This PDF file contains the front matter associated with SPIE Proceedings Volume 7192, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and the Conference Committee listing

Functionalised nanoparticles have been used in a number of studies including detection of DNA at ultra low concentrations, immuno-histochemistry and more recently as substrates for surface enhanced resonance Raman scattering (SERRS) based imaging approaches. The advantages of using metallic nanoparticles are that they are very bright in terms of their optical characteristics and also can be functionalised to provide a SERRS response and hence provide a unique Raman fingerprint. Here we present the functionalisation of gold and silver nanoparticles in such a way that the enhancement effect can be greatly increased through biological interaction and as such effectively turn on the SERRS effect. In an advancement of this nanoparticles have been used as imaging agents for single cells when functionalised with an appropriate antibody and can give information on the expression of specific receptors on cell surfaces as well as sub-cellular compartmentalisation information.

According to the World Health Organization, cardiovascular disease is the most common cause of death in the world. In the US, over 115 million people visit the emergency department (ED), 5 million of which may have acute coronary syndrome (ACS). Cardiac biomarkers can provide early identification and diagnosis of ACS, and can provide information on the prognosis of the patient by assessing the risk of death. In addition, the biomarkers can serve as criteria for admission, indicate possibility of re-infarction, or eliminate ACS as a diagnosis altogether. We propose a SERSbased multi-marker approach towards a point-of-care diagnostic system for ACS. Using a nanofluidic device consisting of a microchannel leading into a nanochannel, we formed SERS active sites by mechanically aggregating gold particles (60 nm) at the entrance to the nanochannel (40nm×1μm). The induced capillary flow produces a high density of aggregated nanoparticles at this precise region, creating areas with enhanced electromagnetic fields within the aggregates, 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 qualitative information of brain natriuretic peptide (biomarker of ventricular dysfunction or pulmonary stress), troponin I (biomarker of myocardial necrosis), and C-reactive protein (biomarker of inflammation potentially caused by atherosclerosis).

Surface enhanced resonance Raman scattering (SERRS) is an analytical technique with several advantages over competitive techniques in terms of improved sensitivity and multiplexing. We have made great progress in the development of SERRS as a quantitative analytical method, in particular for the detection of DNA. SERRS is an extremely sensitive and selective technique which when applied to the detection of labelled DNA sequences allows detection limits to be obtained which rival, and in most cases, are better than fluorescence. Here the conditions are explored which will enable the successful detection of DNA using SERRS. The enhancing surface which is used is crucial and in this case suspensions of nanoparticles were used as they allow quantitative behaviour to be achieved and allow analogous systems to current fluorescence based systems to be made. The aggregation conditions required to obtain SERRS of DNA are crucial and herein we describe the use of spermine as an aggregating agent. The nature of the label which is used, be it fluorescent, positively or negatively charged also effects the SERRS response and these conditions are again explored here. We have clearly demonstrated the ability to identify the components of a mixture of 5 analytes in solution by using two different excitation wavelengths and also of a 6-plex using data analysis techniques. These conditions will allow the use of SERRS for the detection of target DNA in a meaningful diagnostic assay.

The development of an assay for the detection of gene mutations has been attempted based on surface-enhanced Raman scattering (SERS). Using multiplexing property and high sensitivity of SERS technique, the detection of all mutation possibilities on one given spot is achievable. To test the feasibility of approach, SNPs and other types of mutations such as insertion and deletion are investigated. The PCR amplified and isolated genomic DNA without PCR amplification is immobilized on poly-L/D-lysine coated glass surface after denaturing with heating. The SERS probes are prepared by simultaneous attachment of oligonucleotides complementary to the target mutation regions and Raman active dyes to 13 nm gold nanoparticles (GNPs). After the hybridization of SERS probes on the poly-L/D-lysine surfaces, it was stained with silver colloidal nanoparticles for further enhancement of Raman scattering. In the second approach, Raman active dyes are chemically attached on gold nanoparticles and a thin layer of silver film is deposited on top of it to prepare core-shell nanoparticles. The complementary oligonucleotides to the target regions of the gene are chemically attached to silver surfaces of the nanoparticles. The promising results indicate that it is possible to detect certain mutation types without PCR amplification using the approach.

The purpose of this study is to explore the feasibility of using Surface Enhanced Raman Spectroscopy (SERS) to image the distribution of Epidermal Growth Factor Receptor (EGFR) in cells. To accomplish this task, 30 nm gold nanoparticles (AuNPs) tagged with antibodies to EGFR (10¹² per ml) are incubated with cells (10⁶ per ml) of the A431 human epidermoid carcinoma cell line and normal human bronchial epithelial (NHBE) cells. Using the 632.8 nm excitation line of a He-Ne laser, Raman spectroscopy measurements are performed using a point mapping scheme. SERS signals are observed with an overall enhancement of 4-7 orders of magnitude. Raman intensity maps of the 1480 and 1583 cm^-1 peaks correlate well with the expected distribution of AuNPs and EGFR. Normal cells show little to no enhancement. The results therefore present a simple yet effective means to image EGFR over-expression.

In this work we developed new type of biosensors based on nanolayered metal-dielectric structure. It was found that using the combination of nano-size (from 15 to 25 nm) layers of gold and dielectric support a new type of plasmon modes: bulk plasmon-polariton (BPP). In this work the role of corrugation for different nano-layers has been investigated. Thus, it was found that even corrugation of one 25 nm nano-layer results in effective coupling of BPP modes. The coupling efficiency as a function of corrugation depth, corrugation profile has been investigated. Diffraction gratings were fabricated by hot embossing technology.

When a radially polarized beam is focused onto a metal-dielectric interface, the entire beam is TM polarized with respect to the interface. Consequently surface plasmons can be excited at all directions. These surface plasmons will propagate to the geometric center, constructively interfere with each other and generate a strongly focused evanescent nondiffracting Bessel beam. In this paper, we report the experimental results on the direct imaging of such plasmonic focusing. Radially polarized beam is tightly focused onto a silver-glass interface with a high numerical aperture oil immersion objective lens. The intensity distribution at the back focal plane of the objective lens after reflection is captured with a CCD camera. A dark ring corresponding to surface plasmon resonance excitation by a focused radially polarized beam is observed. A collection mode near field scanning optical microscope is applied to map the two-dimensional intensity distributions at different distances from the sample to verify the non-spreading and decaying natures of the evanescent Bessel beam.

This study has developed a surface plasmon resonance (SPR) biosensor with a metal nanoslit structure which couples incident light for real-time analysis of biomolecular interactions. The main advantages of the proposed SPR biosensor are to avoid the disturbance from buffer solution and only need a compact system compared with SPR biosensors with nanostructures. The configuration of the SPR biosensor mainly consists of glass substrate, a metal layer with nanoslits, a biomolecular interaction layer, and buffer solution, sequentially. The incident light first excites the surface plasmons (SPs) which are oscillated on the interface between the glass substrate and the metal layer, and then the emission field of the SPs is coupled via the nanoslits and excites the SPs on the sensing region which is between the metal layer and the buffer solution. The variations of the sensing SPR reflection spectra can be easily detected according to the dynamic biomolecular interaction on the sensing region. The theoretical simulations and the experimental results confirm that the proposed biosensor not only retains the sensitivity based on a grating-coupling SPR biosensor, but also avoids the disturbance from the buffer solution.

Surface plasmon resonance imaging optical biochips have shown promising applications in the field of genetic diagnosis due to their ability to record hundred of different biomolecular interactions without any label and in real time. Single nucleotide polymorphisms can be detected using this technique. To improve the accuracy and the reliability of biochips a quantitative study on the impact of temperature on DNA hybridization and secondary structures is shown for various oligonucleotides sequences. In this proceeding we focus on the impact of temperature T on the binding rate kinetic τ, using the model case of DNA:DNA interactions. We show that SPRI can quantify such characteristics as τ(T) and that they follow the Langmuir model variation law.

Finite-difference time-domain calculations are used to study how fluorescence is modified when fluorophores are located in proximity to various metal nanoparticle systems. The fluorophore is modeled as a radiating point dipole with orientation defined by its polarization. The angle-resolved far-field distributions of the emission in a single plane are computed. The emission patterns show interesting intensity variations and angular profiles depending on the dipole orientation, size of the metal particles and the metal-dipole spacing. We also compute changes in the total radiated power through a closed volume containing the fluorophore and metal nanoparticles relative to an isolated fluorophore. This change in total radiated power is proportional to changes in the relative radiative decay rates of the fluorophore-metal system. Our results suggest a high dependence of the radiated power on the fluorophore orientation, particle size, metalfluorophore distance and particularly the presence of metal nanoparticle dimers. We examined the effect of a fluorophore on the near-fields around silver nanoparticles. The fields can be enhanced compared to the isolated fluorophore and exhibit interesting spatial variations around the nanoparticle that can be useful for applications involving molecular spectroscopy.

We report the influence of organic electroluminescence (OEL) device on the color tunability and emission efficiency enhancement by surface plasmon grating coupled emission (SPGCE). The effect of coupling active surface plasmon polaritons (SPPs) on the metal nanostructure grating with organic material interface was studied. The dispersion relation was obtained from angle-resolved emission measurements. The combination of organic/metal interface SPPs mode allows specific directional emission rather than isotropic emission. Control of light emission angle in the SPGCE is dependent upon the index of refraction at the organic/metal/dielectric interface. Recent experimental results and potential applications of an active plasmonics biosensor with enhanced resonant energy emission due to interactions on the organic/metal nano-grating were presented and discussed.

We have investigated the effects of tuning the localized surface plasmon resonance (LSPR) of a silver film on the extinction spectrum, Raman signal, and fluorescence intensity from nearby fluorophores. We observe the formation of hybridized modes due to strong coupling between the plasmonic and molecular excitations. The Raman spectra of R6G on these films show an enhancement of many orders of magnitude due to surface enhanced scattering mechanisms; we find a maximum signal when a hybridized mode lies in the middle of the Stokes shifted emission band. The effect of fluorophore-film separation on fluorescence intensity has been investigated using an alumina spacer layer. An enhancement in detected signal of up to 18× is observed relative to that detected from a bare Ag film. Overall, we observe a greater than 40× increase in detected intensity from the alumina-coated Ag film relative to fluorophores on glass; this is a result of increased collection efficiency and a greater radiative emission rate.

Metallic nanoparticle clusters coupling strong surface plasmons with a Raman reporter molecule have been developed for application in multiplexed optical imaging. Of interest to our work is the ability of the agents to serve as surface-enhanced Raman spectroscopy (SERS) probes. We present the seed-mediated synthesis and characterization of rhodamine B isothiocyante Au nanoparticle clusters (RhB-AuNPCs). RhB-AuNPCs are anisotropic structures which contain the Raman reporter, RhB, embedded between a gold aggregate core and gold surface layer. In contrast to typical SERS studies, the Raman signal originates from the probe (RhB-AuNPCs) and not from RhB incubated with a noble metal colloid. Characterization of the probes' optical properties is presented. The overall goal of our study is to prepare probes that may be used for the identification and spectroscopic labeling of multiple molecular biomarkers utilizing SERS imaging.

Metal nanoparticles posses the property of changing their optical properties as a function of both internal characteristics (size, shape, dielectric function) and refractive index of the local environment. A special class of applications in the field of biosensing uses the dependency of the nanoparticle's plasmonic peak localization on the local refractive index change. The response of this type of sensors is usually monitored by the change of the extinction spectrum of an ensemble of nanoparticles where analytes interact with functionalized nanoparticles in solution or immobilized at an interface; detection is done with a spectrophotometer. This type of sensors has a limited sensitivity. This can be overcome by using single nanoparticle based biosensors. This type of sensors measures the changes of the scatter spectrum of a collection of individually addressable functionalized nanoparticles in the presence of analytes. Here we report on a new detection method of binding events of analytes to functionalized gold nanoparticle using a standard colour camera attached to a darkfield microscopy setup. This setup is capable of parallel detection of the spectral shifts of thousands of 60 nm antibody-functionalized gold spheres as a result of binding events of protein analyte molecules. This setup can be the basis for multiplexing and quantification.

Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER2) contribute to the regulation of cell proliferation, and when jointly over-expressed are associated with several types of cancer. The ability to monitor both receptors simultaneously results in a more accurate indicator of degree of cancerous activity than either receptor alone. Plasmonic nanoparticles (NPs) show promise as a potential EGFR and HER2 biomarker over alternatives such as fluorophores and quantum dots, which are limited by their cytotoxicity and photobleaching. To observe immunolabeled NPs bound to receptor-expressing cells, our past experiments were conducted using a novel optical darkfield microspectroscopy system. We implemented an epi-illumination darkfield broadband light train, which allows for darkfield analysis of live cells in culture with enhanced NP contrast. Under this setup, molecularly specific binding of NPs immunolabeled with anti-EGFR was confirmed. We have since adapted our darkfield setup, which previously only obtained spectral information from a line imaging spectrometer, to incorporate hyperspectral imaging capabilities, allowing widefield data acquisition within seconds. The new system has been validated through observation of shifts in the peak wavelength of scattering by gold NPs on silanated cover glasses using several immersion media. Peak resonant scattering wavelengths match well with that predicted by Mie theory. We will further demonstrate the potential of the system with simultaneous molecular imaging of multiple receptors in vitro using labeled EGFR+/HER2+ SK-BR-3 human breast cancer cells with anti-EGFR immunolabeled gold nanospheres and anti-HER2 immunolabeled gold nanorods, with each scattering in different spectral windows. Additional trials will be performed to demonstrate molecularly specific binding using EGFR+/HER2- MDA-MB-468 and HER2+/EGFR- MDA-MB-453 breast cancer cells.

We observed spatial and temporal behaviors of surface enhanced Raman scattering (SERS) signals with gold nanoparticles in living cells. Gold nanoparticles with the diameter of 50 nm were introduced into macrophage cells through endocytosis. We performed observation of SERS signals from a macrophage with 785 nm excitation. Strong SERS signal from the particles in the cell was observed, and spectrum from each particle shows difference in Raman peaks and intensity. We also observed time-lapse SERS and dark-field image with a frame rate of 3 min. We confirmed that strong SERS signal from the particle in the cell and the spectral changes with positions of the particles in the cell.

Given their tunable optical properties and high optical absorption and scattering cross sections, gold nanoshells (GNS) have been explored for a number of in vitro and in vivo imaging contrast and cancer therapy agents. While it has been shown that GNSs preferentially accumulate at the tumor site, little is known about the accumulation kinetics within the tumor. We demonstrate accumulation kinetics of GNSs in bulk tumors and histology slides using two-photon induced photoluminescence (TPIP) imaging. We found that GNSs had a heterogeneous distribution with higher accumulation at the tumor cortex. In addition, GNSs were observed in unique patterns surrounding the perivascular region. These results demonstrate that direct luminescence based imaging of metal nanoparticles provides high resolution and molecular specific multiplexed images.

Investigation on the interaction of small particles, e.g. gold nanoparticles with light is a current field of high interest. As light can be absorbed, enhanced or scattered by the nanoparticles a wide variety of possible applications become possible. If the electrons of such a nanoparticles oscillate with the incident light, plasmon resonances occur. Provided that these particles are brought very close to a cell, the cell membrane gets perforated due to the laser induced effect. We investigate nanoparticle mediated laser perforation as an alternative technique for cell transfection. By using weakly focussed femtosecond laser pulses, 150 nm gold particles were stimulated to perforate the cell membrane. Through the perforated area of the membrane macromolecules e.g. DNA are able to enter the cell. By this technique GFSHR-17 rat cells were successfully transfected with GFP vector and the dependence on laser parameters and concentration were studied. Even after 48 hours after manipulation the transfected cells show no indications of apoptosis or necrosis. This technique allows the transfection of cells by opto-perforation without the need of tight focusing conditions and single cell targeting- opening the way for a wide field of applications.

We investigated the effect of rough surface on the performance of extinction-based localized surface plasmon resonance (LSPR) biosensors. The sensor measures resonance wavelength shifts in transmittance due to biomolecular interactions amplified by periodic nanostructures. The numerical calculation was conducted using rigorous coupled-wave analysis with Gaussian random profiles. The results suggest that, when a surface has a roughness smaller than 2 nm, the sensitivity of an LSPR biosensor is not significantly affected regardless of correlation length (CL). However, we found that extinction peak amplitude and curve width are affected substantially with a decrease in CL. At CL less than 100 nm, surface roughness may induce interference between localized surface plasmons excited by the surface and nanowires, which can lead to significant degradation of sensor performance.

In this paper, a new parallel scan spectral surface plasmon resonance (SPR) 2D sensing system is presented. With a lineshaped light illumination, an image acquired with area CCD detector provides both SPR wavelength information and 1D spatial distribution. Thus, 2D distribution of refractive index of the entire sensing plane can be obtained with 1D optical line parallel scan. A refractive index distribution model and a manually dotted DNA array are measured with this system. The technology shows advantages of both high sensitivity and high throughput in these results, and could have potential applications in biochips analysis.

In a SPR sensor, a glass slide is coated with a metal film, whose thicknesses, density and dielectric constant deadly affect the measurement sensitivity and precision in bio-detection. The optimum thickness of gold film used in SPR is between 40nm and 50nm without adhesive film, according to calculations based on multilayer reflection model cited in large numbers of literatures. But experimental study on the optimum film parameters are still lacking attribute to the limitation of film coating technology and high-precision thickness measurement technology. The optimum gold film thickness is not 45nm observed in our SPR bio-detection, and the property of adhesive film which is needed for enhancing the adhesion of metal film affects the SPR responsive bandwidth and minimum reflectivity. The experiment study on the sensor gold film and the property of adhesive film for SPR sensor are described in this paper using high precision SPR detection system, X-ray diffraction and magnetron sputtering technology. The optimum thickness for single gold film is 44nm and is 42nm for gold film with 2.4nm Cr adhesive layer. A new estimating factor is proposed to evaluate the quality and resolution of metallic film.

We report a compact multi-channel biosensor based on diffraction grating-coupled SPR for the most demanding detection applications in the field or home environments. The sensor utilizes special diffraction grating (referred to as surface plasmon coupler and disperser - SPRCD) for coupling light into the surface plasmon and its simultaneous wavelength dispersion through a different diffraction order. This approach combines most of the optical instrumentation on a single SPR chip produced by stamper hot-embossing technique which is fully compatible with mass production. The sensor consists of a disposable cartridge (SPR chip and microfluidics) and a compact SPR instrument with the footprint which includes optical system of SPR sensor, supporting and data acquisition electronics, microfluidics delivering sample into six independent sensing channels in the cartridge, and temperature stabilization. We demonstrate that the sensor is able to measure changes in the refractive index as low as 2x10^-7 refractive index units (RIU) and to detect the binding of antibodies to the antigen-coated sensor surface.