Analyzing the Micro-topography Guidance of Dictyostelium Cells Using Microfabricated Structures.

Over the last decade, the use of microfabricated substrates has proven pivotal for studying the effect of substrate topography on cell deformation and migration. Microfabrication techniques allow one to construct a transparent substrate with topographic features with high designability and reproducibility and thus well suited to experiments that microscopically address how spatial and directional bias are brought about in the cytoskeletal machineries and hence cell motility. While much of the progress in this avenue of study has so far been made in adhesive cells of epithelial and mesenchymal nature, whether related phenomena exist in less adhesive fast migrating cells is relatively unknown. In this chapter, we describe a method that makes use of micrometer-scale ridges to study fast-migrating Dictyostelium cells where it was recently shown that membrane evagination associated with macropinocytic cup formation plays a pivotal role in the topography sensing. The method requires only basic photolithography, and thus the step-by-step protocol should be a good entry point for cell biologists looking to incorporate similar microfabrication approaches.

Fly Me to the Micron: Microtechnologies for Drosophila Research.

Multicellular model organisms, such as Drosophila melanogaster (fruit fly), are frequently used in a myriad of biological research studies due to their biological significance and global standardization. However, traditional tools used in these studies generally require manual handling, subjective phenotyping, and bulk treatment of the organisms, resulting in laborious experimental protocols with limited accuracy. Advancements in microtechnology over the course of the last two decades have allowed researchers to develop automated, high-throughput, and multifunctional experimental tools that enable novel experimental paradigms that would not be possible otherwise. We discuss recent advances in microtechnological systems developed for small model organisms using D. melanogaster as an example. We critically analyze the state of the field by comparing the systems produced for different applications. Additionally, we suggest design guidelines, operational tips, and new research directions based on the technical and knowledge gaps in the literature. This review aims to foster interdisciplinary work by helping engineers to familiarize themselves with model organisms while presenting the most recent advances in microengineering strategies to biologists.

Mechanical Strain Induces and Increases Vesicular Release Monitored by Microfabricated Stretchable Electrodes.

Exocytosis involving the fusion of intracellular vesicles with cell membrane, is thought to be modulated by the mechanical cues in the microenvironment. Single-cell electrochemistry can offer unique information about the quantification and kinetics of exocytotic events; however, the effects of mechanical force on vesicular release have been poorly explored. Herein, we developed a stretchable microelectrode with excellent electrochemical stability under mechanical deformation by microfabrication of functionalized poly(3,4-ethylenedioxythiophene) conductive ink, which achieved real-time quantitation of strain-induced vesicular exocytosis from a single cell for the first time. We found that mechanical strain could cause calcium influx via the activation of Piezo1 channels in chromaffin cell, initiating the vesicular exocytosis process. Interestingly, mechanical strain increases the amount of catecholamines released by accelerating the opening and prolonging the closing of fusion pore during exocytosis. This work is expected to provide revealing insights into the regulatory effects of mechanical stimuli on vesicular exocytosis.

Roadmap for Clinical Translation of Mobile Microrobotics.

Medical microrobotics is an emerging field to revolutionize clinical applications in diagnostics and therapeutics of various diseases. On the other hand, the mobile microrobotics field has important obstacles to pass before clinical translation. This article focuses on these challenges and provides a roadmap of medical microrobots to enable their clinical use. From the concept of a "magic bullet" to the physicochemical interactions of microrobots in complex biological environments in medical applications, there are several translational steps to consider. Clinical translation of mobile microrobots is only possible with a close collaboration between clinical experts and microrobotics researchers to address the technical challenges in microfabrication, safety, and imaging. The clinical application potential can be materialized by designing microrobots that can solve the current main challenges, such as actuation limitations, material stability, and imaging constraints. The strengths and weaknesses of the current progress in the microrobotics field are discussed and a roadmap for their clinical applications in the near future is outlined.

High-aspect-ratio three-dimensional polymer and metallic microstructure microfabrication using two-photon polymerization.

Creating micrometer-resolution high-aspect-ratio three-dimensional (3D) structures remain very challenging despite significant microfabrication methods developed for microelectromechanical systems (MEMS). This is especially the case when such structures are desired to be metallic to support electronic applications. Here, we present a microfabrication process that combines two-photon-polymerization (2PP) printing to create a polymeric high-aspect-ratio three-dimensional structure and electroless metal plating that selectively electroplates only the polymeric structure to create high-aspect-ratio 3D metallic structures having micrometer-resolution. To enable this, the effect of various 2PP processing parameters on SU-8 photoresist microstructures were first systematically studied. These parameters include laser power, slicing/hatching distances, and pre-/post-baking temperature. This optimization resulted in a maximum aspect ratio (height to width) of ~ 12. Following this polymeric structure printing, electroless plating using Tollens' Reagent were utilized to selectively coat silver particles only on the polymeric structure, but not on the silicon substrate. The final 3D metallic structures were evaluated in terms of their resistivity, reproducibly showing resistivity of ~ 10-6 [Ω·m]. The developed 3D metallic structure microfabrication process can be further integrated with conventional 2D lithography to achieve even more complex structures. The developed method overcomes the limitations of current MEMS fabrication processes, allowing a variety of previously impossible metallic microstructures to be created.

Manufacture of Three-Dimensional Optofluidic Spot-Size Converters in Fused Silica Using Hybrid Laser Microfabrication.

We propose a hybrid laser microfabrication approach for the manufacture of three-dimensional (3D) optofluidic spot-size converters in fused silica glass by a combination of femtosecond (fs) laser microfabrication and carbon dioxide laser irradiation. Spatially shaped fs laser-assisted chemical etching was first performed to form 3D hollow microchannels in glass, which were composed of embedded straight channels, tapered channels, and vertical channels connected to the glass surface. Then, carbon dioxide laser-induced thermal reflow was carried out for the internal polishing of the whole microchannels and sealing parts of the vertical channels. Finally, 3D optofluidic spot-size converters (SSC) were formed by filling a liquid-core waveguide solution into laser-polished microchannels. With a fabricated SSC structure, the mode spot size of the optofluidic waveguide was expanded from ~8 µm to ~23 µm with a conversion efficiency of ~84.1%. Further measurement of the waveguide-to-waveguide coupling devices in the glass showed that the total insertion loss of two symmetric SSC structures through two ~50 µm-diameter coupling ports was ~6.73 dB at 1310 nm, which was only about half that of non-SSC structures with diameters of ~9 µm at the same coupling distance. The proposed approach holds great potential for developing novel 3D fluid-based photonic devices for mode conversion, optical manipulation, and lab-on-a-chip sensing.

3D-printed microrobots from design to translation.

Microrobots have attracted the attention of scientists owing to their unique features to accomplish tasks in hard-to-reach sites in the human body. Microrobots can be precisely actuated and maneuvered individually or in a swarm for cargo delivery, sampling, surgery, and imaging applications. In addition, microrobots have found applications in the environmental sector (e.g., water treatment). Besides, recent advancements of three-dimensional (3D) printers have enabled the high-resolution fabrication of microrobots with a faster design-production turnaround time for users with limited micromanufacturing skills. Here, the latest end applications of 3D printed microrobots are reviewed (ranging from environmental to biomedical applications) along with a brief discussion over the feasible actuation methods (e.g., on- and off-board), and practical 3D printing technologies for microrobot fabrication. In addition, as a future perspective, we discussed the potential advantages of integration of microrobots with smart materials, and conceivable benefits of implementation of artificial intelligence (AI), as well as physical intelligence (PI). Moreover, in order to facilitate bench-to-bedside translation of microrobots, current challenges impeding clinical translation of microrobots are elaborated, including entry obstacles (e.g., immune system attacks) and cumbersome standard test procedures to ensure biocompatibility.

Micromolding of Thermoplastic Polymers for Direct Fabrication of Discrete, Multilayered Microparticles.

Soft lithography provides a convenient and effective method for the fabrication of microdevices with uniform size and shape. However, formation of an embossed, connective film as opposed to discrete features has been an enduring shortcoming associated with soft lithography. Removing this residual layer requires additional postprocessing steps that are often incompatible with organic materials. This limits adaptation and widespread realization of soft lithography for broader applications particularly in drug discovery and drug delivery fields. A novel and versatile approach is demonstrated that enables fabrication of discrete, multilayered, fillable, and harvestable microparticles directly from any thermoplastic polymer, even at very high molecular weights. The approach, isolated microparticle replication via surface-segregating polymer blend mold, utilizes a random copolymer additive, designed with a highly fluorinated segment that, when blended with the mold's matrix, spontaneously orients to the surface conferring an extremely low surface energy and nonwetting properties to the template. The extremely nonwetting properties of the mold are further utilized to load soluble biologics directly into the built-in microwells in a rapid and efficient manner using an innovative screen-printing approach. It is believed that this approach holds promise for fabrication of large-array, 3D, complex microstructures, and is a significant step toward clinical translation of microfabrication technologies.

Live single cell imaging assays in glass microwells produced by laser-induced deep etching.

Miniaturization of cell culture substrates enables controlled analysis of living cells in confined micro-scale environments. This is particularly suitable for imaging individual cells over time, as they can be monitored without escaping the imaging field-of-view (FoV). Glass materials are ideal for most microscopy applications. However, with current methods used in life sciences, glass microfabrication is limited in terms of either freedom of design, quality, or throughput. In this work, we introduce laser-induced deep etching (LIDE) as a method for producing glass microwell arrays for live single cell imaging assays. We demonstrate novel microwell arrays with deep, high-aspect ratio wells that have rounded, dimpled or flat bottom profiles in either single-layer or double-layer glass chips. The microwells are evaluated for microscopy-based analysis of long-term cell culture, clonal expansion, laterally organized cell seeding, subcellular mechanics during migration and immune cell cytotoxicity assays of both adherent and suspension cells. It is shown that all types of microwells can support viable cell cultures and imaging with single cell resolution, and we highlight specific benefits of each microwell design for different applications. We believe that high-quality glass microwell arrays enabled by LIDE provide a great option for high-content and high-resolution imaging-based live cell assays with a broad range of potential applications within life sciences.

Microfabrication of polymer microneedle arrays using two-photon polymerization.

Three dimensional (3D) printing technology has pushed state-of-the-art manufacturing towards more advanced processing methods through its ability to produce complex computer-designed 3D structures in a wide range of materials. Two-photon polymerization applied to the fabrication of ultraprecise 3D microstructures is one of the various innovative approaches to cutting-edge 3D printing. The integration of an ultrashort pulsed laser source and an appropriate photoresist has made it an attractive candidate for advanced photonics and biomedical applications. This paper presents the development of 3D solid microneedle arrays as a novel transdermal drug delivery system via two-photon polymerization in a single manufacturing step. Through a series of experiments, the best fabrication parameters are identified. Finite element simulations are then performed to investigate the interaction between a single microneedle and human skin. The results of this study highlight the influence of fabrication parameters such as laser power, scanning speed, hatch distance and layer height on the structural resolution and fabrication time of microneedles, as well as human skin deformation caused through application of force to a single polymer microneedle.

A Primer on Microfluidics: From Basic Principles to Microfabrication.

Microfluidic systems enable manipulating fluids in different functional units which are integrated on a microchip. This chapter describes the basics of microfluidics, where physical effects have a different impact compared to macroscopic systems. Furthermore, an overwiew is given on the microfabrication of these systems. The focus lies on clean-room fabrication methods based on photolithography and soft lithography. Finally, an outlook on advanced maskless micro- and nanofabrication methods is given. Special attention is paid to laser structuring processes.

Fabrication and use of silicon hollow-needle arrays to achieve tissue nanotransfection in mouse tissue in vivo.

Tissue nanotransfection (TNT) is an electromotive gene transfer technology that was developed to achieve tissue reprogramming in vivo. This protocol describes how to fabricate the required hardware, commonly referred to as a TNT chip, and use it for in vivo TNT. Silicon hollow-needle arrays for TNT applications are fabricated in a standardized and reproducible way. In <1 s, these silicon hollow-needle arrays can be used to deliver plasmids to a predetermined specific depth in murine skin in response to pulsed nanoporation. Tissue nanotransfection eliminates the need to use viral vectors, minimizing the risk of genomic integration or cell transformation. The TNT chip fabrication process typically takes 5-6 d, and in vivo TNT takes 30 min. This protocol does not require specific expertise beyond a clean room equipped for basic nanofabrication processes.

Application of armodafinil-loaded microneedle patches against the negative influence induced by sleep deprivation.

Cognition maintenance is essential for healthy and safe life if sleep deprivation happens. Armodafinil is a wake-promoting agent against sleep deprivation related disorders. However, only the tablet formulation is available, which may limit its potential in some circumstances. Here, we report the synthesis of a new formulation of armodafinil, microneedle patches, which can be conveniently used by any individual and removed in time if not wanted. To produce the needles of higher mechanical strength and higher drug loading, polyvinylpyrrolidone (PVP) K90 was used to fabricate armodafinil-loaded microneedles by applying the mold casting method after dissolving in methanol and drying. The higher mechanical strength was validated by COMSOL Multiphysics® software stimulation and universal mechanical testing machines. The obtained armodafinil microneedles can withstand a force of 70 N and penetrate the skin to a depth of 230 µm, and quickly released the drug within 1.5 h in vitro. The pharmacokinetic analysis showed that microneedle administration can maintain a more lasting and stable blood concentration as compared to oral administration. After the treatment of sleep deprived mice with microneedles, the in vivo pharmacodynamics study clearly demonstrated that armodafinil microneedles could eliminate the effects of sleep deprivation and improve the cognitive functions of sleep-deprived mice. A self-administered, high drug-loaded microneedle patch were prepared successfully, which appeared to be highly promising in preserving cognition by transdermal administration.

Growth and site-specific organization of micron-scale biomolecular devices on living mammalian cells.

Mesoscale molecular assemblies on the cell surface, such as cilia and filopodia, integrate information, control transport and amplify signals. Designer cell-surface assemblies could control these cellular functions. Such assemblies could be constructed from synthetic components ex vivo, making it possible to form such structures using modern nanoscale self-assembly and fabrication techniques, and then oriented on the cell surface. Here we integrate synthetic devices, micron-scale DNA nanotubes, with mammalian cells by anchoring them by their ends to specific cell surface receptors. These filaments can measure shear stresses between 0-2 dyn/cm2, a regime important for cell signaling. Nanotubes can also grow while anchored to cells, thus acting as dynamic cell components. This approach to cell surface engineering, in which synthetic biomolecular assemblies are organized with existing cellular architecture, could make it possible to build new types of sensors, machines and scaffolds that can interface with, control and measure properties of cells.

Enzymatic/Magnetic Hybrid Micromotors for Synergistic Anticancer Therapy.

Micro/nanomotors (MNMs), which propel by transforming various forms of energy into kinetic energy, have emerged as promising therapeutic nanosystems in biomedical applications. However, most MNMs used for anticancer treatment are only powered by one engine or provide a single therapeutic strategy. Although double-engined micromotors for synergistic anticancer therapy can achieve more flexible movement and efficient treatment efficacy, their design remains challenging. In this study, we used a facile preparation method to develop enzymatic/magnetic micromotors for synergetic cancer treatment via chemotherapy and starvation therapy (ST), and the size of micromotors can be easily regulated during the synthetic process. The enzymatic reaction of glucose oxidase, which served as the chemical engine, led to self-propulsion using glucose as a fuel and ST via a reduction in the energy available to cancer cells. Moreover, the incorporation of Fe3O4 nanoparticles as a magnetic engine enhanced the kinetic power and provided control over the direction of movement. Inherent pH-responsive drug release behavior was observed owing to the acidic decomposition of drug carriers in the intracellular microenvironment of cancer cells. This system displayed enhanced anticancer efficacy owing to the synergetic therapeutic strategies and increased cellular uptake in a targeted area because of the improved motion behavior provided by the double engines. Therefore, the demonstrated micromotors are promising candidates for anticancer biomedical microsystems.

Stimuli-Responsive Lysozyme Nanocapsule Engineered Microfiltration Membranes with a Dual-Function of Anti-Adhesion and Antibacteria for Biofouling Mitigation.

Biofouling remains as a persistent problem impeding the applications of membranes for water and wastewater treatment. Green anti-biofouling of membranes made of natural and environmentally friendly materials and methods is a promising strategy to tackle this problem. Herein, we have developed a functionalized PVDF membrane with stimuli-responsive lysozyme nanocapsules (NCP). These nanocapsules can responsively release lysozyme according to environmental stimuli (pH and redox) induced by bacteria. Results showed that (i) the surface of the functionalized membrane with NCP had enhanced hydrophilicity, reduced roughness, and negative charge, (ii) a remarkable reduction of adsorption of proteins, polysaccharides, and bacteria was achieved by the functionalized membrane, and (iii) the colony forming unit (CFU) of bacteria on a membrane surface was reduced more than 80% within 24 h of contact. In addition, the NCP membrane showed excellent anti-biofouling activity regarding the bacterial viability being 12.5 and 8.3% on the membrane after filtration with 108 CFU mL-1 Escherichia coli and Staphylococcus aureus solution as feed, respectively. The coating layer and assembled nanocapsules endowed the membrane with improved lysozyme stability, anti-adhesion performance, and antibacterial activity. Stimuli-responsive lysozyme nanocapsule engineered microfiltration membranes show great potential for anti-biofouling in future practical application.

Rapid fabrication of hydrogel micropatterns by projection stereolithography for studying self-organized developmental patterning.

Self-organized patterning of mammalian embryonic stem cells on micropatterned surfaces has previously been established as an in vitro platform for early mammalian developmental studies, complimentary to in vivo studies. Traditional micropatterning methods, such as micro-contact printing (µCP), involve relatively complicated fabrication procedures, which restricts widespread adoption by biologists. Here, we demonstrate a rapid method of micropatterning by printing hydrogel micro-features onto a glass-bottomed culture vessel. The micro-features are printed using a projection stereolithography bioprinter yielding hydrogel structures that geometrically restrict the attachment of cells or proteins. Compared to traditional and physical photomasks, a digitally tunable virtual photomask is used in the projector to generate blue light patterns that enable rapid iteration with minimal cost and effort. We show that a protocol that makes use of this method together with LN521 coating, an extracellular matrix coating, creates a surface suitable for human embryonic stem cell (hESC) attachment and growth with minimal non-specific adhesion. We further demonstrate that self-patterning of hESCs following previously published gastrulation and ectodermal induction protocols achieves results comparable with those obtained with commercially available plates.

Fabricating and printing chemiresistors based on monolayer-capped metal nanoparticles.

Chemiresistors that are based on monolayer-capped metal nanoparticles (MCNPs) have been used in a wide variety of innovative sensing applications, including detection and monitoring of diagnostic markers in body fluids, explosive materials, environmental contaminations and food quality control. The sensing mechanism is based on reversible swelling or aggregation and/or changes in dielectric constant of the MCNPs. In this protocol, we describe a procedure for producing MCNP-based chemiresistive sensors that is reproducible from device to device and from batch to batch. The approach relies on three main steps: (i) controlled synthesis of gold MCNPs, (ii) fabrication of electrodes that are surrounded with a microbarrier ring to confine the deposited MCNP solution and (iii) a tailor-made drying process to enable evaporation of solvent residues from the MCNP sensing layer to prevent a coffee-ring effect. Application of this approach has been shown to produce devices with ±1.5% variance-a value consistent with the criterion for commercial sensors-as well as long shelf life and stability. Fabrication of chemical sensors based on dodecanethiol- or 2-ethylhexanethiol-capped MCNPs with this approach provides high sensitivity and accuracy in the detection of volatile organic compounds (e.g., octane and decane), toxic gaseous species (e.g., HCl and NH3) in air and simulated mixtures of lung and gastric cancer from exhaled breath.

Self-assembled microcage fabrication for manipulating and selectively capturing microparticles and cells.

Single-cell-scale selective manipulation and targeted capture play a vital role in cell behavior analysis. However, selective microcapture has primarily been performed in specific circumstances to maintain the trapping state, making the subsequent in situ characterization and analysis of specific particles or cells difficult and imprecise. Herein, we propose a novel method that combines femtosecond laser two-photon polymerization (TPP) micromachining technology with the operation of optical tweezers (OTs) to achieve selective and targeted capture of single particles and cells. Diverse ordered microcages with different shapes and dimensions were self-assembled by micropillars fabricated via TPP. The micropillars with high aspect ratios were processed by single exposure, and the parameters of the micropillar arrays were investigated to optimize the capillary-force-driven self-assembly process of the anisotropic microcages. Finally, single microparticles and cells were selectively transported to the desired microcages by manipulating the flexibly of the OTs in a few minutes. The captured microparticles and cells were kept trapped without additional forces.

Microtiter plate assays to assess antibiofilm activity against bacteria.

Bacterial biofilms demonstrate high broad-spectrum adaptive antibiotic resistance and cause two thirds of all infections, but there is a lack of approved antibiofilm agents. Unlike the standard minimal inhibitory concentration assay to assess antibacterial activity against planktonic cells, there is no standardized method to evaluate biofilm inhibition and/or eradication capacity of novel antibiofilm compounds. The protocol described here outlines simple and reproducible methods for assessing the biofilm inhibition and eradication capacities of novel antibiofilm agents against adherent bacterial biofilms grown in 96-well microtiter plates. It employs two inexpensive dyes: crystal violet to stain adhered biofilm biomass and 2,3,5-triphenyl tetrazolium chloride to quantify metabolism of the biofilm cells. The procedure is accessible to any laboratory with a plate reader, requires minimal technical expertise or training and takes 4 or 5 d to complete. Recommendations for how biofilm inhibition and eradication results should be interpreted and presented are also described.