Surface enhanced Raman scattering (SERS) has been used to detect biological molecules at a low concentration. We developed a rapid Raman imaging system, which can image dynamic activity of SERS agents, such as gold nanoparticles, in a living cell and the temporal behaviors of SERS spectra. Combination of slit scanning and an EM-CCD camera for measuring SERS spectra enables us to obtain a SERS image in a few seconds. The system can also be used to track a single particle moving in a cell with a laser focus and measure SERS spectra with a temporal resolution of 50 msec. By using the developed microscope systems, we monitored the change of SERS spectra associated cell transportation functions.

Recently there has been tremendous interest about the dynamical sequence of fabrication of the solid state nanopore due to its capability of the nanosize solid state biosensor as a single molecule sensor. Depending upon the instruments such as transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM), the dynamics of nanopore formation present different physical mechanisms. In this report, formation of the nanopores was examined. Metallic nanopores with ~ 50 nm diameter on top of the oxide pyramid were fabricated using conventional Si microfabrication techniques followed by wet isotropic etching of the oxide; sputter metal deposition followed by the focused ion beam (FIB) techniques. No shrinking phenomena were observed for the nanopore diameter greater than 50 nm under electron beam irradiation using TEM. However, for high scanning electron beam irradiation using FESEM, shrinking of the Au nanopore was always observed. We do believe that these phenomena can be attributed to the liquid phase surface modification for TEM electron beam and adiabatic solid state phase surface modification for high scanning FESEM. For a huge amount of energy input from high scan rate and the poor thermal conduction to its surrounding area, the energy spike inside the electron penetration area would occur. However, a TEM electron beam irradiation without repetitive scan can provide the liquid phase surface modification.

In the present work we investigated the properties and behavior of plasmonic modes of silver nanocube monolayers with respect to reflection and transmission of visible radiation. Uniform monolayers of low particle densities were created using the Langmuir-Blodgett technique using the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) as a passive spacer. Dipole-dipole coupling modes were avoided by depositing at low pressures to ensure sufficient spacing between the nanocubes. The refractive index sensitivities of plasmonic modes for monolayers on glass, silicon thin films, and bulk silicon wafers were measured using varying solutions of water and ethylene glycol. By varying the refractive index of the substrates it is possible to investigate the relative contribution of plasmonic modes with respect to absorption of the incident signal.

Plasmonic properties of monolayers of strongly interacting silver nanocubes (AgNC) with controlled interparticle spacing are investigated. Uniform monolayers with controlled particle densities are made using the Langmuir-Blodgett technique with passive phospholipid spacers, such as dioleoyl phosphatidylcholine (DOPC). Both extinction intensity and wavelength of dipole-dipole coupling modes are tuned via particle spacing. The refractive indices of the substrates are used to tune dipolar and interparticle coupling modes via deposition onto thin films of silicon (0 - 25nm). By varying silicon film thickness it is possible to shift and control peak widths and position for both the dipole and interparticle dipole-dipole coupling modes. Control of plasmon shifts and interparticle spacing is applied towards the optimization of SERS substrates. SERS substrates using a Rhodamine B label are tuned at different excitation wavelengths which are in resonance with either the plasmon dipole, fluorescent dye, or interparticle coupling mode. Substrates display reproducible enhancement across multiple sites. This work presents methodology to design and optimize uniform silver nanocube SERS substrates through tuning of plasmon shifts and particle spacing.

Degree of coherence (DOC) of a paired surface plasma waves (PSPWs) in the paired surface plasma waves biosensor (PSPWB) is proposed and discussed in which a paired of surface plasma waves are excited by using a pair of highly spatial and temporal correlated P-polarized waves in a SPR device of the Kretschmann configuration. The heterodyne signal from reflected paired P-polarized laser beam is generated where the visibility of the signal is proportional to DOC of PSPWs in term of the ratio of AC and DC components of the signal. The experimental result shows that the DOC of PSPWs versus incident angle of laser beam which relates to intrinsic phonon distribution in metal film becomes much sensitive than conventional amplitude or intensity sensitive surface plasmon resonance (SPR) biosensor. Finally, the dynamic range of protein-protein interaction at ultralow concentrations is discussed.

One of the In-vivo system's challenge is real time display the sensing information. Usually Ultrasound, CT, MRI, PET are used to get the internal information, this thesis proposed another approach to address the display challenge. Special nano-particles are in-taken or injected to living subject (usually into blood circulation) to sense and collect psychological information when the active particles pass through the tissues of interest. Using the wound healing mechanism, these activated particles (Information collected) can be drifted out to the wound area and adhibited close to the skin, then skin can show different color if the activated particles are concentrated enough in the specific area to create a skin screen. The skin screen can display the blood status, internal organ's temperature, pressure depending the nano-particles' function and their pathway. This approach can also be used to display in-body video if the particles are sensitive and selective enough. In the future, the skin screen can be bio-computer's monitor. The wound healing in an animal model normally divides in four phase: Hemostasis, Inflammation, Proliferation and Maturation. Hemostasis phase is to form a stable clot sealing the damaged vessel. Inflammation phase causes the blood vessels to become leaky releasing plasma and PMN’s (polymorphonucleocytes) into the surrounding tissue and provide the first line of defense against infection. Proliferation phase involves replacement of dermal tissues and sometimes subdermal tissues in deeper wounds as well as contraction of the wound. Maturation phase remodels the dermal tissues mainly by fibroblast to produce greater tensile strength. The skin screen wound will be carefully controlled to be triggered at dermis layer.

We developed a new metrology platform which can detect real-time changes in both a phase-interrogation mode and intensity mode of a SPR (surface plasmon resonance). We integrated a SPR and ellipsometer to a biosensor chip platform to create a new biomolecular interaction measurement mechanism. We adopted a conductive ITO (indium-tinoxide) film to the bio-sensor platform chip to expand the dynamic range and improve measurement accuracy. The thickness of the conductive film and the suitable voltage constants were found to enhance performance. A circularly polarized ellipsometry configuration was incorporated into the newly developed platform to measure the label-free interactions of recombinant human C-reactive protein (CRP) with immobilized biomolecule target monoclonal human CRP antibody at various concentrations. CRP was chosen as it is a cardiovascular risk biomarker and is an acute phase reactant as well as a specific prognostic indicator for inflammation. We found that the sensitivity of a phaseinterrogation SPR is predominantly dependent on the optimization of the sample incidence angle. The effect of the ITO layer effective index under DC and AC effects as well as an optimal modulation were experimentally performed and discussed. Our experimental results showed that the modulated dynamic range for phase detection was 10E-2 RIU based on a current effect and 10E-4 RIU based on a potential effect of which a 0.55 (°/RIU) measurement was found by angular-interrogation. The performance of our newly developed metrology platform was characterized to have a higher sensitivity and less dynamic range when compared to a traditional full-field measurement system.

The concentration ratio of glycated to non-glycated forms of various blood proteins can be used as a diagnostic measure in diabetes to determine a history of glycemic compliance. Depending on a protein’s half-life in blood, compliance can be assessed from a few days to several months in the past, which can then be used to provide additional therapeutic guidance. Current glycated protein detection methods are limited in their ability to measure multiple proteins, and are susceptible to interference from other blood pathologies. In this study, we developed and characterized DNA aptamers for use in Surface Plasmon Resonance (SPR) sensors to assess the blood protein hemoglobin. The aptamers were developed by way of a modified Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process which selects DNA sequences that have a high binding affinity to a specific protein. DNA products resulting from this process are sequenced and identified aptamers are then synthesized. The SELEX process was performed to produce aptamers for a glycated form of hemoglobin. Equilibrium dissociation constants for the binding of the identified aptamer to glycated hemoglobin, hemoglobin, and fibrinogen were calculated from fitted Langmuir isotherms obtained through SPR. These constants were determined to be 94 nM, 147 nM, and 244 nM respectively. This aptamer can potentially be used to create a SPR aptamer based biosensor for detection of glycated hemoglobin, a technology that has the potential to deliver low-cost and immediate glycemic compliance assessment in either a clinical or home setting.

Surface-enhanced Raman spectroscopy (SERS) has been widely used for high-sensitivity molecular detection and identification. It is advantageous for a SERS substrate to have a large surface-to-volume ratio from the standpoint of high density “hot spots” and optical sampling efficiency. In this paper, we show that monolithic porous gold nanostructures such as nanofilms and nanodisks can be effective SERS substrates with large surface area. The average enhancement factor of the nanodisk and nanofilm substrates have been determined using benzenethiol self-assembled monolayers to be ~100 million and 0.5 million, respectively. Variability on the order of 40% has been observed by large area SERS mapping. A single nanodisk coated with benzenethiol self-assembled monolayer (~10 attmoles) can provide SERS spectrum with a signal-to-noise ratio ~400, resulting in an estimated detection limit in the range of zeptomoles.

In this work, we use evaporated gold nanoparticle films (GNPFs) as substrates for plasmon-enhanced imaging of two fluorescent proteins (FPs): mCherry and YFP. Through single-molecule epifluorescence microscopy, we show enhancement of single FP emission in the presence of GNPFs. The gold-coupled FPs demonstrate emission up to four times brighter and seven times longer lived, yielding order-of-magnitude enhancements in total photons detected. Ultimately, this results in increased localization accuracies for single-molecule imaging. Furthermore, we introduce preliminary results for enhancement of mCherry-labeled TcpP membrane proteins inside live Vibrio cholerae cells coupled to GNPFs. Our work indicates that plasmonic substrates are uniquely advantageous for super-resolution imaging and that plasmon-enhanced imaging is a promising technique for improving live cell single-molecule microscopy.

A metallic nano-hole array (NHA) structure with dynamic SP energy matching has been recently introduced, which benefits from a smaller resonance bandwidth, higher resonance transmission efficiency, and higher surface plasmon resonance (SPR) sensitivity compared to a structure without dynamic SP energy matching. Here, we present a more comprehensive study on the effects of SP energy matching on SPR sensitivity of a NHA structure by numerical and experimental means. Both experimental and numerical results were analyzed and compared and demonstrated that structures with dynamic SP energy matching had improved sensitivity over structures without the dynamic SP energy matching property.

We present a new wide-field quantitative photothermal (PT) imaging method of gold nanoparticles (AuNPs), which is suitable for obtaining wide-field holographic molecular specificity in biological samples. To obtain this goal, we built a wide-field interferometric phase microscope and modified it for the excitation of plasmonic resonance in AuNPs, while recording their resultant phase signatures. To check the potential of the AuNPs as interferometric cellular labels, they were conjugated to a glass coverslip and excited with a laser at a wavelength corresponding to their absorption spectral peak. We then acquired an image sequence of the sample phase profile in time without the need for lateral scanning, and analyzed the entire field of view using a Fourier analysis, creating a map of the locations of the AuNPs. We obtained a strong PT signal at AuNPs central locations, exponentially dependent on the distance from their centers. This enabled identification of the central locations of the AuNPs in the chosen field of view. Moreover, these PT signals had shown a linear relation to the illumination intensity, distinguishing them from background noise and out-of-focus particles. To the best of our knowledge, we are the first to record wide-field interferometric PT signals at the subcellular level without the need of total-internal-reflection prisms or scanning.

Conventionally, super-resolution imaging is achieved by manipulating the on/off switching of fluorophores, or by saturation of fluorescence emission. To prevent the photobleaching of fluorophores, we demonstrate novel superresolution imaging based on saturation of scattering from plasmonic particles, for the first time. With spectral studies, we have confirmed the saturation is directly linked to surface plasmon resonance effect. With the aid of saturation excitation microscopy, plus field concentration due to nonlinear plasmon resonance, we have achieved optical resolution below 80-nm based on scattering. Our study will open up a completely new paradigm for super-resolution microscopy.

We report a novel plasmonic tuning technique which allows colorimetric, naked-eye detection of protein-protein binding at extreme sensitivities. Utilizing an engineered approach to molecularly-driven plasmonic-coupling, we construct three-part plasmonic “bowtie” structures within protein nanoarrays using single biomolecular binding events. Precise molecular positioning of single gold nanaoparticles inside plasmonic bowties allows us to shape the plasmon supported by each array element in order to engineer a visible color-shift. By ensuring that only a single binding site is available inside each feature, we ensure plasmon homogeneity across the array, a unique technological solution which is essential to providing the sensitivity and observability we demonstrate here. This work represents a step-change in molecularly-mediated plasmonics and colorimetric biosensing, enabling biologically-controlled nanoengineering at single-protein resolutions. The potential applications of this powerful technique are not limited to biosensing and point-of-care diagnostics, and will also impact the emerging fields of molecularly driven nanoengineering and electronics.

Surface plasmon resonance (SPR) has been applied to sensing biomolecular and drug interactions because it allows real-time monitoring and label-free detection. Traditional thin film based SPR biosensing suffers from moderate detection sensitivity. In this research, we investigate sensitivity enhancement by target colocalized SPR using various subwavelength nanostructures. The nanostructures were designed by calculating near-field distribution based on rigorous coupled-wave analysis. Experimentally, angled shadow evaporation was performed to fabricate the nanostructures for target colocalization and measured resonance shifts using angle scanning SPR. The feasibility was tested by measuring DNA hybridization. Experimental results confirm significantly enhanced detection sensitivity over traditional SPR techniques to be feasible. The results are expected to open a new approach to biomolecular detection based on SPR.

There has been a considerable effort recently in the development of planar chiral metamaterials. Owing to the lack of inversion symmetry, these materials have been shown to display interesting physical properties such as negative index of refraction and giant optical activity. However, the biosensing capabilities of these chiral metamaterials have not been fully explored. Ultrasensitive detection and structural characterization of proteins adsorbed on chiral plasmonic substrates was demonstrated recently using UV-visible circular dichroism (CD) spectroscopy. Second harmonic generation microscopy is an extremely sensitive nonlinear optical probe to investigate the chirality of biomaterials. In this study, we characterize the chiral response of chiral plasmonic metamaterials using second harmonic generation microscopy and CD spectroscopy. These planar chiral metamaterials, fabricated by electron-beam lithography, consist of right-handed and left-handed gold gammadions of length 400 nm and thickness 100nm, deposited on a glass substrate and arranged in a square lattice with a periodicity of 800nm.

Viscoelastic shear waves (VESW) propagation in soft matters such as gelatin under thermal steady state was studied. VESW in a slab of gelatin causes the transverse displacement of the surface in a harmonic wave. The harmonic oscillation frequency of the transverse displacement of gelatin surface was then measured in real time in order to measure the modulus of rigidity of gelatin in terms of the measured oscillation frequency. A polarized heterodyne interferometer (PHI) was setup in this experiment which enables to precisely measure the transverse displacement of surface in real time at 0.3 nm resolution. This results in the proposed VESW method able to characterize gelatin soft material in real time. From the experimental demonstration, the properties of VESW propagation in soft material at thermal steady state potentially can become a novel nano-scale non-intrusion strain-stress sensor able to characterize the modulus rigidity of soft material.

Surface plasmon resonance (SPR) sensing is one of the most widely used methods to implement biosensors due to its sensitivity and capacity for label-free detection. Most conventional SPR sensors measure the change in reflectance at a metal-dielectric interface as a function of either angle or wavelength. However, it has recently been shown that an increase in sensitivity and a greater robustness against noise can be achieved by measuring reflectivity in both domains simultaneously, in a so-called spectro-angular SPR biosensor. This provides a surface plasmon dispersion curve captured on an image sensor that can be tracked in real time. A single value decomposition method is used to project the dispersion curve onto a basis set and allow the image obtained from an unknown refractive index sample to be compared very accurately with a pre-calculated reference set. The objective of the current work is to further improve the detection limit of the spectro-angular biosensor. Simulations have shown that the spatial resolution and numerical precision of the image sensor have a significant impact on the accuracy of the refractive index change measurement. Therefore, upgrading the cameras used for the data acquisition could significantly improve the detection limit of the SPR biosensor. In this work, simulation results are presented to justify the modifications of the experimental system and to estimate the expected improvement in the detection limit of the spectro-angular biosensor by using higher spatial resolution and higher data precision cameras. Experimental results are presented and compared with the previous design.

We present a three-dimensional metallic nano-structure, which consists of a nano-hole array in a gold membrane and a co-registered array of gold nano-disks situated on a substrate below the membrane. This structure provided a transmission resonance due to a localized surface plasmon (LSP) interaction between each hole and co-registered disk. Both numerical and experimental results demonstrated that the position of the LSP resonance transmission depended greatly on hole and disk diameter. Enhanced electric field intensity between each hole and disk was observed in the simulations. The device is expected to be well-suited for optical trapping applications.

This PDF file contains the front matter associated with SPIE Proceedings Volume 8597, including the Title Page, Copyright Information, Table of Contents, and the Conference Committee listing.