This PDF file contains the front matter associated with SPIE Proceedings Volume 6869, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

We propose to integrate the surface-enhanced Raman spectroscopy (SERS) detection capability with a surface plasmon resonance (SPR) biosensor platform. As a demonstration setup, the experimental scheme is built from a Total Internal Reflection Fluorescence (TIRF) microscope. The sample surface is a gold-coated plasmonic crystal substrate. Two oligonucleotide (ODN) probes that have been labeled with two different Raman active dyes are used to achieve a sandwich assay of target ODNs or polynucleotide. Upon complementary hybridizations between the target and probe ODNs, the target can be identified by detecting the narrow-band spectroscopic fingerprints of the Raman tags. This concept has high potential for achieving multiplexed detection of ODN targets because a very large number of probes can be incorporated to the plasmonic crystal substrate, which may find applications in gene based diseases diagnostics. We also explored the detection of single molecules and achieved some preliminary results.

This paper describes the use of plasmonics-based nanoprobes for detection of multidrug-resistant tuberculosis gene. The plasmonics nanoprobe is composed of a silver nanoparticle pre-coated with a stem-loop DNA probe that is tagged with a Raman label at one end of the stem region, while the other end of the probe is covalently conjugated to the nanoparticle via a thiol-silver bond. The loop region is designed to detect a specific target gene sequence. In the absence of target, the Raman label is in close proximity to the metal surface, resulting in an intense SERS signal upon laser excitation. In the presence of the target DNA sequence, hybridization between the target and probe disrupts the stem-loop configuration, separating the Raman label from the metal surface and quenching the SERS signal. In this study, we successfully demonstrated for the first time the feasibility of using plasmonics nanoprobes for the detection of multidrug-resistant tuberculosis gene.

A major limitation of many surfaced enhanced Raman spectroscopy (SERS) approaches is the dependence of the Raman enhancement on the local nanostructure. While these local "hot spots" may provide areas of extremely strong enhancement, which make trace analyte detection possible, they also make quantitative measurements problematic. Gold nanoshells however, with the ratio of the radius of their silica core to gold shell tuned to the near infrared excitation wavelength, have been used as a platform for uniform SERS enhancement. By using nanoshells, the SERS enhancement is dependent on the resonance of single nanoshells, without relying on the uncontrolled contribution from localized "hot spots". The nanoshell platform is functionalized with sialic acid to mimic neuronal cells surfaces to allow for the specific binding of β-amyloid, the primary protein component of the senile plaques found in Alzheimer's disease patients. We ultimately hope that this mechanism will provide insight into the relationship between the progression of Alzheimer's disease and β-amyloid through detection of the toxic form of the protein with structural and concentration information. With this approach, we have obtained concentration dependent spectra, consistent across the platform surface, which indicate the feasibility of detecting β-amyloid oligomers into the picomolar range. Additionally, by monitoring SERS spectra as β-amyloid changes its structural conformation from monomer to fibril, we have demonstrated conformational dependence of the SERS signals.

Hierarchically-ordered Au film-coated polystyrene beads have been well-known to be employed as surface enhanced Raman spectroscopy (SERS) substrate. In this study, we propose a novel and facile method to modify the performance of this SERS substrate and investigate the chemical components in saliva. Results have demonstrated that there is at least a 4-fold increase in SERS signals using the modified substrates compared to the non-modified substrates. Besides, the SERS performances of substrates modified by different preparation conditions are examined and compared. These findings show that our fabricated substrates are effective in further enhancing SERS signals and have potential for biomedical applications in trace analytes analysis.

In the past several years we have demonstrated the metal-enhanced fluorescence (MEF) and the significant changes in the photophysical properties of fluorophores in the presence of metallic nanostructures and nanoparticles using ensemble spectroscopic studies. Here, in the present study, we explored the new insights of these interactions using single-molecule fluorescence spectroscopy. The single molecule study is expected to provide more information, especially on the heterogeneity in the fluorescence enhancement and decrease in lifetimes associated with fluorophore-metal interactions, which is otherwise not possible to observe using ensemble measurements. For the present study, we considered using CdTe nanocrystals (QDots) prepared using modified Weller method as the fluorophores under investigation. QDots having few nanometer sizes, tunable absorption and fluorescence spectral properties, and high photo-stabilities are of important class of fluorescent probes. Because of these unique features Qdots are widely used as probes in various fields, including biological labeling and imaging. These CdTe nanocrystals show characteristic spectral features in solution and on the solid substrate. The CdTe nanocrystals dispersed in PVA and spin-casted on SiFs surface show ~5-fold increase in fluorescence intensity and ~3-fold decrease in lifetimes compared to on glass substrate. The data obtained using ensemble and single molecule spectroscopy are complimentary to each other. Additionally as anticipated we have seen increased heterogeneity in the plasmon induced fluorescence modulations. Moreover single molecule spectroscopic study revealed significant reduction in blinking of CdTe nanocrystals on plasmonic nanostructures. Subsequently, we present these important findings on metal-fluorophore interactions of CdTe nanocrystals (QDots) on plasmonic nanostructures.

Laser therapies can provide a minimally invasive treatment alternative to surgical resection of tumors. However, the effectiveness of these therapies is limited due to nonspecific heating of target tissue which often leads to healthy tissue injury and extended treatment durations. These therapies can be further compromised due to heat shock protein (HSP) induction in tumor regions where non-lethal temperature elevation occurs, thereby imparting enhanced tumor cell viability and resistance to subsequent chemotherapy and radiation treatments. Introducing multi-walled nanotubes (MWNT) into target tissue prior to laser irradiation increases heating selectivity permitting more precise thermal energy delivery to the tumor region and enhances thermal deposition thereby increasing tumor injury and reducing HSP expression induction. This study investigated the impact of MWNT inclusion in untreated and laser irradiated monolayer cell culture and cell phantom model. Cell viability remained high for all samples with MWNT inclusion and cells integrated into alginate phantoms, demonstrating the non-toxic nature of both MWNTs and alginate phantom models. Following, laser irradiation samples with MWNT inclusion exhibited dramatic temperature elevations and decreased cell viability compared to samples without MWNT. In the cell monolayer studies, laser irradiation of samples with MWNT inclusion experienced up-regulated HSP27, 70 and 90 expression as compared to laser only or untreated samples due to greater temperature increases albeit below the threshold for cell death. Further tuning of laser parameters will permit effective cell killing and down-regulation of HSP. Due to optimal tuning of laser parameters and inclusion of MWNT in phantom models, extensive temperature elevations and cell death occurred, demonstrating MWNT-mediated laser therapy as a viable therapy option when parameters are optimized. In conclusion, MWNT-mediated laser therapies show great promise for effective tumor destruction, but require determination of appropriate MWNT characteristics and laser parameters for maximum tumor destruction.

Thermal evaporation was used to deposit particulate aluminum films of varied thicknesses on quartz substrates. These substrates were characterized by scanning electron microscopy (SEM), which reveal that with an increase in aluminum thickness, the films progress from particulate towards smooth surfaces. Until now, metal-enhanced fluorescence (MEF) has primarily been observed in the visible-NIR wavelength region using silver or gold island films and roughened surfaces. We now report that fluorescence can also be enhanced in the ultraviolet-blue region of the spectrum using nano-structured aluminum films. We used two probes, one in the ultraviolet (a DNA base analogue 2-aminopurine: 2- AP) and another one in blue spectral region (a coumarin derivative: 7-HC) for the present study. We observed increased emission, decrease in fluorescence lifetime and increase in photostability of the dyes in a 10 nm spin-casted polyvinyl alcohol film on the Al nanostructured surfaces. We observe that the fluorescence enhancement factor depends on the thickness of the Al films because the size of the nanostructures formed varies with Al thickness. These studies indicate that Al nano-structured substrates can potentially find widespread use in MEF applications particularly in the UV - blue spectral regime. Finite-Difference Time-Domain (FDTD) calculations were performed that revealed enhanced near-fields induced around aluminum nanoparticles by a radiating fluorophore emitting at the emission wavelength of 2-AP. The effect of such enhanced fields on the fluorescence enhancement observed is also discussed.

In this work, we aim at enhancing the sensitivity of surface plasmon resonance sensors towards the detection of biomolecule interactions by means of nanopatterning of the sensor surface. Use of nanostructured interfaces in combination with SPR is a promising step towards realizing biosensors with high efficiency and sensitivity. Nanopatterned surfaces enable multi-dimensional control over the behavior of surface-immobilized probe molecules. By means of a combination of self-assembled monolayer technology, colloidal lithography, and reactive ion etching, nanopatterns with either antibody confining or non-confining characteristics were produced and analyzed via photoelectron spectroscopy and infrared reflection absorption spectroscopy. Antibody immobilization on the patterns and subsequent specific binding of antigen was traced in real time by means of a surface plasmon resonance sensor. It was found that confining nanopatterns yield an increase in antibody activity towards antigen capture on surface of up to 120%, depending on the protocol used for their immobilization.

This paper describes the development of fiber optic sensor probes and planar substrates containing patterned nanostructures such as nanoholes in gold films, as well as gold nanoparticles, nano-pillars, nanorods, and nano-islands. Several methods of producing gold nanofeatures on fiber tips and planar substrates were investigated such as annealing of thin gold films and focused ion beam (FIB) milling. A Hitachi FB-2100 FIB milling machine with a gallium ion source was employed to form the nanoparticles from 20-100 nm gold films deposited on the fiber tip. Nano-engineered gold features were also formed by coating planar substrates and fiber tips with thin gold films (4-10 nm) and annealing these thin films. Excitation of surface plasmons in gold nanostructures leads to substantial enhancement in the Raman scattering signal obtained from molecules attached to the nanostructure surface. In this work, a comparison was made between the SERS signals obtained from the gold substrates developed by employing the different procedures mentioned above. Fiber samples and planar substrates with these nanostructures were coated with SERS active dyes such as pmercaptobenzoic acid (pMBA) and cresyl fast violet (CFV). It was observed that the SERS signal obtained from these gold nanofeatures was much higher than that obtained from a continuous gold film and that the SERS enhancement was shape and size dependent.

Near-field coupling between plasmonic resonant nanoparticles and the associated shifts in scattering spectra enables the accomplishment of unprecedented observation of the co-localization dynamics of in-situ biomolecules on nanometer length-scales. We have recently shown that resonant nanoparticles conjugated to antibodies for cell-surface receptors provide a sensitive probe allowing the unambiguous resolution of not only the time sequence, but also the details of the intracellular pathway, for receptor-mediated endocytosis in live cells. In terms of general principles, the classical electrodynamics determining the scattering cross-section for nanoparticle aggregates is straightforward. However, the specifics of the angular dependence of the differential cross-section at a single wavelength, the wavelength dependence of this cross-section, and the correct implementation and interpretation of statistical averages of cross-section properties over an ensemble of aggregate morphologies are generally quite complicated, and in fact are often misinterpreted in the literature. Despite this complexity, we have constructed a set of few-parameter formulae describing optical scattering from nanoparticle aggregates by judicious combination of experimental results with extensive, near-exact simulation using the T-matrix technique. These phenomenological results facilitate the practical use of nanoparticle aggregates for biological measurement and clinical therapeutic applications.

Noble metal nanoparticles are characterized by a strong peak in the scattering and absorption spectrum, termed the plasmon resonance. Researchers have taken advantage of this to create a new label for biological molecules. A disadvantage of techniques based on scattering and absorption is that the detected signal is at the same wavelength as the incident light, making it more challenging to discriminate between signal and background. Gold nanoparticles also luminesce, suggesting an alternate method for their detection. A tightly focused ultra-short pulse laser beam can be used to achieve multiphoton excitation of the particles; the resulting luminescence exhibits a peak in the same region of the spectrum as the plasmon resonance. Because excitation is nonlinear, significant luminescence is only observed when the particle is in the focus, permitting localization with both high lateral and axial resolution. The physical mechanism underlying multiphoton luminescence in gold is still the subject of debate. Here, we present a systematic study in single gold nanospheres with diameters between 15 nm and 100 nm using peak laser intensities between 10 and 350 GW/cm². A scattering confocal microscope incorporated in the setup was used to distinguish single particles from clusters. We observed that not all gold nanospheres have a detectable multiphoton luminescence signal; however, laser intensities above an exposure-time dependent threshold can alter such particles so that they do. In addition, we found that gold nanoparticles exposed to laser intensities above about 150 GW/cm² can exhibit behavior reminiscent of the bleaching and blinking of conventional fluorophores.

Gold nanoshells are a novel class of hybrid metal nanoparticles whose unique optical properties have spawned new applications including more sensitive molecular assays and cancer therapy. We report a new photo-physical property of nanoshells (NS) whereby these particles glow brightly when excited by near-infrared light. Specifically, we demonstrate NS excited at 780 nm produce strong two-photon induced photoluminescence (TPIP). We characterized the luminescence brightness of NS, comparing to that of fluorescein-labeled fluorescent beads (FB). We find that NS are 140 times brighter than FB. To demonstrate the potential application of this bright TPIP signal for biological imaging, we imaged the 3D distribution of gold nanoshells targeted to murine tumors.

We evaluate the potential ability of c-shaped apertures milled in aluminum thin films to reduce the effective measurement volume and to enhance the fluorescence signal for fluorescence correlation spectroscopy of ATTO655 dye dissolved in a HEPES buffer solution. Previous studies have shown that by morphing a square aperture into a rectangular aperture while holding the cross-sectional area constant will yield strong polarization dependence in the reduction of the effective volume and about a factor of 2-3 enhancement in the fluorescence count rate per molecule. By morphing the rectangular aperture into a c-shaped aperture we gain further reduction in focal volume while maintaining the count rate enhancements. In particular, we compare c-shaped apertures to squares with the same cross-sectional area and show that one can achieve one molecule per focal volume at ~3µM (about a 1000 times reduction in effective volume compared to confocal FCS) while maintaining a fluorescence count rate per molecule of about an order of magnitude higher than for bulk diffusing dyes. Two orthogonal polarizations for the incident field have been studied to explore the effects on the focal volume reduction and fluorescence count rate enhancements.

Now in the leading biomedical centers of the world approved new technology of laser photothermal destruction of cancer cells using plasmon gold nanoparticles. Investigations of influence of gold nanoparticles on white rat platelets aggregative activity in vitro have been made. Platelet aggregation was investigated in platelet rich plasma (PRP) with help of laser analyzer 230 LA <<BIOLA>> (Russia). Aggregation inductor was ADP solution in terminal concentration 2.5 micromole (<<Reanal>>, Russia). Gold nanoshells soluted in salt solution were used for experiments. Samples of PRP were incubated with 50 or 100 μl gold nanoshells solution in 5 minute, after that we made definition ADP induced platelet aggregation. We found out increase platelet function activity after incubation with nanoparticles solution which shown in maximum ADP-induced aggregation degree increase. Increase platelet function activity during intravenous nanoshells injection can be cause of thrombosis on patients. That's why before clinical application of cancer cell destruction based on laser photothermal used with plasmon gold nanoparticles careful investigations of thrombosis process and detail analyze of physiological blood parameters are very necessary.

Alzheimer's disease (AD), a neurodegenerative disease and the most common cause of dementia, affects 4.5 million people according to the 2000 US census and is expected to triple to 13.2 million by the year 2050. Since no definitive pre-mortem tests exist to distinguish AD from mild cognitive impairment due to the natural aging process, we focus on detecting the beta amyloid (Aβ) protein, the primary component of the senile plaques characteristic of AD. We specifically detect cytotoxic species of Aβ by exploiting surface enhanced Raman scattering (SERS). Using a nanofluidic device with a bottleneck shape (a microchannel leading into a nanochannel); we trapped gold colloid particles (60 nm) at the entrance to the nanochannel, with Aβ restricted within the interstices between the aggregated nanoparticles. The continuous flow generated from pumping the solution into the device produced size-dependent trapping of the gold colloid particles, resulting in a high density of aggregated nanoparticles at this precise region, creating localized "hot spots" in the interstitial region between nanoparticles, and shifting the plasmon resonance to the near infrared region, in resonance with incident laser wavelength. With this robust sensing platform, we were able to obtain concentration-dependent SERS spectra of Aβ and of different proteins present in the cerebrospinal fluid of healthy people and people with Alzheimer's disease.

Polarization Modulation Infrared Reflection Absorption Spectroscopy (PM-IRRAS) is a very sensitive imaging technique for the characterization of molecular films. In order to achieve a spatial resolution close to the diffraction limit a very small pinhole which acts as a point-source has to be used. However, such a small pinhole, the typical diameter would be app. 100 μm, may reduces dramatically the intensity of the infrared beam. Using a common FTIR spectrometer the spatial resolution is mainly limited by the brilliance of the globar infrared source. Therefore, an improvement in lateral resolution requires a more brilliant light source. The free electron laser (FEL) is such a high brilliant infrared source. The combination of the FEL with the PM-IRRAS imaging system is a new approach to capture spectroscopic images with an excellent spatial resolution close to the diffraction limit. PM-IRRAS images of a self assembly monolayer of phosphonic acid molecules onto a microstructures gold / aluminum oxide surface where characterized. The spectroscopic image exhibits a spatial resolution of app. 5 μm. An evaluation of characteristic absorbance bands of the phosphate group reveals that phosphonic acid molecules bound with a high degree of orientation but differently at the gold and aluminum oxide surfaces. However, the spectroscopic image reveals also several domains of disordering across the surface. Such domains have a dimension of only few micrometers and can be identified in a high resolved PM-IRRAS image.

Plasmon resonances are computed for prolate spheroidal nanoshells. Both longitudinal and transverse resonances are investigated as a function of aspect ratio. Formulas for the surface charge density on the outside and inside shell surfaces are derived.

A new method for surface-based fluoroimmunoassays that eliminates separation steps while still allowing high sensitivity detection of biomolecular interactions is presented. The capture antibody is electrostatically immobilized on a glass slide coated with a high density silver island film. The metal-enhanced fluorescence generated by the presence of the islands allows the sensitive detection of bound reporter antibodies versus those free in solution. In order to perform the measurement, phase-modulation fluorometry is employed which allows observation of the distinct fluorescence signal of the bound antibodies with a shorter lifetime than unbound antibodies. Here, we show the use of metal-enhanced fluorescence with phase-modulation fluorometry to quantify monoclonal antibody from a cell culture. The results show the new technique produces very similar data upon analysis as measured with ELISA analysis. With further optimization of the procedures, it is forecast that real time monitoring during bioprocessing will be feasible with the described technique.