This PDF file contains the front matter associated with Proceedings of SPIE Volume 6450, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

The Raman cross-section from a molecule is believed to enhance by more than 10 orders of magnitude when it is adsorbed on a cluster of silver nanoparticles. These large enhancements are attributed to the resonant excitation of the surface plasmon modes of the cluster those have very large localized electric field near its surface. The resonant position and the electric field of these modes are very sensitive to the structure of metal particles and the size and shape of the cluster. Using multiple scattering in the wave-vector space between the individual particles in the cluster we have calculated the resonant position of these modes and their enhanced electric field for clusters of different shape formed from two, three, and four nanospheres and nanoshells. We find the maximum enhancement in the cross-section can reach up to 10 orders of magnitude for silver particle clusters. We also find important new results for the chain like clusters of three or more particles where there is a dramatic increase in the enhancement due to very sharp resonant features of the modes. These features may be helpful in identifying the cluster shape and size in the surface enhanced Raman scattering experiments.

Silver nanoparticles (Ag-NPs) functionalized with molecule probes which contains unique Raman vibrational mode as well as a recognition binding site to target specific surface proteins expressed on the transfected human cervical cancel cells was utilized to detect the cell surface protein through surface enhanced Raman scattering (SERS) microscopy. In this study, we demonstrate that only the aggregated Ag-NPs displays detectable SERS signal. We also observed striking polarization anisotropy in many dimer or trimer NP aggregates. This work will impact on the future design of NP clusters for in-vivo cell imaging.

We present details of the development of a optical biochip, with integrated on-chip laser excitation, for fluorescence intensity cell based assays. The biochip incorporates an "active surface" for the control and manipulation of fluorescent species placed directly on the device. The active elements of the biochip are one-dimensional periodic sub-wavelength corrugations fabricated on a thin gold film. We have made fluorescence intensity measurements of both an organic dye (Cy5), and immobilized and fluorescently labeled (with 705 nm emitting quantum dots), mammalian tumor cells in contact with the active surface. Here we show that the presence of the periodic grating can be used to control both the excitation and fluorescence generation process itself. We demonstrate that the gratings convert evanescent surface optical modes into well-defined beams of radiation in the far-field and at the surface of the device this produces highly contrasting regions of fluorescence excitation providing regions of high spatial selectivity.

In recent years our laboratory has described the favorable effects of fluorophores in close proximity to metallic nanostructures (1-6). These include, increased system quantum yields (increased detectability) and much improved fluorophore photostabilities. These effects have led to many applications of metal-enhanced fluorescence (MEF) including, improved DNA detection (7, 8), enhanced ratiometric sensing (5), metal-enhanced phosphorescence (9) and chemiluminescence signatures (10), as well as to the development of nano-rod (6), triangular nano-plate (4) and modified plastic surfaces (1, 3) for their multifarious applications. In all of our applications of MEF to date, we have been able to significantly optically amplify luminescence based signatures, but have been unable to modify the rates of the respective biochemical reactions being either studied or utilized, as these are dependent on the usual solution parameters of temperature, viscosity and their bioaffinity etc. However, our laboratory has recently shown that low power microwaves, when applied to the metallic nanostructures which are suitable for MEF, are preferentially heated, rapidly accelerating local biochemical reactions (11). Subsequently, ultra-fast and ultra-sensitive assays can be realized. We have recently termed the amalgamation of both MEF with microwave heating as "Microwave-Accelerated Metal-Enhanced Fluorescence (MAMEF)." In this conference proceeding, we summarize our MAMEF work on ultra-fast and sensitive myoglobin detection for rapid cardiac risk assessment and DNA detection for bioterrorism applications. In addition we present two new platform technologies, namely, Microwave-Accelerated Surface Plasmon-Coupled Directional Luminescence (MA-SPCL) for ultra fast assays using clinical samples and a Microwave-Accelerated Aggregation Assay (MA-AA) technology, for ultra fast solutionbased nanoparticle aggregation assays.

Surface plasmons are coherent oscillations of the free electrons on metal surface which can be used to improve the excitation efficiency of fluorophores due to increased field enhancement. Surface plasmon resonance fluorescence (SPRF) microscopy is a wide-field optical imaging technique that utilizes the evanescent electromagnetic field of surface plasmons to excite fluorophores near to a surface of a metal film. With the same excitation power, the field enhancement effect of the surface plasmon resonance (SPR) leads to strong fluorescence emission and thus increases the signal to noise ratio of detection. However, there have been few studies on the image formation process for SPRF in terms of its point-spread function. By imaging fluorescent microspheres with size below the diffraction limit, we obtained the point-spread function for SPRF. The SPR enhancement is confirmed by back-focal-plane imaging with various incidence angles of the excitation beam. Furthermore, we will investigate the potential of resolution enhancement by generating standing wave with two symmetric incident excitation beams toward the standing-wave surface plasmon resonance fluorescence (SW-SPRF) microscopy.

Surface plasmon is collective oscillation of free electrons at metal/dielectric interface. As a wave phenomenon, surface plasmon can be focused using appropriate excitation geometry and metallic structures. The strong spatial confinement and high field enhancement make plasmonic lenses very attractive for near-field optical imaging and sensing in biological applications. In this paper, we show that optimal plasmonic focusing can be achieved through a combination of radially polarized illumination and axially symmetric dielectric/metal plasmonic lens structures. As examples, plasmonic lens with planar interface, conical shape and annular rings under radial polarization illumination are studied. The focusing properties and field enhancement effect of these plasmonic lenses are numerically studied with a finite-element- method model. The numerical simulation results show that the field distribution with a full-width-half-maximum of as small as 10 nm and intensity enhancement factor of five orders of magnitude can be achieved with 632.8 nm optical excitation.

This study proposed a novel approach to replace the traditional surface plasmon resonance (SPR) bulk prism by microlens arrays (MLAs). It demonstrated the effect that coupling SPR on the optical response of microlens arrays structure. Surface plasmons are features specific to the interface of metal-dielectric. They are due to charge density oscillations in the metal, accompanied by electromagnetic field dissipation in the metal and in the dielectric. SPR biosensor bulk prism technology has been commercialized and SPR biosensors have become a central tool for characterizing and quantifying biomolecular interactions. We will used this microlens arrays coupling SPP phenomenon, which gives rise to selective spectral response due to a local field enhancement interrelating the optical and biochemical domains.

Here we analyze the influence of 9 nm (mean diameter) silver particles on the nonlinear properties of intrinsic cell molecules. A novel high sensitivity thermal managed eclipse Z-scan technique with a femtosecond laser system was used to analyze the nonlinear susceptibility of water solution of fluorescent and non-fluorescent amino acids (Tryptophan, Tyrosine, Phenylalanine, Proline and Histidine) with different concentration of silver nanoparticles. The generalized Maxwell Garnett model is used to explain the behavior of the measured nonlinear refractive index with the change of the nanoparticles concentration in the sample.

In this study, we experimentally confirmed the sensitivity enhancement by the nanowire-based surface plasmon resonance (SPR) sensor structure. Gold nanowire samples with a period of 500 nm were fabricated by interference lithography on a gold-SF10 glass substrate. Sensitivity enhancement compared to a conventional SPR structure was measured to be 31% when evaluated using a varied concentration of ethanol at a dielectric surrounding layer. This result is consistent with numerical data of rigorous coupled-wave analysis. Rough surfaces of thin gold film and gold nanowires are deemed to induce the sensitivity degradation by more than 10%. More significant sensitivity improvement can be achieved by implementing finer nanowires.

Surface plasmon resonance (SPR) technique is an optical method that allows the real time detection of small changes in the physical properties (in particular the refractive index) of a dielectric medium near a metallic surface. This technique is today applied to the realization of dynamic optical biochips where multiple interactions can be monitored in parallel and in real time. One of the main advantage compared to other techniques as fluorescence detection is that it does not require the presence of labels, which could influence the kinetics or the equilibrium of the biomolecular interactions. However, as the SPR signal amplitude depends on the refractive index shift of the dielectric medium in the contact with the metallic layer, one way to increase the SPR signal shift is to incorporate a substance possessing a strong dispersive refractive index. We present the influence of organic chromophores incorporated in the DNA target molecules on the spectral SPR response of a SPR sensor. Theoretical and experimental results are presented, showing that the DNA target molecules labeled with chromophores presenting strong spectral refractive index variation in the spectral range of the SPR spectrum induce significant spectral SPR response changes. The use of specific chromophores provides a potential way of SPR response enhancement and initial results suggest that this phenomenon can also be used in realtime SPR imaging detection.

Surface plasmon resonance (SPR) sensing is now widely used in biosensing applications. There is significant scope to reduce the cost and complexity of existing commercial devices by increasing the level of optical integration, and also of enhancing the sensitivity through the use of periodic nanostructures to increase the electromagnetic field response. We will present a SPR sensor design that addresses these two issues. This design utilizes a diffractive optical element (DOE) which is integrated directly into the sensor-head and which significantly reduces the optical complexity. It is intended for eventual mass replication via a suitable molding technique. This system is designed to be used within an angular sensing scheme and the DOE delivers the required 15° angular beam divergence. A carefully developed signal processing scheme is then used to extract the maximum possible information from the detected signal. The sensor surface incorporates gold nanogratings and guided molecular self-assembly for the immobilization of ligand-specific probes to achieve a higher sensitivity than can be achieved with a flat surface. The nanostructured surface is also designed for eventual reproduction via molding or imprint approaches. The sensor-head modeling was performed using rigorous coupled-wave analysis (RCWA) and the boundary element method (BEM) whereas the beam-steering optics were modeled using ray tracing. The modeling and experimental results will be presented.

A surface plasmon polariton (SPP) phase imaging microscope with a sub-wavelength grating structure is developed for high-resolution in-plane image measurement, which can be used on biological samples. Conventionally, most of SPP image systems use prism couplers to induce surface plasmons (SPs), but this approach has some drawbacks such as non-normal incident light producing optical aberration in imaging and making the metrology instrument more complicate. It can be improved by utilizing a normal incident light to excite the SPs through subwavelength grating structure, which replaces the prism so that it can observe in-plane sample on the sensing surface and simplify the instrument. Instead of measuring the intensity of the reflectivity, the phase measurement with higher sensitivity is proposed. In this study, the proposed SPP microscope integrates a common-path phase-shift interferometry (PSI) technique to obtain the two-dimensional spatial phase variation caused by biomolecular interactions on the sensing surface without requiring additional labeling. The common-path PSI technique provides long-term stability, even when it is subjected to external disturbances, to match the requirements of biomolecular interaction analysis. The system is presented as a high stability, high sensitivity, and in-plane SPP phase image.

In this study, a surface plasmon resonance (SPR) biosensor with sub-wavelength grating waveguide for the real-time analysis of biomolecular interactions is developed. The conventional SPR has diffractive grating structure to increase the wave vector for exciting the surface plasmons and then detects biomolecular interactions in high order diffraction light. Using this approach has some disadvantages such as the intensity of high order diffraction light is dimmer to be difficult to measure and the measured reflectivity spectrum is too broadened. The proposed SPR biosensor uses a normally incident white light with the help of subwavelength grating structure and provides a sharper reflectivity spectrum according to waveguide interference both to avoid disadvantages of the conventional SPR biosensor with a grating coupler. When the diffraction grating waveguide structure and the condition of SPR are destroyed by external factors such as slight refractive index changes of the buffer or molecule adsorption on the grating surface, the optical path and momentum of the light coupled through the gold grating into the waveguide are changed and a resonance wavelength shift is induced as a result. By detecting this resonance wavelength shift, the SPR biosensor provides the ability to identify the kinetics of the biomolecular interaction on an on-line basis without the need for the extrinsic labeling of the biomolecules. The proposed biosensing metrology system becomes more simply and convenient for real-time biomolecular interaction analysis.

Surface plasmon resonance (SPR) technique has become over the last ten years a powerful tool allowing the analysis and the detection of biomolecular surface interactions in real time without any use of labels. The highest sensitivity is currently obtained using a mono-spot sensor through the measurement of the complete surface plasmon resonance curve (in angular or spectral configuration), since it is inherently more robust than a single intensity variation measurement. However, this last approach is used to perform SPR imaging, allowing parallel monitoring of hundreds of sensing spots onto a camera. We present in this work a SPR spectro-imaging system including dynamical multi-spectral capabilities. The system is based on the illumination over a vertical slit of the biochip (y-dimension) by a white light source. The spectrum of the reflected light obtained through a grating is then imaged on the x dimension of the camera. The complete spectral resonance curve of a full column of sensing spots can be monitored in parallel and in real-time by this simple apparatus. The influence of the main instrumental parameters and of different data processing are investigated. Clear improvements of the sensitivity have been obtained on refractometric tests and preliminary results on DNA:DNA interactions are finally presented.

Surface plasmon resonance (SPR) coupled fluorescence uses an evanescent electromagnetic field to excite fluorophores in the vicinity of surface. We investigated the influence of enhanced evanescent fields at SPR on the induced fluorescence intensity. The system of this study is based on angle scanning with a half-cylinder prism (SF10) and dual motorized rotation stages to observe the correlation between the evanescent fields and fluorescent intensity of microbeads. With this system, emission from fluorophores only exists in close proximity to the surface of the microbead. The results show that evanescent fields produced at SPR provide more sensitive fluorescence images compared to those measured at a total internal reflection angle.

In this study, we investigated the impact of surface roughness on the sensitivity of conventional and nanowire-based surface plasmon resonance (SPR) biosensors. The theoretical research was conducted using rigorous coupled-wave analysis with Gaussian surface profiles of gold films determined by atomic force microscopy. The results suggest that, when surface roughness ranges 1 nm, the sensitivity of a conventional SPR system is not significantly affected regardless of the correlation length. For a nanowire-based SPR biosensor, however, we found that the sensitivity degrades substantially with a decreasing correlation length. Particularly, at a correlation length smaller than 100 nm, random rough surface may induce destructive coupling between excited localized surface plasmons, which can lead to prominent reduction of sensitivity enhancement.

This study utilizes a surface-enhanced Raman spectroscopy (SERS) based on the attenuated-total-reflection (ATR) method to investigate that the structural information of the biomolecular monolayer on sensing surface can be dynamically observed with a higher signal-to-noise ratio signal. The secondary structures of long oligonucleotides and their influence on the DNA hybridization on the sensing surface are investigated. The SERS spectrum provides the structural information of the oligonucleotides with the help of a silver colloidal nanoparticle monolayer by control of the size and distribution of the nanoparticles adapted as a Raman active substrate. It is found that the ring-breathing modes of adenine, thymine, guanine, and cytosine in Raman fingerprint associated with three 60mer oligonucleotides with prominent secondary structures are lower than those observed for the two oligonucleotides with no obvious secondary structures. It is also determined that increasing the DNA hybridization temperature from 35°C to 45°C reduces secondary structure effects. The ATR-SERS biosensing technique will be used to provide valuable structural information regarding the short-term reversible interactions and long-term polymerization events in the A&bgr; aggregates on the sensing surface.

Surface plasmon resonance (SPR) has been used for some time in chemical and biological sensors. Some of the schemes for exciting surface plasmons include prisms and gratings. Grating-based optical SPR sensors have been demonstrated, which use light intensity variations at resonance or wavelength interrogation. Recently, a gold grating made from a commercial recordable compact disk was used for excitation of surface plasmons and SPR imaging. In this paper, we present a new grating configuration that combines the benefits of multi-angle interrogation with interferometric measurement techniques. This gives array sensing capability over a wide refractive index range. The set-up is based on the gold grating of commercially available recordable compact disks, which are mass produced by injection-moulding, resulting in low cost and disposable grating substrates. The potential of using this system for large sample number analysis is demonstrated.

Noble metal nanoparticles arrays are well established substrates for surface enhanced Raman spectroscopy (SERS). Their ability to enhance optical fields is based on the interaction of their surface valence electrons with incident electromagnetic radiation. In the array configuration, noble metal nanoparticles have been used to produce SER spectral enhancements of up to 10⁸ orders of magnitude, making them useful for the trace analysis of physiologically relevant analytes such as proteins and peptides. Electrostatic interactions between proteins and metal surfaces result in the preferential adsorption of positively charged protein domains onto metal surfaces. This preferential interaction has the effect of disrupting the native conformation of the protein fold, with a concomitant loss of protein function. A major historic advantage of Raman microspectroscopy has been is its non-invasive nature; protein denaturation on the metal surfaces required for SER spectroscopy renders it a much more invasive technique. Further, part of the analytical power of Raman spectroscopy lies in its use as a secondary conformation probe. The protein structural loss which occurs on the metal surface results in secondary conformation readings which are not true to the actual native state of the analyte. This work presents a method for chemical fabrication of noble metal SERS arrays with surface immobilized layers which can protect protein native conformation without excessively mitigating the electromagnetic enhancements of spectra. Peptide analytes are used as model systems for proteins. Raman spectra of alpha lactalbumin on surfaces and when immobilized on these novel arrays are compared. We discuss the ability of the surface layer to protect protein structure whilst improving signal intensity.

Gold and silver nanoparticles have been electrodeposited onto fluorine-doped tin oxide by a two pulse method. The statistical distribution of the size and interparticle spacing of nanoparticles can be controlled by altering the overpotential and duration of the nucleation and growth pulses. Isolated gold and silver nanoparticle covered surfaces prepared in this way display a localized surface plasmon absorption. Raman spectra for immobilized trans-1,2-bis(4- pyridyl) ethylene have been recorded from isolated gold and silver nanoparticle surfaces with different mean particle size, and at different excitation wavelengths. The optimal SERS conditions determined for isolated gold and for silver nanoparticles produce enhancement factors of 5.6 x 10² and 4.0 x 10³ , respectively. Reproducibility is typically 20- 30% RSD due to variations in the SERS active area exposed in different measurements and perhaps variations in the enhancement factor at different sites on a single electrodeposited surface.