TiO2 Nano-Biopatterning Reveals Optimal Ligand Presentation for Cell-Matrix Adhesion Formation.

Nanoscale organization of transmembrane receptors is critical for cellular functions, enabled by the nanoscale engineering of bioligand presentation. Previously, a spatial threshold of ≤60 nm for integrin binding ligands in cell-matrix adhesion is demonstrated using monoliganded gold nanoparticles. However, the ligand geometric arrangement is limited to hexagonal arrays of monoligands, while plasmonic quenching limits further investigation by fluorescence-based high-resolution imaging. Here, these limitations are overcome with dielectric TiO2 nanopatterns, eliminating fluorescence quenching, thus enabling super-resolution fluorescence microscopy on nanopatterns. By dual-color super-resolution imaging, high precision and consistency among nanopatterns, bioligands, and integrin nanoclusters are observed, validating the high quality and integrity of both nanopattern functionalization and passivation. By screening TiO2 nanodiscs with various diameters, an increase in fibroblast cell adhesion, spreading area, and Yes-associated protein (YAP) nuclear localization on 100 nm diameter compared with smaller diameters was observed. Focal adhesion kinase is identified as the regulatory signal. These findings explore the optimal ligand presentation when the minimal requirements are sufficiently fulfilled in the heterogenous extracellular matrix network of isolated binding regions with abundant ligands. Integration of high-fidelity nano-biopatterning with super-resolution imaging allows precise quantitative studies to address early signaling events in response to receptor clustering and their nanoscale organization.

Dielectric metasurfaces for next-generation optical biosensing: a comparison with plasmonic sensing.

In the past decades, nanophotonic biosensors have been extended from the extensively studied plasmonic platforms to dielectric metasurfaces. Instead of plasmonic resonance, dielectric metasurfaces are based on Mie resonance, and provide comparable sensitivity with superior resonance bandwidth, Q factor, and figure-of-merit. Although the plasmonic photothermal effect is beneficial in many biomedical applications, it is a fundamental limitation for biosensing. Dielectric metasurfaces solve the ohmic loss and heating problems, providing better repeatability, stability, and biocompatibility. We review the high-Q resonances based on various physical phenomena tailored by meta-atom geometric designs, and compare dielectric and plasmonic metasurfaces in refractometric, surface-enhanced, and chiral sensing for various biomedical and diagnostic applications. Departing from conventional spectral shift measurement using spectrometers, imaging-based and spectrometer-less biosensing are highlighted, including single-wavelength refractometric barcoding, surface-enhanced molecular fingerprinting, and integrated visual reporting. These unique modalities enabled by dielectric metasurfaces point to two important research directions. On the one hand, hyperspectral imaging provides massive information for smart data processing, which not only achieve better biomolecular sensing performance than conventional ensemble averaging, but also enable real-time monitoring of cellular or microbial behaviour in physiological conditions. On the other hand, a single metasurface can integrate both functions of sensing and optical output engineering, using single-wavelength or broadband light sources, which provides simple, fast, compact, and cost-effective solutions. Finally, we provide perspectives in future development on metasurface nanofabrication, functionalization, material, configuration, and integration, towards next-generation optical biosensing for ultra-sensitive, portable/wearable, lab-on-a-chip, point-of-care, multiplexed, and scalable applications.

Gold Nanoparticle Enabled Localized Surface Plasmon Resonance on Unique Gold Nanomushroom Structures for On-Chip CRISPR-Cas13a Sensing.

A novel localized surface plasmon resonance (LSPR) system based on the coupling of gold nanomushrooms (AuNMs) and gold nanoparticles (AuNPs) is developed to enable a significant plasmonic resonant shift. The AuNP size, surface chemistry, and concentration are characterized to maximize the LSPR effect. A 31 nm redshift is achieved when the AuNMs are saturated by the AuNPs. This giant redshift also increases the full width of the spectrum and is explained by the 3D finite-difference time-domain (FDTD) calculation. In addition, this LSPR substrate is packaged in a microfluidic cell and integrated with a CRISPR-Cas13a RNA detection assay for the detection of the SARS-CoV-2 RNA targets. Once activated by the target, the AuNPs are cleaved from linker probes and randomly deposited on the AuNM substrate, demonstrating a large redshift. The novel LSPR chip using AuNP as an indicator is simple, specific, isothermal, and label-free; and thus, provides a new opportunity to achieve the next generation multiplexing and sensitive molecular diagnostic system.

Quality Characteristics and Acceptance Intention for Healthcare Kiosks: Perception of Elders from South Korea Based on the Extended Technology Acceptance Model.

This study aimed to perform a path analysis to understand the effects of quality characteristics on perceived usefulness, perceived ease to use, involvement, and acceptance intention of healthcare kiosks in elderly using the extended technology acceptance model. We performed structural equation modeling (SEM) with data from 300 elderly. The following results were obtained. Firstly, elderly's perceived quality characteristics of healthcare kiosks had a partial positive effect on perceived usefulness. Secondly, elderly's perceived quality characteristics of healthcare kiosks had a partial positive effect on perceived ease to use. Thirdly, elderly's perceived ease to use healthcare kiosks had a partial positive effect on perceived usefulness. In addition, elderly's perceived usefulness of healthcare kiosks had a positive effect on acceptance intention. Lastly, elderly's perceived ease to use healthcare kiosks had a positive effect on acceptance intention.

The obligate intracellular bacterium Orientia tsutsugamushi differentiates into a developmentally distinct extracellular state.

Orientia tsutsugamushi (Ot) is an obligate intracellular bacterium in the family Rickettsiaceae that causes scrub typhus, a severe mite-borne human disease. Its mechanism of cell exit is unusual amongst Rickettsiaceae, as Ot buds off the surface of infected cells enveloped in plasma membrane. Here, we show that Ot bacteria that have budded out of host cells are in a distinct developmental stage compared with intracellular bacteria. We refer to these two stages as intracellular and extracellular bacteria (IB and EB, respectively). These two forms differ in physical properties: IB is both round and elongated, and EB is round. Additionally, IB has higher levels of peptidoglycan and is physically robust compared with EB. The two bacterial forms differentially express proteins involved in bacterial physiology and host-pathogen interactions, specifically those involved in bacterial dormancy and stress response, and outer membrane autotransporter proteins ScaA and ScaC. Whilst both populations are infectious, entry of IB Ot is sensitive to inhibitors of both clathrin-mediated endocytosis and macropinocytosis, whereas entry of EB Ot is only sensitive to a macropinocytosis inhibitor. Our identification and detailed characterization of two developmental forms of Ot significantly advances our understanding of the intracellular lifecycle of an important human pathogen.

Spread spectrum SERS allows label-free detection of attomolar neurotransmitters.

The quantitative label-free detection of neurotransmitters provides critical clues in understanding neurological functions or disorders. However, the identification of neurotransmitters remains challenging for surface-enhanced Raman spectroscopy (SERS) due to the presence of noise. Here, we report spread spectrum SERS (ss-SERS) detection for the rapid quantification of neurotransmitters at the attomolar level by encoding excited light and decoding SERS signals with peak autocorrelation and near-zero cross-correlation. Compared to conventional SERS measurements, the experimental result of ss-SERS shows an exceptional improvement in the signal-to-noise ratio of more than three orders of magnitude, thus achieving a high temporal resolution of over one hundred times. The ss-SERS measurement further allows the attomolar SERS detection of dopamine, serotonin, acetylcholine, γ-aminobutyric acid, and glutamate without Raman reporters. This approach opens up opportunities not only for investigating the early diagnostics of neurological disorders or highly sensitive biomedical SERS applications but also for developing low-cost spectroscopic biosensing applications.

Kinetics of lipid raft formation at lipid monolayer-bilayer junction probed by surface plasmon resonance.

A label-free, non-dispruptive, and real-time analytical device to monitor the dynamic features of biomolecules and their interactions with neighboring molecules is an essential prerequisite for biochip- and diagonostic assays. To explore one of the central questions on the lipid-lipid interactions in the course of the liquid-ordered (lo) domain formation, called rafts, we developed a method of reconstituting continuous but spatially heterogeneous lipid membrane platforms with molayer-bilayer juntions (MBJs) that enable to form the lo domains in a spatiotemporally controlled manner. This allows us to detect the time-lapse dynamics of the lipid-lipid interactions during raft formation and resultant membrane phase changes together with the raft-associated receptor-ligand binding through the surface plasmon resonance (SPR). For cross-validation, using epifluorescence microscopy, we demonstrated the underlying mechanisms for raft formations that the infiltration of cholesterols into the sphingolipid-enriched domains plays a crucial roles in the membrane phase-separation. Our membrane platform, being capable of monitoring dynamic interactions among lipids and performing the systematic optical analysis, will unveil physiological roles of cholesterols in a variety of biological events.

Ag/Au Alloyed Nanoislands for Wafer-Level Plasmonic Color Filter Arrays.

Alloyed metals in nanoscale exhibit some intriguing features that are absent in mono-metallic nanostructures. Here we report silver and gold alloyed nanoislands with high tunability of localized surface plasmon resonance (LSPR) wavelength in the visible range for wafer-level plasmonic color filter arrays. The nanofabrication includes two simple steps of concurrent thermal evaporation of Ag and Au grains and solid-state dewetting of the as-deposited nanocomposite thin film. The alloy ratio during the evaporation precisely tunes the LSPR wavelengths within 415-609 nm spectrum range. The elemental composition map reveals that alloyed nanoislands are completely miscible while preserving uniform size, regardless of the alloy ratio. Besides, the multiple lift-off processes and thermal dewetting of Ag/Au nanocomposite thin films successfully demonstrate the wafer-level nanofabrication of plasmonic color filter mosaic. Each plasmonic color pixel comprises different alloy ratio and efficiently transmits colors ranging from cyan, yellow, and magenta. The transmission spectra transposed onto a CIE 1931 color map show comparable color diversity to the plasmonic color filters fabricated by conventional e-beam lithographic techniques. This novel method provides a new direction for large-scale and visible plasmonic color filter arrays in advanced display or imaging applications.

Nanoislands as plasmonic materials.

Subwavelength metal nanoislands thermally dewetted from a thin film emerge as a powerful and cost-effective photonic material, due to the formation of substantially strong nano-gap-based plasmonic hot spots and their simple large-area nanofabrication. Unlike conventional nanostructures, nanoislands dewetted from thin metal films can be formed on a large scale at the wafer level and show substrate-dependent plasmonic phenomena across a broad spectral range from ultraviolet to infrared. Substrate-selective dewetting methods for metal nanoislands enable diverse nanophotonic and optoelectronic technologies, underlining mechanical, structural, and material properties of a substrate. Emerging bioplasmonic technology using metal nanoislands also serves as a high-throughput and surface-sensitive analytical technique with wide-ranging application in rapid, real-time, and point-of-care medical diagnostics. This review introduces an assortment of dewetting fabrication methods for metal nanoislands on distinct substrates from glass to cellulose fibers and provides novel findings for metal nanoislands on a substrate by three-dimensional numerical modeling. Furthermore, the plasmonic properties of metal nanoislands and recent examples for their photonic applications, in particular, biological sensing, are technically summarized and discussed.

Structural coloration of transmission light through self-aligned and complementary plasmonic nanostructures.

Structural coloration of natural surfaces often originates from the change of reflected colors depending on the viewing or illumination angle. Recently, the structural coloration of nanoplasmonic structures has attracted a great deal of attention due to high compactness, robust stability and high color-tunability, as well as high sensitivity to the incidence angle. Here we report complementary plasmonic structures (CPS) for transmission structural coloration by tailoring a single spectral peak depending on the incidence angle of light. The CPS features self-aligned silver nanohole and nanodisk arrays, supported by dielectric nanopillar arrays of hydrogen silsesquioxane. Unlike the conventional hybridized nanostructures of plasmonic nanohole and nanodisk arrays, the nanodisks of CPS effectively attenuate undesired spectral peaks of nanoholes by exploiting an extinction peak of nanodisks, serving as a spectral suppressor. As a result, a single transmission spectral peak becomes red-shifted from 736 nm to 843 nm as the incidence angle varies from 0° to 30°. This unique configuration provides a new direction for tunable filters that can be utilized for compact multispectral or hyperspectral imaging applications.

Cell-Shaping Micropatterns for Quantitative Super-Resolution Microscopy Imaging of Membrane Mechanosensing Proteins.

Patterning cells on microcontact-printed substrates is a powerful approach to control cell morphology and introduce specific mechanical cues on a cell's molecular organization. Although global changes in cellular architectures caused by micropatterns can easily be probed with diffraction-limited optical microscopy, studying molecular reorganizations at the nanoscale demands micropatterned substrates that accommodate the optical requirements of single molecule microscopy techniques. Here, we developed a simple micropatterning strategy that provides control of cellular architectures and is optimized for nanometer accuracy single molecule tracking and three-dimensional super-resolution imaging of plasma and nuclear membrane proteins in cells. This approach, based on fibronectin microcontact printing on hydrophobic organosilane monolayers, allows evanescent wave and light-sheet microscopy of cells whilst fulfilling the stringent optical demands of point reconstruction optical microscopy. By imposing steady-state mechanical cues on cells grown in these micropatterns, we reveal nanoscale remodeling in the dynamics and the structural organizations of the nuclear envelope mechanotransducing protein emerin and of the plasma membrane mechanosensing protein caveolin-1 using single particle tracking photoactivated localization microscopy and direct stochastic optical reconstruction microscopy imaging. In addition to allowing quantitative biophysical studies of mechanoresponsive membrane proteins, this approach provides an easy means to probe mechanical regulations in cellular membranes with high optical resolution and nanometer precision.

Split-GFP: SERS Enhancers in Plasmonic Nanocluster Probes.

The assembly of plasmonic metal nanoparticles into hot spot surface-enhanced Raman scattering (SERS) nanocluster probes is a powerful, yet challenging approach for ultrasensitive biosensing. Scaffolding strategies based on self-complementary peptides and proteins are of increasing interest for these assemblies, but the electronic and the photonic properties of such hybrid nanoclusters remain difficult to predict and optimize. Here, split-green fluorescence protein (sGFP) fragments are used as molecular glue and the GFP chromophore is used as a Raman reporter to assemble a variety of gold nanoparticle (AuNP) clusters and explore their plasmonic properties by numerical modeling. It is shown that GFP seeding of plasmonic nanogaps in AuNP/GFP hybrid nanoclusters increases near-field dipolar couplings between AuNPs and provides SERS enhancement factors above 108 . Among the different nanoclusters studied, AuNP/GFP chains allow near-infrared SERS detection of the GFP chromophore imidazolinone/exocyclic CC vibrational mode with theoretical enhancement factors of 108 -109 . For larger AuNP/GFP assemblies, the presence of non-GFP seeded nanogaps between tightly packed nanoparticles reduces near-field enhancements at Raman active hot spots, indicating that excessive clustering can decrease SERS amplifications. This study provides rationales to optimize the controlled assembly of hot spot SERS nanoprobes for remote biosensing using Raman reporters that act as molecular glue between plasmonic nanoparticles.

A compact light concentrator by the use of plasmonic faced folded nano-rods.

We propose a compact nano-metallic structure for enhancing and concentrating far-field transmission: a faced folded nano-rod (FFR) unit, composed of two folded metallic nano-rods placed facing each other in an aperture. By analyzing local charge, field, and current distributions in the FFR unit using three-dimensional finite difference time domain (FDTD) calculation results, we show that although charge and field configurations become somewhat different depending on the polarization states of the illumination, similar current flows are formed in the FFR unit, which entail similar far-field radiation patterns regardless of the polarization states, making the FFR unit a quasi-polarization-insensitive field concentrator. We demonstrate this functionality of the FFR unit experimentally using the holographic microscopy which provides us a three-dimensional map of the complex wavefronts of optical fields emanating from the FFR unit.

Overview of the characteristics of micro- and nano-structured surface plasmon resonance sensors.

The performance of bio-chemical sensing devices has been greatly improved by the development of surface plasmon resonance (SPR) based sensors. Advancements in micro- and nano-fabrication technologies have led to a variety of structures in SPR sensing systems being proposed. In this review, SPR sensors (from typical Kretschmann prism configurations to fiber sensor schemes) with micro- or nano-structures for local light field enhancement, extraordinary optical transmission, interference of surface plasmon waves, plasmonic cavities, etc. are discussed. We summarize and compare their performances and present guidelines for the design of SPR sensors.

Plasmonic nanostructures for nano-scale bio-sensing.

The optical properties of various nanostructures have been widely adopted for biological detection, from DNA sequencing to nano-scale single molecule biological function measurements. In particular, by employing localized surface plasmon resonance (LSPR), we can expect distinguished sensing performance with high sensitivity and resolution. This indicates that nano-scale detections can be realized by using the shift of resonance wavelength of LSPR in response to the refractive index change. In this paper, we overview various plasmonic nanostructures as potential sensing components. The qualitative descriptions of plasmonic nanostructures are supported by the physical phenomena such as plasmonic hybridization and Fano resonance. We present guidelines for designing specific nanostructures with regard to wavelength range and target sensing materials.