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This thesis deals with the study of microbial pigment violacein in the real sample of lyophilized microorganisms. The sample was investigated by using methods of vibrational spectroscopy with focusing on the applicability of surface enhanced and resonance micro-Raman spectroscopy. For this purpose several different systems for enhancing Raman intensity together with the set of excitation lasers emitting in the visible light region were used. The conclusion of this thesis are the recommendations connected with the appropriateness of using each amplifying systems and excitation wavelengths for the successful identification of violacein pigment in the sample. Powered by TCPDF (www.tcpdf.org)

Title: Optical Responses of Biomolecules on Regular Metal Plasmonic Nanostructures Author: Martin Šubr Department: Institute of Physics of Charles University Supervisor of the doctoral thesis: prof. RNDr. Marek Procházka, Dr., Institute of Physics of Charles University Abstract: Adsorption of molecules on metal plasmonic nanostructures leads to significant enhancement of many optical processes, such as Raman scattering (surface-enhanced Raman scattering - SERS) or fluorescence (surface-enhanced fluorescence - SEF). Two groups of substrates were tested within this thesis: (i) Silver nanorods prepared by oblique angle vapor deposition, and (ii) silver and gold nanoislands growing on magnetron-sputtered polytetrafluoroethylene film. Step-by- step optimization process was performed on the nanoislands in order to obtain optimum SERS sensitivity and reproducibility. Detailed SERS intensity profiles were obtained using gradient nanostructures with the localized surface plasmon resonance (LSPR) condition varying across the sample and three different excitation wavelengths. It was also found that spectral position and height of the LSPR band can be controlled simultaneously using mixed gold/silver nanoislands. Detailed investigation of polarization- and angular- dependences of anisotropic silver nanorods was...

The first thermal cycling experiments of ionic self-assembled multilayer (ISAM) films have been reported examining their survivability through repeated thermal cycles from -20° C to 120° C in ambient atmospheric conditions. The films were constructed from alternating layers of Nile Blue A and gold nanoparticles which provided a strong absorbance in the optical wavelength range. No degradation of the optical characteristics of the ISAM films was observed [1]. Techniques for measuring the capacitance and resistivity of various ISAM films have also been developed allowing for a more complete electrical characterization of ISAM films. Capacitance measurements enabled a calculation of the dielectric function and breakdown field strength of the ISAM films. The capacitance measurement technique was verified by measuring the dielectric function of a spin-coated thin film PMMA, which has a well characterized dielectric function [2]. Surface-enhanced Raman spectroscopy (SERS) has been studied as a possible detection method for malignant melanoma revealing spectral differences in blood sera from healthy horses and horses with malignant melanoma. A SERS microscope system was constructed with the capability of resolving the Raman signal from biologically important molecules such as beta-carotene and blood sera. The resulting Raman signals from sera collected from horses with malignant melanoma were found to have additional peaks not found in the Raman signals obtained from sera collected from healthy horses. A systematic analysis of the combination of absorbance and fluorescence signals of blood sera collected from populations of healthy dogs and dogs with cancer has resulted in a rapid and cost-effective method for monitoring protein concentrations that could possibly be used as part of a cancer screening process. This method was developed using the absorbance and fluorescence signals from known serum proteins, the combinations of which were used to match the absorbance and fluorescence signals of blood sera allowing for an accurate determination of protein concentrations in blood sera [3]. Finally, a novel method for measuring the melting point of DNA in solution using capacitance measurements is presented. This method allows for the determination of the melting temperature as well as the melting entropy and melting enthalpy of DNA strands. Two different short strands of DNA, 5'-CAAAATAGACGCTTACGCAACGAAAAC-3' along with its complement and 5'-GGAAGAGACGGAGGA-3' along with its complement were used to validate the technique as the characteristics of these strands could be modeled using theoretical methods. This experimental technique allows for the precise determination of the melting characteristics of DNA strands and can be used to evaluate the usefulness of theoretical models in calculating the melting point for particular strands of DNA. Additionally, a micro-fluidic device has been proposed that will allow for a rapid and cost-effective determination of the melting characteristics of DNA [4]. / Ph. D.

The propagation of contaminants in the environment continues to threaten public health and safety. Conventional analytical techniques for environmental detection require centralized facilities and intensive resources for operation. An effective implementation of a wide network of field deployable point-of-use (POU) sensors can potentially enable real-time monitoring of water quality parameters and inform decision making on public health outbreaks. The use of nanotechnology and field-deployable analytical tools can potentially advance the development of POU sensors for future field application. In this dissertation, we developed environmental sensing techniques that utilize nanocomposites made of low-cost, biocompatible, and sustainable nanomaterials combined with Raman spectroscopy. First, a technology pre-assessment was performed that included a comprehensive evaluation of cellulose-derived nanocomposites and nanobiotechnology enabled techniques for their sustainable long-term environmental application. Furthermore, to contribute to the better understanding of the potential environmental implications of nanomaterial production and application, life cycle assessment (LCA) was used to evaluate the environmental impacts of six iron precursors and seven iron oxide nanoparticle synthesis methods. Secondly, in the technology development step, gold (Au) and iron oxide (Fe3O4) nanoparticles were incorporated onto bacterial cellulose nanocrystals and nanoscale magnetite were synthesized. As proof-of-concept environmental applications, the Au@Fe3O4@BCNCs were applied for the magnetic separation and surface-enhanced Raman scattering (SERS) detection of malachite green isothiocyanate (MGITC), and nanoscale magnetite were applied for phosphate (PO43-) removal and recovery from synthetic urine matrices. Finally, in the technological application step, three environmental sensing applications are presented that use nanomaterial-based sensor platforms and/or Raman spectroscopic techniques. The first application involved using Lectin-modified BCNCs coupled SERS and machine learning for discrimination of bacterial strains. The second application presents a simple Raman-stable isotope labeling approach for the study of viral infection of bacteria. The third application involved use of SERS pH nanoprobes for measuring pH in droplets of complex matrices (e.g., DMEM cell culture media, human saliva). / Doctor of Philosophy / The current generation of analytical tools for environmental detection rely upon centralized facilities and intensive resources for operation. The combination of nanotechnology and field deployable analytical tools can aid in the development of point-of-use (POU) sensors for field monitoring of environmental contaminants. In this dissertation, we combined low-cost, biocompatible, and sustainable nanomaterials with Raman spectroscopy-based techniques to develop potentially field-deployable environmental sensing techniques. First, a technology pre-assessment was performed which involved a comprehensive evaluation of cellulose-derived nanocomposites and nanobiotechnology enabled techniques for their sustainable long-term environmental application. Furthermore, life cycle assessment (LCA) was used to evaluate the environmental impacts of iron oxide nanoparticle synthesis methods to better understand environmental impacts of nanoparticle production. Secondly, in the technology development step, we developed the nanocomposites: Au and Fe3O4 nanoparticles incorporated bacterial cellulose nanocrystals and nanoscale magnetite. As proof-of-concept environmental applications, the Au@Fe3O4@BCNCs were used for the detection of malachite green isothiocyanate (MGITC), and the nanoscale magnetite were used for phosphate (PO43-) removal and recovery from synthetic urine. Finally, in the technological application step, (1) selective detection of bacteria was performed using lectin-modified BCNCs as SERS biosensors coupled with SERS and machine learning. (2) Viral infection of bacteria was evaluated using Raman spectroscopy and Deuterium isotope labeling, and (3) pH in micro-droplets of DMEM cell culture media and human saliva were observed using SERS pH nanoprobes.

Surface enhanced Raman spectroscopy (SERS) is highly promising analytical technique for trace detection of analytes. It is particularly well suited for environmental analyses due to its high sensitivity, specificity, ease of operation and rapidity. The detection and characterization of environmental contaminants, using SERS is highly related to the uniformity, activity and reproducibility of the SERS substrate. In this thesis, SERS substrates were produced by gold nanoparticle formation on wax patterned chromatography paper. In situ reduction of hydrogen tetrachloroaurate (gold precursor) by trisodium citrate dihydrate (reducing agent) was used to produce gold nanoparticles within a paper matrix. These gold nanoparticle based SERS substrates were analyzed by FE-SEM, UV-Vis and Raman spectroscopy. This work discusses the SERS signal enhancements for Raman active MGITC dye for a series of substrates prepared by in situ reduction of gold salt and pre-produced gold nanoparticles. UV-Vis analysis was performed to understand the effect of different molar ratio (reducing agent to gold precursor) and reaction time on the size and shape of the localized surface plasmon resonance (LSPR) band that dictates the SERS enhancements. It was concluded that lower molar ratio (1:1 and 2:1) of citrate-to gold produced better SERS signal enhancements and broader LSPR band. Therefore, use of lower molar ratio (MR) was recommended for paper-based substrates using in situ-based reduction approach. / Master of Science

Corneal cross-linking (CXL) with riboflavin and ultraviolet A light is a therapeutic procedure to restore the mechanical stability of corneal tissue. The treatment method is applied to pathological tissue, such as keratoconus and induces the formation of new cross-links. At present, the molecular mechanisms of induced cross-linking are still not known exactly. In this study, we investigated molecular alterations within porcine cornea tissue after treatment with riboflavin and ultraviolet A light by surface enhanced Raman spectroscopy (SERS). For that purpose, after CXL treatment a thin silver layer was vapor-deposited onto cornea flaps. To explore molecular alterations induced by the photochemical process hierarchical cluster analysis (HCA) was used. The detailed analysis of SERS spectra reveals that there is no general change in collagen secondary structure while modifications on amino acid side chains are the most dominant outcome. The formation of secondary and aromatic amine groups as well as methylene and carbonyl groups were observed. Even though successful cross-linking could not be registered in all treated samples, Raman signals of newly formed chemical groups are already present in riboflavin only treated corneas.

Raman spectroscopy is a powerful tool in the field of biomedicine for disease diagnosis owing to its potential to provide the molecular fingerprint of biological samples. However due to the inherent weak nature of the Raman process, there is a constant quest for enhancing the sensitivity of this technique for enhanced diagnostic efficiency. This thesis focuses on achieving this goal by integrating advanced methods with Raman spectroscopy. Firstly this thesis explores the applicability of a laser based fluorescence suppression technique – Wavelength Modulated Raman Spectroscopy (WMRS) - for suppressing the broad luminescence background which often obscure the Raman peaks. The WMRS technique was optimized for its applications in single cell studies and tissue studies for enhanced sensing without compromising the throughput. It has been demonstrated that the optimized parameter would help to chemically profile single cell within 6 s. A two fold enhancement in SNR of Raman bands was demonstrated when WMRS was implemented in fiber Raman based systems for tissue analysis. The suitability of WMRS on highly sensitive single molecule detection techniques such as Surface Enhanced Raman Spectroscopy (SERS) and Surface Enhanced Resonance Raman Spectroscopy (SERRS) was also explored. Further this optimized technique was successfully used to address an important biological problem in the field of immunology. This involved label-free identification of major immune cell subsets from human blood. Later part of this thesis explores a multimodal approach where Raman spectroscopy was combined with Optical Coherence Tomography (OCT) for enhanced diagnostic sensitivity (>10%). This approach was used to successfully discriminate between ex-vivo adenocarcinoma tissues and normal colon tissues. Finally this thesis explores the design and implementation of a specialized fiber Raman probe that is compatible with surgical environments. This probe was originally developed to be compatible with Magnetic Resonance Imaging (MRI) environment. It has the potential to be used for performing minimally invasive optical biopsy during interventional MRI procedures.

Protein structures (cytochrome c) were visualized by TERS measurements on whole mitochondria referring to specific spectral features describing the electronic state of the heme moiety. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

The adsorbate formed by adsorption of thiophenol on a polycrystalline gold electrode and brought into contact with aqueous solutions of 1 M HClO4 and 0.1 M KClO4 has been studied using cyclic voltammetry and surface-enhanced Raman spectroscopy. A strong adsorption is deduced from observations made using cyclic voltammetry. From the SER spectra, interactions of thiophenol with the gold surface via a gold–sulfur bond with the aromatic ring pointing away from the surface is concluded for both electrolyte solutions. / Dieser Beitrag ist aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.