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.

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.

Intracellular redox potential (IRP) is a measure of how oxidising or reducing the environment is within a cell. It is a function of numerous factors including redox couples, antioxidant enzymes and reactive oxygen species. Disruption of the tightly regulated redox status has been linked to the initiation and progression of cancer. However, there is very limited knowledge about the quantitative nature of the redox potential and pH gradients that exist in cancer tumour models. Multicellular tumour spheroids (MTS) are three-dimensional cell cultures that possess their own microenvironments, similar to those found in tumours. From the necrotic core to the outer proliferating layer there exist gradients of oxygen, lactate, pH and drug penetration. Tumours also have inadequate vasculature resulting in a state of hypoxia. Hypoxia is a key player in metabolic dysregulation but can also provide cells with resistance against cancer treatments, particularly chemotherapy and radiation therapy. The primary hypoxia regulators are HIFs (Hypoxia Inducible Factors) which under low O2 conditions bind a hypoxia response element, inhibiting oxidative phosphorylation and upregulating glycolysis which has two significant implications: the first is an increase in levels of NADPH/NADH, the main electron donors found in cells which impacts the redox state, whilst the second is a decrease in intracellular pH (pHi) because of increased lactate production. Thus, redox state and intracellular pHi can be used as indicators of metabolic changes within 3D cultures and provide insight into cellular response to therapy. Surface-Enhanced Raman Spectroscopy (SERS) provides a real-time, high resolution method of measuring pHi and IRP in cell culture. It allows for quick and potentially portable analysis of MTS, providing a new platform for monitoring response to drugs and therapy in an unobtrusive manner. Redox and pH-active probes functionalised to Au nanoshells were readily taken up by prostate cancer cell lines and predominantly found to localise in the cytosol. These probes were characterised by density functional theory and spectroelectrochemistry, and their in vitro behaviour modelled by the chemical induction of oxidative and reductive stress. Next, targeting nanosensors to different zones of the MTS allowed for spatial quantification of redox state and pHi throughout the structure and the ability to map the effects of drug treatments on MTS redox biology. The magnitude of the potential gradient can be quantified as free energy (ΔG) and used as a measurement of MTS viability. Treatment of PC3 MTS with staurosporine, an apoptosis inducer, was accompanied by a decrease in free energy gradients over time, whereas treatment of MTS with cisplatin, a drug to which they are resistant, showed an increase in viability indicating a compensatory mechanism and hence resistance. Finally, using this technique the effects of ionising radiation on IRP and pHi in the tumour model was explored. Following exposure to a range of doses of x-ray radiation, as well as single and multi-fractionated regimes, IRP and pHi were measured and MTS viability assessed. Increased radiation dosage diminished the potential gradient across the MTS and decreased viability. Similarly, fractionation of a single large dose was found to enhance MTS death. This novel SERS approach therefore has the potential to not only be used as a mode of drug screening and tool for drug development, but also for pre-clinical characterisation of tumours enabling clinicians to optimise radiation regimes in a patient-specific manner.

This thesis reports the investigation of spontaneous Raman Spectroscopy (RS) for its applicability in early drug discovery. A key focus has been to develop an understanding of the applicability of RS for the quantification and localisation of compound concentration inside mammalian cells. Further investigation into the use of Surface Enhanced Raman Spectroscopy (SERS) for research on Visceral Leishmaniasis (VL) and Leishmania donovani as well as investigating applicability for cancer research are decisive parts of this work. The key work described in this thesis is the investigation of whole cell concentration of compounds inside THP-1 and Madin Darby Canine Kidney (MDCK) cells. For true quantification the Cell Silent Region (CSR) is used to measure without interference from cellular background signal. The model compound is erlotinib, an anti-cancer drug with an alkyne group expressing a peak in the CSR. The developed RS system is calibrated using the current gold standard technique Ultra Performance Liquid Chromatography tandem Mass Spectrometry (UPLC-MS/MS). However, because of the single cell nature of the RS information on inter cell variability can be extracted. The RS measurements suggest that there is a large variation of concentration within single cell populations. The RS measurements can therefore give insight in single cell behaviour within a large cell population. Findings shows that washing cycles, before fixation, alter the intra-cellular concentrations significantly. This is hypothesised to be caused by the sudden change in concentration on the outside of the cell that applies an osmotic pressure, leading to loss of substance from inside the cell wall. Localisation of erlotinib is shown within THP-1 cells and points towards an accumulation inside the cell nucleus. Later, internalised Au nano-particles in the range of 30 nm to 80 nm have been investigated for their enhancement effects and localisation inside THP-1 cells. Au nano-particles are found to be internalised easily by differentiated THP-1 cells and accumulate in lysosomes. This allows for a high local enhancement of the spontaneous Raman signal. However, no advantage for the detection of lysosomally trapped compounds (chloroquine, chlorpromazine) was achieved. The detection of substances without a signal in the CSR was achieved without enhancement. Nonetheless, compounds with intrinsic peaks in the CSR could benefit from this enhancement. Lastly the RS system is explored for alternative uses in early drug discovery. This includes the detection of toxicity as well as the discrimination of cell types. Toxicity has been detected using optically trapped THP-1 cells and doxorubicin. Utilising Principal Component Analysis (PCA) combined with Linear Discriminant Analysis (LDA) on these measured spectra, allowed for a clear discrimination of toxically influenced from healthy cells. Differences mainly show up in DNA content caused by the mode of action of doxorubicin and caused by the trapping, which generates most of the signal within the nucleus of the cell. Discriminating cancerogenic (DU145) from healthy prostate cells (PNT2) has been achieved by probing fixed cells and evaluating the acquired Raman spectra with a PCA/LDA combination. The accuracy of separation of these cells when tested with a 10-fold cross-validation technique, is above 98 %, allowing a good discrimination.

ABSTRACT PREPARATION AND CHARACTERIZATION OF SILVER SERS NANOTAGS Kibar, Seda M.S., Department of Chemistry Supervisor: Prof. Dr. M&uuml / rvet Volkan December 2010, 88 pages Tags are materials used for labeling substances and so make possible the qualitative and quantitative analysis both in macroscopic and microscopic world. Nowadays, surface enhanced Raman spectroscopy became the favored one among the optical based-tag detection systems. Progress in surface enhanced Raman detection and imaging technologies depends on the availability of Raman labels with strong light scattering characteristics. In this study various SERS nanotags were prepared. An ideal SERS nanotag consists of three parts, core nanoparticle for enhancement, Raman active molecule for signature and a shell for protection and further functionalization. As a core material, silver nanoparticles were prepared using the chemical reduction method with sodium citrate as reductant. SERS enhancement provided by Ag particles prepared was examined. For colloidal stabilization and further surface modifications, silica with a controlled thickness was deposited on Ag nanoparticles. Three single-dye doped nanotags, Ag-BCB@SiO2 Ag-CFV@SiO2 and Ag-CV@SiO2 were prepared using positively charged dyes, brilliant cresyl blue (BCB), cresyl fast violet (CFV) and cresyl violet (CV). The effects of silica thickness and dye concentration in the reaction medium were examined. Stability of prepared nanotags and repeatability of the method were investigated. Multi-dye doped nanotags were prepared using BCB and CFV solutions mixed at various concentration ratios. Resulting Raman spectra Ag-BCB-CFV@SiO2 nanotags successfully exhibited characteristic peaks of each dye with a good resolution. In addition, the molar ratio between dyes BCB and CFV was reflected on the related spectra. A linear correlation was observed between the molar ratio of the dyes and their Raman intensity ratio.

The objectives of the work presented were to 1) fabricate reproducible nanorod array SERS substrates, 2) detection of bacteria using nanorod substrates, 3) detection of DNA hybridization using nanorod substrates and 4) critically evaluate the sensing method. Important findings from this work are as follows. A novel method for batch fabrication of substrates for surface enhanced Raman scattering (SERS) has been developed using a modified platen machined to fit in a commercial electron beam evaporator. The use of this holder enables simultaneous deposition of silver nanorod (AgNR) arrays onto six microscope slide substrates utilizing glancing angle deposition. In addition to multiple substrate fabrication, patterning of the AgNR substrates with 36 wells allows for physical isolation of low volume samples. The well-to-well, slide-to-slide, and batch-to-batch variability in both physical characteristics and SERS response of substrates prepared via this method was nominal. A critical issue in the continued development of AgNR substrates is their stability over time, and the potential impact on the SERS response. The thermal stability of the arrays was investigated and changes in surface morphology were evaluated using scanning electron microscopy and x-ray diffraction and correlated with changes in SERS enhancement. The findings suggest that the shelf-life of AgNR arrays is limited by migration of silver on the surface. Continued characterization of the AgNR arrays was carried out using fluorescent polystyrene microspheres of two different sizes. Theory suggests that enhancement between nanorods would be significantly greater than at the tops due to contributing electromagnetic fields from each nanostructure. In contrast to the theory, SERS response of microspheres confined to the tops of the AgNR array was significantly greater than that for beads located within the array. The location of the microspheres was established using optical fluorescence and scanning electron microscopy. The application of SERS to characterizing pathogens such as bacteria and viruses is an active area of investigation. AgNR array-based SERS substrates have enabled detection of pathogens present in biofluids. Specifically, several publications have focused on determining the spectral bands characteristic of bacteria from different species and cell lines. Studies were carried out on three strains of bacteria as well as the medium in which the bacteria were grown. The spectra of the bacteria and medium were surprisingly similar, so additional spectra were acquired for commonly used bacterial growth media. In many instances, these spectra were similar to published spectra purportedly characteristic of specific bacterial species. In addition to bacterial samples, nucleic acid hybridization assays were investigated. Oligonucleotide pairs specifically designed to detect respiratory syncytial virus (RSV) in nasal fluids were prepared and evaluated. SERS spectra acquired on oligos, alone or in combination, contain the known spectral signatures of the nucleosides that comprise the oligo. However, spectra acquired on an oligo with a 5'- or 3' thiol were distinctly different from that acquired on the identical oligo without a thiol pendant group suggesting some control over the orientation of the oligo on the nanorod surface. The signal enhancement in SERS depends markedly upon the location of the probe relative to the substrate surface. By systematic placement of nucleotide markers along the oligo chain, the point at which the nucleotide disappears from the spectrum was identified. The overall findings for AgNR SERS substrates suggest that the applicability of SERS for detecting nucleic acid hybridization is limited. The strong distance dependence coupled with the lack of substrate stability at temperatures required for annealing oligos during hybridization suggest that AgNRs are not the platform to use for hybridization assays.

Raman spectroscopy is a promising technique because it contains abundant vibrational chemical information. However, Raman spectroscopy is restricted by its small scattering cross section, and many techniques have been developed to amplify Raman scattering intensity. In this dissertation, I study two of these techniques, coherent Raman scattering and surface enhanced Raman scattering and discuss their properties. In the first part of my dissertation, I investigate two coherent Raman processes, coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS). In CARS project, I mainly focus on the molecular resonance effect on detection sensitivity, and I find the detection sensitivity can be pushed into 10 [micromolar] with the assistance of molecular resonance. Also, I am able to retrieve background-free Raman spectra from nonresonant signals. For SRS, we develop a new SRS system by applying spectral focusing mechanism technique. We examine the feasibility and sensitivity of our SRS system. The SRS spectra of standards obtained from our system is consistent with literature, and the sensitivity of our system can achieve 10 times above shot-noise limit. In second part of this dissertation, I study surface enhanced Raman scattering (SERS) and related plasmonic effects. I synthesize different shapes of nanoparticles, including nanorod, nanodimer structure with gap and pyramids by template method, and study how electric field enhancement effects correlate to SERS by two photon luminescence (TPL). Also, I build an optical system to study optical image, spectra and particle morphology together. I find that SERS intensity distribution is inhomogeneous and closely related to nanoparticle shape and polarization direction. However, TPL and SERS are not completely correlated, and I believe different relaxation pathways of TPL and SERS and coupling of LSPR and local fields at different frequencies cause unclear correlation between them. / text