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This PDF file contains the front matter associated with SPIE Proceedings Volume 9550, including the Title Page, Copyright information, Table of Contents, Invited Panel Discussion, and Conference Committee listing.
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Efficient and specific delivery of particles and drugs intracellularly is a critical area of research which can allow for further understanding of cellular processes and increased efficacy of therapeutic treatments. Visualizing these delivery mechanisms occurs through use of florescent molecules, some of which are bulky and can change the processes being studied. Alternatively, semiconductor nanocrystals called quantum dots (QDs) are nanoscale particles that provide a scaffold for drugs or targeting moieties while providing superior optical properties that include size-tunable photoluminescence and resistance to photobleaching. Utilizing these platforms, delivery methods and intracellular assembly can be studied. Potential delivery mechanisms include either specified delivery through attenuation to targeting ligands or systemic delivery through microinjection or electroporation. Once within the cytosol, the QDs can assemble to express proteins, a process that can be visualized through the use of FRET. The effectiveness of various delivery methods and QD surface chemistry has been analyzed and is described here.
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Osteosarcoma (OS) is amongst the most commonly diagnosed bone tumors occurring in adolescence, young adults and
adults over the age of 65. Current treatment is based on a combination of surgery and chemotherapy. Chemotherapy has
improved the survival rate, however it is associated with severe side effects due to the use of high dosages, nonspecific
uptake and poor bone blood supply. At present bisphosphonates (BP) are widely used in the treatment of bone disorders
including OS. We have engineered a unique biodegradable BP nanoparticle that possesses a dual functionality: 1)
covalent attachment of a dye (e.g., NIR dye) or drug to the nanoparticles through the primary amine groups on the
surface of the nanoparticle; 2) chelation to the bone mineral hydroxyapatite through the BP on the surface of the
nanoparticle. Due to a high concentration of PEG in the BP nanoparticles they possess a relatively long plasma half-life
time. Therefore, the nanoparticle has potential for use both in diagnosis and therapy of OS. Doxorubicin was conjugated
to the free amine on the surface of the BP nanoparticles. In vitro experiments on osteosarcoma cells demonstrated that
the doxorubicin-conjugated BP nanoparticles possess a higher efficacy than the free doxorubicin. Further investigation in
vivo in a chicken embryo model confirmed that the doxorubicin-conjugated nanoparticle was significantly more effective
in inhibiting tumor growth compared to free doxorubicin at a similar concentration. Additionally, we have shown that
these BP nanoparticles preferentially target OS tumor tissue, thus increasing anti-cancer drug bioavailability at targeted
site.
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Near-infrared optical coherence tomography (OCT) has gained a lot of attention due to the fact that it is relatively cheap, non-invasive and provides high resolution and fast method of imaging. However the main challenge of this technique is the poor signal to noise ratio of the images of the tissue at large depths due to optical scattering. The signal to noise ratio can be improved by increasing the source power, however the laser safety standards (ANSI Z136.1) restricts the maximum amount of power that can be used safely to characterize the biological tissue. In this talk, we discuss the advantage of implanting a micro-lens inside the tissue to have a higher signal to noise ratio for confocal and OCT measurements. We explain the theoretical background, experimental setup and the method of implanting the micro lens at arbitrary depths within a live mouse brain. The in-vivo 3D OCT and two-photon microscopy images of live mouse with implanted micro-lens are presented and significant enhancement of signal to noise ratio is observed. The confocal and OCT measurements have been performed with super-luminescent LEDs emitting at 1300 nm. We believe that the high resolution and high sensitivity of this technique is of fundamental importance for characterization of neural activity, monitoring the hemodynamic responses, tumors and for performing image guided surgeries.
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Our aim is to develop quantitative single-molecule assays to study when and where molecules are interacting inside living cells and where enzymes are active. To this end we present a three-camera imaging microscope for fast tracking of multiple interacting molecules simultaneously, with high spatiotemporal resolution. The system was designed around an ASI RAMM frame using three separate tube lenses and custom multi-band dichroics to allow for enhanced detection efficiency. The frame times of the three Andor iXon Ultra EMCCD cameras are hardware synchronized to the laser excitation pulses of the three excitation lasers, such that the fluorophores are effectively immobilized during frame acquisitions and do not yield detections that are motion-blurred. Stroboscopic illumination allows robust detection from even rapidly moving molecules while minimizing bleaching, and since snapshots can be spaced out with varying time intervals, stroboscopic illumination enables a direct comparison to be made between fast and slow molecules under identical light dosage. We have developed algorithms that accurately track and co-localize multiple interacting biomolecules. The three-color microscope combined with our co-movement algorithms have made it possible for instance to simultaneously image and track how the chromosome environment affects diffusion kinetics or determine how mRNAs diffuse during translation. Such multiplexed single-molecule measurements at a high spatiotemporal resolution inside living cells will provide a major tool for testing models relating molecular architecture and biological dynamics.
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We present a novel approach to the detection of cancerous kidney tissue areas by measuring vibrational spectra (IR absorption or SERS) of intercellular fluid taken from the tissue. The method is based on spectral analysis of cancerous and normal tissue areas in order to find specific spectral markers. The samples were prepared by sliding the kidney tissue over a substrate - surface of diamond ATR crystal in case of IR absorption or calcium fluoride optical window in case of SERS. For producing the SERS signal the dried fluid film was covered by silver nanoparticle colloidal solution. In order to suppress fluorescence background the measurements were performed in the NIR spectral region with the excitation wavelength of 1064 nm. The most significant spectral differences – spectral markers - were found in the region between 400 and 1800 cm-1, where spectral bands related to various vibrations of fatty acids, glycolipids and carbohydrates are located. Spectral markers in the IR and SERS spectra are different and the methods can complement each other. Both of them have potential to be used directly during surgery. Additionally, IR absorption spectroscopy in ATR mode can be combined with waveguide probe what makes this method usable in vivo.
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Pseudomonas aeruginosa (PA), a biofilm forming bacterium, commonly affects cystic fibrosis, burn victims, and immunocompromised patients. PA produces pyocyanin, an aromatic, redox active, secondary metabolite as part of its quorum sensing signaling system activated during biofilm formation. Surface enhanced Raman scattering (SERS) sensors composed of Au nanospheres chemically assembled into clusters on diblock copolymer templates were fabricated and the ability to detect pyocyanin to monitor biofilm formation was investigated. Electromagnetic full wave simulations of clusters observed in scanning electron microcopy images show that the localized surface plasmon resonance wavelength is 696 nm for a dimer with a gap spacing of 1 nm in an average dielectric environment of the polymer and analyte; the local electric field enhancement is on the order of 400 at resonance, relative to free space. SERS data acquired at 785 nm excitation from a monolayer of benzenethiol on fabricated samples was compared with Raman data of pure benzenethiol and enhancement factors as large as 8×109 were calculated that are consistent with simulated field enhancements. Using this system, the limit of detection of pyocyanin in pure gradients was determined to be 10 parts per billion. In SERS data of the supernatant from the time dependent growth of PA shaking cultures, pyocyanin vibrational modes were clearly observable during the logarithmic growth phase corresponding to activation of genes related to biofilm formation. These results pave the way for the use of SERS sensors for the early detection of biofilm formation, leading to reduced healthcare costs and better patient outcomes.
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Bacteria colonize plant roots to form a symbiotic relationship with the plant and can play in important role in promoting plant growth. Raman spectroscopy is a useful technique to study these bacterial systems and the chemical signals they utilize to interact with the plant. We present a Raman study of Pantoea YR343 that was isolated from the rhizosphere of Populus deltoides (Eastern Cottonwood). Pantoea sp. YR343 produce yellowish carotenoid pigment that play a role in protection against UV radiation, in the anti-oxidative pathways and in membrane fluidity. Raman spectroscopy is used to non-invasively characterize the membrane bound carotenoids. The spectra collected from a mutant strain created by knocking out the crtB gene that encodes a phytoene synthase responsible for early stage of carotenoid biosynthesis, lack the carotenoid peaks. Surface Enhanced Raman Spectroscopy is being employed to detect the plant phytoharmone indoleacetic acid that is synthesized by the bacteria. This work describes our recent progress towards utilizing Raman spectroscopy as a label free, non-destructive method of studying plant-bacteria interactions in the rhizosphere.
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Keynote Joint Session with Conferences 9550 and 9568
20 years after their first reference interband cascade lasers (ICLs) have become a mature and competitive semiconductor laser source in the mid-infrared region. The carrier rebalancing concept that was introduced in 2011 drastically improved the performance. As a consequence the wavelength window that is accessible for ICLs operating at ambient temperatures could be extended. For GaSb based ICLs continuous wave (cw) emission at room temperature could be achieved up to a wavelength of 5.6 μm. As the need for thicker claddings at longer wavelengths makes the growth of the superlattice claddings increasingly difficult and limits the heat dissipation, a plasmon waveguide structure with highly doped InAslayers grown on InAs-substrates is typically used for ICLs emitting up to ~7 μm in pulsed mode at room temperature. With regard to the short wavelength limit of ICLs, we present cw emission of a GaSb based ICL emitting at 2.8 μm and with regard to the long wavelength limit we present room temperature pulsed operation of an InAs based plasmonic waveguide ICL up to 7.1 μm. Furthermore, we show single mode emitting ICLs with distributed feedback gratings emitting between 2.8 and 5.2 μm.
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An important field of application of lasers is biomedical optics. Here, they offer great utility
for diagnosis, therapy and surgery. For the development of novel methods of laser-based
biomedical diagnostics careful study of light propagation in biological tissues is necessary to
enhance our understanding of the optical measurements undertaken, increase research and
development capacity and the diagnostic reliability of optical technologies.
Ultimately, fulfilling these requirements will increase uptake in clinical applications of laser
based diagnostics and therapeutics. To address these challenges informative biomarkers
relevant to the biological and physiological function or disease state of the organism must be
selected. These indicators are the results of the analysis of tissues and cells, such as blood.
For non-invasive diagnostics peripheral blood, cells and tissue can potentially provide
comprehensive information on the condition of the human organism. A detailed study of the
light scattering and absorption characteristics can quickly detect physiological and
morphological changes in the cells due to thermal, chemical, antibiotic treatments, etc [1-5].
The selection of a laser source to study the structure of biological particles also benefits from
the fact that gross pathological changes are not induced and diagnostics make effective use of
the monochromatic directional coherence properties of laser radiation.
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Smart and flexible bioelectronics on graphene have emerged as a new frontier in the field of biosensors and bioelectronics. Graphene has begun to be seen as an ideal signal transducer and a promising alternative for the production of low-cost bioelectronic devices.1-2 However, biological systems used in these devices suffer from a lack of control and regulation. There is an obvious need to develop “switchable” and “smart” interfaces for both fundamental and applied studies. Here, we report the fabrication of a stimuli-responsive graphene interface, which is used to regulate biomolecular reactions.
The present study aims to address the design and development of a novel auto-switchable graphene bio-interface that is capable of positively responding, by creating smart nanoarchitectures. By changing the external conditions such as temperature, light and pH of the medium, we acheived control of the biochemical interactions. In the negative mode, access of an associated enzyme to its substrate is largely restricted, resulting in a decrease in the diffusion of reactants and the consequent activity of the system. In contrast, the biosubstrate could freely access the enzyme facilitating bioelectrocatalysis in a positive response.
Using electrochemical techniques, we demonstrated that interfacial bio-electrochemical properties can be tuned by modest changes in conditions. Such an ability to independently regulate the behaviour of the interface has important implications for the design of novel bioreactors, biofuel cells and biosensors with inbuilt self-control features.
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In breast cancer, certain types of circulating immune cells respond to long-range chemical signals from tumors by leaving the blood stream to actively infiltrate tumor tissue. The aim of this study was to evaluate whether immune cells could be used to deliver therapeutic nanoparticles into breast tumors in mice. Mononuclear splenocytes (MS) were harvested from donor mice, labeled with Indium-111, injected intravenously into immune-competent recipient mice (3 tumor-bearing and 3 control), and imaged longitudinally by SPECT/CT. For comparison, the biodistribution of bonemarrow derived macrophages (BMDM) in one pair of mice was also imaged. Quantitative analysis of the SPECT images demonstrates that, after 24 hours, the concentration of MS detected in mammary tumors is more than 3-fold higher than the concentration detected in normal mammary glands. The ratio of MS concentration in mammary tissue to MS concentration in non-target tissues (muscle, lung, heart, liver, spleen, and kidney) was enhanced in tumor-bearing mice (compared to controls), with statistical significance achieved for mammary/muscle (p<0.01), mammary/lung (p<0.05), and mammary/kidney (p<0.05). By contrast, BMDM did not show a different affinity for tumors relative to normal mammary tissue. MS were incubated with 100 nm red fluorescent nanoparticles, and flow cytometry demonstrated that ~35% of the MS population exhibited strong phagocytic uptake of the nanoparticles. After intravenous injection into tumor-bearing mice, fluorescence microscopy images of tumor sections show qualitatively that nanoparticle-loaded MS retain the ability to infiltrate mammary tumors. Taken together, these results suggest that MS carriers are capable of actively targeting therapeutic nanoparticles to breast tumors.
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Low density lipoproteins (LDL) are considered as suitable natural in vivo delivery system for hydrophobic photosensitizers (pts) such as hypericin (Hyp) and it was shown that over expression of LDL-receptors in tumor cells can be used for specific targeting. Activation of pts by irradiation results in a formation of reactive oxygen species (ROS) at the place of light application and starts destructive mechanism. PKCα plays a key role in the cell survival and its overexpression was observed in glioma cell lines. In the present study we aim to present the effectivity of the pts delivery in the glioma cells and consequences of silencing pkcα gene on cell death/survival after Hyp photo-activation. Pts can be delivered through two pathways: endocytosis - when cells are incubated with LDL/Hyp complex and Hyp transport through cellular membrane without any carrier. Preliminary results show that incubation of cells with or without LDL leads to PKCα activation. Photo-activated Hyp seems to be more effective in terms of apoptosis induction when compared to photo-activated LDL/Hyp complex. We have evaluated the influence of photo-activated Hyp on cell death in non-transfected and transfected (PKCα-) human glioma cells (U87-MG). Level of ROS production and type of cell death was notably affected by silencing pkca gene resulting in significant increase of necrosis after Hyp photo-activation.
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Enzymes are important players in multiple applications, be it bioremediation, biosynthesis, or as reporters. The business of catalysis and inhibition of enzymes is a multibillion dollar industry and understanding the kinetics of commercial enzymes can have a large impact on how these systems are optimized. Recent advances in nanotechnology have opened up the field of nanoparticle (NP) and enzyme conjugates and two principal architectures for NP conjugate systems have been developed. In the first example the enzyme is bound to the NP in a persistent manner, here we find that key factors such as directed enzyme conjugation allow for enhanced kinetics. Through controlled comparative experiments we begin to tease out specific mechanisms that may account for the enhancement. The second system is based on dynamic interactions of the enzymes with the NP. The enzyme substrate is bound to the NP and the enzyme is free in solution. Here again we find that there are many variables , such as substrate positioning and NP selection, that modify the kinetics.
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We demonstrate that patches of two dimensional arrays of circular plasmonic nanoholes patterned on gold-titanium thin film enables subwavelength focusing of visible light in far field region. Efficient coupling of the light with the excited surface plasmon at metal dielectric interface results in strong light transmission. As a result, surface plasmon plays an important role in the far field focusing behavior of the nanohole-aperture patches device. Furthermore, the focal length of the focused beam was found to be predominantly dependent on the overall size of the patch, which is in good agreement with that calculated by Rayleigh-Sommerfield integral formula. The focused light beam can be utilized to separate bio-particles in the dynamic range from 0.1 μm to 1 μm through mainly overcoming the drag force induced by fluid flow. In our proposed model, focused light generated by our plasmonic lenses will push the larger bio-particles in size back to the source of fluid flow and allow the smaller particles to move towards the central aperture of the patch. Such a new kind of plasmonic lenses open up possibility of sorting bacterium-like particles with plasmonic nanolenses, and also represent a promising tool in the field of virology.
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Tissue engineering and regenerative medicine have been constantly developing of late due to the major progress in cell and organ transplantation, as well as advances in materials science and engineering. Although stem cells hold great potential for the treatment of many injuries and degenerative diseases, several obstacles must be overcome before their therapeutic application can be realized. These include the development of advanced techniques to understand and control functions of micro environmental signals and novel methods to track and guide transplanted stem cells. A major complication encountered with stem cell therapies has been the failure of injected cells to engraft to target tissues. The application of nanotechnology to stem cell biology would be able to address those challenges. Combinations of stem cell therapy and nanotechnology in tissue engineering and regenerative medicine have achieved significant advances. These combinations allow nanotechnology to engineer scaffolds with various features to control stem cell fate decisions. Fabrication of Nano fiber cell scaffolds onto which stem cells can adhere and spread, forming a niche-like microenvironment which can guide stem cells to proceed to heal damaged tissues. In this paper, current and emergent approach based on stem cells in the field of liver tissue engineering is presented for specific application. The combination of stem cells and tissue engineering opens new perspectives in tissue regeneration for stem cell therapy because of the potential to control stem cell behavior with the physical and chemical characteristics of the engineered scaffold environment.
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Regular check of blood creatinine level is very important as it is a measurement of renal function. Therefore, the objective of this study is to develop a simple and reliable creatinine biosensor based on admittance measurement for precise determination of creatinine. The creatinine biosensor was fabricated with creatinine deiminase immobilized on screen-printed carbon electrodes. Admittance measurement at a specific frequency ranges (22.80 - 84.71 Hz) showed that the biosensor has an excellent linear (r2 > 0.95) response range (50 - 250 uM), which covers the normal physiological and pathological ranges of blood creatinine levels. Intraclass correlation coefficient (ICC) showed that the biosensor has excellent reliability and validity (ICC = 0.98). In conclusion, a simple and reliable creatinine biosensor was developed and it is capable of precisely determining blood creatinine levels in both the normal physiological and pathological ranges.
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Cancer is a major health threat worldwide. Options for targeted cancer therapy, however, are often limited, in a large part due to our incomplete understanding of how key processes including oncogenesis and drug response are mediated at the molecular level. New imaging techniques for visualizing biomolecules and their interactions at the nanometer and single molecule scales, collectively named fluorescence nanoscopy, hold the promise to transform biomedical research by providing direct mechanistic insight into cellular processes. We discuss the principles of quantitative single-molecule localization microscopy (SMLM), a subset of fluorescence nanoscopy, and their applications to cancer biomedicine. In particular, we will examine oncogenesis and drug resistance mediated by mutant Ras, which is associated with ~1/3 of all human cancers but has remained an intractable drug target. At ~20 nm spatial and single-molecule stoichiometric resolutions, SMLM clearly showed that mutant Ras must form dimers to activate its effector pathways and drive oncogenesis. SMLM further showed that the Raf kinase, one of the most important effectors of Ras, also forms dimers upon activation by Ras. Moreover, treatment of cells expressing wild type Raf with Raf inhibitors induces Raf dimer formation in a manner dependent on Ras dimerization. Together, these data suggest that Ras dimers mediate oncogenesis and drug resistance in tumors with hyperactive Ras and can potentially be targeted for cancer therapy. We also discuss recent advances in SMLM that enable simultaneous imaging of multiple biomolecules and their interactions at the nanoscale. Our work demonstrates the power of quantitative SMLM in cancer biomedicine.
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Resonant waveguide grating (RWG) systems illuminate an array of diffractive nanograting waveguide structures in microtiter plate to establish evanescent wave for measuring tiny changes in local refractive index arising from the dynamic mass redistribution of living cells upon stimulation. Whole-plate RWG imager enables high-throughput profiling and screening of drugs. Microfluidics RWG imager not only manifests distinct receptor signaling waves, but also differentiates long-acting agonism and antagonism. Spatially resolved RWG imager allows for single cell analysis including receptor signaling heterogeneity and the invasion of cancer cells in a spheroidal structure through 3-dimensional extracellular matrix. High frequency RWG imager permits real-time detection of drug-induced cardiotoxicity. The wide coverage in target, pathway, assay, and cell phenotype has made RWG systems powerful tool in both basic research and early drug discovery process.
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The motion behavior of mammalian adipose tissue derived stem cells (AT-SCs) on an integrated channel waveguide under the evanescent field illumination is demonstrated and analyzed. The AT-SCs, suspended to a concentration of 1 x 105 cells per ml, are deposited in a reservoir over a copper ion-exchanged channel waveguide. Light from a HeNe laser operating at 632.8nm was coupled into the waveguide, causing the cells under the illumination of evanescent field and moved in a skewed stochastic motion in accordance to the laser power. The trajectory angle of the motion of the cells towards the illuminated channel waveguide was investigated and analyzed to distinguish the factors that affect such behavior. The cells reach a position relative to the illuminated channel, which is dictated by the compounded effect of the convectional current and evanescent field. The observations deduced that motion due to the optical field exists and were more pronounced when considering the trajectory angle towards the output facet. However, the optical forces are not significantly large enough to counter the motion due to the convection current. The results are discussed in light of the potential application of optical channel waveguides for bioanalytical applications, namely in the identification, sorting and analysis of differently sized mammalian cells without recourse to fluorescence or antibody staining.
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Lattice plasmon resonances (LPRs), which originate from the plasmonic-photonic coupling in gold or silver nanoparticle arrays, possess ultra-narrow linewidth by suppressing the radiative damping and provide the possibility to develop the plasmonic sensors with high figure of merit (FOM). However, the plasmonic-photonic coupling is greatly suppressed when the nanoparticles are immobilized on substrates because the diffraction orders are cut off at the nanoparticle-substrate interfaces. Here, we develop the rational design of LPR structures for the high-performance, on-chip plasmonic sensors based on both orthogonal and parallel coupling. Our finite-difference time-domain simulations in the core/shell SiO2/Au nanocylinder arrays (NCAs) reveal that new modes of localized surface plasmon resonances (LSPRs) show up when the aspect ratio of the NCAs is increased. The height-induced LSPRs couple with the superstrate diffraction orders to generate the robust LPRs in asymmetric environment. The high wavelength sensitivity and narrow linewidth in these LPRs lead to the plasmonic sensors with high FOM and high signal-to-noise ratio (SNR). Wide working wavelengths from visible to near-infrared are also achieved by tuning the parameters of the NCAs. Moreover, the wide detection range of refractive index is obtained in the parallel LPR structure. The electromagnetic field distributions in the NCAs demonstrate the height-enabled tunability of the plasmonic “hot spots” at the sub-nanoparticles resolution and the coupling between these “hot spots” with the superstrate diffraction waves, which are responsible for the high performance LPRs-based on-chip refractive index sensors.
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Although Raman spectroscopy has been commercialized, low-cost and large-area surface enhanced Raman spectroscopy (SERS) substrates with localized enhanced field are heavily required. However, currently dominant manufacturing techniques are expensive and complicated for large-area fabrication. Furthermore, most SERS substrates can only be used for individual excitation wavelengths. In this work, we will report an ultra-broadband super absorbing metasurface to enhance SERS signals in a broadband region (i.e. from 450 nm to 1000 nm). The design consisting of an Ag ground plate, a SiO2 spacer, and a layer of Ag nanoparticles was fabricated using simple film deposition and thermal annealing techniques. A broadband absorption over 80% from 414 nm to 956 nm was obtained, resulting in localized field enhancement between adjacent nanoparticles. We employed this metasurface to test its broadband SERS signal by adsorbing 1,2-Bis(4-pyridyl)-ethylene (BPE) molecules on top of it. We employed 5 laser lines (i.e., 514, 532, 633, 671 and 785 nm) to excite the sample and observed fingerprint signature of BPE molecules under all 5 excitation wavelengths with the average enhancement factor up to 5.3×107. Therefore, the designed SERS substrate can work for almost “all” available excitation wavelengths over a broadband, which is particularly useful for sensing a broad spectrum of chemicals on the same chip.
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Surface Plasmon Resonance (SPR) in metallic nanostructures is an optical effect that can be exploited for the detection of small molecules. There is a broad range of metallic nanostructures supporting different SPR modes, and nanostructures can be even geometrically combined leading to the creation of new hybridised SPR modes. In our study, we investigated the properties of a hybridised SPR mode (gap modes GM) created by the placement of metallic nanoparticles onto metallic layers and its use as a sensitive sensor. A tunneling current passing through a metal-insulator-semiconductor structure can generate supported SPR modes that can be scattered through GM, which was experimentally confirmed. Moreover, we were able to experimentally follow the degradation of anisotropic (silver nanoprism) nanoparticles under ambient conditions in real time. Using atomic force microscopy and optical spectroscopy we observed an anisotropic corrosion that is starting from the tips of the nanoparticles.
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Moxtek has leveraged existing capabilities in wafer-scale patterning of sub-wavelength wire grid polarizers into the fabrication of 1D and 2D periodic aluminum plasmonic structures. This work will discuss progress in 200 mm diameter wafer-scale fabrication, with detailed emphasis within the realm of microarray based fluorescence detection. Aluminum nanohole arrays in a hexagonal lattice are first numerically investigated. The nanohole array geometry and periodicity are specifically tuned to coincide both with the excitation of the fluorophore Cy3, and to provide a high field enhancement within the nanoholes where labeled biomolecules are captured. This is accomplished through numerical modelling, nanofabrication, SEM imaging, and optical characterization. A 200mm diameter wafer, patterned with the optically optimized nanohole array, is cut into standard 1x3 inch microscope slide pieces and then subsequently printed with various antigens at 9 different concentrations. A sandwich bioassay is then carried out, using the corresponding conjugate antibodies in order to demonstrate specificity. The nanohole array exhibit a 3-4 times total fluorescence enhancement of Cy3, when compared to a leading commercial microarray glass slide.
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We present a study of the dynamics of protein aggregation using a Bloch surface wave (BSW) label-free sensing scheme. In a previous work, we demonstrated the ability to detect the early dynamic events of fibrillogenesis of amyloid betapeptides (Aβ), linked to Alzheimer’s Disease. Here, we demonstrate the efficacy of the BSW sensor by describing a simultaneous light scattering measurement, with the purpose of real-time monitoring the size change of the Aβ aggregates, throughout fibrillization.
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Cancer is a leading cause of death worldwide, with metastasis responsible for the majority of cancer-related deaths. Circulating tumour cells (CTCs) play a central role in metastasis. Fluorescent silica particles (NPs), of diameter ~50 nm which contain a large concentration of Cy5 dye molecules and are extremely bright, have been developed to detect these rare CTCs. Due to this brightness, the particles have superior performance compared to single Cy5 dye molecule labels, for detecting cancer cells. Fluorescence measurements show that the NPs are almost 100 times brighter than the free dye. They do not photo bleach as readily and, due to the biocompatible silica surface, they can be chemically modified, layer-by-layer, in order to bind to cells. The choice of these chemical layers, in particular the NP to antibody linker, along with the incubation period and type of media used in the incubation, has a strong influence on the specific binding abilities of the NPs. In this work, NPs have been shown to selectively bind to the MCF-7 cell line by targeting epithelial cellular adhesion molecule (EpCAM) present on the MCF-7 cell membrane by conjugating anti-EpCAM antibody to the NP surface. Results have shown a high signal to noise ratio for this cell line in comparison to a HeLa control line. NP attachment to cells was verified qualitatively with the use of fluorescence microscopy and quantitatively using image analysis methods. Once the system has been optimised, other dyes will be doped into the silica NPs and their use in multiplexing will be investigated.
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Alzheimer’s disease (AD), an age-related neurodegenerative disorder, is the seventh leading cause of death in the United
States. One strong pathological indicator of AD is senile plaques, which are aggregates of fibrils formed from amyloid β
(Aβ) peptides. Thus, detection and inhibition of Aβ aggregation are critical for the prevention and treatment of AD.
Congo red (CR) is one of the most widely used dye molecules for probing as well as inhabiting Aβ aggregation.
However, the nature of interaction between CR and Aβ is not well understood. In this research, we systematically
studied the interaction between CR and Aβ using a combination of optical techniques, including electronic absorption,
fluorescence, Raman scattering, and circular dichroism, to provide detailed information with molecular specificity and
high sensitivity. Compared to CR alone, interaction of the dye with Aβ results in a new absorption peak near 540 nm and
significantly enhanced photoluminescence as well as Raman signal. Our results led us to propose a new model
suggesting that CR exists primarily in a micellar form, resembling H-aggregates, in water and dissociates into monomers
upon interaction with Aβ. This model has significant implications for the development of new strategies to detect and
inhibit brain plaques for treatment of neurological diseases like AD.
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In respiratory-gated radiotherapy of patients with lung or liver cancer, the patient’s respiratory pattern and repeatability are important factors affecting therapy accuracy; it has been reported that these factors can be controlled if patients undergo respiration training. As such, this study evaluates the feasibility of micro-electro-mechanical-system (MEMS) in radiotherapy by investigating the effect of radiation on a miniature portable respiratory monitoring system based on the MEMS system, which is currently under development. Using a patient respiration simulation phantom, the time-acceleration graph measured by a normal sensor according to the phantom’s respiratory movement before irradiation and the change in this graph with accumulated dose were compared using the baseline slope and the change in amplitude and period of the sine wave. The results showed that with a 400Gy accumulated dose in the sensor, a baseline shift occurred and both the amplitude and period changed. As a result, if the MEMS is applied in respiratory-gated radiotherapy, the sensor should be replaced after use with roughly 6-10 patients so as to ensure continued therapy accuracy, based on the characteristics of the sensor itself. In the future, a more diverse range of sensors should be similarly evaluated.
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