Raman spectroscopy, together with multivariate statistical analyses, has proven to be a near real-time analytical technique capable of phenotyping cells, tissues and organs. This dissertation will show exclusively the application of the Raman spectroscopy phenotypic profiling method to; (i) microbial toxicity, (ii) ex-vivo organ perfusion, and (iii) subcellular location targeting. Real-time analytical methods for monitoring living biological systems will enable study of the physiological changes associated with growth, genetic manipulations, and adverse environmental conditions. Most existing analytical methods (NMR exempt), though highly accurate, must be performed off-line and most require destruction of the studied sample. These attributes make these methodologies less desirable to the study of physiological changes of cells, tissues, and organs. In this work, Raman spectroscopy has been identified and shown to be a good candidate for real-time analysis mainly because it can be performed: (i) in near real-time, (ii) non-destructively and with minimal sample preparation, (iii) through a glass barrier (i.e., can be performed in situ), and (iv) with minimal spectral interference from water. Here, Raman spectroscopy was used in combination with multivariate statistics to analyze the differing toxic effects of 4-C chain alcohols on E. coli. Good correlations were established between Raman spectra and off-line analytical techniques used to measure: (i) saturated, unsaturated, and cyclopropane fatty acids; (ii) amino acid composition of total protein; and (iii) cell membrane fluidity. Also, Raman 'fingerprint' analysis was used to discriminate among different phenotypic responses of cells. In addition, this methodology was applied to analyze perfusates of organs maintained by the VasoWave® organ perfusion system. Raman fingerprints can be used to assess organ health, and it is believed this data can be used to inform decisions such as whether or not to transplant an organ. Finally, molecular biology techniques were used to design and produce specific protein targets harboring a silver binding domain fusion, which upon release migrate to specific subcellular locations. By employing the related technique of surface-enhanced Raman scattering (SERS), which produces a highly amplified Raman signal in the presence of metallic nanoparticle substrates (e.g., silver nanoparticles), different regions of the E. coli cell structure were studied. The target regions studied by the technique included: (i) outer cell membrane, (ii) periplasm, and the (iii) cytoplasm. / Ph. D.

Due to an ever-increasing global population and limited resource availability, there is a constant need for detection of both natural and anthropogenic hazards in water, air, food, and material goods. Traditionally a different instrument would be used to detect each class of contaminant, often after a concentration or separation protocol to extract the analyte from its matrix. Raman spectroscopy is unique in its ability to detect organic or inorganic, airborne or waterborne, and embedded or adsorbed analytes within environmental systems. This ability comes from the inherent abilities of the Raman spectrometer combined with concentration, separation, and signal enhancement provided by drop coating deposition Raman (DCDR) and surface-enhanced Raman spectroscopy (SERS). Herein the capacity of DCDR to differentiate between cyanotoxin variants in aqueous solutions was demonstrated using principal component analysis (PCA) to statistically demonstrate spectral differentiation. A set of rules was outlined based on Raman peak ratios to allow an inexperienced user to determine the toxin variant identity from its Raman spectrum. DCDR was also employed for microcystin-LR (MC-LR) detection in environmental waters at environmentally relevant concentrations, after pre-concentration with solid-phase extraction (SPE). In a cellulose matrix, SERS and normal Raman spectral imaging revealed nanoparticle transport and deposition patterns, illustrating that nanoparticle surface coating dictated the observed transport properties. Both SERS spectral imaging and insight into analyte transport in wax-printed paper microfluidic channels will ultimately be useful for microfluidic paper-based analytical device (𝜇PAD) development. Within algal cells, SERS produced 3D cellular images in the presence of intracellularly biosynthesized gold nanoparticles (AuNP), documenting in detail the molecular vibrations of biomolecules at the AuNP surfaces. Molecules involved in nanoparticle biosynthesis were identified at AuNP surfaces within algal cells, thus aiding in mechanism elucidation. The capabilities of Raman spectroscopy are endless, especially in light of SERS tag design, coordinating detection of analytes that do not inherently produce strong Raman vibrations. The increase in portable Raman spectrometer availability will only facilitate cheaper, more frequent application of Raman spectrometry both in the field and the lab. The tremendous detection power of the Raman spectrometer cannot be ignored. / Ph. D.

Increased proliferation of nanotechnology has led to concerns regarding its implication to the water environment. Gold nanoparticles (AuNP) were used as a model nanomaterial to investigate the fate and dynamics of nanoparticles in the complex water environment. A column study was performed to examine the fate and transport of gold nanoparticles with two different coatings in porous media. The resulting data suggested that gold nanoparticles aggregate significantly in the porespace of the column interior, a finding that is not predicted by traditional colloidal filtration theory or Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. Surface-enhanced Raman spectroscopy (SERS) was developed as a new technique to investigate AuNP aggregation in water with varying salt levels. The SERS technique proved valuable as an analytical technique, elucidating information about aggregation as well as AuNP surface interactions with dissolved halides in water. A thorough investigation examining Aunt aggregation with monovalent and divalent salts utilizing SERS, ultraviolet-visible light (UV-Vis) spectroscopy, and dynamic light scattering (DLS) was conducted. Each technique provided data describing different aspects of the dynamic behavior of AuNPs in complex water environments. Results suggest that in addition to attractive and repulsive interactions described by DLVO theory, chemical interactions between the AuNP surface and dissolved halides were also a significant driving force for aggregation and other transformative behaviors of AuNPs in water. The SERS technique developed in this work was shown to be a viable tool to help unveil the vastly complex dynamics of nanomaterial in the water environment. / Ph. D.

Hyper-Raman-Streuung folgt anderen Symmetrieauswahlregeln als Raman-Streuung und profitiert als nicht-linearer Zweiphotonenprozess noch mehr von verstärkten elektromagnetischen Feldern an der Oberfläche plasmonischer Nanostrukturen. Damit könnte die oberflächenverstärkte Hyper-Raman-Streuung (SEHRS) praktische Bedeutung in der Spektroskopie erlangen. Durch die Kombination von SEHRS und oberflächenverstärkter Raman-Streuung (SERS) können komplementäre Strukturinformationen erhalten werden. Diese eignen sich aufgrund der Lokalisierung der Verstärkung auf die unmittelbare Umgebung der Nanostrukturen besonders für die Charakterisierung der Wechselwirkung zwischen Molekülen und Metalloberflächen. Ziel dieser Arbeit war es, ein tieferes Verständnis des SEHRS-Effekts zu erlangen und dessen Anwendbarkeit für analytische Fragestellungen einzuschätzen. Dazu wurden SEHRS-Experimente mit Anregung bei 1064 nm und SERS-Experimente mit Anregung bei derselben Wellenlänge sowie mit Anregung bei 532 nm - für eine Detektion von SEHRS und SERS im gleichen Spektralbereich - durchgeführt. Als Beispiel für nicht-resonante Anregung wurden die vom pH-Wert abhängigen SEHRS- und SERS-Spektren von para-Mercaptobenzoesäure untersucht. Mit diesen Spektren wurde die Wechselwirkung verschiedener Silbernanostrukturen mit den Molekülen charakterisiert. Anhand von beta-Carotin wurden Einflüsse von Resonanzverstärkung im SEHRS-Experiment durch die gleichzeitige Anregung eines molekularen elektronischen Übergangs untersucht. Dabei wurde durch eine Thiolfunktionalisierung des Carotins eine intensivere Wechselwirkung mit der Silberoberfläche erzielt, sodass nicht nur resonante SEHRS- und SERS-Spektren, sondern auch nicht-resonante SERS-Spektren von Carotin erhalten werden konnten. Die Anwendbarkeit von SEHRS für hyperspektrale Kartierung in Verbindung mit Mikrospektroskopie wurde durch die Untersuchung von Verteilungen verschiedener Farbstoffe auf strukturierten plasmonischen Oberflächen demonstriert. / Hyper-Raman scattering follows different symmetry selection rules than Raman scattering and, as a non-linear two-photon process, profits even more than Raman scattering from enhanced electromagnetic fields at the surface of plasmonic nanostructures. Surface-enhanced hyper-Raman scattering (SEHRS) could thus gain practical importance for spectroscopy. The combination of SEHRS and surface-enhanced Raman scattering (SERS) offers complementary structural information. Specifically, due to the localization of the enhancement to the close proximity of the nanostructures, this information can be utilized for the characterization of the interaction between molecules and metal surfaces. The aim of this work was to increase the understanding of the SEHRS effect and to assess its applicability to answer analytical questions. For that purpose, SEHRS experiments with excitation at 1064 nm and SERS experiments with excitation at the same wavelength, as well as with excitation at 532 nm - to detect SEHRS and SERS in the same spectral region - were conducted. As an example for non-resonant excitation, pH-dependent SEHRS and SERS spectra of para-mercaptobenzoic acid were examined. Based on these spectra, the interaction of different silver nanostructures with the molecules was characterized. beta-Carotene was used to study the influence of resonance enhancement by the excitation of a molecular electronic transition during SEHRS experiments. By the thiol-functionalization of carotene, a more intense interaction with the silver surface was achieved, which enables to obtain not only resonant SEHRS and SERS but also non-resonant SERS spectra of carotene. Hyperspectral SEHRS imaging in combination with microspectroscopy was demonstrated by analyzing the distribution of different dyes on structured plasmonic surfaces.

In this diploma thesis, SERRS(Surface - enhanced resonance Raman Scattering) and SERS (Surface - enhanced Raman Scattering), surface of plasmon extinction spectra and TEM images of systems with tetracationic zinc porphyrin ZnTMPyP and silver nanoparticles (Ag NPs) were studied and interpreted. The systems with isolated nanoparticles and the systems with compact aggregates were investigated. The systems with isolated nanoparticles were prepared by addition of ZnTMPyP to hydrosol of Ag NPs. NaCl was added to this system and so compact aggregates were prepared. SERRS and SERS spectra of ZnTMPyP were studied at excitation wavelengths λexc= 441.6 nm a 532 nm. Firstly, stationary systems and secondly dynamic development of hydrosol Ag NPs → Ag NPs/ ZnTMPyP → Ag NPs/ ZnTMPyP/ NaCl systems for 680 s was investigated. Time evolution of SERRS and SERS spectra were analyzed by factor analysis (FA) and time evolution of spectra extinction plasmon were measured. FA showed a strong increase of the signal of ZnTMPyP after the addition of NaCl, i.e. after conversion of isolated nanoparticles to compact aggregates. In these systems with high concentration of ZnTMPyP the increase of signal was succeeded by its decreased. With help of FA the limit of SERRS spectral detection (λexc= 441,6 nm) and limit of SERS spectral...

This work describes a master’s thesis in engineering physics at Umeå University carried out during the spring semester of 2014. In the thesis the student has constructed and tested a temperature controlled cell for cooling/heating of surface-enhanced-Raman-spectroscopy (SERS) substrates for rapid detection of volatile substances. The thesis was carried out at the Swedish Defence Research Agency (FOI) in Umeå, Sweden. A Linkam Scientific Instruments TS1500 cell was equipped with a Peltier element for cooling/heating and a thermistor temperature sensor. A control system was constructed, based on an Arduino Uno microcontroller board and a pulse-width-modulated (PWM) H-bridge motor driver to control the Peltier element using a proportional-integral (PI) control algorithm. The temperature controlled cell was able to regulate the temperature of a SERS substrate within -15 to +110 °C and maintain the temperature over prolonged periods at ±0.22 °C of the set point temperature. Gas phase of 2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane (isoflurane) was flowed through the cell and SERS spectra were collected at different temperatures and concentrations. This test showed that the signal is increased when the substrate is cooled and reversibly decreased when the substrate was heated. Keywords: temperature control, Raman scattering, surface enhanced Raman spectroscopy SERS, SERS substrate, volatile substances, Peltier module, thermistor, PWM, H-bridge, PI(D) control. / Detta dokument beskriver ett examensarbete för civilingenjörsexamen i teknisk fysik vid Umeå Universitet som utförts under vårterminen 2014. I examensarbetet har en kyl-/värmecell för temperaturkontroll av substratytor för ytförstärkt ramanspektroskopi (SERS) för snabb detektion av farliga flyktiga ämnen konstruerats och testats. Arbetet utfördes vid Totalförsvarets forskningsinstitut (FOI) i Umeå, Sverige. Utgångspunkten var ett Linkam Scientific Instruments TS1500 mikroskopsteg, vilket utrustades med ett Peltierelement för kylning/värmning och en termistor för temperaturövervakning. Ett styrsystem baserat på ett Arduino Uno mikrostyrenhetskort konstruerades med ett motordrivkort (H-brygga) vilket använder pulsbreddsmodulering (PWM) för att reglera spänningen till Peltierelementet utifrån en PI-regulator. Den färdiga cellen klarade att reglera temperaturen på ett SERS-substrat i ett temperaturspann på ungefär -15 till +110 °C med en temperaturstabilitet på ±0.22 °C av måltemperaturen. Cellen testades sedan på flyktiga ämnen för att visa dess funktion. Difluorometyl-2,2,2-trifluoro-1-kloroetyleter (isofluran) i gasfas, med instrumentluft som bärargas, flödades genom cellen och SERS-spektra erhölls vid olika koncentrationer och temperaturer. Vid samtliga koncentrationer visades att lägre temperatur ger ökad signalstyrka. När ytan sedan värmdes upp sjönk signalen reversibelt tillbaka till ursprungsvärdet. Nyckelord: temperaturkontroll, ytförstärkt ramanspektroskopi, SERS, flyktiga ämnen, Peltierelement, thermistor, PWM, H-brygga, PI(D)-regulator.

Surface- and tip-enhanced resonant Raman scattering (resonant SERS and TERS) by optical phonons in a monolayer of CdSe quantum dots (QDs) is demonstrated. The SERS enhancement was achieved by employing plasmonically active substrates consisting of gold arrays with varying nanocluster diameters prepared by electron-beam lithography. The magnitude of the SERS enhancement depends on the localized surface plasmon resonance (LSPR) energy, which is determined by the structural parameters. The LSPR positions as a function of nanocluster diameter were experimentally determined from spectroscopic micro-ellipsometry, and compared to numerical simulations showing good qualitative agreement. The monolayer of CdSe QDs was deposited by the Langmuir–Blodgett-based technique on the SERS substrates. By tuning the excitation energy close to the band gap of the CdSe QDs and to the LSPR energy, resonant SERS by longitudinal optical (LO) phonons of CdSe QDs was realized. A SERS enhancement factor of 2 × 10³ was achieved. This allowed the detection of higher order LO modes of CdSe QDs, evidencing the high crystalline quality of QDs. The dependence of LO phonon mode intensity on the size of Au nanoclusters reveals a resonant character, suggesting that the electromagnetic mechanism of the SERS enhancement is dominant. Finally, the resonant TERS spectrum from CdSe QDs was obtained using electrochemically etched gold tips providing an enhancement on the order of 10⁴. This is an important step towards the detection of the phonon spectrum from a single QD. / Dieser Beitrag ist aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Cette thèse considère de nouvelles architectures prometteuses des métamatériaux plasmoniques pour biosensing, comprenant: (I) des réseaux périodiques 2D de nanoparticules d'Au, qui peuvent supporter des résonances des réseaux de surface couplées de manière diffractive; (II) Reseaux 3D à base de cristaux plasmoniques du type d'assemblage de bois. Une étude systématique des conditions d'excitation plasmonique, des propriétés et de la sensibilité à l'environnement local dans ces géométries métamatérielles est présentée. On montre que de tels réseaux peuvent combiner une très haute sensibilité spectrale (400 nm / RIU et 2600 nm / RIU, ensemble respectivement) et une sensibilité de phase exceptionnellement élevée (> 105 deg./RIU) et peuvent être utilisés pour améliorer l'état actuel de la technologie de biosensing the-art. Enfin, on propose une méthode de sondage du champ électrique excité par des nanostructures plasmoniques (nanoparticules uniques, dimères). On suppose que cette méthode aidera à concevoir des structures pour SERS (La spectroscopie du type Raman à surface renforcée), qui peut être utilisée comme une chaîne d'information supplémentaire à un biocapteur de transduction optique. / This thesis consideres novel promissing architechtures of plasmonic metamaterial for biosensing, including: (I) 2D periodic arrays of Au nanoparticles, which can support diffractively coupled surface lattice resonances; (II) 3D periodic arrays based on woodpile-assembly plasmonic crystals, which can support novel delocalized plasmonic modes over 3D structure. A systematic study of conditions of plasmon excitation, properties and sensitivity to local environment is presented. It is shown that such arrays can combine very high spectral sensitivity (400nm/RIU and 2600 nm/RIU, respectively) and exceptionally high phase sensitivity (> 105 deg./RIU) and can be used for the improvement of current state-of-the-art biosensing technology. Finally, a method for probing electric field excited by plasmonic nanostructures (single nanoparticles, dimers) is proposed. It is implied that this method will help to design structures for SERS, which will later be used as an additional informational channel for biosensing.

<p>Chromatography is a powerful technique, essential in chemical analyses and preparative separation in industry and research. Many different kinds of chromatographic material are needed, due to the large variety of applications. Detailed methods of characterisation are needed to design new chromatographic materials and understand their properties. In this thesis, confocal Raman spectroscopy and surface enhanced Raman spectroscopy (SERS) have been applied to micrometer-size chromatographic beads, for which these techniques have not been used earlier. New methodology, optimized for use with the chromatographic beads, has been developed and evaluated. </p><p>Confocal spectroscopy has been used to determine distributions of functional groups within single chromatographic beads. This distribution is of great importance in determining the chromatographic properties, since the material is porous and the solute molecules can diffuse inside the beads. Most of the confocal experiments have been performed with Raman spectroscopy; fluorescence spectroscopy, using Nd³⁺ ions or dye-labelled proteins as fluorescence probes, has been used for comparison. </p><p>The concentration of adsorbed analytes is very low within the beads. SERS was therefore used to enhance the Raman signal. SERS-active surfaces were prepared by incorporating gold nano-particles into the interior of the bead. TEM measurements showed that the gold nano-particles could be observed throughout, and it was possible to record analyte spectra from different positions within the bead. Enhanced spectra could be obtained both for small test molecules and for larger bio-molecules, although the spectra for the smaller analytes were much more intense.</p>

Chromatography is a powerful technique, essential in chemical analyses and preparative separation in industry and research. Many different kinds of chromatographic material are needed, due to the large variety of applications. Detailed methods of characterisation are needed to design new chromatographic materials and understand their properties. In this thesis, confocal Raman spectroscopy and surface enhanced Raman spectroscopy (SERS) have been applied to micrometer-size chromatographic beads, for which these techniques have not been used earlier. New methodology, optimized for use with the chromatographic beads, has been developed and evaluated. Confocal spectroscopy has been used to determine distributions of functional groups within single chromatographic beads. This distribution is of great importance in determining the chromatographic properties, since the material is porous and the solute molecules can diffuse inside the beads. Most of the confocal experiments have been performed with Raman spectroscopy; fluorescence spectroscopy, using Nd3+ ions or dye-labelled proteins as fluorescence probes, has been used for comparison. The concentration of adsorbed analytes is very low within the beads. SERS was therefore used to enhance the Raman signal. SERS-active surfaces were prepared by incorporating gold nano-particles into the interior of the bead. TEM measurements showed that the gold nano-particles could be observed throughout, and it was possible to record analyte spectra from different positions within the bead. Enhanced spectra could be obtained both for small test molecules and for larger bio-molecules, although the spectra for the smaller analytes were much more intense.