This dissertation comprises experimental studies on the synthesis and applications of metallic nanoshells. These are a class of nanoparticles composed of a dielectric core and a thin metallic shell. Metallic nanoshells play an important role in nanotechnology, particularly in nanomedicine, due to their peculiar optical properties. The overall objectives of the dissertation were to improve the fabrication of these nanoparticles, and to demonstrate new applications of these materials in cancer research and spectroscopy. The fabrication of nanoshells is a multi-step process. Previously to our work, the procedures for the synthesis of nanoshells reported in the literature lacked systematic characterization of the various steps. The procedure was extremely time-consuming and the results demonstrated a high degree of size variation. In Chapter 3, we have developed characterization tools that provide checkpoints for each step of the synthesis. We demonstrated that it is possible to control the degree of coverage on the shell for a fixed amount of reagents, and also showed important differences on the shell growth phase for gold and silver. The synthetic optimization presented in Chapter 3 led to an overall faster protocol than those previously reported. Although the improvements presented in Chapter 3 led to a higher degree of control on the synthesis of nanoshells, the variations in the resulting particle population were still too large for applications in single particle spectroscopy and imaging. In Chapter 4, the synthesis was completely reformulated, aiming to narrow the size distribution of the nanoshell colloids. Through the use of a reverse microemulsion, we were able to fabricate ultramonodisperse silica (SiO2) cores, which translate into nanoshell colloids with narrow extinction bands that are comparable to those of a single nanoshell. We then fabricate a library of colloids with different core sizes, shell thicknesses and composition (gold or silver). The localized surface plasmon resonance (LSPR) of these colloids span across the visible range. From this library, two nanoshells (18nm silver on a 50nm SiO2 core, and 18nm gold on a 72nm SiO2 core) were selected for a proof of principle cell imaging experiment. The silver nanoshells were coated with a nuclear localization signal, allowing it to target the nuclear membrane. The gold nanoshells were coated with an antibody that binds to a receptor on the plasma membrane of MCF-7 human breast cancer cells. The nanoshells were easily distinguishable by eye in a dark field microscope and successful targeting was demonstrated by hyperspectral dark field microscopy. A comparison was made between fluorescent phalloidin and nanoshells, showing the superior photostability of the nanoparticles for long-term cell imaging. The results from Chapter 4 suggest that the nanoshells obtained by our new synthetic route present acceptable particle-to-particle variations in their optical properties that enables single particle extinction spectroscopy for cell imaging. In Chapter 5 we explored the use of these nanoshells for single-particle Surface-enhanced Raman spectroscopy (SERS). Notice that particle-to-particle variations in SERS are expected to be more significant than in extinction spectroscopy. This is because particle-to-particle SERS variabilities are driven by subtle changes in geometric parameters (particle size, shape, roughness). Two types of gold nanoshells were prepared and different excitation wavelengths (λex) were evaluated, respective to the LSPR of the nanoshells. Individual scattering spectra were acquired for each particle, for a total of 163 nanoshells, at two laser excitation wavelengths (632.8 nm and 785 nm). The particle-to-particle variations in SERS intensity were evaluated and correlated to the efficiency of the scattering at the LSPR peak. Chapter 6 finally shows the application of gold nanoshells as a platform for the direct visualization of circulating tumor cells (CTCs). 4T1 breast cancer cells were transduced with a non-native target protein (Thy1.1) and an anti-Thy1.1 antibody was conjugated to gold nanoshells. The use of a transduced target creates the ideal scenario for the assessment of nonspecific binding. On the in vitro phase of the study, non-transduced cells were used as a negative control. In this phase, parameters such as incubation times and nanoshell concentration were established. A murine model was then developed with the transduced 4T1 cells for the ex vivo portion of the work. Non-transduced cells were implanted in a control group. Blood was drawn from mice in both groups over the course of 29 days. Antibody-conjugated nanoshells were incubated with the blood samples and detection of single CTCs was achieved in a dark field microscope. Low levels of nonspecific binding were observed in the control group for non-transduced cells and across different cell types normally found in peripheral blood (e.g. lymphocytes). All positive and negative subjects were successfully identified. Chapter 7 provides an outlook of the work presented here and elaborates on possible directions to further develop the use of nanoshells in bioapplications and spectroscopy. / Graduate / 2019-05-03

Através da modificação adequada da superfície de nanopartículas de ouro com uma molécula sulfurada que apresenta vários sítios de coordenação, foi possível desenvolver um conceito interessante de sensoriamento quantitativo conjugado ao efeito SERS. Estudos com sistemas desse tipo permitem explicar não apenas como uma molécula se liga a uma superfície, mas também como aproveitar essas informações para elaborar sensores de alta sensibilidade. Entretanto, as teorias eletrodinâmico-quânticas que regem as interações dos plasmons de superfície ainda estão sendo trabalhadas. Dessa forma, o presente trabalho procura explorar as teorias atuais sobre a origem dos plasmons e as interações plasmônicas entre as partículas. Essas interações promovem fenômenos de intensificação do espalhamento Raman por superfície e também foram discutidas. Visando conhecer e compreender os fenômenos envolvidos, foram sintetizadas nanopartículas de ouro estabilizadas com 2,4,6-trimercapto-1,3,5-triazina e estudada a química do ligante na superfície das nanopartículas de ouro. Além disso, foi estudada a influência dos equilíbrios ácido-base nos espectros SERS e monitorou-se as bandas mais diretamente sensíveis ao pH, associando as mudanças observadas aos diversos equilíbrios das espécies presentes em solução. Por fim, explorou-se o conceito de sensoriamento através das mudanças nos modos vibracionais dos espectros SERS, na formação de complexos em superfície. / Surface modification of gold nanoparticles with a molecule exhibiting several coordination points, allowed to elaborate an interesting concept for quantitative sensing conjugated with the SERS effect. In such systems it is important to know how the molecule binds to the surface, since, based on those informations one can elaborate sensors displaying high sensibility.. However, in dealing with the interaction of nanoparticles with light, the electrodynamic-quantum theories related to the interactions of the surface plasmons are not yet completely developed. In this way, in this t dissertation we made use of the current theories to understand the origin of the plasmons and of how they interact with the the nanoparticles. We have synthesized gold nanoparticles stabilized with 2,4,6-trimercapto-1,3,5-triazine and studied the ligand chemistry at the particles surface.. The influence of acid-base equilibria was studied based on the changes of the SERS spectra SERS with the pH, and interpreted in terms of the several equilibria of the species in solution. Finally, a new sensing concept, based on the observed changes in the SERS spectra in the presence of metal ions, was successfully demonstrated.

abstract: Proteins play a central role to human body and biological activities. As powerful tools for protein detections, many surface plasmon resonance based techniques have been developed to enhance the sensitivity. However, sensitivity is not the only final goal. As a biosensor, four things really matter: sensitivity, specificity, resolution (temporal/spatial) and throughput. This dissertation presents several works on developing novel plasmonic based techniques for protein detections on the last two aspects to extend the application field. A fast electrochemically controlled plasmonic detection technique is first developed with the capability of monitoring electrochemical signal with nanosecond response time. The study reveals that the conformational gating of electron transfer in a redox protein (cytochrome c) takes place over a broad range of time scales (sub-µs to ms). The second platform integrates ultra-low volume piezoelectric liquid dispensing and plasmonic imaging detection to monitor different protein binding processes simultaneously with low sample cost. Experiment demonstrates the system can observe binding kinetics in 10×10 microarray of 6 nL droplet, with variations of kinetic rate constants among spots less than ±5%. A focused plasmonic imaging system with bi-cell algorithm is also proposed for spatial resolution enhancement. The two operation modes, scanning mode and focus mode, can be applied for different purposes. Measurement of bacterial aggregation demonstrates the higher spatial resolution. Detections of polystyrene beads binding and 50 nm gold nanoparticles oscillation show a high signal to noise ratio of the system. The real properties of protein rely on its dynamic personalities. The above works shed light upon fast and high throughput detection of protein kinetics, and enable more applications for plasmonic imaging techniques. It is anticipated that such methods will help to invoke a new surge to unveil the mysteries of biological activities and chemical process. / Dissertation/Thesis / Doctoral Dissertation Electrical Engineering 2018

Luk, Wai Chun = / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 101-108). / Abstracts in English and Chinese. / Luk, Wai Chun = / Chapter 1 --- Introduction --- p.1 / Chapter 2 --- Theoretical Background --- p.5 / Chapter 2.1 --- Maxwell´ةs equations in matter --- p.5 / Chapter 2.2 --- Dielectric constant of materials --- p.8 / Chapter 2.3 --- Dispersion relation of surface plasmon polaritons --- p.10 / Chapter 2.4 --- Excitation of surface plasmon polaritons --- p.16 / Chapter 2.4.1 --- Prism coupling --- p.17 / Chapter 2.4.2 --- Grating coupling --- p.21 / Chapter 2.5 --- Diffraction of light in gratings --- p.26 / Chapter 2.6 --- Applications --- p.27 / Chapter 3 --- Analysis Methods --- p.29 / Experimental Section --- p.29 / Chapter 3.1 --- Interference Lithography --- p.29 / Chapter 3.2 --- Gold grating fabrication --- p.32 / Chapter 3.2.1 --- Substrate preparation --- p.33 / Chapter 3.2.2 --- Photoresist preparation --- p.34 / Chapter 3.2.3 --- Spin coating of omnicoat and photoresist --- p.35 / Chapter 3.2.4 --- Interference lithography set-up and procedures --- p.35 / Chapter 3.2.5 --- The post-exposed treatments --- p.37 / Chapter 3.2.6 --- The optimal exposure time calibration --- p.37 / Chapter 3.2.7 --- Gold thin film deposition --- p.39 / Chapter 3.2.8 --- Typical gold grating sample --- p.41 / Chapter 3.3 --- Measurement system --- p.41 / Chapter 3.3.1 --- The angle dependent reflectivity measurement --- p.42 / Chapter 3.3.2 --- Data presentation of a typical band structure --- p.45 / Chapter 3.3.3 --- Periodicity measurement of the grating samples --- p.48 / Chapter 3.3.4 --- Diffracted intensity measurement of gratings --- p.52 / Chapter 3.3.5 --- Data presentation of the angle dependent diffracted intensity measurement --- p.53 / Calculation Section --- p.54 / Chapter 3.4 --- RCWA simulations --- p.54 / Chapter 3.4.1 --- The dispersion relation --- p.56 / Chapter 3.4.2 --- The diffracted intensity --- p.56 / Chapter 3.4.3 --- The field pattern graphs --- p.57 / Chapter 4 --- Resonance modes in one-dimensional gold gratings --- p.60 / Chapter 4.1 --- Structure of the gold grating samples --- p.61 / Chapter 4.2 --- Results of angle dependent reflectivity --- p.63 / Chapter 4.2.1 --- Surface Plasmon Polaritons (SPPs) --- p.65 / Chapter 4.2.2 --- Wood´ةs anomaly --- p.65 / Chapter 4.2.3 --- Waveguide Resonance (WG) --- p.67 / Chapter 4.2.4 --- Coupling of SPPs and WG --- p.67 / Chapter 4.3 --- Results of angle dependent diffracted intensity measurement --- p.68 / Chapter 4.4 --- Basic properties of SPPs and WG modes by RCWA --- p.73 / Chapter 4.4.1 --- Sample 1 (D = 40 nm) --- p.74 / Chapter 4.4.1(a) --- λ = 980 nm of Sample 1 --- p.75 / Chapter 4.4.1(b) --- λ = 633 nm of Sample 1 --- p.81 / Chapter 4.4.2 --- Sample 2 (D = 390 nm) --- p.83 / Chapter 4.4.2(a) --- λ = 980 nm of Sample 2 --- p.85 / Chapter 4.4.2(b) --- λ = 725 nm of Sample 2 --- p.87 / Chapter 4.4.2(c) --- λ = 633 nm of Sample 2 --- p.92 / Chapter 4.5 --- Summary --- p.97 / Chapter 5 --- Conclusions --- p.99 / Bibliography --- p.101

Sham, Chun Hong = 金屬光栅光學特性的實驗和理論研究 / 沈鎮康. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 109-111). / Abstract also in Chinese. / Sham, Chun Hong = Jin shu guang shan guang xue te xing de shi yan he li lun yan jiu / Shen Zhenkang. / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Background --- p.1 / Chapter 1.2 --- Overview of the Thesis --- p.2 / Chapter 2 --- Basic Theory --- p.4 / Chapter 2.1 --- Dielectric Constant of Metals --- p.5 / Chapter 2.2 --- The Maxwell´ةs Equations --- p.7 / Chapter 2.3 --- Scaling Properties of the Maxwell´ةs Equations --- p.9 / Chapter 2.4 --- Translational Symmetry and the Bloch´ةs Theorem --- p.10 / Chapter 2.4.1 --- Continuous Translational Symmetry --- p.11 / Chapter 2.4.2 --- Discrete Translational Symmetry --- p.12 / Chapter 2.4.3 --- Photonic Bloch´ةs Theorem --- p.13 / Chapter 3 --- Principles of Rigorous Coupled Wave Analysis --- p.14 / Chapter 3.1 --- Mathematical Formulation --- p.15 / Chapter 3.2 --- One-layer systems --- p.15 / Chapter 3.3 --- Layered Systems --- p.19 / Chapter 3.3.1 --- Matching Boundary Conditions --- p.19 / Chapter 3.3.2 --- The Transfer Matrices --- p.21 / Chapter 3.3.3 --- Scattering Matrices --- p.22 / Chapter 3.4 --- Calculation of Reflection and Transmission --- p.24 / Chapter 3.5 --- Calculation of Field Pattern --- p.26 / Chapter 3.5.1 --- Finding the Coefficients --- p.26 / Chapter 3.5.2 --- Summing to Get the Field --- p.27 / Chapter 3.6 --- 5-polarization --- p.27 / Chapter 3.7 --- Analogy to mechanics --- p.29 / Chapter 3.8 --- Conclusion --- p.30 / Chapter 4 --- Numerical Implementation of Rigorous Coupled Wave Analysis --- p.31 / Chapter 4.1 --- Finite Number of Terms --- p.31 / Chapter 4.2 --- Fourier Factorization Rule --- p.32 / Chapter 4.3 --- Calculation of Field Pattern --- p.34 / Chapter 4.4 --- Transfer Matrix for Forward Deduction --- p.36 / Chapter 4.5 --- Calculation of Time-Averaged Poynting Vector --- p.36 / Chapter 4.6 --- Convergence of RCWA --- p.37 / Chapter 4.7 --- Simple Examples --- p.40 / Chapter 4.7.1 --- Oblique Incidence on Vacuum --- p.40 / Chapter 4.7.2 --- Oblique Incidence on Semi-Infinite Glass --- p.41 / Chapter 4.7.3 --- Normal Incidence on a Thin Gold Film --- p.41 / Chapter 5 --- A Tunable All-Direction Light Absorber --- p.43 / Chapter 5.1 --- Description of the Absorber --- p.44 / Chapter 5.2 --- Tunability --- p.45 / Chapter 5.3 --- Theoretical Understanding on the Results --- p.46 / Chapter 5.4 --- Other EM modes Involved --- p.49 / Chapter 5.5 --- Structural Flexibility --- p.54 / Chapter 6 --- Sample Preparation Techniques --- p.57 / Chapter 6.1 --- Interference Lithography --- p.57 / Chapter 6.1.1 --- Basic Principle of IL --- p.58 / Chapter 6.1.2 --- Experimental Setup for IL --- p.59 / Chapter 6.1.3 --- Experimental Procedures for IL --- p.60 / Chapter 6.1.4 --- Tuning the Period --- p.61 / Chapter 6.1.5 --- Tuning Grating Width --- p.62 / Chapter 6.1.6 --- Tuning the grating height --- p.64 / Chapter 6.2 --- Sputtering --- p.65 / Chapter 6.2.1 --- Description of Sputtering System --- p.65 / Chapter 6.2.2 --- Effect of Varying the Position on Sample Holder --- p.66 / Chapter 6.3 --- Chemical deposition of silver --- p.69 / Chapter 7 --- Sample Characterization Techniques --- p.72 / Chapter 7.1 --- Scanning Electron Microscope --- p.72 / Chapter 7.1.1 --- Grating width measurement --- p.72 / Chapter 7.1.2 --- Sidewall Coverage Measurement --- p.73 / Chapter 7.2 --- Thickness Measurement --- p.74 / Chapter 7.3 --- Reflectance Measurement --- p.75 / Chapter 7.3.1 --- Experimental Setup --- p.76 / Chapter 7.3.2 --- Use of Prism --- p.79 / Chapter 8 --- Experimental Realization of All-Direction Light Absorber --- p.82 / Chapter 8.1 --- Reflectance of Glass Substrate --- p.82 / Chapter 8.2 --- Planar Metal-SU8-Metal Systems --- p.87 / Chapter 8.3 --- SU8 Grating on Glass Substrate --- p.89 / Chapter 8.4 --- SU8 Grating on Gold Substrate --- p.92 / Chapter 8.4.1 --- Grating on a Thick Layer of Gold --- p.93 / Chapter 8.4.2 --- Grating on a Thin Layer of Gold --- p.93 / Chapter 8.5 --- Cavities-Embedded Systems and Future Work --- p.96 / Chapter 8.6 --- Summary and Future Experimental Work --- p.100 / Chapter 9 --- Conclusion --- p.102 / Chapter A --- Notes on Glass Cleaning --- p.104 / Chapter B --- An Experiment on Sidewall Coverage --- p.107 / Bibliography --- p.109

Através da modificação adequada da superfície de nanopartículas de ouro com uma molécula sulfurada que apresenta vários sítios de coordenação, foi possível desenvolver um conceito interessante de sensoriamento quantitativo conjugado ao efeito SERS. Estudos com sistemas desse tipo permitem explicar não apenas como uma molécula se liga a uma superfície, mas também como aproveitar essas informações para elaborar sensores de alta sensibilidade. Entretanto, as teorias eletrodinâmico-quânticas que regem as interações dos plasmons de superfície ainda estão sendo trabalhadas. Dessa forma, o presente trabalho procura explorar as teorias atuais sobre a origem dos plasmons e as interações plasmônicas entre as partículas. Essas interações promovem fenômenos de intensificação do espalhamento Raman por superfície e também foram discutidas. Visando conhecer e compreender os fenômenos envolvidos, foram sintetizadas nanopartículas de ouro estabilizadas com 2,4,6-trimercapto-1,3,5-triazina e estudada a química do ligante na superfície das nanopartículas de ouro. Além disso, foi estudada a influência dos equilíbrios ácido-base nos espectros SERS e monitorou-se as bandas mais diretamente sensíveis ao pH, associando as mudanças observadas aos diversos equilíbrios das espécies presentes em solução. Por fim, explorou-se o conceito de sensoriamento através das mudanças nos modos vibracionais dos espectros SERS, na formação de complexos em superfície. / Surface modification of gold nanoparticles with a molecule exhibiting several coordination points, allowed to elaborate an interesting concept for quantitative sensing conjugated with the SERS effect. In such systems it is important to know how the molecule binds to the surface, since, based on those informations one can elaborate sensors displaying high sensibility.. However, in dealing with the interaction of nanoparticles with light, the electrodynamic-quantum theories related to the interactions of the surface plasmons are not yet completely developed. In this way, in this t dissertation we made use of the current theories to understand the origin of the plasmons and of how they interact with the the nanoparticles. We have synthesized gold nanoparticles stabilized with 2,4,6-trimercapto-1,3,5-triazine and studied the ligand chemistry at the particles surface.. The influence of acid-base equilibria was studied based on the changes of the SERS spectra SERS with the pH, and interpreted in terms of the several equilibria of the species in solution. Finally, a new sensing concept, based on the observed changes in the SERS spectra in the presence of metal ions, was successfully demonstrated.

Candida albicans, endo-saprophyte du tube digestif, est responsable d’infections superficielles ou invasives. Les candidoses invasives (CI) constituent un problème persistant de santé publique avec une mortalité attribuable de 40% en réanimation et une morbidité exponentielle liée à la consommation d’antifongiques ou aux séjours prolongés des patients. Cet impact médico-économique est principalement lié aux difficultés diagnostiques. En effet, ni la symptomatologie, ni l’imagerie ne sont spécifiques des CI. Le diagnostic biologique demeure complexe et souvent tardif. Les tests conventionnels (hémoculture) sont peu sensibles, et les tests alternatifs à la culture manquent souvent de sensibilité ou de spécificité. Dans ce contexte, il est nécessaire de développer de nouvelles stratégies diagnostiques en mesure d’améliorer la précocité du diagnostic, d’identifier les sujets à haut risque d’infection et de permettre la mise en place d’un traitement antifongique approprié. Notre travail a porté sur deux médiateurs circulants de l’immunité anti-Candida, la mannose-binding lectin (MBL) et les anticorps anti- proteines (Ab) en relation le diagnostic des CI.En ce qui concerne la détection d’anticorps, nous avons produit 6 protéines recombinantes (Hwp1, Eno1, Hsp90, Fba1, Mp65 et Sod5), surexprimées pendant la phase pathogène de C. albicans et développés 5 prototypes de tests EIA. L’association de ces biomarqueurs avec la détection du mannane circulant (Mn) par EIA a permis d’identifier 3 protéines à fort potentiel diagnostique. Pour une spécificité fixée arbitrairement à 80%, la sensibilité des associations Fba1-Ab/Mn, Hwp1-Ab/Mn et Hsp90-Ab/Mn étaient de 84%, 84% et 81% respectivement. Le délai moyen de positivité était de 5 jours avant l’isolement de Candida d’hémoculture. Les Odds ratios associés aux trois combinaisons de marqueurs étaient de 108, 65 et 77 pour Fba1-Ab/Mn, Hwp1-Ab/Mn et Hsp90-Ab/Mn.En ce qui concerne la MBL, nous avons montré i) une évolution dynamique de la MBL au cours des CI, ii) une interaction directe avec le mannane pariétal de levure ainsi qu’une iii) prédisposition à la colonisation chez les patients déficients en MBL. Outre les aspects quantitatifs, nous avons mis en place un modèle d’évaluation fonctionnels de la MBL par resonance plasmonique de surface (SPR) en impliquant des cellules entières ou des glycannes fongiques issus de la paroi de C. albicans ou des oligommanosides de synthèse. Ce modèle nous a permis de mieux caractériser les épitopes reconnus par la MBL et de montrer l’interaction de cette lectine avec un panel d’espèces Candida communément isolées en pathologie humaine.Ces médiateurs de l’immunité humorale ou innée permettent d’envisager des perspectives cliniques en termes diagnostique ou pronostique. Ils permettent également d’améliorer notre compréhension de la physiopathologie des CI et d’optimiser la prise en charge des patients. / Candida albicans, digestive tract endo-saprophyte, is responsible of superficial and invasive infections. Invasive candidiasis (IC) is a persistent public health with an attributable mortality of 40% in intensive care unit and an exponential morbidity related to the antifungals consumption and extended hospital stays of patients. This medico-economic impact is mainly related to diagnostic difficulties. Indeed, neither clinical symptoms nor the imaging are specific of IC. Biological diagnosis remains complex and often late. Conventional tests (blood culture) have a low sensitivity, and non-culture based methods often lack sensitivity or specificity. In this context, it’s necessary to develop new diagnostic strategies to improve the early diagnosis, to identify patients with high risk of infection and to lead the prescription of an appropriate antifungal therapy. Our work has focused on two circulating anti-Candida immunity mediators, mannose-binding lectin (MBL) and anti-protein antibodies (Ab) in relation to IC diagnosis. Regarding the detection of antibodies, we produced six recombinant proteins (Hwp1, Eno1, Hsp90, FBA1, Mp65 and Sod5) overexpressed during the C. albicans pathogen phase and developed five prototypes of EIA. The association of these biomarkers with the detection of circulating mannan (Mn) revealed three proteins with high diagnostic performances. For a specificity fixed arbitrarily at 80%, the sensitivity of combinations of Fba1-Ab/Mn, Hwp1-Ab/Mn and Hsp90-Ab/Mn were 84%, 84% and 81%, respectively. The average delay of positivity was 5 days before isolation of Candida from blood culture. Odds ratios associated with these three combinations of markers were 108, 65 and 77 for Fba1-Ab/Mn, Hwp1-Ab/Mn and Hsp90-Ab/Mn, respectively. Our findings on serum MBL have shown i) dynamic evolution of MBL levels during IC, ii) direct interaction with circulating cell wall mannan and iii) susceptibility to colonization in MBL deficient patients. Besides quantitative aspects, we have developed a model for assessing the MBL functionality by surface plasmon resonance (SPR) involving whole cells, fungal cell wall glycans from C. albicans cell wall or synthetic oligommanosides. This model was used to better characterize the epitopes recognized by MBL and showed the interaction of this lectin with a panel of Candida species commonly isolated in human pathology.These mediators of innate or humoral immunity open new clinical perspectives in terms of diagnostic or prognosis of IC. These findings may participate to improve our understanding of IC pathophysiology and to optimize management of patients.

The bacterial meningitis caused by Neisseria meningitidis is responsible for considerable morbidity and mortality throughout the world. Given the limitations of existing diagnostic tests and the severity of the illness associated with the disease, there is a clear requirement for a rapid and specific diagnostic assay. This thesis describes the development of nanoparticle based tests for the detection of Neisseria meningitidis specific cell surface markers. As an initial target antigen, a recombinant form of highly conserved outer membrane protein, OMP85 was used. Within the OMP85 protein sequence, a predicted antigenic sequence between residues 720 and 745 was identified and found to be unique to this organism. This amino acid sequence was synthesised as peptide (SR1) with a gly-gly-cysteine spacer sequence at the N-terminus using t-boc chemistry. Also, the major virulence factor, capsular polysaccharide of N. meningitidis serogroup B bacteria was purified. Polyclonal antibodies were raised against purified OMP85 antigen in rabbits and against SR1 peptide and also against formalin inactivated N. meningitidis serogroup B whole cell bacteria in sheep. This panel of different antibodies including the commercial anti-capsular monoclonal antibodies were examined for cross reactivity against a range of closely related Gram negative bacteria. Based on these cross-reactivity studies, a highly specific anti-NM antibody was developed following purification of the anti-SR1 antiserum by immuno-affinity chromatography. Purified OMP85 antigen and anti-OMP85 antibody were successfully conjugated on 13, 30, 40, 50 and 60 nm gold nanoparticles by an electrostatic adsorption method. Coupling of the gold nanoparticles results in a shift of the respective surface plasmon peak toward longer wavelengths (in the range of 600-800 nm) resulting in a change of the colour of the colloidal suspension from red to purple to blue. An attempt was made to develop a rapid diagnostic assay based on gold nanoparticle induced colour shift assay for N. meningitidis by utilising the specific interaction of OMP85 and anti-OMP85 antibody conjugated to gold nanoparticles as a model system. However, this system was not reproducible and is likely to be due to problems with stability of gold nanoparticles during the conjugation process. As an alternative approach, a highly selective quartz crystal microbalance (QCM)-based immunosensor was designed using the same OMP85/anti-OMP85 antibody system. A method was developed using polyvinylidene fluoride (PVDF) coated QCM crystals with protein A for the directional orientation of the antibodies. To further enhance the sensitivity of the test, OMP85-conjugated gold nanoparticles were used as signal amplification probes for the reproducible detection of the target down to 300 ng/mL, corresponding to a five fold increase in sensitivity compared to detection of OMP85 antigen alone. Also, this sensor has successfully been employed to detect whole cell bacteria at a concentration as low as 100 cfu/mL. Thus, in this study using the real-time QCM measurements, a novel strategy has been developed for the sensitive detection of both N. meningitidis bacteria and the protein antigen at very low concentrations, using gold nanoparticles as signal amplification probes.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 361-380).

<p>Immobilization of different bio- and organic molecules on solid supports is fundamental within many areas of science. Sometimes, it is desirable to obtain a directed orientation of the molecule in the immobilized state. In this thesis, the copper (I) catalyzed Huisgen 1,3-dipolar cycloaddition, referred to as a “click chemistry” reaction, was explored as a means to perform directed immobilization of small molecule ligands on gold surfaces. The aim was to synthesize alkyne- and azide-terminated alkanethiols that would form well-organized self assembled monolayers (SAMs) on gold from the commercially available substances orthoethylene glycol and bromo alkanoic acid. N-(23-azido-3,6,9,12,15,18,21-heptaoxatricosyl)-n-mercaptododekanamide/hexadecaneamide (n = 12, 16) were successfully synthesized and allowed to form SAMs of different compositions to study how the differences in density of the functional groups on the surface would influence the structure of the monolayer and the click chemistry reaction. The surfaces were characterized by different optical methods: ellipsometry, contact angle goniometry and infrared reflection-absorption spectroscopy (IRAS). The click reaction was found to proceed at very high yields on all investigated surfaces. Finally, the biomolecular interaction between a ligand immobilized by click chemistry on the gold surfaces and a model protein (bovine carbonic anhydrase) was demonstrated by surface plasmon resonance using a Biacore system.</p>