Surface-enhanced Raman scattering (SERS) has long been the interest of researchers in chemistry, physics and engineering, especially since the discovery that SERS can probe into the system down to the single molecule (SM) level. Despite the large number of publications regarding the fabrication of SERS substrates, it has been a challenge in the field to quantify the SERS signal and universally compare substrates. Traditionally, enhancement factor (EF) is used as an indicator of substrate quality, but the EF calculation is hugely dependent on the estimation of the surface coverage and other factors that are determined largely subjectively. Therefore, this thesis aims at discussing other parameters that can also be used to evaluate different substrates. Six different SERS substrates of Ag or Au nanoparticles of different sizes were fabricated by nanosphere lithography (NSL) and characterized by electron microscopy and UV-vis spectroscopy. SERS substrates were mapped for different concentrations of a probe molecule. Through subsequent baseline correction and principle component analysis (PCA), the "intensity" of individual spectrum was obtained and the shapes of intensity histograms of each substrate were acquired. Instead of calculating EF, five criteria (six quantification methods in total) were employed to comprehensively evaluate the six substrates. These were density of hot spots (characterized by the number of zero-intensity events), enhancement (represented by mean intensity), spatial variation (calculated by RSD of intensity), repeatability (realized by cross correlation) and histogram shape (quantified by skewness and kurtosis). These new methods provide insights to the understanding of the properties of SERS substrates in terms of hot spots. Different substrates may exhibit better performance in terms of one criterion but worse in terms of others. Those variations in performance can be explained by their surface morphology. These more elaborated methods are believed to provide a more comprehensive approach to evaluate and compare substrates than the traditional EF values. The thesis also paves the way for future study on SM-SERS and fabricating better SERS substrates. / Graduate

Surface enhanced Raman scattering (SERS) has achieved much attention since its conception in 1974. The analytical technique overcomes many difficulties associated with conventional Raman whilst also increasing sensitivity. However, the increased interest and work in the field has also identified flaws, many of which are centred on the irreproducibility of the SERS enhancement effect. The majority of the work described in this thesis focusses on the ‘optimisation’ of solid-state and solution based SERS systems. Optimisation plays a crucial role in maximising both enhancement effects and reproducibility. Here criteria are outlined for the synthesis of high performance solid-state SERS substrates and the synthesis of a range of substrates is assessed, each with associated pros and cons. The most successful substrate was synthesised by exploiting redox potentials which allow for the direct deposition of silver onto copper foil. The deposition times and temperatures were optimised sequentially to generate a high performance substrate capable of detecting Rhodamine 6G at trace levels. Reproducibility comparisons of the silver on copper (SoC) substrate were carried out against commercial substrates: Klarite and QSERS, multiple univariate and multivariate methods were used to assess the substrates performance. The results confirmed that the SoC substrate performed better than both the commercial substrates. The work also highlights the importance of using multiple data analysis methods in order to assess the performance of a solid-state SERS substrate. Deposition of the silver surface was also successful on British 2p coins allowing the for the detection and discrimination of illegal and legal drugs when coupled with multivariate data analysis methods such as PCA and PLS. Solution based SERS analyses were also carried out successfully using different optimisation strategies. The initial investigation involved careful control of the individual components of a SERS system (nanoparticles, aggregating agents and analyte) in order to establish a low limit of detection for the increasingly abused ‘legal high’ MDAI. The use of a reduced factorial design was then successfully employed to explore a greater number of SERS variables and define a low limit of detection for the class B drug mephedrone. The robust experimental design also allowed an insight into the importance of each of the individual components within a solution based SERS system. The final piece of work carried out was the SERS discrimination of antibiotics: ampicillin, ticarcillin and carbenicillin. Optimisation of the solution based experiment allowed the in-situ hydrolysis of the β-lactam moiety present in ampicillin rendering it pharmacologically inactive to be followed under acidic conditions at concentrations of 10 ppm.

Thesis (Ph.D.)--Boston University / The large electromagnetic field enhancement provided by nanostructured noble metal surfaces forms the foundation for a series of enabling optical analytical techniques, such as surface enhanced Raman spectroscopy (SERS), surface enhanced IR absorption spectroscopy (SEIRA), surface enhanced fluorescent microscopy (SEF), to name only a few. Critical sensing applications have, however, other substrate requirements than mere peak signal enhancement. The substrate needs to be reliable, provide reproducible signal enhancements, and be amenable to a combination with microfluidic chips or other integrated sensor platforms. These needs motivate the development of engineerable SERS substrate "chips" with defined near- and far-field responses. In this dissertation, two types of rationally designed SERS substrates - nanoparticle cluster arrays (NCAs) and SERS stamp - will be introduced and characterized. NCAs were fabricated through a newly developed template guided self-assembly fabrication approach, in which chemically synthesized nanoparticles are integrated into predefined patterns using a hybrid top-down/bottom-up approach. Since this method relies on chemically defined building blocks, it can overcome the resolution limit of conventional lithographical methods and facilitates higher structural complexity. NCAs sustain near-field interactions within individual clusters as well as between entire neighboring clusters and create a multi-scale cascaded E-field enhancement throughout the entire array. SERS stamps were generated using an oblique angle metal deposition on a lithographically defined piston. When mounted on a nanopositioning stage, the SERS stamps were enabled to contact biological surfaces with pristine nanostructured metal surfaces for a label-free spectroscopic characterization. The developed engineered substrates were applied and tested in critical sensing applications, including the ultratrace detection of explosive vapors, the rapid discrimination of bacterial pathogens, and the label-free monitoring of the enzymatic degradation of pericellular matrices of cancer cells.

Plasmonic nanoparticles, specifically gold and silver nanoparticles, exhibit unique optical, physical, and chemical properties that are exploited in many biomedical applications. Due to their nanometer size, facile surface functionalization and enhanced optical performance, gold and silver nanoparticles can be used to investigate cellular biology. The work herein highlights a new methodology that has exploited these remarkable properties in order to probe various aspect of cancer cell biology, such as cell cycle progression, drug delivery, and cell death. Cell death mechanisms due to localized gold and silver nanoparticle exposure were also elucidated in this work. Chapter 1 introduces the reader to the synthesis and functionalization of gold and silver nanoparticles as well as reviews their implementation in biodiagnostic and therapeutic applications to provide a foundation for Chapters 3 and 4, where their use in spectroscopic and cytotoxic studies are presented. Chapter 2 provides the reader with detailed explanations of experimental protocols for nanoparticle synthesis and functionalization, in vitro cellular biology experiments, and live-cell Raman spectroscopy experiments that were utilized throughout Chapters 3 and 4. Chapter 3 presents the use of nuclear-targeted gold nanoparticles in conjunction with a Raman microscope modified to contain a live-cell imaging chamber to probe cancer cell cycle progression (Chapter 3.1), examine drug efficacy (Chapter 3.2), monitor drug delivery (Chapter 3.3), and detect apoptotic molecular events in real-time (Chapter 3.4). In Chapter 4, the intracellular effects of gold and silver nanoparticles are explored through live-cell Rayleigh imaging, cell cycle analysis and DNA damage (Chapter 4.1), as well as through the elucidation of cytotoxic cell death mechanisms after nanoparticle exposure (Chapter 4.2) and live cell imaging of silver nanoparticle treated cancer cell communities (Chapter 4.3).

The purpose of this study is to demonstrate the ability of surface enhanced Raman scattering (SERS) active particles to enable multiplexed immunoassays in a lateral flow format for point of care (POC) testing. The SERS particles used for this study are chemically active glass coated gold particles, containing tracer molecules which in principle can be chosen to provide Raman Spectra with unique features allowing multiple tracers to be simultaneously measured and distinguished without interference between each other. Lateral flow immunoassay technology is the important part of this study and can be conveniently packaged for the use of other than highly skilled technicians outside of the laboratory. A well-known (single channel - simplex) device for the pregnancy test is a typical example of the lateral flow assay. Similar formats have been/are being developed by others for a range of POC applications – but most diagnostic applications require simultaneous determination of a range of biomarkers and multiplexed assays are difficult to achieve without significant interference between the individual assays. This is where SERS particles may provide some advantages over existing techniques. Cardiac markers are the growing market for point of care technology therefore biomarkers of cardiac injury (Troponin, myoglobin and CRP) have been chosen as a model. The object of the study is to establish the proof of concept multiplexing assay using these chosen biomarkers. Thus, initially all different particles were characterised in single and mixture form. Also development of conjugate chemistry between antibodies for each analyte that have been purchased from commercial sources and SERS particles were analysed using different conditions like buffer, pH and antibody loading concentration to get the optimum intensity. The selected SERS particles and their conjugates were tested for size, aggregation and immune quality using a range of techniques: ultraviolet-visible (UV/Vis) absorption spectroscopy, dynamic light scattering (DLS) and lateral flow assay. These characterisations methodologies gave the understanding of optimum conditions of the each conjugates and individual’s behaviour in mixture conditions as well. After the characterisation all conjugates were tested singularly on the lateral flow assay using buffers and serum. The results of this single analyte immunoassay explained the individual’s bioactivity on the lateral flow strip. Further in study, multiplex assay have been demonstrated in serum. These outcomes have described each candidate characteristic in a mixture form on the lateral flow strip. In order to get the optimum Raman intensity from multiplex assay, the detection and capture antibodies loading concentrations were tuned in the assay. Later on different combinations (high, medium and low concentrations) of all three analytes were analysed and has found some interferences in multiplex assay. To investigate these issues various aspect were considered. First of all, different possibilities of non-specific interactions between the co-analytes and antibodies were tested. In addition, steric hindrance and optical interference investigations were performed via several assays and analysis using Scanning electron microscopy. The outcomes have confirmed related optical interferences. Therefore other assay (wound biomarkers) established to eliminate the interferences. In summary, the works reported here have built and test the equipment and necessary reagents for individual assays before moving on the more complicated task. In addition, the entire study has given a deep knowledge of multiplex assay on a single test line including the investigation of the issues for selected cardiac biomarkers and their applications in the future.

The Warburg effect describes the survival advantage of cancer cells in that they can proliferate under low oxygen/hypoxic conditions via a less efficient pathway known as glycolysis. It has not yet been documented at which point, in an oxygen gradient, phenotypic changes occur. Measuring the intracellular redox potential (IRP) and its impact on cellular dynamics would provide greater insight into how disruption of redox homeostasis caused by changes in oxygen concentration leads to aberrant cell signalling and diseases such as cancer. Current techniques in measuring IRP include redox-sensitive fluorescent proteins such as roGFP which is glutathione-specific. Measuring the concentration of one redox couple is, however, not an accurate representation of IRP as it does not necessarily inform about the state of other redox couples. Furthermore, fluorescent biosensors can suffer from photobleaching and may interact with other oxidants. The IRP was measured, in this work, using our newly developed novel-class of surface enhanced Raman scattering nanoparticles which can quantitatively measure the redox potential of cells in vitro. A 'homemade' device was created to keep the cells under fixed pO2 whilst obtaining measurements. The IRP was correlated with the transcriptomic and downstream metabolic profiles of MCF7 breast cancer cells, under perturbed pO2, using 1H NMR spectroscopy (NMR), mass spectrometry (MS) and RNA-sequencing. Discriminatory metabolites were all associated with energy and glucose metabolism. Discriminatory microRNAs were all affiliated with the hallmarks of cancer; the regulation of some is controlled by transcription factors containing redox-sensitive motifs in their DNA binding domains. Multivariate analysis techniques were used to analyse the different data streams in a holistic way that allows the correlation of redox potential, metabolism and transcription.

Protein engineering strategies, such as directed evolution, generate large libraries of enzyme variants, typically in the range of 106-108 variants. However, the availability of rapid, robust high-throughput screening methods has often limited the impact of directed evolution in discovering enzymes with enhanced catalyst performance. Raman spectroscopy is an established analytical technique, providing molecular specific information, permitting analysis in aqueous solutions and as such is an attractive, alternative screening method for biological systems. Although an inherently weak physical phenomenon, enhanced Raman scattering techniques, such as surface enhanced Raman scattering (SERS) and ultraviolet resonance Raman (UVRR) spectroscopy, can be used to overcome the associated sensitivity issues. Herein, we successfully monitored xanthine oxidase (XO) catalysed conversions of xanthine to uric acid, before extending to hypoxanthine, using two contrasting Raman scattering enhanced approaches. Firstly, a SERS-based assay was developed utilising silver nanoparticles to measure analytes directly and quantitatively on micromolar scale, in the absence of chromogenic substrates or lengthy chromatography. Secondly, a UVRR approach was developed enabling monitoring of the XO-mediated reaction in real-time and without the need to quench the system. Significantly, both methods demonstrated over &gt;30 fold reduction in acquisition times (when compared to conventional HPLC analysis), and offered excellent medium-term reproducibility and accuracy of results over significant time periods. Furthermore, investigations were made into developing this SERS-based assay into an enantiomeric screen using another vibrational spectroscopy approach, Raman optical activity (ROA), along with circular dichroism (CD). Successful chiral reduced nanoparticles were synthesised, with multiple characterisation techniques employed, affording enantiopure Au-cysteine and Ag-tyrosine colloids. However, it was not possible to generate consistent and reproducible SEROA responses, with these techniques ultimately being unsuccessful in analysing these chiral sensitive nanoprobes, and thus differentiating between the D- and L- forms. Finally, a novel SERS-based approach, in combination with the standard addition method (SAM), was developed for the routine analysis of uric acid (end product in XO catalysed reaction(s) and biomarker for various diseases), at clinically relevant levels in urine samples from patients. Results were highly comparable and in very good agreement with HPLC analyses, with an average < 9% difference in predictions between the two analytical approaches across all samples analysed, and a 60-fold reduction in acquisition time (when compared with HPLC). Together, the research presented in this thesis demonstrates the suitability of Raman enhanced techniques for quantitative analysis, measuring the analytes directly using a portable Raman instrument and, most importantly, offering significant reductions in acquisition times when compared to established analytical techniques.

The main theme of this thesis is to study effects of different environments on geometric and electronic structures, as well as optical responses, of molecules using time-(in)dependent density functional theory. Theoretical calculations have been carried out for properties that can be measured by conventional and advanced experimental techniques, including one-photon absorption (OPA), two-photon absorption (TPA), surface-enhanced Raman scattering (SERS) and second order nonlinear optical (NLO) response. The obtained good agreement between the theory and the experiment allows to further extract useful information about inter- and intra-molecular interactions that are not accessible experimentally. By comparing calculated one-photon absorption spectra of aluminum phthalocyanine chloride (AlPcCl) and AlPcCl -water complexes with the corresponding experiments, detailed information about the interaction between water molecules and AlPcCl, and geometric changes of AlPcCl molecule has been obtained. Effects of aggregation on two-photon absorption spectra of octupolar molecules have been examined. It is shown that the formation of clusters through inter-molecular hydrogen bonding can drastically change profiles of TPA spectra. It has also demonstrated that a well designed molecular aggregate/cluster, dendrimer, can enhance the second order nonlinear optical response of the molecules. In collaboration with experimentalists, a series of end-capped triply branched dendritic chromophores have been characterized, which can lead to large enhancement of the second order NLO property when the dipoles of the three branches in the dendrimers are highly parallelized. Surface-enhanced Raman scattering has made the detection of single molecules on metal surface become possible. Chemically bonded molecule-metal systems have been extensively studied. We have shown in a joint experimental and theoretical work that stable Raman spectra of a non-bonding molecule, perylene, physically adsorbed on Ag nano-particles can also be observed at low temperature. It is found that the local enhanced field has a tendency to drive molecule toward a gap of two closely lying nano-particles. The trapped molecule can thus provide a stable Raman spectrum with high resolution when its thermal motion is reduced at low temperature. For the ever growing size of molecular complexes, there is always the need to develop new computational methods. A conceptually simple but computationally efficient method, named as central insertion scheme (CIS), is proposed that allows to calculate electronic structure of quasi-periodic system containing more than 100,000 electrons at density functional theory levels. It enables to monitor the evolution of electronic structure with respect to the size of the system. / QC 20100823

<p>The electromagnetic properties of various plasmonic nanostructures are investigated. These nanostructures, which include random clusters, controlled clusters and particle-film hybrids are applied to surface-enhanced Raman scattering (SERS). A variety of techniques are utilized to fabricate, characterize, and model these SERS-active structures, including nanoparticle functionalization, thin film deposition, extinction spectroscopy, elastic scattering spectroscopy, Raman scattering spectroscopy, single-assembly scattering spectroscopy, transmission electron microscopy, generalized Mie theory, and finite element method. </p><p>Initially, the generalized Mie theory is applied to calculate the near-field of the small random clusters to explain their SERS signal distribution. The nonlinear trend of SERS intensity versus size of clusters is demonstrated in experiments and near-field simulations. </p><p>Subsequently, controlled nanoparticle clusters are fabricated for quantitative SERS. A 50 nm gold nanoparticle and 20nm gold nanoparticles are tethered to form several hot spots between them. The SERS signal from this assembly is compared with SERS signals from single particles and the relative intensities are found to be consistent with intensity ratios predicted by near-field calculation.</p><p>Finally, the nanoparticle/film hybrid structure is studied. The scattering properties and SERS activity are observed from gold nanoparticles on different substrates. The gold nanoparticle on gold film demonstrates high field enhancement. Raman blinking is observed and implies a single molecule signal. Furthermore, the doughnut shape of Raman images indicates that this hybrid structure serves as nano-antenna and modifies the direction of molecular emission. </p><p>In additional to the primary gap dipole utilized for SERS, high order modes supported by the nanoparticle/film hybrid also are investigated. In experiments, the HO mode show less symmetry compared to the gap dipole mode. The simulation indicates that the HO modes observed may be comprised of two gap modes. One is quadrupole-like and the other is dipole-like in terms of near-field profile. The analytical treatment of the coupled dipole is performed to mimic the imaging of the quadrupole radiation.</p> / Dissertation

In this research, two major experiments, including the self-assembly of silica spheres, were performed by using a physical confinement method with an attractive capillary force. The silica spheres were dragged and aggregated as results of the evaporation of the solvent. In the first experiment, silica spheres were assembled into the two-dimensional pattered substrate, constructed by the photo-resist film formed under a lithography process. Several patterned substrates could work as a physical trap during the flow of the silica spheres. The ordered arrangement of the silica spheres was controlled by the concentration and the size of the silica spheres, the thickness of the photo-resist film, and the titled angle of the substrate. In our conditions, the silica spheres could orderly arrange in larger area of the substrate. In the second experiment, a surface-enhanced Raman scattering (SERS) enhancement was observed from a chain of silica spheres with silver nanoparticles, which worked as a excitation source to provid a strong local electromagnetic fields exciting the crystal violet (CV) dye coated on the silica spheres. We found that the CV molecules has a strong SERS intensity due to the refraction and reflection of the incident light within the silica spheres. When the silica spheres were linearly arranged, longer length of the chained silica spheres would lead to a maximum value of the SERS intensity.