This PDF file contains the front matter with SPIE Proceedings Volume 10894, including the Title Page, Copyright information, Table of Contents, and Author and Conference Committee lists.

In order to allow for a molecular specific as well as trace level sensitive detection in biosensing, vibrational spectroscopy is applied in combination with plasmonic active materials. Within this contribution, we report on the surface-enhanced Raman spectroscopy (SERS) technique, the fabrication of powerful SERS substrates and finally illustrate the potential in biosensing. Mannose-modified SERS tags are applied to be taken up by macrophages in a large amount which serves as a model for the detection of vulnerable atherosclerotic plaques in an early stage. Moreover, to allow for an improved quantification SERS active surfaces are equipped with an internal standard. Finally, a newly developed fabrication strategy of SERS substrate templates will be introduced forming 3D hierarchical nanostructures by metastable state assisted atomic layer deposition. Thus, a detailed overview on innovative plasmonic nanostructures for biosensing developed in our labs is given.

Understanding how gold nanoparticles interact with liposomes is important for biotechnology and drug delivery. The characterization of liposome structure and composition using gold nanoparticles by surface-enhanced Raman scattering (SERS) has show that the composition of the lipid bilayers influences the interaction of the gold nanoparticles with the lipid structures. Here, vesicles composed of phosphatidylcholine, sphingomyelin, and cholesterol in different proportions reveal that very small changes in the lipid composition can alter the contact between liposomes and gold nanostructures. The SERS spectra of phosphatidylcholine and phosphatidylcholine / cholesterol liposomes indicate that cholesterol has strong effects on the contacts of the vesicles with the nanoparticles. Moreover, the interaction of citrate-stabilized gold nanoparticles varies depending on the preparation protocol, and the presence of organic solvent during preparation of the gold nanoparticle-liposome composites. In a model system where the charge of the lipid bilayers is varied, the influence of negative charge of the lipids on liposome structure and their contact with the nanoparticles is discussed. The results have implications for the development of new gold nanoparticle and gold nanoparticle-liposome-based drug delivery systems.

Cancer is the most common cause of death in people aged less than 85 years and it is estimated that in 2012 there were more than 14.1 million new cancer cases worldwide and 8.2 million deaths that resulted [1]. Nanomedicine has contributed to important advances in health care over the past few decades. In particular, the use of nanoparticles in medicine has attracted increased attention for their unique efficacy and specificity in therapy. A special type of metallic nanoparticles, called “plasmonic” nanoparticles, has received great interest because they exhibit enhanced optical and electromagnetic properties. Plasmonic nanoparticles have unique properties that allow them to amplify the optical properties of the excitation light and thus increase the effectiveness of light-based photothermal tumor ablation. Among nanoparticles used for light-induced photothermal therapy, gold nanostars (GNS) are of particular interest because their structure has multiple sharp branches that produce the numerous curvatures responsible for the ‘lightning rod’ effect that strongly enhances the local electromagnetic field when subject to light stimulation. As with other nanoparticles, GNS sizes can be controlled so that they passively accumulate in tumors due to the enhanced permeability and retention (EPR) effect of tumor vasculature. The combination of the EPR effect and the capacity for efficient photon to heat conversion, make GNS an ideal photothermal transducer for selective cancer therapy at the nanoscale level, as we have demonstrated both in vitro and in vivo. The unique properties of GNS which contribute to plasmonics-amplified immune nanotherapy for cancer, include: (1) plasmonic nano-enhancers of light, (2) nano-targeting of tumor cells, (3) nano-sources for heating tumor cells from the inside (4) nano-activators of the immune system, and (5) synergistic amplification of immunomodulation. These elements will be discussed in detail. We demonstrated that the use of plasmonic nanoparticles in combination with immunotherapy—a treatment we referred to as Synergistic Immuno Photothermal Nanotherapy (SYMPHONY)—can dramatically enhance the efficacy of immunotherapy [2]. Remarkably, we have found that SYMPHONY not only eradicates primary “treated” tumors but also has resulted in the immune-mediated destruciton of distant “untreated” metastatic tumors. This abscopal effect occurred uniquely when nanoparticle tumor heating was used in combination with immunotherapy. This strategy could lead to an entirely new treatment paradigm that challenges traditional surgical resection approaches for many cancers and metastases. Of great importance is the possibility that such an approach can induce long-term immunological memory that can provide protection against tumor recurrence long after treatment of the initial tumors similar to an “anti-cancer vaccine”. [1] World Health Organization, Latest world cancer statistics: Global cancer burden rises to 14.1 million new cases in 2012: Marked increase in breast cancers must be addressed [2] Liu Y, Maccarini P, Palmer GM, Etienne W, Zhao Y, Lee C, Ma X, Inman BA, Vo-Dinh T. Synergistic Immuno Photothermal Nanotherapy (SYMPHONY) for the Treatment of Unresec-table and Metastatic Cancers. Scientific Reports. 7, 8606 (2017).

Extracellular vesicles are nanoscale and microscale biological vesicles actively released by nearly all cell types within the body. These small vesicles have been shown to play important biological roles, including cell-to-cell communication, coagulation and signal transduction. They have also been shown to play oncogenic roles in cancer metastasis and progression. Extracellular vesicles are composed of an aqueous cytosolic core and a phospholipid membrane, and exhibit variability in their internal and external cargoes. Developing a better understanding of the structure and diversity of the components of extracellular vesicles may hold promise in uncovering the pathways involved in the formation and progression of various cancers and diseases. Current studies of extracellular vesicles focus on bulk analysis, whereas variability amongst individual extracellular vesicles has been minimally reported in literature. In this study, we propose the use of a surface-enhanced Raman spectroscopy platform in movement towards trapping extracellular vesicles secreted from a mesenchymal stem cell line followed by probing their individual spectral signatures. Here, we propose the use of plasmonic-well based structures as a means of isolating, trapping and controlling the position of biologically relevant vesicles on plasmonic platforms. Trapping and identification of extracellular vesicles occurs without use of labelling agents, allowing for characterization of the intrinsic molecular information of individual extracellular vesicles.

The knowledge over gene expression dynamics and location in plants is crucial for applications ranging from basic biological research to agricultural biotechnology (e.g., biofuel development). However, current methods cannot provide in vivo dynamic detection of genomic targets in plants. This limitation is due to the complex sample preparation needed by current methods for nucleic acids detection, which disrupt spatial and temporal resolution. We report the development of a unique multimodal method based on plasmonics-active nanoprobes, referred to inverse molecular sensitnels capable of in vivo imaging and biosensing of microRNA biotargets within whole plant using surface-enhanced Raman scattering (SERS) detection. This work lays the foundations for in vivo functional imaging of RNA biotargets in plants with previously unmet spatial and temporal resolution for many applications ranging from agricultural biotechnology to biofuel research.

We report on the development of a set of Raman based techniques to monitor a large variety of biological and chemical analytes, such as various microorganisms, gels of hyaluronic acid and selected halogenated hydrocarbons using Raman spectroscopy, Raman tweezers and surface-enhanced Raman spectroscopy (SERS). We analyzed individual microbial cells with Raman tweezers to provide solutions for fast and label-free identification of specific bacterial or yeast species. We designed an optofluidic SERS platform for quantification of sub-millimolar concentrations of halogenated environmental pollutants such as 1,2,3-trichloropropane and chloroform. We also examined the gel structure of hyaluronic acid by Raman spectroscopy.

The misuse or abuse of over-the-counter (OTC) drugs has a significant impact on patient health and if used without caution can lead to serious health issues. The prevalence of OTC drugs due to a well-developed market and the lack of patient knowledge about the drug safety has led to the increase in the number of hospitalizations related to the drug missuses. In order to choose a correct treatment procedure a fast and precise identification of the misused drug is necessary. Various accurate drug-screening methods are clinically available, however, they are rather slow or too complicated and more efficient method would be beneficial. In this work we present the first results of application of SERS spectroscopy for a fast screening of salicylic acid (metabolite of aspirin) and paracetamol directly in human blood. Conventional Raman spectroscopy is known to be a sensitive technique for identification and quantification of molecular substances in various molecular mixtures without the need of a large amount of the sample. However, when dealing with low concentrations of molecules under study in some biological fluids (like drugs or their metabolites in blood) unconventional sensitive spectral methods like surface enhanced Raman scattering (SERS) spectroscopy must be used. The extremely high sensitivity of the SERS allows identification of the blood metabolites in micromole or even in lower concentrations by analyzing the spectra of thin films of dried blood samples formed on the metal colloidal film. Additionally, the possibility to tailor the metal nanoparticles applied in colloidal SERS can be utilized to increase selectivity and sensitivity of the method. The SERS methodology proposed in this work allows fast and accurate identification of the aspirin with the detection limit – standard 500 mg one and half pills, while usage of up to 2 pills of paracetamol at once cannot be detected due to extremely low concentration of this drug in the blood.

In this study, a variety of direct laser written surface-enhanced Raman spectroscopy (SERS) micro-structures, designed for bacteria detection, are presented. Various SERS micro-structures were designed to achieve both a high density of plasmonic hot spots and a strong probability of interaction between the hot spots and the target bacterial cells. Twophoton polymerization was used for initial fabrication of the polymeric skeletons of the SERS micro-structures, which were then coated with a 50 nm-thick gold layer via e-beam evaporation. The micro-structures were fabricated on glass coverslips and were assessed using a confocal Raman microscope. To this end, Rhodamine 6G was used as an analyte under 785 nm laser illumination. The optimal SERS micro-structures showed approximately 7×10³ enhancement in Raman signal (analytical enhancement factor, AEF) at a wavenumber of 600 cm^-1. Real-time detection of E. coli in solution was demonstrated using the fabricated SERS platform with low laser powers and a short acquisition time (785 nm, 5 mW, 50 ms).

The interactions of programmed death protein-1 (PD-1) on T cells with its ligand, PD-L1, strongly contributes to an immunosuppressive microenvironment. Blockades of PD-L1 have shown long-term survival in patients. However, <30% of patients respond to PD-L1 blockade, in part due to inaccurate identification of PD-L1 expression. An unmet clinical need exists for noninvasive detection techniques. Surface-enhanced Raman spectroscopy (SERS) imaging mediated by gold nanostructures has gained interest as a pre-clinical noninvasive diagnostic tool due to the high spatial resolution, portability, low cost, and rapid analysis. In this work, we demonstrate the use of gold nanostars conjugated with Raman tags for multiplexed SERS and simultaneous diagnosis of PD-L1 and epidermal growth factor receptor (EGFR) in vivo. Nanostars conjugated with anti-PDL1 antibodies/DTNB Raman molecule, and nanostars conjugated with anti-EGFR antibodies/pMBA Raman molecules were concurrently introduced to mice. Longitudinal Raman analysis demonstrated maximum accumulation of nanostars occurred 6h post IV delivery when strong increases in SERS signals for both Raman tags were observed. Raman signals decreased by 30% when both targeted receptors were pre-blocked with antibody (IP), indicating both the sensitivity and specificity of our platform in distinguishing tumors with varied expression of PD-L1 and EGFR. Furthermore, ex-vivo Raman maps provided assessment of biomarker status with cellular-resolution, and nanostars distribution in tumors. Finally, gold contents in organs were quantified with inductively coupled plasma mass spectrometry to evaluate their pharmacokinetics and biodistribution. This work shows gold nanostars-mediated SERS imaging provides a quantitative measure of PD-L1 to allow predictive and personalized immunotherapies with minimal toxicities.

In this work, we present a digital micro-mirror device (DMD)-enabled real-time biosensing array based on angular interrogation surface plasmon resonance (SPR) with a single point photodetector. It has been studied that the performance of SPR sensors are predominantly limited by photon statistics, which can be achieved by increasing the photon capture efficiency and dynamic range of the detector. Such attributes uniquely put the single point photodetector as a potential light detecting element in high performance SPR sensors. In our DMD-based-SPR system, angular scanning is achieved by the DMD which facilitates SPR measurements with a single point photodetector, leading to a high-performance system with user-selectable interrogation range, enhanced S/N ratio and fast data throughput. Experimental results demonstrate a system resolution of 3.54×10−6 in terms of refractive index unit (RIU). Using a 4-channel array setup, we have performed real-time monitoring of bovine serum albumin (BSA)/anti-BSA binding interactions at various concentration levels. The limit of detection (LOD) is 27 ng/mL. DMD-based SPR interrogation opens up a new design route for practical solid-state SPR biosensors.

Bacterial pathogen contamination is the leading cause of both foodborne and hospital-acquired diseases. Therefore, there is a constant need for more effective, reliable and easy-to-use microbiology study techniques and detection systems. This is critical, as pathogenic contamination has become a central issue in the food industry and healthcare. This paper describes the novel use of resolution-optimized prism-based surface plasmon resonance imaging (resolution around the size of a bacteria) and data processing to further understand the behavior of individual bacteria near specifically engineered surfaces. We show that our technique is effective for both the dynamic study of individual bacteria behavior near interface on a statistically representative sample, and their interactions with chemically functionalized surfaces.

In the last decades, the plasmonic effect of metallic nanoparticles (NPs) has been broadly exploited for label-free optical sensing. To analyze the scattered light from NPs, dark-field microscopy is the most employed technique, which typically requires complex and expensive set-ups. To overcome these limitations, here, we propose a new methodology to develop plasmonic sensors. In our approach, gold nanoparticles (AuNPs) are bonded to the end-face of convectional multimode optical fibers (MMFs). The measuring set-up is as follows: light is launched from a white light source to the end of the MMF where AuNPs are located. The guided light interacts with the AuNPs where localized surface plasmons are excited. The absorption and reflection spectra are analyzed with a miniature spectrometer. Our system is robust, portable, cost effective, and operates in the 250-1200 nm wavelength range. Moreover, the acquisition of data is in real time. Instead of monitoring the conventional shift in the plasmon resonance, our strategy relies on the plasmon resonance energy transfer (PRET) from functionalized AuNPs to metal ion complexes build on top of the AuNPs surface. Our methodology facilitates the detection of copper ions (Cu2+) in water (<10^(-9) M) by the formation of conjugated resonant complexes of N-[3-(trimethoxysilyl)propyl]ethylenediamine (TMSen). By applying our technology, new emerging, fast, and cheaper devices with intrinsic high sensitivity can be developed for the detection of different heavy metal ions in water, which are harmful to the environment and human health.

Plasmonic structures for biomedical sensing are in use for a long time. However, there is a fundamental limitation of their sensitivity due to low effective refractive index of layered plasmonic structures. We are proposing a hyperbolic metamaterial (HMM) structure which is a combination of surface plasmon Polaritons (SPPs) and long-range surface plasmon Polaritons (LRSPPs) modes. The result of the interaction between these modes leads to plasmonic modes with ultra-high effective refractive index. We calculated and optimized plasmonic HMM structure with effective refractive index equal to 8.1, i.e. twice as much as that of germanium, a natural material with the highest refractive index. We simulated these structures for gold, silver, copper and aluminum. The best way to use these structures for protein sensing is to use diffraction gratings – there is no natural material which can be used as a prism. By optimizing layer parameters and diffraction grating we were able to build a model of the structure with sensitivity as 10^-9 for refractive index. We are hoping to achieve sensitivity up to 10^-11, so this structure can be used for different protein sensing application including detection of metastatic cells spreading the human body.

We present nanostructured gold films ⎯ with complex plasmonic nanostructures present on the surface of the films ⎯ for enhanced SPR-based sensing and imaging of biomolecules attached to the surface of these films. We employed rigorous coupled wave analysis (RCWA) for simulating the nanostructured plasmonic gold films. In our simulations, surface plasmon polaritons were excited on the surface of the nanostructured gold films using the Kretschmann configuration. We observe that these nanostructured gold films show a significant enhancement in the sensitivity of SPR sensing and imaging of biomolecules as compared to planar gold films when optimal geometries and sizes of the plasmonic nanostructures (present on the surface of the gold film) are employed.

Development of label-free, highly sensitive, miniaturized surface plasmon resonance sensors enables real-time quantification of biomolecule interactions at atomic-levels, desirable for medical diagnostics and which will allow rapid clinical decisions. However, multi-target diagnostic assays require skilled labor, expensive materials, lengthy manual steps, as well as complicated analysis steps. Here, we develop a microfluidic-integrated digital optical disc (DVD) grating as a metasurface, which is coated with titanium-silver-gold (Ti-Ag-Au, 10, 30, and 15nm) for real-time monitoring of biomolecular interactions and binding affinities. Device fabrication process consists of poly (methyl methacrylate) (PMMA) microfluidic channel assembly on nanoplasmonic DVD surface gratings via double side adhesive (DSA) layers. Compared with other nano- and micro-fabrication methods, DVD-based sensor fabrication is relatively simple, cost-effective, and enables large-scale fabrication with minimum efforts. The plasmonic microfluidic chip surface was illuminated with a broadband light source and the normal reflection signal was monitored using a customized optical-setup. Maximum bulk sensitivity (337 nm/RIU) was observed with 30 seconds of etching period and low glycerol concentration (5%, v/v). Red-shifts of peak-wavelength (~16 nm) upon glycerol concentrations were observed as a function of time (seconds). A 0.6 nm peak-wavelength shift was observed in the step of EDC/NHS coupling and continuous protein A/G and G binding resulted in 0.353 ± 0.211 nm and 0.667 ± 0.116 nm (n=3, p<0.05). The presented platform could be potentially applicable to detect and real-time monitor of various biotargets including bacteria, cells, viruses, and proteins.

Surface Plasmon Resonance (SPR) is a very powerful optical sensing technique that detects bimolecular binding interactions and it has turned out to be a suitable platform for clinical analysis. In biological and chemical sensing applications, SPR is used to monitor molecular binding real-time and it also promotes epitope mapping for determining biomolecular structures such as the interactions of proteins, DNA and viruses. This sensing technique also provides sensitive, label free and real-time monitoring of reactions. In this study we have built, characterized and optimized the SPR system for biosensing applications. Spectroscopy and scanning electron microscopy were used to characterize the surface of the SPR biosensor chip functionalized with antibodies. The home-built SPR system was successful in detecting biological analytes thereby paving a way into designing a label-free point-of-care (POC) diagnostic tool.

In this study, we report the design of a 2D nanomaterial-enhanced biosensor by integrating both the 2D nanomaterials and immunoassay sensing techniques. A phase interrogation surface plasmon resonance (SPR) system was used for detecting antigen with a concentration ranging from nanomolar to femtomolar level. Our work has shown that the evanescent field generated from the Au film to 2D perovskite could lead to a significant sensitivity improvement. This specially antibody-functionalized sensing substrate, embedded with plasmonic metasurface structure, exhibited strong plasmonic coupling effect. And this optimized nanostructure could be engineered as a powerful and ultrasensitive platform for cancer diagnostics. The thickness of the sensing substrate is tuned in an atomic scale and optimized to obtain an enhanced sensing effect. More specifically, a sharp phase signal change and phase-related Goos-Hänchen signal shift was achieved that results from the strong resonance. The improved sensitivities of 2D Perovskite nanostructures were investigated. It is worth noting that the atomic layer design led to the sensing substrate optimized with a tuning scale less than 1 nm. Through a precise engineering of the metasurface substrates, 3 orders of magnitude improvement of the sensitivity (800,000 um/RIU) were demonstrated compared to the one with pure gold sensing substrate (300 um/RIU). This hybrid 2D nanomaterial-based metasurfaces would provide a good opportunity for the development of integrated cancer theranostic devices.

We will present surface enhanced Raman spectroscopy (SERS) and localized surface plasmon resonance (LSPR) detection of hydrogen peroxide (H₂O₂) using plasmonic gels. It has been known that reactive oxygen species (ROS), such as hydrogen peroxide, are involved in various biological processes, including metabolism, cell signaling, protein folding, biosynthesis, and host defense. Therefore, developing a simple and sensitive method for monitoring ROS levels is very important for live cell studies. Nevertheless, a challenge of utilizing SERS-based or LSPR-based method for molecular detection in complex fluids, such as cell culture media, is that a variety of molecules in sample solutions could interfere with detection results. In addition, when using SERS-based methods, the chance of having the target molecules at the SERS hot spots is reduced when other molecules are present in the solution. To enable detection of H₂O₂ in cell culture media, we have developed SERS and LSPR detection methods based on gels containing plasmonic nanoparticles. Since gels are filter-like materials, H₂O₂ can penetrate through the gels, but cells and large molecules such as proteins are blocked. We have successfully utilized these two methods to detect H₂O₂ in cell culture media without any sample pretreatment.

Rapid and adjustable shifted excitation Raman difference spectroscopy at 785 nm is presented. Here, a dual-wavelength diode laser is used for the excitation. This monolithic device contains two laser resonators and two distributed Bragg reflector (DBR) gratings which provide the two excitation lines for SERDS. The diode laser shows an optical power of 170 mW for each emission line. A flexible spectral distance up to 36 cm^-1 is realized using two separate heater elements which are implemented close to the DBR gratings. An alternating operation with modulation frequencies up to 100 Hz is performed without changing the spectral properties including a narrow linewidth of 11 pm. Raman experiments are carried out for validation. Here, Irish Cream is used as the test sample. Raman and SERDS spectra at the selected spectral distance at 15 cm^-1 are presented and discussed as an example. Rapid SERDS is demonstrated with an alternating operation and integration times of 50 ms for a single Raman spectrum. A discrimination of closely neighboured Raman bands of different components of Irish Cream and a 15-fold improvement of the signal-tobackground noise in the reconstructed SERDS spectrum is achieved. The results demonstrate rapid and adjustable SERDS using a dual-wavelength diode laser as a potential tool for Raman applications such as real-time measurements which require quick on-site decisions and experiments under strong and varying background interferences, e.g., from laser induced fluorescence and ambient light.

Plasmonic and metasurface structures offer unique optical features such as sub-wavelength field confinement, unusual optical nonlinear/quantum properties and wavefront shaping for advanced light manipulation and development of novel and ultracompact optical devices [1]. On the other hand, optical fiber is very efficient in transmitting light, however, its functionality is somewhat limited by the dielectric material of the core, which has poor electronic, magneto-optical, and nonlinear-optical responses and has the dielectric diffraction limit. In this talk, I will review our recent research efforts on the study of “meta”-optical fiber by integrating metasurface and plasmonic nanostructures with optical fibers. I will discuss our development of novel and ultracompact in-fiber optical meta-devices such as an optical fiber color filter and metalens [2, 3]. I will also present the opportunity on using plasmonic nanowire probe on optical fiber for tip-enhanced Raman spectroscopy for bio/chemical sensing. These advanced “meta”-optical fibers open the path to revolutionary in-fiber optical imaging and sensing applications [4]. 1. Y. W. Huang, H. W. Lee, R. Sokhoyan, R. Pala, K. Thyagarajan, S. Han, D. P. Tsai, and H. A. Atwater, “Gate-tunable conducting oxide metasurfaces,” Nano Lett. 16, 5319-5325 (2016). 2. J. Yang, I Ghirmire, P. C. Wu, S. Gurung, C. Arndt, D. P. Tsai, H. W. Lee, “Photonic crystal fiber metalens”, submitted (2018). 3. Indra Ghimire, Jingyi Yang, Sudip Gurung, Satyendra K. Mishra and Ho Wai Howard Lee,” Polarization dependent photonic crystal fiber color filter using asymmetric metasurfaces,” Submitted (2018) 4. K. Minn, A. Anopchenko, J. Yang, H. W. Lee, “Excitation of epsilon-near-zero resonance in ultra-thin indium tin oxide shell embedded nanostructured optical fiber,” Nature Scientific Reports 8, 2342 (2018).

We present a four-wave mixing interferometry technique recently developed by us, whereby single non-fluorescing gold nanoparticles are imaged background-free even inside highly heterogeneous cellular environments, owing to their specific nonlinear plasmonic response. The set-up enables correlative four-wave mixing/confocal fluorescence imaging, opening the prospect to study the fate of nanoparticle-biomolecule-fluorophore conjugates and their integrity inside cells. Beyond imaging, the technique features the possibility to track single particles with nanometric position localization precision in 3D from rapid single-point measurements at 1 ms acquisition time, by exploiting the optical vortex field pattern in the focal plane of a high numerical aperture objective lens. These measurements are also uniquely sensitive to the particle in-plane asymmetry and orientation. The localization precision in plane is found to be consistent with the photon shot-noise, while axially it is limited to about 3nm by the nano-positioning sample stage, with an estimated photon shot-noise limit of below 1 nm. As a proof-of- principle, the axial localization is exploited to track single gold nanoparticles of 25nm radius while diffusing across aqueous pockets in a dense agarose gel, mimicking a relevant biological environment.

Recently there have been significant interests about fabrication of optical nanopore for single molecule analysis and manipulation. However, due to very small amount of the optical intensity through the tiny size of the nano-aperture, optical intensity enhancement via plasmonic effect by using pore array or periodic groove patterns have been tried. In addition, the double slits with nanoscale width is reported to provide the constructive interference of the surface plasmonic wave. In this report, the nanoscale double slits with Au aperture array has been fabricated and optically characterized.

Raman spectroscopy is a well-established tool for material analysis, investigation of biological targets, food control, and medical applications. Analyzing real-world samples, fluorescence and intense background light can superimpose the weak Raman signals. Due to a different spectral behavior of these interferences compared to the Raman effect, experimental methods have been demonstrated to overcome this drawback. The so-called shifted excitation Raman difference spectroscopy (SERDS) utilizes shifts of the Raman lines with respect to shifts in the excitation wavelengths by a distance comparable to the width of the Raman lines under study. Disturbing background light remains spectrally constant and subsequent signal subtraction separates the Raman signal from the background. This technique relies on dual-wavelength light sources as key elements. In this contribution, tailored diode laser based light sources suitable for SERDS will be presented. First, we present hybrid and monolithic devices in the spectral range between 457 nm and 830 nm with output powers up to the watt range. This includes dual-wavelength lasers as well as DBR lasers with an adjustable emission wavelength, e.g. at 785 nm, enabling SERDS and related methods separating Raman signals from background interferences. Second, we present a compact UV light source at 222.5 nm with 160 μW output power suitable for UV Raman spectroscopy. This light source enables detecting Raman signals in a fluorescence-free spectral region.

The possibility of investigating small amounts of molecules, moieties, or nano-objects dispersed in solution constitutes a central step for various application areas in which high sensitivity is necessary. Here, we show that the rapid expansion of a water bubble can act as a fastmoving net for molecules or nano-objects, collecting the floating objects in the surrounding medium in a range up to 100 μm. Thanks to an engineered 3D patterning of the substrate, the collapse of the bubble could be guided toward a designed area of the surface with micrometric precision. Thus, a locally confined high density of particles is obtained, ready for evaluation by most optical/spectroscopic detection schemes. One of the main relevant strengths of the long-range capture and delivery method is the ability to increase, by a few orders of magnitude, thelocal density of particles with no changes in their physiological environment. The bubble is generated by an ultrafast IR laser pulse train focused on a resonant plasmonic antenna; due to the excitation process, the technique is trustworthy and applicable to biological samples. We have tested the reliabilities of the process by concentrating highly dispersed fluorescence molecules and fluorescent beads. Lastly, as an ultimate test, we have applied the bubble clustering method on nanosized exosome vesicles dispersed in water; due to the clustering effect, we were able to effectively perform Raman spectroscopy on specimens that were otherwise extremely difficult to measure

The plasmonic nanostructures required for the SERS are commonly in the form of solid substrates, or as colloidal solutions, both of them are not very useful to detect the biomarkers directly on human skins. Gel-based SERS substrates, into which the plasmonic nanostructures are incorporated, will be helpful for the direct collection of the biomarkers from secretions such as sweat. To elucidate these points, we studied the diffusion of Raman probe 4, 4’-Bipyridine (BPY) in the cetyltrimethylammonium bromide:sodium salicylate (CTAB:NaSal) gel. Au nano-island SERS chip was coated with a 1:1 complex of CTAB:NaSal . Then, the diffusion of the probe was studied by SERS spectra as a function of time. The SERS signal intensity increases gradually with increasing time. Highly porous gel rapidly absorbed aqueous analyte solutions generating large SERS signals. The subsequent increase in signal could arise from the diffusion of the analyte molecule into the gel and onto the Au aggregates. Importantly, this gel-based SERS sensor did not significantly compromise the SERS performance of the analyte. We propose that this gel-based SERS sensor can be smeared directly onto the skin surface to absorb the body fluids from sweat, enabling the detection of biomarkers.

We developed an optofluidic device containing a nanostructured substrate for surface enhanced Raman spectroscopy (SERS). The device is based on a silicon chip, on which structures were fabricated using electron lithography and wet etching to achieve a pattern of inverted pyramids on the surface, which was then covered by gold layer of defined thickness and roughness. The geometry of the surface allows localized plasmon oscillations to give rise to the SERS effect, in which the Raman spectral lines are intensified by the interaction of the plasmonic field with the electrons in the molecular bonds. The SERS substrate was enclosed in a microfluidic system from silicone polymer and glass, which allowed transport and precise mixing of fluids entering the chip, while preventing contamination or abrasion of the highly sensitive substrate. We used this device as a platform for quantitative detection of halogenated hydrocarbons such as 1,2,3-trichloropropane (TCP) in water in submillimolar concentrations. TCP is used in industry and it is a persistent environmental pollutant. The presented sensor allows fast and simple quantification of such molecules and it could contribute to environmental monitoring disciplines as well as enzymologic experiments with genetically engineered dehalogenases, which are potentially useful for bioremediation. This research is supported by Czech Science Foundation (CSF) 16-07965S, infrastructure was supported by MEYS (LO1212, LM2015055) and EC (CZ.1.05/2.1.00/01.0017).

The development of dynamic DNA nanostructures has opened the door to a wide variety of applications including sensing and information processing. DNA based molecular logic devices (MLDs) are DNA structures that have the ability to sense multiple inputs or “targets”, autonomously process the absence or presence of targets, and provide an output signal indicating the logic state of the system. As DNA is readily functionalized with fluorescent molecules, fluorophores can be strategically placed on MLDs so that the Förster resonance energy transfer efficiencies between the fluorophores are modulated when the DNA structure undergoes rearrangement. Consequently, the fluorescent signal of the dyes can be used as an output that provides the current logic state of the system. Although still in their elementary phase, MLDs have proved to be a promising modality for sensing multiple nanoscale targets, especially nucleic acids. Here, we review the development of multifluorophore MLDs and utilize examples from the literature and our own work to highlight their potential capabilities.

Here, we propose easy and robust strategies for the versatile integration 2D material flakes on plasmonic nanoholes by means of site selective deposition of MoS₂. The methods can be applied both to simple metallic flat nanostructures and to complex 3D metallic structures comprising nanoholes. The deposition methods allow the decoration of large ordered arrays of plasmonic structures with single or few layers of MoS2. We show that the plasmonic field generated by the nanohole can interact significantly with the 2D layer, thus representing an ideal system for hybrid 2DMaterial/ Plasmonic investigation. The controlled/ordered integration of 2D materials on plasmonic nanostructures opens a pathway towards new investigation of the following: enhanced light emission; strong coupling from plasmonic hybrid structures; hot electron generation; and sensors in general based on 2D materials.

Bridged-bowtie nanohole arrays and cross bridged-bowtie nanohole arrays in a gold film are presented as surfaceenhanced Raman scattering (SERS) substrates. We employed the numerical FDTD method to calculate the maximum electromagnetic SERS enhancement factor (EF) as a function of wavelength. It is found that the proposed nanohole arrays do not only display an extremely large enhancement factor but also have the hotspot spread over a larger area compared to the various other nanopillar structures. The calculation of electromagnetic SERS enhancement factor reveals that the cross bridged-bowtie nanohole arrays exhibit the maximum electromagnetic SERS EF of ~ 10⁹ spreading over an area of 100 nm². In addition, the electromagnetic SERS EF of ~ 10⁸ is spread over 500 nm² area which is higher than hotspot area in case of nanopillar structures. The resonance wavelength of the nanohole array can be tuned by varying the size of the nanoholes. These nanohole arrays can be employed both in transmission as well as in reflection mode as effective SERS substrates. In addition, bridged-bowtie and cross bridged-bowtie nanohole arrays show the significantly high electromagnetic SERS EF at more than one wavelength and therefore are useful for application involving multiple wavelength SERS response. Furthermore, the cross bridged-bowtie nanohole array exhibit the spatial tunability of hotspot by rotating the direction of polarization of incident field.

In this article, we present an innovative SPR sensor containing Au-TiO₂-Ti planar comb-structure Schottky diodes based on Kretschmann’s configuration, and discussed the feasibility of collecting plasmon-induced hot electrons as the signal of SPR sensor instead of traditional optical measurement. Taking advantage of the intrinsic energy transition process between electromagnetic waves and electrons, i.e., Landau damping, the hot electron-hole pairs (EHPs) are excited directly where the surface plasmon waves decay into. Theoretically, the amount of EHPs is determined by the resonance state of surface plasmon, and further determined by the refractive index change in the sensing area. In this device, the effective sensing area, which is critical and limited by the propagating characters of surface plasmon, and the mean free path of EHPs, is enlarged by intensively distributed micro comb structures. We fabricated the devices on 4-inch quartz wafer with photolithography, electron-beam evaporation (EBE), and lift-off process. These fabricated devices exhibited rectified I-V relations in electrical characterization experiments. The evaluated barrier height is 0.73 eV, but series resistance and ideality factor were not ideal as expected due to fabrication defects. We measured the responsivity of 0.75 uA/mW, under illumination of a 850nm infrared laser beam through a N-BK7 prism coupler. The current response from detection of standard solutions indicated a sensitivity of 1.87×10^-4 RIU/nA and a limit of detection (LoD) of 4.13×10^-3 RIU. In conclusion, this article provides a feasible method to drastically simplify the conventional SPR sensing configuration with mass-produced, small, and economical comb-structure Schottky diode sensor.

Now that a fully-implantable, continuous glucose monitor has received FDA approval, optical techniques other than fluorescence will seek to overcome the limited lifetimes resulting from photobleaching. Using plasmonic nanoparticles, we present the potential of reversible SERS-active sensing assays to function as long-term implantable sensors. The assays offer high selectivity and specificity of analyte detection and concentration without loss of emission intensity over time due to photodestruction. These assays are encapsulated in microdomains bounded by polyelectrolyte multilayers (PEMs), permeable to the target but impermeable to proteins. The microdomains are stabilized in hydrogels for biocompatibility and longevity. This study characterizes the performance of pH-sensitive Raman probes in three different hydrogels in a simulated in vivo environment with changing pH over time.

Over the past few years gold nanoparticle (AuNPs) have become extremely interesting because they possess enhanced optical, electrical and chemical properties. AuNPs have the ability to form robust conjugates with biomolecules such as antibodies and can enhance optical signals making them suitable for a variety of diagnostic applications including the surface plasmon resonance (SPR) technique. SPR is a highly sensitive and label free optical technique which is widely used for detecting biological analytes and analysing the interaction between different types of biomolecules. In this study, bioconjugation was achieved by covalently attaching antibodies to AuNPs and gold coated slides were used as SPR sensor chips in Kretschmann configuration. Several UV/VIS excitation spectra were collected before and after AuNPs were conjugated to antibodies. The results showed that the sensitivity of the SPR system significantly increased because of the bioconjugation of antibodies to AuNPs and this is a promising approach for biosensing applications.

Gold thin metal layers have been seen to be the most important signal amplification components in electrochemical and optical sensor applications. In surface plasmon resonance (SPR) applications, gold thin metal film possess electron densities that have the plasmon frequencies in the visible light range. In this study, gold thin film layer coating was deposited onto a glass substrate by using the ebeam physical vapour deposition technique. The structural and morphological investigations of the thin film layer coating were investigated using the X-ray diffraction system (XRD) while the scanning electron microscope (SEM) was used to investigate the morphology of the thin film layer coating. The optical analysis using different thicknesses, showed good absorption and transmission spectra that is required in determining the appropriate layer to be used for surface plasmon resonance. Using the XRD, SEM as well as transmission and absorption spectroscopy, the findings indicated that gold thin film layer deposition using the ebeam evaporation system is perfect for thin film layer coating for surface plasmon resonance applications.

We investigate switching near-field distribution on metal random nanoislands by changing the direction and the angle of light incidence in 14 channel modes. Distribution of the near-fields induced by different channel modes was calculated by finite difference time domain method. The size of near-fields under oblique channel modes ranges 48 - 77 nm in contrast to 127 - 145 nm with normal incidence. Quantitative analysis of near-field position was performed relative to nanoislands. Near-field position was largely well aligned with the direction of incident channel modes. Switching near fields was experimentally confirmed in two ways, first by measurement of fluorescence intensity and by NSOM. Fluorescence experiment was conducted by using bare glass substrate and gold nanoislands in seven channel modes. Fluorescence intensity on bare glass substrate shows symmetric intensity changes with channel modes. However, fluorescence intensity on gold nanoislands was found to be asymmetric. For quantitative analysis, mean-squared error (MSE) was calculated by defining fluorescence intensity as a 7D vector. Distribution of MSE in case of gold nanoislands was broader than on bare glass substrate. In other words, near fields induced on gold nanoislands were switched more strongly than bare glass substrate. Also, near fields induced on nanoislands were measured directly using NSOM in two channel modes. It was confirmed that spatial positions of near-fields depend on channel modes. The results of this study suggest that the near fields can be controlled by adjusting channel modes, which opens possibilities of highly sensitive and super-resolved detection and imaging.

Although nanotechnology has led to important advances in in vitro diagnostics, the development of nanosensors for in vivo molecular detection remains very challenging. Here, we demonstrated the proof‐of‐principle of in vivo detection of nucleic acid targets using a promising type of surface‐enhanced Raman scattering (SERS) nanosensor implanted in the skin of a large animal model (pig). The in vivo “smart tattoo” nanosensor used in this study employs the “inverse molecular sentinel” (iMS) detection scheme, which is a label-free homogeneous biosensing system based on a non-enzymatic DNA strand-displacement process and conformational change of stem-loop (hairpin) oligonucleotide probes upon target binding. In this study, plasmonics‐active nanostar was utilized as an efficient in vivo SERS sensing platform due to their tunable absorption bands in the near infrared region of the “tissue optical window. The results of this study illustrate the usefulness of SERS iMS nanosensors as an implantable skin‐based in vivo biosensing platform, providing a foundation for developments in continuous health status sensing, disease biomarker monitoring, and other clinical translation applications.

We fabricated hollow nanoantennas with varying inner channels sizes on a gold-covered silicon nitride membrane. Our fabrication technique allowed us to narrow the size of the inner channels down to 15nm. We managed to exclusively decorate the tips of the antennas with thiol-conjugated dyes by creating a concentration gradient through the nanoantennas. Finally, we characterized the antennas in terms of their effect on the lifetime of dyes. We used Atto 520 and Atto 590 for the experiments. We carried out experiments with the antennas decorated with Atto 520, with Atto 590 as well as with the two Atto dyes at the same time. The experiments carried out with the antennas decorated with Atto 520 only and Atto 590 only yielded a lifetime reduction with respect to the confocal case. Interestingly, their lifetime reductions were significantly different. Then, we decorated the antennas with the two dyes at the same time. Even though we could not control the distance between the two dyes, FRET effects were clearly observed. The FRET effects were found to be dependent on the size of the inner channel. We believe that our tip decorated hollow nanoantennas could find application in FRET-based single molecule nanopore technologies.