This PDF file contains the front matter associated with SPIE Proceedings Volume 7911, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Detection and identification of biomacromolecules is of critical importance in many fields ranging from biotechnology to medicine. Surface-enhanced Raman scattering (SERS) is an emerging technique for the label-free detection and identification of biological molecules and structures with its fingerprinting properties and high sensitivity. However, there are a number of obstacles for its applications for biological macromolecules due to their complexity. In this report, manipulation of microscopic processes in play during the drying of a sessile droplet as a tool to influence the nanoparticle-macromolecule packing, which has dramatic effect on SERS performance, before the SERS acquisition is demonstrated. A process known as the coffee ring phenomenon jams all particles and molecular species to the edges of the droplet during drying. This uncontrolled process has dramatic effects on a SERS experiment, using colloidal metal nanoparticles as substrates, by sweeping everything to the edges and influencing the packing of nanoparticles in the droplet area. A plastic tip was dipped into a drying sample droplet to influence the uncontrolled piling up. A negatively-charged protein, BSA, a positively-charged protein, cytochrom c, and a 20-base long oligonucleotide, were used as model biomacromolecules in this study. While a minimum of one order of magnitude lower concentration improvement in detection limit was observed with negatively-charged biomacromolecules, no significant improvement was observed with positively-charged ones compared to a sample droplet left on the surface without any interference. With the demonstrated approach, picomolar-level biomolecular detection using SERS is possible.

A plasmonic Raman sensor using periodic hole arrays was investigated numerically and experimentally. In previous work, we fabricated a hole array in a thin metal film on a dielectric substrate using focused ion beam lithography and succeeded in observing surface plasmon resonance. Those experimental results agreed well with simulation results (for an array of cylindrical holes) obtained using the finite-difference time-domain method. However, a cylindrical hole array provides insufficient sensitivity (i.e., electric field enhancement) for measuring surface-enhanced Raman scattering (SERS). Therefore, we enhanced the electric field by using focusing holes (tapered structure), which we expected to would give us a larger electric field than the cylindrical holes. Furthermore, for a hole array, we optimized the structural design in terms of metal film thickness, hole diameter, and hole period on the basis of theoretical predictions. We successfully designed and fabricated an arbitrary localized surface plasmon resonance for the optimized array for the excitation wavelength (λ= 632.8 nm) for the target molecule rhodamine 6G for SERS.

We report the development of a novel, low-cost surface enhanced Raman spectroscopy (SERS) substrate that is fabricated by ink-jet-printing silver nanostructures into cellulose paper. Analysis of a liquid sample is performed by spotting a 1 microliter droplet onto the printed SERS substrate. The droplet is contained within a small area of the SERS substrate by ink-jet-printing hydrophobic barriers to define microfluidic boundaries. Using Rhodamine 6G as the analyte, we are able to measure a strong SERS fingerprint signal when only 10 femtomoles of analyte are applied to the paper.

Urinary tract infection (UTI) diagnosis requires an overnight culture to identify a sample as positive or negative for a UTI. Additional cultures are required to identify the pathogen responsible for the infection and to test its sensitivity to antibiotics. A rise in ineffective treatments, chronic infections, rising health care costs and antibiotic resistance are some of the consequences of this prolonged waiting period of UTI diagnosis. In this work, Surface Enhanced Raman Spectroscopy (SERS) is used for classifying bacterial samples as positive or negative for UTI. SERS spectra of serial dilutions of E.coli bacteria, isolated from a urine culture, were classified as positive (10⁵-10⁸ cells/ml) or negative (10³-10⁴ cells/ml) for UTI after mixing samples with gold nanoparticles. A leave-one-out cross validation was performed using the first two principal components resulting in the correct classification of 82% of all samples. Sensitivity of classification was 88% and specificity was 67%. Antibiotic sensitivity testing was also done using SERS spectra of various species of gram negative bacteria collected 4 hours after exposure to antibiotics. Spectral analysis revealed clear separation between the spectra of samples exposed to ciprofloxacin (sensitive) and amoxicillin (resistant). This study can become the basis for identifying urine samples as positive or negative for a UTI and determining their antibiogram without requiring an overnight culture.

In order to increase agricultural productivity, several countries heavily rely on deadly insecticides, known to be toxic to most living organisms and thus significantly affect the food chain. The most obvious impact is to human beings who come into contact, or even consume, pesticide-exposed crops. This work hence focused on an alternative method for insecticide detection at trace concentration under field tests. We proposed a compact Raman spectroscopy system, which consisted of a portable Raman spectroscope, and a surface-enhanced Raman scattering (SERS) substrate, developed for the purpose of such application, on a chip. For the selected portable Raman spectroscope, a laser diode of 785 nm for excitation and a thermoelectric-cooled CCD spectrometer for detection were used. The affordable SERS substrates, with a structure of distributed silver nanorods, were however fabricated by a low-energy magnetron sputtering system. Based on an oblique-angle deposition technique, several deposition parameters, which include a deposition angle, an operating pressure and a substrate rotation, were investigated for their immediate effects on the formation of the nanorods. Trace concentration of organophosphorous chemical agents, including methyl parathion, chlorpyrifos, and malathion, adsorbed on the fabricated SERS substrates were analyzed. The obtained results indicated a sensitive detection for the trace organic analyses of the toxic chemical agents from the purposed portable SERS system.

Micro-patterned thin films interrogated in the Kretschmann configuration of SPR could extend the detection range to lower concentrations and small biomolecules due to a greater sensitivity. This was achieved with the same instrumentation and analysis methodologies developed for SPR with continuous films. The plasmonic properties of micro-patterned thin films composed of various layers of Ag and Au were investigated to find an optimal structure for biosensing application. The analytical parameters and biosensing performances were also evaluated for analysis of biological samples. Au microhole arrays of 3.2 μm periodicity and 1.6 μm hole diameter were prepared using a modified nanosphere lithography (NSL) technique. These microstructures showed optimal plasmonic properties for biosensing applications as they exhibit a 50% increase in sensitivity to refractive index changes compared to continuous thin films of the same thickness. Moreover, microhole arrays presented a faster response time to refractive index changes while analytical parameters such as the resolution and the noise in biosensing measurement were comparable to continuous films. When combined to the appropriate surface chemistry, a greater SPR response was measured for proteins using microhole arrays. Although microhole arrays required an additional preparation step, a cleaning step using oxygen plasma allowed multiple measurements with the same metallic surface with great repeatability. Hence, microhole arrays proved to be a simple approach to improve current SPR biosensing technique. Further investigations to understand the plasmonic properties of microhole were performed using an angle scanning SPR instrument.

A novel surface plasmon resonance (SPR) sensor based on differential spectral phase interferometry is introduced. Our scheme incorporates a broadband white-light emitting diode (WLED) with double-pass Michelson interferometer for highly sensitive Kretschmann SPR phase detection over the visible spectrum. Superior to laser based SPR interferometer which is vulnerable to nonlinear phase saturation and conventional spectroscopic SPR sensor which only measures the spectral intensity, the proposed spectral phase interferometer directly acquires the optimal SPR phase response of every spectral component which is equivalent to having infinitely many SPR laser interferometers operating simultaneously at fixed angle of incidence. Therefore the inherent phase saturation problem due to monochromatic laser source could be readily addressed. As the result, our system prevail over existing phase detection schemes by (1) achieving comparable ultimate detection limit as good as 10^-7 refractive index unit (RIU), (2) extending the phase measurement range as far as 10^-2 RIU, (3) simplifying the phase modulation scheme by directly acquiring the spectral oscillation instead of adding a temporal carrier. Experimental verification with BSA-aBSA interaction demonstrates that our system is capable of achieving ultimate sensitivity of 0.5ng·ml^-1 (3.3pM) for ultra-sensitive aBSA detection which is among the best reported in literature. Yet such sensitivity is extended over a wide range of measurement as each wavelength specific SPR phase jump is monitored over the entire visible spectrum. Further biosensing application such as detection of cytochrome-c with aptamer immobilized on the SPR sensing surface is currently under investigation. We believe that by combination of high sensitivity, wide dynamic range and simplicity of operation, our SPR system would be truly applicable to complicated real-life biosensing.

The peak extinction wavelength of the nano-size noble metal localized surface plasmon resonance (LSPR) spectrum is unexpectedly sensitive to nanoparticle size, shape, and local external dielectric environment. This sensitivity to the environment has enabled the development of a new class of nanoscale affinity biosensors. Aptamer (single strand DNA) based gold nanorods (Au NRs) and magnetic beads (MBs) combined LSPR biosensor has been developed for the rapid and label-free detection of glycated proteins in small solution volumes. An aptamer self-assembly monolayer (SAM) functionalized surface plasmon resonance sensor has also been developed for comparison purposes. For demoonstration purposes, albumin and thrombin are used initially as the target proteins. The ability to monitor such molecules in the body could facilitate the diagnosis and treatment of diabetic patients.

Molecularly imprinted polymer (MIP) thin films and surface plasmon resonance (SPR) sensing technologies were combined to develop a novel sensing platform for monitoring real-time theophylline concentration, which is a compound of interest in environmental monitoring and a molecular probe for phenotyping certain cytochrome P450 enzymes. The MIPs hydrogel is easy to synthesize and provides shape-selective recognition with high affinity to specific target molecules. Different polymerization formulas were tested and optimized. The influence of the monomer sensitive factors were addressed by SPR. SPR is an evanescent wave optics based sensing technique that is suitable for real-time and label free sensing purposes. Gold nanorods (Au NRs) were uniformly immobilized onto a SPR sensing surface for the construction of a fiber optics based prism-free localized SPR (LSPR) measurement. This technique can be also applied to assess the activities of other small organic molecules by adjusting the polymerization formula, thus, this approach also has many other potential applications.

Gold nanorods (GNRs) show enhanced absorption in the near infrared (NIR) region and can be conveniently employed for selective conversion of light into heat. We have succeeded in fabricating hybrid GNRs-polysaccharides composites in the form of semi-solid formulations, films or hydrogels, with good biocompatibility, enhanced stabilization in physiological environment and under laser irradiation and high versatility. Thanks to these properties, these materials are good candidates for applications in many biomedical methodologies including laser-mediated tissue repair and drug delivery. The present results are encouraging toward the development of a novel minimally-invasive technology based on the introduction of bio-inspired nanoplasmonic materials into photothermal applications.

Liposomes show great promise as intravenous drug delivery vehicles, but it is difficult to combine stability in the circulation, extended drug retention and rapid, targeted release at the site of interest. Accessorizing conventional and multicompartment liposomes with photo-activated hollow gold nanoshells (HGN) provides a convenient method to initiate drug release with spatial and temporal control. HGN efficiently absorb near infrared (NIR) light and rapidly convert the absorbed optical energy into heat. Femto- to nano-second NIR light pulses cause the HGNs to rapidly heat, creating large temperature gradients between the HGNs and surrounding fluid. The formation and collapse of unstable vapor bubbles transiently rupture liposome and other bilayer membranes to trigger contents release. Near-complete contents release occurs when the nanoshells are encapsulated within the liposome or tethered to the outer surface of the liposome, with no chemical damage to the contents. Release is achieved by focusing the laser beam at the target, eliminating the need for highly specific targeting ligands or antibodies. Although HGN heating can be intense, the overall energy input is small causing minimal heating of the surroundings. To ensure that drugs are retained within the liposomes until delivery in a physiological environment, we have made novel multicompartment carriers called vesosomes, which consist of an outer lipid bilayer shell that encloses and protects the drug-carrying liposomes. The second bilayer increases the serum half-life of ciprofloxacin from <10 minutes in liposomes to 6 hours in vesosomes and alters the release kinetics. The enhanced drug retention is due to the outer membrane preventing enzymes and proteins in the blood from breaking down the drug-carrying interior compartments.

Here we are representing the results of the tight-binding molecular modeling of the process of the synthesis of the dimer (C₂₈)₂ and the retinoid-C₆₀ linked molecules inside a carbon nanotube. The nanotube is located between two electrodes connected with a power source. The positively charged fullerene C₆₀ moves from one end of the tube to the other. The motion of C₆₀ is controlled by an external electric field. Moving fullerene C₆₀ compresses the molecules located in one of the nanotube ends. The located molecules undergone axial compression moves toward each other. When the pressure created in the tube provides both the overlap of π-electrons of the C₂₈ fullerenes and the covalent bonds formation, the intermediate phase of the (C₂₈)₂ dimer is synthesized: (C₂₈)₂ [6 + 6]. The pressure becomes equal to ~35 TPa. After returning the fullerene C₆₀ to the initial state, the (C₂₈)₂ dimer is izomerized with the reorientation in the tube field. So, (C₂₈)₂ transfers to the stable phase (C₂₈)₂ [1+1]. If the moving fullerene C₆₀ compresses the retinol-molecule then synthesis of retinoid-C₆₀ linked molecules takes place. In conclusion, the dimer/polymer synthesis inside the carbon nanotube is real, the dimers and polymers are stable and may be synthesized in the field of the holding potential nanotube, and the fullerene polymerization in the nanotube guarantees the absence of any additives in the final product. The motion of the atoms is determined by the classical molecular modeling method where Newton's equations of motion are integrated with a third-order Nordsieck predictor corrector. Time steps of 0.15-0.25 fs were used in the simulations.

This work presents simultaneous imaging and detection of three types of cell receptors using three types of plasmonic nanoparticles. The size, shape, and composition-dependent scattering profiles of these particles allow for a system of multiple distinct molecular markers using a single optical source. With this goal in mind, a system of tags consisting of anti-EGFR gold nanorods, anti-IGF1R silver nanospheres, and anti-HER-2 gold nanospheres was developed for monitoring the expression of three commonly overexpressed receptors in cancer cells. These labels were chosen because they each scatter strongly in a distinct spectral window. A hyperspectral dark-field microscope was developed to record the scattering spectra of cells labeled with these molecular tags. The ability to monitor multiple tags simultaneously may lead to applications such as profiling the immunophenotype of cell lines and gaining better knowledge of receptor signaling pathways. Single, dual, and triple tag experiments were performed to analyze the specificity of the nanoparticle tags as well as their effect on one another. While distinct resonance peaks in these studies show the ability to characterize cell lines using conjugated nanoparticles, shifts in these peaks also indicate changes in the cellular dielectric environment which may not be distinct from plasmon coupling between nanoparticles bound to proximal receptors.

We report on a highly sensitive plasmon-based nanosensor. This hybrid sensor is composed of a glass tip with a set of gold nanoparticles grafted at its apex. It allows to optimize the excitation and detection of the localized surface plasmon resonance of metal nanoparticles and thus to increase dramatically the sensitivity. First results show the possibility to detect a concentration as low as 10^-10 M on a single gold dimer. Furthermore, promising SERS measurements are reported.

The on-chip detection of a weak optical signal in biological experiments can easily be complicated by the presence of an overwhelming background signal, and as such, pre-detection background suppression is substantively important for weak signal detection. In this paper, we report a structure that can be directly incorporated onto optical sensors to accomplish background suppression prior to detection. This structure, termed surface-wave-enabled darkfield aperture (SWEDA), consists of a central sub-wavelength hole surrounded by concentric grooves that are milled onto a gold layer. Incoming light can be collected and converted into surface waves (SW) by the concentric grooves and then be recoupled into propagating light through the central hole. We show that the SW-assisted optical component and the direct transmission component of the central hole can cancel each other, resulting in near-zero transmission under uniform illumination (observed suppression factor of 1230). This structure can therefore be used to suppress a light field's bright background and allow sensitive detection of localized light field non-uniformity (observed image contrast enhancement of 27dB). We also show that under a coherent background illumination, a CMOS pixel patterned with the proposed structure achieves better SNR performance than an un-patterned single pixel.

We have developed a novel multiphoton microscopy technique not relying on (and hence not limited by) fluorescence emission, which exploits the third-order nonlinearity called four-wave mixing of gold nanoparticles in resonance with their surface Plasmon. The coherent, transient and resonant nature of this signal allows its detection free from backgrounds that limit other contrast methods for gold nanoparticles. We show detection of single 10nm gold nanoparticles with low excitation intensities, corresponding to negligible average thermal heating. Owing to the the third-order nonlinearity we measure a transversal and axial resolution of 140nm and 470nm respectively, better than the one-photon diffraction limit. We also show high-contrast imaging of gold-labels down to 5nm size in Golgi structures of HepG2 cells at useful imaging speeds (10 kHz pixel rate). Thermal dissociation of gold nanoparticles from their bonding sites when varying the excitation intensity is also investigated.

The distance dependent interactions between individual noble metal nanoparticles enable active plasmonic nanostructures with applications in microscopy, sensing and imaging. If two nanoparticles approach each other close enough for their plasmons to couple, the resonance wavelength of the dimer red-shifts. This effect is utilized in plasmon coupling microscopy (PCM) to resolve subdiffraction limit contacts. We apply polarization resolved PCM to monitor changes in the orientation and interparticle separation of individual silver plasmon rulers during their lateral diffusion on a plasma membrane. The ability to track the position of individual silver plasmon rulers and to simultaneously monitor their rotation and separation in PCM makes plasmon ruler probes for the local structure of the supporting membrane.

Various gold nanostructures are being investigated for medical and biological uses. For many medical applications, it would be beneficial to use near infrared (NIR) excitation as well as small gold nanospheres which can easily reach the cytoplasm and cell nucleus. For that purpose, we propose a novel nanostructure, the "shell aggregate," which consists of small nanospheres aggregated around a core such as an intracellular organelle. The extinction efficiency of such monolayfer and bilayer shell aggregates is thoroughly investigated with appropriate simulations using the Descrete Dipole Approximation (DDA) method. This technique can deal with any arbitrary size, shape, synthesis and external environment. The effect of parameters such as the overall radius of the nanostructure, the small nanosphere radius, and the distance between the nanospheres, on the extinction efficiency factor of the nanostructures was examined. The results indicate that the extinction spectra appear to depend heavily on the distance between the small nanospheres. Finally, the monolayer shell aggregate could be a suitable candidate for use in various biological, intracellular, applications since it provides a reasonably tunable plasmon resonance wavelength while the small size of its components can be exploited for intracellular distribution.

In this paper, we present detailed experimental analysis on the optical resonance transmission properties of nano-hole arrays in metallic films. Arrays of sub-wavelength holes with different periodicity (spacing between adjacent holes) in a square lattice arrangement were fabricated in optically thick metal films (Au, Ag, and Al) on a Pyrex substrate using Electron Beam Lithography. The optical transmission spectra of the nano-hole arrays were characterized in the visible and near infrared regime. The optical resonance transmission properties were observed to depend on the type of metal film and the periodicity in the lattice arrangement.

In this paper, we present experimental analysis on the effect of composition of the adhesion layer (chromium or titanium) between gold and a Pyrex substrate on the optical resonance transmission properties of nano-hole arrays in an optically thick gold film. Nano-hole arrays of different hole periodicities in a square lattice arrangement were fabricated with three types of adhesion layer (5 nm Cr, 5 nm Ti or 10 nm Ti). The optical transmission of each nano-hole array was measured and the optical resonance transmission properties were analyzed and compared as a function of hole periodicity and type of adhesion layer.

We microfabricated the plasmonic nanopore with ~ 1 nm on top of the pyramid for single molecule dynamics. This plasmonic micro device provides huge photon transmission through the fabricated nanochannel on the top of the pyramidal structure. This can generate the huge photonic pressure gradient between the free space and nanopore inside. The huge pressure gradient can be attributed to the resonance transmission between the fabricated V groove cavity and the nanosize waveguide formed during the metal deposition. This fabricated huge photonic device can be utilized as biomolecule translocation and single molecule dynamics.

We investigated the combination of near infrared (NIR) photothermolysis and photodynamic therapy against different models of bacteria (S. aureus, S. epidermidis both methicillin susceptible and resistant), in order to discover possible synergistic pathways in the fight against cancer. Photothermolysis was mediated by NIR light absorption from gold nanorods, which were coated with polyethylene glycol to gain biocompatibility and provide for a convenient interface with the bacterial cell walls. At the same time photodynamic therapy was delivered by administration of Indocyanine Green (ICG), whose spectrum of molecular excitation overlaps the plasmonic oscillations of gold nanorods (~ 800 nm). Therefore irradiation with NIR light from a low power diode laser resulted into simultaneous photothermolysis and generation of reactive oxygen species and cytotoxic byproducts of ICG. We assessed the inhibition of the bacterial colony forming ability under different NIR light exposures, and compared the performance of the combined treatment (gold nanorods plus ICG) with the projected addition of the separate treatments (either gold nanorods or ICG). Our preliminary results may originate from the interplay of synergistic and conflicting interactions, which may include e.g. the enhanced intake of cytotoxic species due to permeabilization of the bacterial cell walls, quenching of ICG and modification of the bleaching of ICG due to the noble metal surface.

Optical techniques towards the realisation of sensitive and selective biosensing platforms have received a considerable amount of attention in recent times. Techniques based on interferometry, surface plasmon resonance, field-effect transistors and waveguides have all proved popular, and in particular, spectroscopy offers a large range of options. Raman spectroscopy has always been viewed as an information rich technique in which the vibrational frequencies reveal a lot about the structure of a compound. The issue with Raman spectroscopy has traditionally been that its rather low cross section leads to poor limits-of-detection. In response to this problem, Surface-enhanced Raman Scattering (SERS), which increases sensitivity by bringing the sample in contact with many types of enhanceing substrates, has been developed. Here we discuss a facile and rapid technique for the detection of pterins using colloidal silver suspensions. Pteridine compounds are a family of biochemicals, heterocyclic in structure, and employed in nature as components of colour pigmentation and also as facilitators for many metabolic pathways, particularly those relating to the amino acid hydroxylases. In this work, xanthopterin, isoxanthopterin and 7,8- dihydrobiopterin have been examined whilst absorbed to SERS-active silver colloids. SERS, while far more sensitive than regular Raman spectroscopy, has its own issues relating to the reproducibility of substrates. In order to obtain quantitative data for the pteridine compounds mentioned above, exploratory studies of methods for introducing an internal standard for normalisation of the signals have been carried out.e

Localized Surface Plasmon Resonance (LSPR) is based on the electromagnetic-field enhancement of metallic nanoparticles. It is observed at the metal-dielectric interface and the resonance wavelength can be tuned by the size, shape, and periodicity of the metallic nanoparticles and the surrounding dielectric environment. This makes LSPR a powerful candidate in bio-sensing. In the present work, the size and period dependency of the LSPR wavelength was studied through simulations and fabrications. The surface functionalization, that transforms the surface into a sensing platform was done and verified. Finally, the concentration dependency of the LSPR shifts was observed. All the measurements were done by a transmission set-up. The study is at an early stage, however results are promising. The detection of specific bacteria species can be made possible with such a detection method.