandes / Banca: Maria Del Pilar Taboada Sotomayor / Banca: Paulo Inácio da Costa / Resumo: O sucesso no tratamento de muitos tipos doenças passa pela detecção seletiva e sensível de biomarcadores proteicos que permitam um diagnóstico precoce. A dengue é uma doença infecciosa de diagnóstico clínico impreciso e diagnóstico laboratorial demorado e custoso, a qual não possui tratamento ou vacina efetivos. Portanto se requer de ferramentas diagnósticas precisas, baratas e portáveis que permitam o diagnóstico rápido para realizar um tratamento adequado de sintomas e identificar os focos infecciosos para prevenir o espalhamento da doença. Um biomarcador útil na detecção da dengue, é a proteína NS1 que vem sendo utilizada com sucesso em diferentes plataformas de diagnóstico. Porém, nenhuma das plataformas oferecidas a nível comercial, consegue combinar a precisão, portabilidade, baixo custo e facilidade de manuseio. Portanto, o melhoramento de ditas ferramentas é o foco de bastantes pesquisas. Neste trabalho se apresenta uma plataforma que se amostra útil para a detecção de diferentes biomarcadores, incluindo a proteína NS1. Esta plataforma combina o uso de uma técnica eletroquímica como é a Espectroscopia de Capacitância Eletroquímica (ECE), com o uso de peptídeos redox e está baseada na funcionalização de eletrodos de ouro mediante formação de monocamadas auto-organizadas (SAM) confeccionadas com um peptídeo redox (Fc-Glu-Gli-Ser-Gli-Ser-Cys) desenhado para ser ancorado em superfícies metálicas, ao mesmo tempo que tem capacidade de ancorar uma sonda redox e um biorecepto... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Success in the treatment of many kinds of illnesses depends on the selective and sensitive detection of protein biomarkers that allow an early diagnosis. Dengue is and infectious disease of imprecise clinical diagnostic and delayed and expensive laboratorial diagnostic. This disease does not have an effective vaccine or treatment. Therefore, precise, cheap and portable diagnostic tools are necessary to allow a fast diagnostic in order to treat the symptoms, identify focuses of infection, and thus prevent the spreading of the disease. A useful biomarker in the detection of dengue is the protein NS1, which has been successfully used in different diagnostic platforms. However, none of the commercially available platforms combines precision, portability, low cost and user friendliness. Consequently, the improvement of such tools is object of ample research. This work, introduces a platform, which is useful for the detection of various biomarkers, including the protein NS1. This platform combines the usage of an electrochemical technique such as Electrochemical Capacitance Spectroscopy (ECS) and the use of redox peptides. It is based in the functionalization of gold electrodes through formation of Self Assembled Monolayers (SAM) formed by a redox peptide (Fc-Glu-Gli-Ser-Gli-Ser-Cys) designed to bind to metallic surfaces as well as to anchor a redox probe and a bioreceptor in the same structure/molecule. It presents the additional advantage of forming anti-fowling SAMs, which is a ... (Complete abstract click electronic access below) / Mestre