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.

A universal metasurface transfer technique for heterogeneous integration.

Metasurfaces offer a versatile platform for engineering the wavefront of light using nanostructures with subwavelength dimensions and hold great promise for dramatically miniaturizing conventional optical elements due to their small footprint and broad functionality. However, metasurfaces so far have been mainly demonstrated on bulky and planar substrates that are often orders of magnitude thicker than the metasurface itself. Conventional substrates not only nullify the reduced footprint advantage of metasurfaces, but also limit their application scenarios. The bulk substrate also determines the metasurface dielectric environment, with potentially undesired optical effects that undermine the optical performance. Here we develop a universal polymer-assisted transfer technique to tackle this challenge by decoupling the substrate employed on the fabrication of metasurfaces from that used for the target application. As an example, Huygens' metasurfaces with 120 nm thickness in the visible range (532 nm) are demonstrated to be transferred onto a 100 nm thick freestanding SiNx membrane while maintaining excellent structural integrity and optical performance of diffraction-limited focusing. This transfer method not only enables the thinnest dielectric metalens to the best of our knowledge, but also opens up new opportunities in integrating cascaded and multilayer metasurfaces, as well as the heterogeneous integration with nonconventional substrates and various electronic/photonic devices.

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.

On-Demand Fully Enclosed Superhydrophobic-Optofluidic Devices Enabled by Microstereolithography.

Superhydrophobic surface-based optofluidics have been introduced to biosensors and unconventional optics with unique advantages, such as low light loss and power consumption. However, most of these platforms were made with planar-like microstructures and nanostructures, which may cause bonding issues and result in significant waveguide loss. Here, we introduce a fully enclosed superhydrophobic-based optofluidics system, enabled by a one-step microstereolithography procedure. Various microstructured cladding designs with a feature size down to 100 µm were studied and a "T-type" overhang design exhibits the lowest optical loss, regardless of the excitation wavelength. Surprisingly, the optical loss of superhydrophobic-based optofluidics is not solely decided by the solid area fraction at the solid/water/air interface, but also the cross-section shape and the effective cladding layer composition. We show that this fully enclosed optofluidic system can be used for CRISPR-labeled quantum dot quantification, intended for in vitro and in vivo CRISPR therapeutics.

Ligand functionalization of titanium nanopattern enables the analysis of cell-ligand interactions by super-resolution microscopy.

The spatiotemporal aspects of early signaling events during interactions between cells and their environment dictate multiple downstream outcomes. While advances in nanopatterning techniques have allowed the isolation of these signaling events, a major limitation of conventional nanopatterning methods is its dependence on gold (Au) or related materials that plasmonically quench fluorescence and, thus, are incompatible with super-resolution fluorescence microscopy. Here we describe a novel method that integrates nanopatterning with single-molecule resolution fluorescence imaging, thus enabling mechanistic dissection of molecular-scale signaling events in conjunction with nanoscale geometry manipulation. Our method exploits nanofabricated titanium (Ti) whose oxide (TiO2) is a dielectric material with no plasmonic effects. We describe the surface chemistry for decorating specific ligands such as cyclo-RGD (arginine, glycine and aspartate: a ligand for fibronectin-binding integrins) on TiO2 nanoline and nanodot substrates, and demonstrate the ability to perform dual-color super-resolution imaging on these patterns. Ti nanofabrication is similar to other metallic materials like Au, while the functionalization of TiO2 is relatively fast, safe, economical, easy to set up with commonly available reagents, and robust against environmental parameters such as humidity. Fabrication of nanopatterns takes ~2-3 d, preparation for functionalization ~1.5-2 d, and functionalization 3 h, after which cell culture and imaging experiments can be performed. We suggest that this method may facilitate the interrogation of nanoscale geometry and force at single-molecule resolution, and should find ready applications in early detection and interpretation of physiochemical signaling events at the cell membrane in the fields of cell biology, immunology, regenerative medicine, and related fields.

Kirigami Engineering-Nanoscale Structures Exhibiting a Range of Controllable 3D Configurations.

Kirigami structures provide a promising approach to transform flat films into 3D complex structures that are difficult to achieve by conventional fabrication approaches. By designing the cutting geometry, it is shown that distinct buckling-induced out-of-plane configurations can be obtained, separated by a sharp transition characterized by a critical geometric dimension of the structures. In situ electron microscopy experiments reveal the effect of the ratio between the in-plane cut size and film thickness on out-of-plane configurations. Moreover, geometrically nonlinear finite element analyses (FEA) accurately predict the out-of-plane modes measured experimentally, their transition as a function of cut geometry, and provide the stress-strain response of the kirigami structures. The combined computational-experimental approach and results reported here represent a step forward in the characterization of thin films experiencing buckling-induced out-of-plane shape transformations and provide a path to control 3D configurations of micro- and nanoscale buckling-induced kirigami structures. The out-of-plane configurations promise great utility in the creation of micro- and nanoscale systems that can harness such structural behavior, such as optical scanning micromirrors, novel actuators, and nanorobotics. This work is of particular significance as the kirigami dimensions approach the sub-micrometer scale which is challenging to achieve with conventional micro-electromechanical system technologies.

Effect of Brisk Walking on Health-Related Physical Fitness Balance and Life Satisfaction Among the Elderly: A Systematic Review.

BACKGROUND: Although the elderly frequently engages in brisk walking as a form of exercise, little has been reported in the literature about the effect of brisk walking on health-related physical fitness, balance, and overall life satisfaction. OBJECTIVES: The purpose of this systematic review is to determine the effect of brisk walking on the elderly's health-related physical fitness, balance, and life satisfaction. DESIGN: We conducted a comprehensive search from the PubMed, Web of Science, Scopus, and SPORTDiscus databases from January to September 2021. We selected studies through PICOS and conducted a systematic literature review according to the PRISMA guidelines. RESULTS: Thirteen studies met all criteria; 11 were classed as low risk of bias, while two were classified as high risk of bias. Generally, brisk walking has been shown to improve cardiorespiratory fitness, muscular strength, and body composition. Limited evidence was presented on flexibility, muscular endurance and development and life satisfaction, and there was conflicting evidence on balance. Moreover, evidence of restriction proves that high-intensity (80-85%) brisk walking is more effective than moderate-intensity (60-75%) brisk walking on the aerobic capacity of the elderly. Furthermore, there was less research conducted on males. CONCLUSION: Brisk walking has been shown to improve cardiorespiratory fitness, muscular strength, and body composition. Other outcomes (balance, flexibility, muscular endurance, and life satisfaction) and the impact of the intensity of brisk walking on the elderly should be confirmed. Therefore, there remains insufficient research on brisk walking, while single brisk walking cannot meet requirements of elderly in terms of their health-related physical fitness, balance, and life satisfaction. Future research should aim to examine the effectiveness of combining several types of exercises to promote general health in the elderly, as the World Health Organization recommends. Unintelligible FITT (frequency, intensity, time, type) principles of brisk walking training should be trenched for the results of scientific and effective physical exercise.

The CAR T-Cell Mechanoimmunology at a Glance.

Chimeric antigen receptor (CAR) T-cell transfer is a novel paradigm of adoptive T-cell immunotherapy. When coming into contact with a target cancer cell, CAR T-cell forms a nonclassical immunological synapse with the cancer cell and dynamically orchestrates multiple critical forces to commit cytotoxic immune function. Such an immunologic process involves a force transmission in the CAR and a spatiotemporal remodeling of cell cytoskeleton to facilitate CAR activation and CAR T-cell cytotoxic function. Yet, the detailed understanding of such mechanotransduction at the interface between the CAR T-cell and the target cell, as well as its molecular structure and signaling, remains less defined and is just beginning to emerge. This article summarizes the basic mechanisms and principles of CAR T-cell mechanoimmunology, and various lessons that can be comparatively learned from interrogation of mechanotransduction at the immunological synapse in normal cytotoxic T-cell. The recent development and future application of novel bioengineering tools for studying CAR T-cell mechanoimmunology is also discussed. It is believed that this progress report will shed light on the CAR T-cell mechanoimmunology and encourage future researches in revealing the less explored yet important mechanosensing and mechanotransductive mechanisms involved in CAR T-cell immuno-oncology.

Approaching the Strain-Free Limit in Ultrathin Nanomechanical Resonators.

Ultrathin mechanical structures are ideal building platforms to pursue the ultimate limit of nanomechanical resonators for applications in sensing, signal processing, and quantum physics. Unfortunately, as the thickness of the vibrating structures is reduced, the built-in strain of the structural materials plays an increased role in determining the mechanical performance of the devices. As a consequence, it is very challenging to fabricate resonators working in the modulus-dominant regime, where their dynamic behavior is exclusively determined by the device geometry. In this Letter, we report ultrathin doubly clamped nanomechanical resonators with aspect ratios as large as L/t ∼5000 and working in the modulus-dominant regime. We observed room temperature thermomechanically induced motion of multiple vibration modes with resonant frequencies closely matching the predicted values of Euler-Bernoulli beam theory under an axial strain of 6.3 × 10-8. The low strain of the devices enables a record frequency tuning ratio of more than 50 times. These results illustrate a new strategy for the quantitative design of nanomechanical resonators with unprecedented performance.

Integrin nanoclusters can bridge thin matrix fibres to form cell-matrix adhesions.

Integrin-mediated cell-matrix adhesions are key to sensing the geometry and rigidity of extracellular environments and influence vital cellular processes. In vivo, the extracellular matrix is composed of fibrous arrays. To understand the fibre geometries that are required for adhesion formation, we patterned nanolines of various line widths and arrangements in single, crossing or paired arrays with the integrin-binding peptide Arg-Gly-Asp. Single thin lines (width ≤30 nm) did not support cell spreading or formation of focal adhesions, despite the presence of a high density of Arg-Gly-Asp, but wide lines (>40 nm) did. Using super-resolution microscopy, we observed stable, dense integrin clusters formed on parallel (within 110 nm) or crossing thin lines (mimicking a matrix mesh) similar to those on continuous substrates. These dense clusters bridged the line pairs by recruiting activated but unliganded integrins, as verified by integrin mutants unable to bind ligands that coclustered with ligand-bound integrins when present in an active extended conformation. Thus, in a fibrous extracellular matrix mesh, stable integrin nanoclusters bridge between thin (≤30 nm) matrix fibres and bring about downstream consequences of cell motility and growth.

Full control of ligand positioning reveals spatial thresholds for T cell receptor triggering.

Elucidating the rules for receptor triggering in cell-cell and cell-matrix contacts requires precise control of ligand positioning in three dimensions. Here, we use the T cell receptor (TCR) as a model and subject T cells to different geometric arrangements of ligands, using a nanofabricated single-molecule array platform. This comprises monovalent TCR ligands anchored to lithographically patterned nanoparticle clusters surrounded by mobile adhesion molecules on a supported lipid bilayer. The TCR ligand could be co-planar with the supported lipid bilayer (2D), excluding the CD45 transmembrane tyrosine phosphatase, or elevated by 10 nm on solid nanopedestals (3D), allowing closer access of CD45 to engaged TCR. The two configurations resulted in different T cell responses, depending on the lateral spacing between the ligands. These results identify the important contributions of lateral and axial components of ligand positioning and create a more complete foundation for receptor engineering for immunotherapy.

Spatial Control of Biological Ligands on Surfaces Applied to T Cell Activation.

In this chapter, we present techniques, based on molecular-scale nanofabrication and selective self-assembly, for the presentation of biomolecules of interest (ligands, receptors, etc.) on a surface with precise spatial control and arbitrary geometry at the single-molecule level. Metallic nanodot arrays are created on glass coverslips and are then used as anchors for the immobilization of biological ligands via thiol linking chemistry. The nanodot size is controlled by both lithography and metallization. The reagent concentration in self-assembly can be adjusted to ensure single-molecule occupancy for a given dot size. The surrounding glass is backfilled by a protein-repellent layer to prevent nonspecific adsorption. Moreover, bifunctional surfaces are created, whereby a second ligand is presented on the background, which is frequently a requirement for simulating complex cellular functions involving more than one key ligand. This platform serves as a novel and powerful tool for molecular and cellular biology, e.g., to study the fundamental mechanisms of receptor-mediated signaling.

Improved Glass Surface Passivation for Single-Molecule Nanoarrays.

Single-molecule fluorescence techniques provide a critical tool for probing biomolecular and cellular interactions with unprecedented resolution and precision. Unfortunately, many of these techniques are hindered by a common problem, namely, the nonspecific adsorption of target biomolecules. This issue is mostly addressed by passivating the glass surfaces with a poly(ethylene glycol) (PEG) brush. This is effective only at low concentrations of the probe molecule because there are defects inherent to polymer brushes formed on glass coverslips due to the presence of surface impurities. Tween-20, a detergent, is a promising alternative that can improve surface passivation, but it is incompatible with living cells, and it also possesses limited selectivity for glass background over metallic nanoparticles, which are frequently used as anchors for the probe molecules. To address these issues, we have developed a more versatile method to improve the PEG passivation. A thin film of hydrogen silsesquioxane (HSQ) is spin-coated and thermally cured on glass coverslips in order to cover the surface impurities. This minimizes the formation of PEG defects and reduces nonspecific adsorption, resulting in an improvement comparable to Tween-20 treatment. This approach was applied to single-molecule nanoarrays of streptavidin bound to AuPd nanodots patterned by e-beam lithography (EBL). The fluorescence signal to background ratio (SBR) on HSQ-coated glass was improved by ∼4-fold as compared to PEG directly on glass. This improvement enables direct imaging of ordered arrays of single molecules anchored to lithographically patterned arrays of metallic nanodots.

Molecular Occupancy of Nanodot Arrays.

Single-molecule nanodot arrays, in which a biomolecule of choice (protein, nucleic acid, etc.) is bound to a metallic nanoparticle on a solid substrate, are becoming an increasingly important tool in the study of biomolecular and cellular interactions. We have developed an on-chip measurement protocol to monitor and control the molecular occupancy of nanodots. Arrays of widely spaced nanodots and nanodot clusters were fabricated on glass surfaces by nanolithography and functionalized with fluorescently labeled proteins. The molecular occupancy was determined by monitoring individual fluorophore bleaching events, while accounting for fluorescence quenching effects. We found that the occupancy can be interpreted as a packing problem, and depends on nanodot size and binding ligand concentration, where the latter is easily adjusted to compensate the flexibility of dimension control in nanofabrication. The results are scalable with nanodot cluster size, extending to large area close packed arrays. As an example, the nanoarray platform was used to probe the geometric requirement of T-cell activation at the single-molecule level.

Directed Assembly of End-Functionalized Single Wall Carbon Nanotube Segments.

A key impediment to the implementation of a nanoelectronics technology based on single wall carbon nanotubes (SWCNTs) is the inability to arrange them in a manner suitable for integration into complex circuits. As a step toward addressing this problem, we explore the binding of fixed-length, end-functionalized SWCNT segments to lithographically defined nanoscale anchors, such that individual SWCNTs can be placed with control over position and orientation. Both monovalent and bivalent bindings are explored using covalent and noncovalent binding chemistries. Placement efficiency is assessed in terms of overall yield of SWCNT binding, as well as binding specificity and the degree of nonspecific binding. Placement yields as high as 93% and 79% are achieved, respectively, for covalent binding and for binding through DNA hybridization. Orientational control of the SWCNT segments is achieved with 95% and 51% efficiency for monovalent and bivalent bindings, respectively. This represents a new approach that could pave the way toward complex SWCNT devices and circuits.

Bifunctional nanoarrays for probing the immune response at the single-molecule level.

Bifunctional nanoarrays were created to simulate the immunological synapse and probe the T-cell immune response at the single-molecule level. Sub-5 nm AuPd nanodot arrays were fabricated using both e-beam and nanoimprint lithography. The nanoarrays were then functionalized by two costimulatory molecules: antibody UCHT1 Fab, which binds to the T-cell receptor (TCR) and activates the immune response, bound to metallic nanodots; and intercellular adhesion molecule-1, which enhances cell adhesion, on the surrounding area. Initial T-cell experiments show successful attachment and activation on the bifunctional nanoarrays. This nanoscale platform for single-molecule control of TCR in living T-cells provides a new approach to explore how its geometric arrangement affects T-cell activation and behavior, with potential applications in immunotherapy. This platform also serves as a general model for single-molecule nanoarrays where more than one molecular species is required.

A MEMS differential viscometric sensor for affinity glucose detection in continuous glucose monitoring.

Micromachined viscometric affinity glucose sensors have been previously demonstrated using vibrational cantilever and diaphragm. These devices featured a single glucose detection module that determines glucose concentrations through viscosity changes of glucose-sensitive polymer solutions. However, fluctuations in temperature and other environmental parameters might potentially affect the stability and reliability of these devices, creating complexity in their applications in subcutaneously implanted continuous glucose monitoring (CGM). To address these issues, we present a MEMS differential sensor that can effectively reject environmental disturbances while allowing accurate glucose detection. The sensor consists of two magnetically driven vibrating diaphragms situated inside microchambers filled with a boronic-acid based glucose-sensing solution and a reference solution insensitive to glucose. Glucose concentrations can be accurately determined by characteristics of the diaphragm vibration through differential capacitive detection. Our in-vitro and preliminary in-vivo experimental data demonstrate the potential of this sensor for highly stable subcutaneous CGM applications.