Cette thèse se concentre sur la caractérisation d'assemblages organisés de nanoparticules plasmoniques appelés supracristaux. Les nanoparticules utilisées mesurent entre 5 et 11 nm de diamètre et possèdent une distribution en taille étroite permettant leur organisation à trois dimensions. Une fois les supracristaux obtenus, nous avons mesuré les spectres d'absorbance de supracristaux individuels constitués de différentes nanoparticules : cuivre, argent et or. Un modèle théorique simple a été utilisé pour calculer les spectres d'absorbance à partir de données disponibles dans la littérature. Nous avons ensuite montré que les supracristaux de nanoparticules d'argent ou d'or constituent de nouveaux substrats présentant un grand intérêt pour des applications en Spectroscopie Raman Exaltée de Surface (SERS). Les tailles des nanoparticules utilisées sont en effet beaucoup plus petites que celles reportées dans la littérature, d'où un nombre plus important de points chauds et donc une grande sensibilité. En vue d'applications couplées électrochimie/Raman, nous avons également mesuré les spectres de réflectance sous potentiel. De plus, des mesures par AFM conducteur et par microscopie électrochimique montrent que la conductivité des assemblages est faible mais autorise néanmoins des transferts d'électrons entre les supracristaux les plus fins et une sonde redox en solution. Enfin, des expériences de microscopie holographique ont permis de suivre la formation de ces édifices en solution. / This thesis focuses on the characterization of organized assemblies of plasmonic nanoparticles called supracrystals. The nanoparticles have a diameter ranging from 5 to 11 nm diameter and a narrow size distribution allowing their organization at three dimensions. Once the supracrystals were obtained, we measured the absorbance spectrum of individual supracrystals made of various nanoparticles: copper, silver or gold. A simple theoretical model was used to calculate the absorbance spectrum from available data in the literature. We then demonstrated that the supracrystals made of silver or gold nanoparticles are suitable substrates for Surface Enhanced Raman Spectroscopy applications. The sizes of the nanoparticles we use are indeed much smaller than in the literature, so that the number of hot spots and thus the sensitivity are increased. In view of coupling electrochemistry and Raman spectroscopy for future applications, we also measured the reflectance spectrum under potential control. Moreover, conductive AFM and Scanning ElectroChemical Microscopy measurements demonstrate that even if the conductivity of the supracrystals is weak, electron transfers between thin supracrystals and a redox probe in solution are nevertheless possible. Finally, holographic microscopy experiments allowed to follow the formation of these structures inside the solution.

Raman spectroscopy is an established analytical technique for determining molecular structure, whose major drawback is lack of sensitivity. Enhanced Raman scattering techniques, such as surface-enhanced Raman scattering (SERS) and tip-enhanced Raman scattering (TERS), utilise nanoscale substrates to enhance the Raman signal through the interaction of surface charges with the incident electromagnetic radiation. Here, nanoparticle-based SERS was used to detect dipicolinic acid (DPA), a biomarker for bacterial spores. Whilst this has been demonstrated previously, the use of a different nanoparticle aggregation mechanism and the inclusion of an internal standard has enabled a SERS detection method to be developed that is quantitative to almost an order of magnitude lower than previously reported. Moreover, for the first time, a nanoparticle-based SERS method was applied to the detection of viable Bacillus spores. Investigations were made into the possibility of SERS enhancement using deep UV laser excitation at 244 nm using a novel boron nitride surface material. This semiconductor has a band gap of comparable magnitude to the laser excitation wavelength and therefore had the potential to impart a SERS enhancement via a chemical enhancement mechanism. Whilst initial results looked promising using Rhodamine 6G as a test analyte, it was not possible to demonstrate reproducibly and no enhancement was observed on other analytes that were tested. TERS was shown to be able to discriminate between glycosylated and non-glycosylated forms of protein molecules, based on the measurement of just a few molecules at a time. This was achieved even without control of the protein interaction with the TERS substrate. The vibrational peak positions in TERS experiments were shown to be highly dependent on the analyte’s orientation relative to the TERS tip, giving variable and complex spectral data. As such, the data processing and analysis methods had to be carefully considered in order to eliminate bias. Lastly, a novel SERS detector for high-performance liquid chromatography (HPLC) was built and tested. It was shown to be able to quantify purine bases from mixtures in tandem with, and in lower amounts than the conventionally used UV absorbance detection, even when the analyte peaks were co-eluting. This quantitative analysis is conducted on-line and in real-time, making it applicable to high throughput applications. Together the four research projects presented in this thesis make a significant contribution to the field of enhanced Raman scattering and promote its sensitivity and reproducibility as a quantitative analytical technique for the trace analysis of biomolecules.

This PhD thesis focuses on improving the limit of detection (LOD) of Raman biosensors by using surface enhanced Raman scattering (SERS) and/or hollow core photonic crystal fibers (HC-PCF), in conjunction with statistical methods. Raman spectroscopy is a multivariate phenomenon that requires statistical analysis to identify the relationship between recorded spectra and the property of interest. The objective of this research is to improve the performance of Raman biosensors using SERS techniques and/or HC-PCF, by applying partial least squares (PLS) regression and principal component analysis (PCA). I began my research using Raman spectroscopy, PLS analysis and two different validation methods to monitor heparin, an important blood anti-coagulant, in serum at clinical levels. I achieved lower LOD of heparin in serum using the Test Set Validation (TSV) method. The PLS analysis allowed me to distinguish between weak Raman signals of heparin in serum and background noise. I then focused on using SERS to further improve the LOD of analytes, and accomplished simultaneous detection of GLU-GABA in serum at clinical levels using the SERS and PLS models. This work demonstrated the applicability of using SERS in conjunction with PLS to measure properties of samples in blood serum. I also used SERS with HC-PCF configuration to detect leukemia cells, one of the most recurrent types of pediatric cancers. This was achieved by applying PLS regression and PCA techniques. Improving LOD was the next objective, and I was able to achieve this by improving the PLS model to decrease errors and remove outliers or unnecessary variables. The results of the final optimized models were evaluated by comparing them with the results of previous models of Heparin and Leukemia cell detection in previous sections. Finally, as a clinical application of Raman biosensors, I applied the enhanced Raman technique to detect polycystic ovary syndrome (PCOS) disease, and to determine the role of chemerin in this disease. I used SERS in conjunction with PCA to differentiate between PCOS and non-PCOS patients. I also confirmed the role of chemerin in PCOS disease, measured the level of chemerin, a chemoattractant protein, in PCOS and non-PCOS patients using PLS, and further improved LOD with the PLS regression model, as proposed in previous section.

Metabolomics is a post genomic field of research concerned with the study of low molecular weight compounds within a biological system permitting the investigation of the metabolite differences between natural and perturbed systems (such as cells, organs and tissues). Rapid identification and discrimination of biological samples based upon metabolic differences and physiological status in microbiology, mammalian systems (particularly for disease diagnosis), plants and food science is highly desirable. Volatile organic compound (VOC) profiling is a novel area of research where the composition of the VOCs emitted by the biological samples can be correlated to its origin and physiological status. The aim of this project was to investigate the applicability of VOC profiling as a potential complementary tool within metabolomics.In this project the discrimination of bacteria using a novel gas phase separation method was investigated and the development of VOC-based profiling tools for the collections of VOCs emitted from biological samples was also studied. The optimisation and validation of a high throughput method for VOC analysis was achieved and this was used to assess wound healing.VOC metabolite profiling was further extended to the discrimination of S. typhimurium contaminated meat; the study was conducted in parallel with metabolite profiling analysis for the analysis of non-volatile small molecules. Finally, enhanced vibrational spectroscopic techniques were applied to the characterisation and screening of dye molecules in contaminated foodstuffs using Raman spectroscopy. This thesis clearly demonstrates that VOC metabolic profiling is a complementary tool within the metabolomics toolbox, one of its great attractions is that it permits the characterisation of biological samples in a rapid and non-invasive manner. The technique provides detailed chemical information regarding the VOC composition present above the headspace of the sample and can be used to understand its physiological status and biological origin. VOCs metabolite profiling will become a valuable tool for non-invasive analysis of many biological systems. Raman spectroscopy is a sensitive and non-destructive technique which can generate detailed chemical and structural information regarding the analyte under investigation with little or no sample preparation needed. The effect of the weak Raman signal can be significantly amplified by coupling the analyte molecule to surfaces of nanoparticles and demonstrated that it is ideal for analysing aqueous dye solutions in a quantitative manner.

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.

This thesis explores new analytical advances using compact disk biosensing technologies, and comprises six scientific publications distributed along four chapters. Special attention is herein payed to Thermochromic Etching Disks (TED) technology (Chapter 1), rational design of disk-based biorecognition assays (Chapter 2), and label-free detection systems for point-of-need analysis (Chapters 3 and 4). First, insights into a novel light-mediated signal developing system for biorecognition assays (based on TED disks and drives) are provided together with an overview of the state-of-the art and future trends in photo- and thermochromic biosensing. This signal developing approach exploits photo- and thermochromism for biosensing in an original manner and represents a potential strategy to simplify signaling processes in bioanalytical systems. Then, how to transform TED technology into lab-on-a-disk systems is addressed. TED has proven to be a very versatile tool to perform sensitive analysis of biorecognition assays, using platforms and scanners easily obtained from regular disks and drives, respectively. Biologically relevant assays of different nature (microarray, cell culture, immunofiltration, turbidimetry, etc.) have been arrayed in a single disk and sensitively analyzed by imaging. Regarding rational design, a theoretical-experimental method (INSEL) based on kinetics and mass-transport modelling for optimizing biorecognition assays and exploring their behavior is presented. INSEL has been implemented as an in silico tool that enables to characterize biointeractions with minimal experimentation, to perform optimizations directed towards custom objectives defined by the user, and to easily compute the effect of critical variables without further experiments. In another study included in this thesis, polycarbonate grooved structures obtained from standard recordable disks (CD-R and DVD-R) were coated with silver and tailored to become SERS-active. This strategy represents a cost-effective and industrially scalable alternative to the SERS substrates typically used for bioanalysis. These disk-based materials have presented tunable plasmonic responses, significant Raman enhancement, and have allowed complex biological targets (such as proteins and exosomes) to be analyzed by SERS without using labeled reagents as tracers. In addition to introduce inexpensive and large-scale SERS substrates for biosensing, this study also suggests the development of prospective Raman scanners based on disk drives. Another approach herein presented addresses the implementation of diffraction-based sensing (DBS) in TED technology in order to conceive disk-based label-free biosensors based on standard disks and drives. At first, a comprehensive experimental assessment of the analytical possibilities offered by DBS is presented. Then, the fabrication of arrays of diffractive protein networks on TED disks is investigated, with which sensitive analysis of antibodies in label-free conditions has been demonstrated, using adapted drives as scanners. This investigation provides important insights into cost-effective and industrially scalable functional materials and detection setups that exploit consumer electronics for label-free biosensing. / Esta tesis explora nuevos avances en química analítica usando tecnologías de biosensado basadas en sistemas de disco compacto y comprende seis publicaciones científicas distribuidas a lo largo de cuatro capítulos. Los estudios se han centrado en la tecnología Thermochromic Etching Disks (TED) (Capítulo 1), el diseño racional de ensayos de bioreconocimiento en discos compactos (Capítulo 2), y la detección sin marcaje para realizar análisis in situ (Capítulos 3 y 4). Primero, enmarcado en una discusión del estado del arte y futuras tendencias en biosensado foto y termocrómico, se presenta un nuevo sistema (basado en discos y lectores TED) mediado por luz para el desarrollo de señales en ensayos de bioreconocimiento. Ésta constituye una estrategia novedosa para aprovechar el foto y termocromismo en biosensado, y presenta un gran potencial para simplificar los procesos de desarrollo de señal en sistemas bioanalíticos. A continuación, se aborda cómo transformar la tecnología TED en sistemas analíticos integrados basados en discos compactos. TED ha demostrado ser una herramienta muy versátil para analizar, de forma sensible, ensayos de bioreconocimiento usando plataformas y escáneres fácilmente obtenidos a partir de discos y lectores convencionales, respectivamente. Un único disco ha mostrado poder albergar varios ensayos biológicos importantes y de distinta naturaleza (micromatriz, cultivos celulares, inmunofiltración, turbidimetría, etc.), para ser analizados de forma sensible a través de imágenes En cuanto al diseño racional, se presenta un método teórico-experimental (INSEL), basado en modelos cinéticos y de transporte de masa, para optimizar ensayos de bioreconocimiento y explorar su comportamiento. INSEL se ha implementado como una herramienta in silico que permite caracterizar biointeracciones mediante mínima experimentación, realizar optimizaciones dirigidas a objetivos particulares definidos por el usuario, y computar el efecto de variables críticas de forma sencilla y sin experimentos adicionales. En otro estudio incluido en esta tesis, nanoestructuras en forma de surco obtenidas a partir de discos regrabables convencionales (CD-R y DVD-R) fueron recubiertas con plata y adaptadas para ser activas en SERS. Esta estrategia supone una alternativa, económicamente efectiva e industrialmente escalable, a los sustratos SERS típicamente usados en bioanálisis. Estos materiales han mostrado respuestas plasmónicas sintonizables, una amplificación Raman significativa, y han permitido analizar muestras biológicas complejas (como proteínas y exosomas) mediante SERS sin usar marcadores. Además de introducir sustratos SERS grandes y baratos, este trabajo también sugiere el desarrollo de escáneres Raman basados en lectores de disco. Otra aproximación presentada en esta tesis aborda la implementación de DBS (diffraction-based sensing) en tecnologías TED, con el fin de desarrollar biosensores para detección sin marcaje basados en discos y lectores convencionales. Primero, se presenta una amplia evaluación experimental de las posibilidades analíticas ofrecidas por DBS. A continuación, se investiga la fabricación de multitud de redes difractivas de proteínas sobre discos TED, con las que se ha demostrado la determinación sensible y sin marcaje de anticuerpos, usando lectores adaptados como escáneres analíticos. Esta investigación introduce avances importantes que apuntan al desarrollo de materiales funcionales y sistemas de detección, baratos e industrialmente escalables, que aprovechen las tecnologías de consumo para realizar biosensado sin marcaje. / Aquesta tesi explora nous avanços en la química analítica usant tecnologies de biosensat basades en sistemes de disc compacte, i comprèn sis publicacions científiques distribuïdes en quatre capítols. Els estudis s'han centrat en la tecnologia Thermochromic Etching Disks (TED) (Capítol 1), el disseny racional d'assajos de bioreconeixement en discos compactes (Capítol 2), i la detecció sense marcatge per realitzar anàlisi in situ (Capítols 3 i 4). Primer, dins del marc d'una discussió de l'estat de l'art i tendències futures en biosensat foto i termocròmic, es presenta un nou sistema (basat en discos i lectors TED) per al desenvolupament de senyals mitjançant llum, en assajos de bioreconeixement. Aquesta constitueix una nova estratègia per aprofitar el foto i termocromisme en biosensat, mentre que també presenta una gran potencial per simplificar els processos de desenvolupament de senyal en sistemes bioanalítics. Tot seguit, s'aborda com transformar la tecnologia TED en sistemes analítics integrats basats en discos compactes. TED ha demostrat ser una eina molt versàtil per analitzar, de forma sensible, assajos de bioreconeixement usant plataformes i escàners fàcilment obtinguts a partir de discos i lectors convencionals, respectivament. Un únic disc ha mostrat poder albergar diversos assajos biològicament importants i de distinta naturalesa (micromatrius, cultius cel·lulars, immunofiltració, turbidimetria, etc.), per a ser analitzats de forma sensible a través d'imatges. Pel que fa al disseny racional, es presenta un mètode teòric-experimental (INSEL), basat en models cinètics i de transport de massa, per optimitzar assajos de bioreconeixement i explorar el seu comportament. INSEL s'ha implementat com a una eina in silico que permet caracteritzar biointeraccions amb mínima experimentació, realitzar optimitzacions dirigides cap a objectius particulars definits per l'usuari, i computar l'efecte de variables crítiques de forma senzilla i sense experiments addicionals. En un altre estudi inclòs en aquesta tesi, nanoestructures en forma de solc obtingudes a partir de discos compactes regravables convencionals (CD-R i DVD-R) van ser recobertes amb plata i adaptades per a ser actives en SERS. Aquesta estratègia suposa una alternativa, econòmicament efectiva i industrialment escalable, als substrats SERS típicament usats en bioanàlisi. Aquests materials han mostrat respostes plasmòniques sintonitzables, una amplificació Raman significativa, i han permès analitzar mostres biològiques complexes (com proteïnes i exosomes) mitjançant SERS sense usar marcadors. A més d'introduir substrats SERS grans i barats, aquest treball també suggereix el desenvolupament d'escàners Raman basats en lectors de disc. Una altra aproximació presentada en aquesta tesi aborda la implementació de DBS (diffraction-based sensing) en tecnologies TED, per tal de desenvolupar biosensors basats en discos i lectors convencionals que permeten detecció sense marcatge. Primer, es presenta una amplia avaluació experimental de les possibilitats analítiques que ofereix aquesta tècnica. A continuació, s'investiga la fabricació de multitud de xarxes difractives de proteïnes sobre discos TED, amb les quals s'ha demostrat la determinació sensible i sense marcatge d'anticossos, usant lectors adaptats com a escàners analítics. Aquesta investigació introdueix avanços importants que apunten cap al desenvolupament de materials funcionals i sistemes de detecció, barats i industrialment escalables, que aprofiten les tecnologies de consum per dur a terme bioanàlisi sense marcatge. / Avellà Oliver, JM. (2017). Towards label-free biosensing in compact disk technologies f or point-of-need analysis [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/86128 / TESIS

Tato diplomova prace se zabyva prpravou a charakterizac GaN nanokrystalu s kovovym jadrem. V teoreticke casti teto prace je predstaven material GaN se svymi vlastnos- tmi a aplikacemi. Dale jsou uvedeny substraty pro rust a jednotlive mechanismy rustu GaN nanokrystalu. V dalsm jsou popsany kovove nanocastice a jejich opticke vlastnosti umoznujc zesilovan fotoluminiscence na zaklade interakce plasmonu a GaN. Experi- mentaln cast se zabyva prpravou GaN nanokrystalu s Ag jadrem ve ctyrech krocch. Prvne jsou Ag nanocastice naneseny na substrat Si(111). Nasledne se nechaj zoxidovat. Tretm krokem je depozice Ga a poslednm je nitridace. Jednotlive kroky byly opti- malizovany a analyzovany ruznymi metodami, jako je XPS, SEM, fotoluminiscence a Ramanova spektroskopie.

In this work we studied gallium on graphene. Depositions were done by Molecular beam epitaxy. We observed Raman enhancement and peak shifts by individual Ga islands. Simulation confirmed our assumption, that the enhancement is based on plasmonics effect that is also the main contribution of Surface-enhanced Raman spectroscopy. Another result is hydrogenation of graphene before deposition does have an effect on Ga structure and reduces diffusion length of Ga atoms.

Two types of 3-dimensional (3D) Ag nanosponge aggregates were prepared and tested as samples for surface-enhanced Raman scattering (SERS) and as active surfaces for surface- enhanced luminescence. 3D Ag nanosponge aggregates were assembled from 2D fused fractal aggregates (D = 1.87 ± 0.02) prepared by modification of Ag nanoparticle (NP) hydrosol resulting from the reduction of AgNO3 by NH2OH·HCl. For SERS measurements, 3D Ag nanosponge aggregates with incorporated [Ru(bpy)3]2+ cations and chloride anions were prepared and overlayed by a thin layer of aqueous phase. For SEL measurements, the 3D Ag nanosponge aggregates were assembled from fused fractal aggregates of chloride- modified Ag NPs. After preparation the active surface was overlayed by a 1×10-5 M aqueous solution of [Ru(bpy)3]2+ . The SERRS (1×10-15 M) and SER(R)S (1×10-14 M) limits of detection of [Ru(bpy)3]2+ determined at 445 and 532 nm excitations, respectively, correspond to the single molecule level of the complex detection. Its achievement is attributed to a large electromagnetic mechanism enhancement experienced by [Ru(bpy)3]2+ incorporated in "hot spots", an efficient localization of "hot spots" in the 3D aggregate to the focus of the laser beam in micro-Raman spectral measurements and to a molecular resonance contribution to the...

Title: SERS spectroscopy of model biomolecules for SERS biosensing Author: Martin Šubr Department: Institute of Physics of Charles University Supervisor: doc. RNDr. Marek Procházka, Dr., Institute of Physics of Charles University Abstract: The main requirement for surface enhanced Raman scattering (SERS)-based biomolecular sensing is high sensitivity and spectral reproducibility. For this purpose, ordered silver and gold nanostructures fabricated by magnetron sputtering and lithography methods at cooperating institutes were tested in this work. Reproducible SERS spectra of employed model biomolecules (amino acids, lysozyme and albumin) were obtained on ordered silver surfaces at concentrations 10-4 M - 10-6 M and as low as ≈ 10-7 M in the case of porphyrins. SERS spectra of certain biomolecules were also compared to spectra measured on silver colloid. The limit of detection provided by hydroxylamine-reduced silver colloid, using KCl as an aggregating agent, is substantially lower (on the order of 10-8 M for cysteine), but with lower spectral reproducibility. The main drawback of SERS spectra measured on silver surfaces was the occurrence of spurious bands resulting from the preparation procedure. In the case of sputter-deposited silver surfaces, it was found that keeping the substrates several hours in...