Cell adhesion and migration on nanopatterned substrates and their effects on cell-capture yield

With scanning electron microscopy analysis, we investigated the role of nanoscale topography on cellular activities; e.g.?cell adhesion and spreading by culturing A549 cells (human lung carcinoma cell line cells) for 1-48?h on three sets of nanostructures; quartz nanopillars (QNPs), silicon nanopillars and silicon nanowire (SiNW) arrays, along with planar glass substrates. We found that cells on QNP arrays developed a longer shape than those on SiNW arrays. In addition, we studied how cell morphologies influence the cell-capture yield on the three sets of nanostructures. This research showed that the filopodial formations were directing the cell-capture yield on nanostructured substrates. This finding implies the possibility of using nanoscale topography features to control the filopodial formation including extension and migration from the cells. Using streptavidin-functionalized SiNW substrate, we further demonstrated a substantially higher yield (～91.8?±?5.9%) than the planar glass wafers (～24.1?±?7.5%) in the range of 200-3000 cells.     

A Combinatorial Library of Micro‐Topographies and Chemical Compositions for Tailored Surface Wettability

Surface modification of topography and chemistry in order to achieve a specific water contact angle (CA) has been explored by using a novel combinatorial screening platform. The screening arrays consisted of 507 distinct combinations of micro‐topographies and chemical compositions. By performing chemical modifications with 1H, 1H, 2H, 2H perfluoroethyltriethoxy‐silane (PFS) and n‐octadecyltriethoxysilane (ODS) on standard silicon wafers it was possible to include both superhydrophobic and very hydrophilic pad arrays in the same screening platform. Surfaces modified with PFS were more hydrophobic than surfaces modified with ODS, while the unmodified silicon surfaces were hydrophilic. For the PFS modified surfaces the largest CAs were achieved with a small pillar size of X = 1 µm and an intermediate inter‐pillar gap size of Y = 4 µm with superhydrophobic CAs over 170°. Surface analysis with X‐ray photoelectron spectroscopy (XPS) revealed that CF₃ groups were present at the surface, contributing to the superhydrophobic effect. The ODS modified surfaces had intermediate wettabilities with CAs between 100 and 150°, which were dependent on the pillar size, the inter‐pillar gap size, and the specific pillar pattern. The unmodified silicon topographical surfaces were very hydrophilic with CAs below 20° independent of specific topography. With this approach we have managed to fabricate 507 distinct surface areas covering a range of wettabilities, which is useful when screening these effects in several different applications. The measured CAs did not follow the simple Wenzel model. Furthermore, the adaptation of the Cassie model introduces Φ_s, the fraction of solid surface in contact with the liquid, which is difficult to estimate, thereby emphasizing the need for an experimental determination.     

Photographing impact of plasma-sprayed particles on rough substrates

Plasma-sprayed nickel and molybdenum particles (∼55 μm diameter) were photographed during spreading on silicon wafers that were patterned with micron-sized columns to make a textured rough surface. Impact on grit-blasted glass was also studied. The surfaces were maintained at either room temperature or at 350 °C. As the droplets approached the surface they were sensed by a photodetector and, after a known delay, a fast charge-coupled device (CCD) camera was triggered to capture time-integrated images of the spreading splat. A rapid two-color pyrometer was used to collect the thermal radiation from the spreading particles to record the evolution of their temperature and calculate splat cooling rates. It was found that micron-sized columns on the textured surfaces impeded fluid flow during spreading of splats, promoting splashing. When the column height was on the order of the splat thickness, increasing the space between each column increased the splat cooling rate as the columns penetrated into the liquid splat, providing a larger surface area for heat transfer. On the grit-blasted glass surfaces it was found that as the surface roughness increased, the maximum spread diameters of the molten droplets decreased, while the splat cooling rates increased. Impact on non-heated and heated roughened glass with similar roughness values produced splats with approximately the same maximum spread diameters, skewed morphologies, and cooling rates. On smooth glass, the splat morphologies were circular, with large maximum spread diameters and smaller cooling rates on non-heated smooth glass. An established model was used to estimate the splat-substrate thermal contact resistances. On highly roughened glass, the thermal contact resistance decreased as the glass roughness increased, suggesting that splat-substrate contact was improved as the molten metal penetrated the spaces between the large asperities.     

Microscale Electro‐Hydrodynamic Cell Printing with High Viability

Cell printing has gained extensive attentions for the controlled fabrication of living cellular constructs in vitro. Various cell printing techniques are now being explored and developed for improved cell viability and printing resolution. Here an electro‐hydrodynamic cell printing strategy is developed with microscale resolution (<100 µm) and high cellular viability (>95%). Unlike the existing electro‐hydrodynamic cell jetting or printing explorations, insulating substrate is used to replace conventional semiconductive substrate as the collecting surface which significantly reduces the electrical current in the electro‐hydrodynamic printing process from milliamperes (>0.5 mA) to microamperes (<10 µA). Additionally, the nozzle‐to‐collector distance is fixed as small as 100 µm for better control over filament deposition. These features ensure high cellular viability and normal postproliferative capability of the electro‐hydrodynamically printed cells. The smallest width of the electro‐hydrodynamically printed hydrogel filament is 82.4 ± 14.3 µm by optimizing process parameters. Multiple hydrogels or multilayer cell‐laden constructs can be flexibly printed under cell‐friendly conditions. The printed cells in multilayer hydrogels kept alive and gradually spread during 7‐days culture in vitro. This exploration offers a novel and promising cell printing strategy which might benefit future biomedical innovations such as microscale tissue engineering, organ‐on‐a‐chip systems, and nanomedicine.     

Drop‐on‐Demand Inkjet Printing of Thermally Tunable Liquid Crystal Microlenses

In this letter, the authors demonstrate Drop‐on‐Demand printing of variable focus, polarization‐independent, liquid crystal (LC) microlenses. By carefully selecting the surface treatment applied to a glass substrate, the authors are able to deposit droplets with a well‐defined curvature and contact angle, which result in micron‐sized lenses with focal lengths on the order of 300–900 µm. Observations with an optical polarizing microscope confirm the homeotopic alignment of the LC director in the droplets, which is in accordance with the polarization independent focal length. Results show that microlenses of different focal lengths can be fabricated by depositing successive droplets onto the same location on the substrate, which can then be used to build up programmable and arbitrary arrays of microlenses of various lens sizes and focal lengths. Finally, the authors utilize the thermal dependency of the order parameter of the LC to demonstrate facile tuning of the focal length. This technique has the potential to offer a low‐cost solution to the production of variable focus, arbitrary, microlens arrays.

     

Moving Water Droplets Over Nanoscaled (Super) hydrophobic Wettability Contrasts: Experimental Test of a Simple Model Describing Driving Forces

Applying advanced nanolithography techniques, various arrays of nanopillars on top of Si‐wafers are fabricated with all geometric parameters on the nanoscale. Additional chemical functionalization together with control over areal pillar density, height, and diameter allows the preparation of superhydrophobic surfaces exhibiting a wide range of contact angles (CA). Further improvement of this approach enables the production of step‐like wettability contrasts involving various CB–CB (Cassie‐Baxter) and CB–S (Smooth substrate)‐transitions. Such samples in combination with a high‐speed camera allow studying under optimized conditions quantitatively additional driving forces acting on a water droplet due to CA gradients. Experimentally it turns out that the maximum driving force on the droplet is well predicted by a simple model assuming circularly‐shaped base lines during the passage of a step‐like gradient of wettability. The provided study permits a comparison between maximum retention forces when tilting the substrate up to a critical angle and the presently determines maximum driving forces acting on a droplet due to a step‐like CA gradient. Both situations can be nicely described by a joint linear relation between normalized forces and CA hysteresis values with a slope close to theoretical values.     

Surface second-harmonic generation from vertical GaP nanopillars

We report on the experimental observation and analysis of second-harmonic generation (SHG) from vertical GaP nanopillars. Periodic arrays of GaP nanopillars with varying diameters ranging from 100 to 250 nm were fabricated on (100) undoped GaP substrate by nanosphere lithography and dry etching. We observed a strong dependence of the SHG intensity on pillar diameter. Analysis of surface and bulk contributions to SHG from the pillars including the calculations of the electric field profiles and coupling efficiencies is in very good agreement with the experimental data. Complementary measurements of surface optical phonons by Raman spectroscopy are also in agreement with the calculated field intensities at the surface. Finally, polarization of the measured light is used to distinguish between the bulk and surface SHG from GaP nanopillars.     

Rapid,Controllable Fabrication of Regular Complex Microarchitectures by Capillary Assembly of Micropillars and Their Application in Selectively Trapping/Releasing Microparticles

A simple strategy to realize new controllable 3D microstructures and a novel method to reversibly trapping and releasing microparticles are reported. This technique controls the height, shape, width, and arrangement of pillar arrays and realizes a series of special microstructures from 2‐pillar‐cell to 12 cell arrays, S‐shape, chain‐shape and triangle 3‐cell arrays by a combined top down/bottom up method: laser interference lithography and capillary force‐induced assembly. Due to the inherent features of this method, the whole time is less than 3 min and the fabricated area determined by the size of the laser beam can reach as much as 1 cm², which shows this method is very simple, rapid, and high‐throughput. It is further demonstrated that the ‘mechanical hand’‐like 4‐cell arrays could be used to selectively trap/release microparticles with different sizes, e.g., 1.5, 2, or 3.5 μm, which are controlled by the period of the microstructures from 2.5 to 4 μm, and 6 μm. Finally, the ‘mechanical hand’‐like 4‐cell arrays are integrated into 100 μm‐width microfluidic channels prepared by ultraviolet photolithography, which shows that this technique is compatible with conventional microfabrication methods for on‐chip applications.     

Fibroblasts Cultured on Nanowires Exhibit Low Motility,Impaired Cell Division,and DNA Damage

Nanowires are commonly used as tools for interfacing living cells, acting as biomolecule‐delivery vectors or electrodes. It is generally assumed that the small size of the nanowires ensures a minimal cellular perturbation, yet the effects of nanowires on cell migration and proliferation remain largely unknown. Fibroblast behaviour on vertical nanowire arrays is investigated, and it is shown that cell motility and proliferation rate are reduced on nanowires. Fibroblasts cultured on long nanowires exhibit failed cell division, DNA damage, increased ROS content and respiration. Using focused ion beam milling and scanning electron microscopy, highly curved but intact nuclear membranes are observed, showing no direct contact between the nanowires and the DNA. The nanowires possibly induce cellular stress and high respiration rates, which trigger the formation of ROS, which in turn results in DNA damage. These results are important guidelines to the design and interpretation of experiments involving nanowire‐based transfection and electrical characterization of living cells.     

Nanostructured gold microelectrodes for extracellular recording from electrogenic cells

We present a new biocompatible nanostructured microelectrode array for extracellular signal recording from electrogenic cells. Microfabrication techniques were combined with a template-assisted approach using nanoporous aluminum oxide to develop gold nanopillar electrodes. The nanopillars were approximately 300-400 nm high and had a diameter of 60 nm. Thus, they yielded a higher surface area of the electrodes resulting in a decreased impedance compared to planar electrodes. The interaction between the large-scale gold nanopillar arrays and cardiac muscle cells (HL-1) was investigated via focused ion beam milling. In the resulting cross-sections we observed a tight coupling between the HL-1 cells and the gold nanostructures. However, the cell membranes did not bend into the cleft between adjacent nanopillars due to the high pillar density. We performed extracellular potential recordings from HL-1 cells with the nanostructured microelectrode arrays. The maximal amplitudes recorded with the nanopillar electrodes were up to 100% higher than those recorded with planar gold electrodes. Increasing the aspect ratio of the gold nanopillars and changing the geometrical layout can further enhance the signal quality in the future.     

Bottom-up photonic crystal lasers

The directed growth of III-V nanopillars is used to demonstrate bottom-up photonic crystal lasers. Simultaneous formation of both the photonic band gap and active gain region is achieved via catalyst-free selective-area metal-organic chemical vapor deposition on masked GaAs substrates. The nanopillars implement a GaAs/InGaAs/GaAs axial double heterostructure for accurate, arbitrary placement of gain within the cavity and lateral InGaP shells to reduce surface recombination. The lasers operate single-mode at room temperature with low threshold peak power density of ～625 W/cm2. Cavity resonance and lasing wavelength is lithographically defined by controlling pillar pitch and diameter to vary from 960 to 989 nm. We envision this bottom-up approach to pillar-based devices as a new platform for photonic systems integration.     

3D,Reconfigurable, Multimodal Electronic Whiskers via Directed Air Assembly

A batch‐assembly technique for forming 3D electronics on shape memory polymer substrates is demonstrated and is used to create dense, highly sensitive, multimodal arrays of electronic whiskers. Directed air flow at temperatures above the substrate's glass transition temperature transforms planar photolithographically defined resistive sensors from 2D precursors into shape‐tunable, deterministic 3D assemblies. Reversible 3D assembly and flattening is achieved by exploiting the shape memory properties of the substrate, enabling context‐driven shape reconfiguration to isolate/enhance specific sensing modes. In particular, measurement schemes and device configurations are introduced that allow for the sensing of temperature, stiffness, contact force, proximity, and surface texture and roughness. The assemblies offer highly spatiotemporally resolved, wide‐range measurements of surface topology (50 nm to 500 µm), material stiffness (200 kPa to 7.5 GPa), and temperature (0–100 °C), with response times of <250 µs. The development of a scalable process for 3D assembly of reconfigurable electronic sensors, as well as the large breadth and sensitivity of complex sensing modes demonstrated, has applications in the growing fields of 3D assembly, electronic skin, and human–machine interfaces.     

Optical,Thermal, and Mechanical Characterization of Ga2Se3‐Added GLS Glass

Gallium lanthanum sulfide glass (GLS) has been widely studied in the last 40 years for middle‐infrared applications. In this work, the results of the substitution of selenium for sulphur in GLS glass are described. The samples are prepared via melt‐quench method in an argon‐purged atmosphere. A wide range of compositional substitutions are studied to define the glass‐forming region of the modified material. The complete substitution of Ga₂S₃ by Ga₂Se₃ is achieved by involving new higher quenching rate techniques compared to those containing only sulfides. The samples exhibiting glassy characteristics are further characterized. In particular, the optical and thermal properties of the sample are investigated in order to understand the role of selenium in the formation of the glass. The addition of selenium to GLS glass generally results in a lower glass transition temperature and an extended transmission window. Particularly, the IR edge is found to be extended from about 9 µm for GLS glass to about 15 µm for Se‐added GLS glass defined by the 50% transmission point. Furthermore, the addition of selenium does not affect the UV edge dramatically. The role of selenium is hypothesized in the glass formation to explain these changes.     

The structure of Ta nanopillars grown by glancing angle deposition

Regular arrays of Ta nanopillars, 200 nm wide and 500 nm tall, were grown on SiO₂ nanosphere patterns by glancing angle sputter deposition (GLAD). Plan-view and cross-sectional scanning electron microscopy analyses show dramatic changes in the structure and morphology of individual nanopillars as a function of growth temperature T_s ranging from 200 to 700 °C. At low temperatures, T_s ≤ 300 °C, single nanopillars develop on each sphere and branch into subpillars near the pillar top. In contrast, T_s ≥ 500 °C leads to branching during the nucleation stage at the pillar bottom. The top branching at low T_s is associated with surface mounds on a growing pillar that, due to atomic shadowing, develop into separated subpillars. At high T_s, the branching occurs during the nucleation stage where multiple nuclei on a single SiO₂ sphere develop into subpillars during a competitive growth mode which, in turn, leads to intercolumnar competition and the extinction of some nanopillars.     

Microfluidic Contact Lenses

Contact lens is a ubiquitous technology used for vision correction and cosmetics. Sensing in contact lenses has emerged as a potential platform for minimally invasive point‐of‐care diagnostics. Here, a microlithography method is developed to fabricate microconcavities and microchannels in a hydrogel‐based contact lens via a combination of laser patterning and embedded templating. Optical microlithography parameters influencing the formation of microconcavities including ablation power (4.3 W) and beam speed (50 mm s^?1) are optimized to control the microconcavity depth (100 µm) and diameter (1.5 mm). The fiber templating method allows the production of microchannels having a diameter range of 100–150 µm. Leak‐proof microchannel and microconcavity connections in contact lenses are validated through flow testing of artificial tear containing fluorescent microbeads (Ø = 1–2 µm). The microconcavities of contact lenses are functionalized with multiplexed fluorophores (2 µL) to demonstrate optical excitation and emission capability within the visible spectrum. The fabricated microfluidic contact lenses may have applications in ophthalmic monitoring of metabolic disorders at point‐of‐care settings and controlled drug release for therapeutics.     

Configurable Low‐Cost Plotter Device for Fabrication of Multi‐Color Sub‐Cellular Scale Microarrays

The construction and operation of a low‐cost plotter for fabrication of microarrays for multiplexed single‐cell analyses is reported. The printing head consists of polymeric pyramidal pens mounted on a rotation stage installed on an aluminium frame. This construction enables printing of microarrays onto glass substrates mounted on a tilt stage, controlled by a Lab‐View operated user interface. The plotter can be assembled by typical academic workshops from components of less than 15 000 Euro. The functionality of the instrument is demonstrated by printing DNA microarrays on the area of 0.5 cm² using up to three different oligonucleotides. Typical feature sizes are 5 μm diameter with a pitch of 15 μm, leading to densities of up to 10⁴–10⁵ spots/mm². The fabricated DNA microarrays are used to produce sub‐cellular scale arrays of bioactive epidermal growth factor peptides by means of DNA‐directed immobilization. The suitability of these biochips for cell biological studies is demonstrated by specific recruitment, concentration, and activation of EGF receptors within the plasma membrane of adherent living cells. This work illustrates that the presented plotter gives access to bio‐functionalized arrays usable for fundamental research in cell biology, such as the manipulation of signal pathways in living cells at subcellular resolution.     

Electrohydrodynamic Printing of Microfibrous Architectures with Cell-Scale Spacing for Improved Cellular Migration and Neurite Outgrowth

Electrohydrodynamic (EHD) printing provides unparalleled opportunities in fabricating microfibrous architectures to direct cellular orientation. However, it faces great challenges in depositing orderly microfibers with cell-scale spacing due to inherent fiber–fiber electrostatic interactions. Here a finite element method is established to analyze the electrostatic forces induced on the EHD-printed microfibers and the relationship between the fiber diameter and spacing for parallel deposition of EHD-printed microfibers is revealed theoretically and experimentally. It is found that uniform fiber arrangement can be achieved when the fiber spacing is five times larger than the fiber diameter. This finding enables the successful printing of parallel fibrous architectures with a fiber diameter of 4.9 ± 0.1 µm and a cell-scale fiber spacing of 25.6 ± 1.9 µm. The resultant microfibrous architectures exhibit unique capability to direct cellular alignment and enhance cellular density and migration as the fiber spacing decreases from 100 to 25 µm. The EHD-printed parallel microfibers with cell-scale spacing are found to improve the outgrowth length of neurites and accelerate the migration of Schwann cells from Dorsal Root Ganglion spheres, which facilitate the formation of densely-arranged and highly-aligned cellular constructs. The presented method is promising to produce biomimetic microfibrous architectures for functional nerve regeneration.     

Dimension‐Controllable Microtube Arrays by Dynamic Holographic Processing as 3D Yeast Culture Scaffolds for Asymmetrical Growth Regulation

Transparent microtubes can function as unique cell culture scaffolds, because the tubular 3D microenvironment they provide is very similar to the narrow space of capillaries in vivo. However, how to realize the fabrication of microtube‐arrays with variable cross‐section dynamically remains challenging. Here, a dynamic holographic processing method for producing high aspect ratio (≈20) microtubes with tunable outside diameter (6–16 µm) and inside diameter (1–10 µm) as yeast culture scaffolds is reported. A ring‐structure Bessel beam is modulated from a typical Gaussian‐distributed femtosecond laser beam by a spatial light modulator. By combining the axial scanning of the focused beam and the dynamic display of holograms, dimension‐controllable microtube arrays (straight, conical, and drum‐shape) are rapidly produced by two‐photon polymerization. The outside and inside diameters, tube heights, and spatial arrangements are readily tuned by loading different computer‐generated holograms and changing the processing parameters. The transparent microtube array as a nontrivial tool for capturing and culturing the budding yeasts reveals the significant effect of tube diameter on budding characteristics. In particular, the conical tube with the inside diameter varying from 5 to 10 µm has remarkable asymmetrical regulation on the growth trend of captured yeasts.     

Fabrication of Hierarchical Pillar Arrays from Thermoplastic and Photosensitive SU‐8

By exploiting the thermoplastic and photosensitive nature of SU‐8 photoresists, different types of hierarchical pillar arrays with variable aspect ratios are fabricated through capillary force lithography (CFL), followed by photopatterning. The thermoplastic nature of SU‐8 enables the imprinting of micropillar arrays with variable aspect ratios by CFL using a single poly(dimethylsiloxane) mold, simply by tuning the initial film thickness of SU‐8 on a substrate. The pillar array is subsequently photopatterned through a photomask, followed by post‐exposure baking above the glass transition temperature (T_g) of SU‐8. The pillars in the exposed region become highly crosslinked and, therefore, neither soluble nor able to reflow above T_g, whereas the pillars in the unexposed regions can reflow and flatten out. Two developing strategies are investigated after UV exposure of the SU‐8 pillar arrays including i) solvent development and drying and ii) thermal reflow to create bilevel hierarchical structures with short pillars and single‐level, dual‐scaled, high‐aspect‐ratio (up to 7.7) pillars in a microdot array, respectively.     

Dry Etching with Nanoparticles: Formation of High Aspect‐Ratio Pores and Channels Using Magnetic Gold Nanoclusters

Methods for generating nanopores in substrates typically involve one or more wet‐etching steps. Here a fundamentally different approach to produce nanopores in sheet substrates under dry, ambient conditions, using nanosecond‐pulsed laser irradiation and magnetic gold nanoclusters (MGNCs) as the etching agents is described. Thermoplastic films (50–75 µm thickness) are coated with MGNCs then exposed to laser pulses with a coaxial magnetic field gradient, resulting in high‐aspect ratio channels with tapered cross sections as characterized by confocal fluorescence tomography. The dry‐etching process is applicable to a wide variety of substrates ranging from fluoropolymers to borosilicate glass, with etch rates in excess of 1 µm s^–1. Finite‐element modeling suggests that the absorption of laser pulses by MGNCs can produce temperature spikes of nearly 1000 °C, which is sufficient for generating photoacoustic responses that can drive particles into the medium, guided by magnetomotive force.