Structure of Adenine on Metal Nanoparticles: pH Equilibria and Formation of Ag+ Complexes Detected by Surface-Enhanced Raman Spectroscopy

The SERS spectra of adenine recorded under a broad range of pH values and concentrations using both silver and gold colloids provided evidence for the existence of several distinct species. At high concentration (0.5-10 ppm), the spectra recorded between pH 1 and 11 showed only two distinct spectra, rather than the three forms that would be expected for a compound with two pK(a) values of 4.2 and 9.8. The spectra at neutral and alkaline pH were identical and assigned to the deprotonated form of adenine on the basis of DFT calculations, isotope shifts, and comparison with the normal Raman spectra of neutral and deprotonated adenine. The spectra at acidic pH were different, consistent with adenine protonation. Neutral adenine was not detected at any pH studied. At low adenine concentration (

DNA Reorientation on Au Nanoparticles: Label-Free Detection of Hybridization by Surface Enhanced Raman Spectroscopy

DNA sequences attached to Au nanoparticles via thiol linkers stand up from the surface, giving preferential enhancement of the adenine ring breathing SERS band. Non-specific binding via the nucleobases reorients the DNA, reducing this effect. This change in intensity on reorientation was utilised for label-free detection of hybridization of a molecular beacon.

Label-Free Detection of Single-Base Mismatches in DNA by Surface-Enhanced Raman Spectroscopy

Singles only: DNA sequences can be induced to spontaneously adsorb to the surfaces of Ag colloids through their nucleotide side chains (see picture). The SERS spectra of these nonspecifically bound strands are sufficiently reproducible that they can be used to identify single-base mismatches in short (25-mer and 23-mer) strands. Subtracting the spectra of different DNA sequences results in difference spectra that contain features corresponding to the exchanged nucleotides.

RAMAN-SCATTERING AND SCANNING ELECTRON-TUNNELING STUDIES OF METAL LIQUID-LIKE FILMS PRODUCED FROM SILVER AND GOLD SOLS

Surface-enhanced Raman scattering (SERS) excited at several visible wavelengths and recorded using a cooled charged-coupled device detector is reported from the mobile, interfacial, liquid-like metal films (MELLFs) formed when solutions of metal complexes or pyridine in chlorocarbon solvents are mixed with aqueous sols of silver or gold. MELLF formation has not previously been reported for gold sols or for pyridine as stabilizer. Comparison of the spectra for the MELLFs formed from individual metal complexes and from 50:50 mixtures show that the spectral patterns observed for the latter are distinctive and are not generally equivalent to the sum of the spectra associated with the individual complexes, in contrast to the situation observed for sols where the individual spectra do appear to be additive. Raman scattering from both gold and silver MELLFs is readily observed at excitation wavelengths in the red, around 750 nm, but at 514 nm only that from silver films is detectable. These findings are considered in terms of particle size and absorption band intensities. A preliminary study of the film surface topography and particle size was carried out by scanning tunnelling electron microscopy (STM) of Ag MELLFs deposited on gold-coated mica substrates. Computer-processed images of the STM data show the presence on the film surface of finger-like bars, 200-400 nm long with approximately square cross-section, 40-60 nm side, together with other smaller cuboid features. The implications of these findings in relation to SERS are briefly considered.

Label-free detection of nanomolar unmodified single- and double-stranded dna by using surface-enhanced raman spectroscopy on Ag and Au colloids

Unlabelled single- and double-stranded DNA (ssDNA and dsDNA, respectively) has been detected at concentrations =10-9?M by surface-enhanced Raman spectroscopy. Under appropriate conditions the sequences spontaneously adsorbed to the surface of both Ag and Au colloids through their nucleobases; this allowed highly reproducible spectra with good signal-to-noise ratios to be recorded on completely unmodified samples. This eliminated the need to promote absorption by introducing external linkers, such as thiols. The spectra of model ssDNA sequences contained bands of all the bases present and showed systematic changes when the overall base composition was altered. Initial tests also showed that small but reproducible changes could be detected between oligonucleotides with the same bases arranged in a different order. The spectra of five ssDNA sequences that correspond to different strains of the Escherichia coli bacterium were found to be sufficiently composition-dependent so that they could be differentiated without the need for any advanced multivariate data analysis techniques.

Highly Sensitive, Uniform, and Reproducible Surface-Enhanced Raman Spectroscopy from Hollow Au-Ag Alloy Nanourchins

A hierarchical nanoparticle strategy to simultaneously gain super Raman signal amplification, high uniformity, and reproducibility is presented. Using hollow Au-Ag alloy nanourchins, an ultrahigh sensitivity, e.g., down to 1 fM concentrations for DEHP molecule is obtained. A small standard deviation of <10% is achieved by simply dropping and evaporating sub-100 nm nanourchins onto a substrate.

Preaggregated Ag Nanoparticles in Dry Swellable Gel Films for Off-the-Shelf Surface-Enhanced Raman Spectroscopy

Large, thin (50 mu m) dry polymer sheets containing numerous surface-enhanced Raman spectroscopy (SERS) active Ag nanopartide aggregates have been prepared by drying aqueous mixtures of hydroxyethylcelloulose (HEC) and preaggregated Ag colloid in 10 x 10 cm molds. In these dry films, the particle aggregates are protected from the environment during storage and are easy to handle; for example, they can be cut to size with scissors. When in use, the highly swellable HEC polymer allowed the films to rapidly absorb aqueous analyte solutions while simultaneously releasing the Ag nanoparticle aggregates to interact with the analyte and generate large SERS signals. Either the films could be immersed in the analyte solution or 5 mu L droplets were applied to the surface; in the latter method, the local swelling caused the active area to dome upward, but the swollen film remained physically robust and could be handled as required. Importantly, encapsulation and release did not significantly compromise the SERS performance of the colloid; the signals given by the swollen films were similar to the very high signals obtained from the parent citrate-reduced colloid and were an order of magnitude larger than a commercially available nanoparticle substrate. These "Poly-SERS" films retained 70% of their SERS activity after being stored for 1 year in air. The films were sufficiently homogeneous to give a standard deviation of 3.2% in the absolute signal levels obtained from a test analyte, primarily due to the films' ability to suppress "coffee ring" drying marks, which meant that quantitative analysis without an internal standard was possible. The majority of the work used aqueous thiophenol as the test analyte; however, preliminary studies showed that the Poly-SERS films could also be used with nonaqueous solvents and for a range of other analytes including theophylline, a therapeutic drug, at a concentration as low as 1.0 x 10(-5) mol dm(-3) (1.8 mg/dm(3)), well below the sensitivity required for theophylline monitoring where the target range is 10-20 mg/dm(3).

The Surface Plasmon Resonance of Supported Noble Metal Nanoparticles: Characterization, Laser Tailoring, and SERS Application

This work deals with the optical properties of supported noble metal nanoparticles, which are dominated by the so-called Mie resonance and are strongly dependent on the particles’ morphology. For this reason, characterization and control of the dimension of these systems are desired in order to optimize their applications. Gold and silver nanoparticles have been produced on dielectric supports like quartz glass, sapphire and rutile, by the technique of vapor deposition under ultra-high vacuum conditions. During the preparation, coalescence is observed as an important mechanism of cluster growth. The particles have been studied in situ by optical transmission spectroscopy and ex situ by atomic force microscopy. It is shown that the morphology of the aggregates can be regarded as oblate spheroids. A theoretical treatment of their optical properties, based on the quasistatic approximation, and its combination with results obtained by atomic force microscopy give a detailed characterization of the nanoparticles. This method has been compared with transmission electron microscopy and the results are in excellent agreement. Tailoring of the clusters’ dimensions by irradiation with nanosecond-pulsed laser light has been investigated. Selected particles are heated within the ensemble by excitation of the Mie resonance under irradiation with a tunable laser source. Laser-induced coalescence prevents strongly tailoring of the particle size. Nevertheless, control of the particle shape is possible. Laser-tailored ensembles have been tested as substrates for surface-enhanced Raman spectroscopy (SERS), leading to an improvement of the results. Moreover, they constitute reproducible, robust and tunable SERS-substrates with a high potential for specific applications, in the present case focused on environmental protection. Thereby, these SERS-substrates are ideally suited for routine measurements.

Advanced schemes for surface plasmon resonance and plasmon-enhanced fluorescence biosensors

Advanced optical biosensor platforms exploiting long range surface plasmons (LRSPs) and responsive N-isopropylacrylamide (NIPAAm) hydrogel binding matrix for the detection of protein and bacterial pathogen analytes were carried out. LRSPs are optical waves that originate from coupling of surface plasmons on the opposite sites of a thin metallic film embedded between two dielectrics with similar refractive indices. LRSPs exhibit orders of magnitude lower damping and more extended profile of field compared to regular surface plasmons (SPs). Their excitation is accompanied with narrow resonance and provides stronger enhancement of electromagnetic field intensity that can advance the sensitivity of surface plasmon resonance (SPR) and surface plasmon-enhanced fluorescence spectroscopy (SPFS) biosensors. Firstly, we investigated thin gold layers deposited on fluoropolymer surface for the excitation of LRSPs. The study indicates that the morphological, optical and electrical properties of gold film can be changed by the surface energy of fluoropolymer and affect the performance of a SPFS biosensor. A photo-crosslinkable NIPAAm hydrogel was grafted to the sensor surface in order to serve as a binding matrix. It was modified with bio-recognition elements (BREs) via amine coupling chemistry and offered the advantage of large binding capacity, stimuli responsive properties and good biocompatibility. Through experimental observations supported by numerical simulations describing diffusion mass transfer and affinity binding of target molecules in the hydrogel, the hydrogel binding matrix thickness, concentration of BREs and the profile of the probing evanescent field was optimized. Hydrogel with a up to micrometer thickness was shown to support additional hydrogel optical waveguide (HOW) mode which was employed for probing affinity binding events in the gel by means of refractometric and fluorescence measurements. These schemes allow to reach limits of detection (LODs) at picomolar and femtomolar levels, respectively. Besides hydrogel based experiments for detection of molecular analytes, long range surface plasmon-enhanced fluorescence spectroscopy (LRSP-FS) was employed for detection of bacterial pathogens. The influence of capture efficiency of bacteria on surfaces and the profile of the probing field on sensor response were investigated. The potential of LRSP-FS with extended evanescent field is demonstrated for detection of pathogenic E. coli O157:H7 on sandwich immunoassays . LOD as low as 6 cfu mL-1 with a detection time of 40 minutes was achieved.rn

Plasmon-enhanced dynamic light scattering of gold nanorods in bulk and near a wall

Transportprozesse von anisotropen metallischen Nanopartikeln wie zum Beispiel Gold-Nanostäbchen in komplexen Flüssigkeiten und/oder begrenzten Geometrien spielen eine bedeutende Rolle in einer Vielzahl von biomedizinischen und industriellen Anwendungen. Ein Weg zu einem tiefen, grundlegenden Verständnis von Transportmechanismen ist die Verwendung zweier leistungsstarker Methoden - dynamischer Lichtstreuung (DLS) und resonanzverstärkter Lichtstreuung (REDLS) in der Nähe einer Grenzfläche. In dieser Arbeit wurden nanomolare Suspensionen von Gold-Nanostäbchen, stabilisiert mit Cetyltrimethylammoniumbromid (CTAB), mit DLS sowie in der Nähe einer Grenzfläche mit REDLS untersucht. Mit DLS wurde eine wellenlängenabhängige Verstärkung der anisotropen Streuung beobachtet, welche sich durch die Anregung von longitudinaler Oberflächenplasmonenresonanz ergibt. Die hohe Streuintensität nahe der longitudinalen Oberflächenplasmonenresonanzfrequenz für Stäbchen, welche parallel zum anregenden optischen Feld liegen, erlaubte die Auflösung der translationalen Anisotropie in einem isotropen Medium. Diese wellenlängenabhängige anisotrope Lichtstreuung ermöglicht neue Anwendungen wie etwa die Untersuchung der Dynamik einzelner Partikel in komplexen Umgebungen mittels depolarisierter dynamischer Lichtstreuung. In der Nähe einer Grenzfläche wurde eine starke Verlangsamung der translationalen Diffusion beobachtet. Hingegen zeigte sich für die Rotation zwar eine ausgeprägte aber weniger starke Verlangsamung. Um den möglichen Einfluss von Ladung auf der festen Grenzfläche zu untersuchen, wurde das Metall mit elektrisch neutralem Polymethylmethacrylat (PMMA) beschichtet. In einem weiteren Ansatz wurde das CTAB in der Gold-Nanostäbchen Lösung durch das kovalent gebundene 16-Mercaptohexadecyltrimethylammoniumbromid (MTAB) ersetzt. Daraus ergab sich eine deutlich geringere Verlangsamung.

Localized surface plasmon resonance biosensors for real-time biomolecular binding study

Surface Plasmon Resonance (SPR) and localized surface plasmon resonance (LSPR) biosensors have brought a revolutionary change to in vitro study of biological and biochemical processes due to its ability to measure extremely small changes in surface refractive index (RI), binding equilibrium and kinetics. Strategies based on LSPR have been employed to enhance the sensitivity for a variety of applications, such as diagnosis of diseases, environmental analysis, food safety, and chemical threat detection. In LSPR spectroscopy, absorption and scattering of light are greatly enhanced at frequencies that excite the LSPR, resulting in a characteristic extinction spectrum that depends on the RI of the surrounding medium. Compositional and conformational change within the surrounding medium near the sensing surface could therefore be detected as shifts in the extinction spectrum. This dissertation specifically focuses on the development and evaluation of highly sensitive LSPR biosensors for in situ study of biomolecular binding process by incorporating nanotechnology. Compared to traditional methods for biomolecular binding studies, LSPR-based biosensors offer real-time, label free detection. First, we modified the gold sensing surface of LSPR-based biosensors using nanomaterials such as gold nanoparticles (AuNPs) and polymer to enhance surface absorption and sensitivity. The performance of this type of biosensors was evaluated on the application of small heavy metal molecule binding affinity study. This biosensor exhibited ∼7 fold sensitivity enhancement and binding kinetics measurement capability comparing to traditional biosensors. Second, a miniaturized cell culture system was integrated into the LSPR-based biosensor system for the purpose of real-time biomarker signaling pathway studies and drug efficacy studies with living cells. To the best of our knowledge, this is the first LSPR-based sensing platform with the capability of living cell studies. We demonstrated the living cell measurement ability by studying the VEGF signaling pathway in living SKOV-3 cells. Results have shown that the VEGF secretion level from SKOV-3 cells is 0.0137 ± 0.0012 pg per cell. Moreover, we have demonstrated bevacizumab drug regulation to the VEGF signaling pathway using this biosensor. This sensing platform could potentially help studying biomolecular binding kinetics which elucidates the underlying mechanisms of biotransportation and drug delivery.