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.

Silicon microfabrication technologies for biology integrated advance devices and interfaces.

Miniaturization is the trend to manufacture ever smaller devices and this process requires knowledge, experience, understanding of materials, manufacturing techniques and scaling laws. The fabrication techniques used in semiconductor industry deliver an exceptionally high yield of devices and provide a well-established platform. Today, these miniaturized devices are manufactured with high reproducibility, design flexibility, scalability and multiplexed features to be used in several applications including micro-, nano-fluidics, implantable chips, diagnostics/biosensors and neural probes. We here provide a review on the microfabricated devices used for biology driven science. We will describe the ubiquity of the use of micro-nanofabrication techniques in biology and biotechnology through the fabrication of high-aspect-ratio devices for cell sensing applications, intracellular devices, probes developed for neuroscience-neurotechnology and biosensing of the certain biomarkers. Recently, the research on micro and nanodevices for biology has been progressing rapidly. While the understanding of the unknown biological fields -such as human brain- has been requiring more research with advanced materials and devices, the development protocols of desired devices has been advancing in parallel, which finally meets with some of the requirements of biological sciences. This is a very exciting field and we aim to highlight the impact of micro-nanotechnologies that can shed light on complex biological questions and needs.

Vat photopolymerization 3D printed microfluidic devices for organ-on-a-chip applications.

Organs-on-a-chip, or OoCs, are microfluidic tissue culture devices with micro-scaled architectures that repeatedly achieve biomimicry of biological phenomena. They are well positioned to become the primary pre-clinical testing modality as they possess high translational value. Current methods of fabrication have facilitated the development of many custom OoCs that have generated promising results. However, the reliance on microfabrication and soft lithographic fabrication techniques has limited their prototyping turnover rate and scalability. Additive manufacturing, known commonly as 3D printing, shows promise to expedite this prototyping process, while also making fabrication easier and more reproducible. We briefly introduce common 3D printing modalities before identifying two sub-types of vat photopolymerization - stereolithography (SLA) and digital light processing (DLP) - as the most advantageous fabrication methods for the future of OoC development. We then outline the motivations for shifting to 3D printing, the requirements for 3D printed OoCs to be competitive with the current state of the art, and several considerations for achieving successful 3D printed OoC devices touching on design and fabrication techniques, including a survey of commercial and custom 3D printers and resins. In all, we aim to form a guide for the end-user to facilitate the in-house generation of 3D printed OoCs, along with the future translation of these important devices.

Construction of intelligent moving micro/nanomotors and their applications in biosensing and disease treatment.

Micro/nanomotors are containers that pass through liquid media and carry cargo. Because they are tiny, micro/nanomotors exhibit excellent potential for biosensing and disease treatment applications. However, their size also makes overcoming random Brownian forces very challenging for micro/nanomotors moving on targets. Additionally, to achieve desired practical applications, the expensive materials, short lifetimes, poor biocompatibility, complex preparation methods, and side effects of micro/nanomotors must be addressed, and potential adverse effects must be evaluated both in vivo and in practical applications. This has led to the continuous development of key materials for driving micro/nanomotors. In this work, we review the working principles of micro/nanomotors. Metallic and nonmetallic nanocomplexes, enzymes, and living cells are explored as key materials for driving micro/nanomotors. We also consider the effects of exogenous stimulations and endogenous substance conditions on micro/nanomotor motions. The discussion focuses on micro/nanomotor applications in biosensing, treating cancer and gynecological diseases, and assisted fertilization. By addressing micro/nanomotor shortcomings, we propose directions for further developing and applying micro/nanomotors.

Rapid prototyping of microfluidic chips enabling controlled biotechnology applications in microspace.

Rapid prototyping of microfluidic chips is a key enabler for controlled biotechnology applications in microspaces, as it allows for the efficient design and production of microfluidic systems. With rapid prototyping, researchers and engineers can quickly create and test new microfluidic chip designs, which can then be optimized for specific applications in biotechnology. One of the key advantages of microfluidic chips for biotechnology is the ability to manipulate and control biological samples in a microspace, which enables precise and controlled experiments under well-defined conditions. This is particularly useful for applications such as cell culture, drug discovery, and diagnostic assays, where precise control over the biological environment is crucial for obtaining accurate results. Established methods, for example, soft lithography, 3D printing, injection molding, as well as other recently highlighted innovative approaches, will be compared and challenges as well as limitations will be discussed. It will be shown that rapid prototyping of microfluidic chips enables the use of advanced materials and technologies, such as smart materials and digital sensors, which can further enhance the capabilities of microfluidic systems for biotechnology applications. Overall, rapid prototyping of microfluidic chips is an important enabling technology for controlled biotechnology applications in microspaces, as well as for upscaling it into macroscopic bioreactors, and its continued development and improvement will play a critical role in advancing the field. The review will highlight recent trends in terms of materials and competing approaches and shed light on current challenges on the way toward integrated microtechnologies. Also, the possibility to easy and direct implementation of novel functions (membranes, functionalization of interfaces, etc.) is discussed.

Multicellular dynamics on structured surfaces: Stress concentration is a key to controlling complex microtissue morphology on engineered scaffolds.

Tissue engineers have utilised a variety of three-dimensional (3D) scaffolds for controlling multicellular dynamics and the resulting tissue microstructures. In particular, cutting-edge microfabrication technologies, such as 3D bioprinting, provide increasingly complex structures. However, unpredictable microtissue detachment from scaffolds, which ruins desired tissue structures, is becoming an evident problem. To overcome this issue, we elucidated the mechanism underlying collective cellular detachment by combining a new computational simulation method with quantitative tissue-culture experiments. We first quantified the stochastic processes of cellular detachment shown by vascular smooth muscle cells on model curved scaffolds and found that microtissue morphologies vary drastically depending on cell contractility, substrate curvature, and cell-substrate adhesion strength. To explore this mechanism, we developed a new particle-based model that explicitly describes stochastic processes of multicellular dynamics, such as adhesion, rupture, and large deformation of microtissues on structured surfaces. Computational simulations using the developed model successfully reproduced characteristic detachment processes observed in experiments. Crucially, simulations revealed that cellular contractility-induced stress is locally concentrated at the cell-substrate interface, subsequently inducing a catastrophic process of collective cellular detachment, which can be suppressed by modulating cell contractility, substrate curvature, and cell-substrate adhesion. These results show that the developed computational method is useful for predicting engineered tissue dynamics as a platform for prediction-guided scaffold design. STATEMENT OF SIGNIFICANCE: Microfabrication technologies aiming to control multicellular dynamics by engineering 3D scaffolds are attracting increasing attention for modelling in cell biology and regenerative medicine. However, obtaining microtissues with the desired 3D structures is made considerably more difficult by microtissue detachments from scaffolds. This study reveals a key mechanism behind this detachment by developing a novel computational method for simulating multicellular dynamics on designed scaffolds. This method enabled us to predict microtissue dynamics on structured surfaces, based on cell mechanics, substrate geometry, and cell-substrate interaction. This study provides a platform for the physics-based design of micro-engineered scaffolds and thus contributes to prediction-guided biomaterials design in the future.

Bubble-Based Microrobots with Rapid Circular Motions for Epithelial Pinning and Drug Delivery.

Remotely powered microrobots are proposed as next-generation vehicles for drug delivery. However, most microrobots swim with linear trajectories and lack the capacity to robustly adhere to soft tissues. This limits their ability to navigate complex biological environments and sustainably release drugs at target sites. In this work, bubble-based microrobots with complex geometries are shown to efficiently swim with non-linear trajectories in a mouse bladder, robustly pin to the epithelium, and slowly release therapeutic drugs. The asymmetric fins on the exterior bodies of the microrobots induce a rapid rotational component to their swimming motions of up to ≈150 body lengths per second. Due to their fast speeds and sharp fins, the microrobots can mechanically pin themselves to the bladder epithelium and endure shear stresses commensurate with urination. Dexamethasone, a small molecule drug used for inflammatory diseases, is encapsulated within the polymeric bodies of the microrobots. The sustained release of the drug is shown to temper inflammation in a manner that surpasses the performance of free drug controls. This system provides a potential strategy to use microrobots to efficiently navigate large volumes, pin at soft tissue boundaries, and release drugs over several days for a range of diseases.

A Universal Microscopic Patterned Doping Method for Perovskite Enables Ultrafast, Self-Powered, Ultrasmall Perovskite Photodiodes.

Novel metal halide perovskite is proven to be a promising optoelectronic material. However, fabricating microscopic perovskite devices is still challenging because the perovskite is soluble with the photoresist, which conflicts with conventional microfabrication technology. The size of presently reported perovskite devices is about 50 µm. Limited by the large size of perovskite optoelectronic devices, they cannot be readily adopted in the fields of imaging, display, etc. Herein a universal microscopic patterned doping method is proposed, which can realize microscale perovskite devices. Rather than by the conventional doping method, in this study the local Fermi level of perovskite is modulated by the redistributing intrinsic ion defects via a polling voltage. A satisfactorily stable polarized ion distribution can be achieved by optimization of the perovskite material and polling voltage, resulting in ultrafast (40 µs), self-powered microscale (2 µm) photodiodes. This work sheds light on a route to fabricate integrated perovskite optoelectronic chips.

Xenon Difluoride Dry Etching for the Microfabrication of Solid Microneedles as a Potential Strategy in Transdermal Drug Delivery.

Although hypodermic needles are a "gold standard" for transdermal drug delivery (TDD), microneedle (MN)-mediated TDD denotes an unconventional approach in which drug compounds are delivered via micron-size needles. Herein, an isotropic XeF2 dry etching process is explored to fabricate silicon-based solid MNs. A photolithographic process, including mask writing, UV exposure, and dry etching with XeF2 is employed, and the MN fabrication is successfully customized by modifying the CAD designs, photolithographic process, and etching conditions. This study enables fabrication of a very dense MNs (up to 1452 MNs cm-2 ) with height varying between 80 and 300 µm. Geometrical features are also assessed using scanning electron microscopy (SEM) and 3D laser scanning microscope. Roughness of the MNs are improved from 0.71 to 0.35 µm after titanium and chromium coating. Mechanical failure test is conducted using dynamic mechanical analyzer to determine displacement and stress/strain values. The coated MNs are subjected to less displacement (≈15 µm) upon the applied force. COMSOL Multiphysics analysis indicates that MNs are safe to use in real-life applications with no fracture. This technique also enables the production of MNs with distinct shape and dimensions. The optimized process provides a wide range of solid MN types to be utilized for epidermis targeting.

Photolithographic microfabrication of hydrogel clefts for cell invasion studies.

Invasion of migrating cells into surrounding tissue plays a key role in cancer metastasis and immune response. In order to assess invasiveness, most in vitro invasion assays measure the degree to which cells migrate between microchambers that provide a chemoattractant gradient across a polymeric membrane with defined pores. However, in real tissue cells experience soft, mechanically deformable microenvironments. Here we introduce RGD-functionalized hydrogel structures that present pressurized clefts for invasive migration of cells between reservoirs maintaining a chemotactic gradient. Using UV-photolithography, equally spaced blocks of polyethylene glycol-norbornene (PEG-NB) hydrogels are formed, which subsequently swell and close the interjacent gaps. The swelling ratio and final contours of the hydrogel blocks were determined using confocal microscopy confirming a swelling induced closure of the structures. The velocity profile of cancer cells transmigrating through the clefts, which we name 'sponge clamp', is found to depend on the elastic modulus as well as the gap size between the swollen blocks. The 'sponge clamp' discriminates the invasiveness of two distinct cell lines, MDA-MB-231 and HT-1080. The approach provides soft 3D-microstructures mimicking invasion conditions in extracellular matrix.

Organs-on-a-chip: a union of tissue engineering and microfabrication.

We review the emergence of the new field of organ-on-a-chip (OOAC) engineering, from the parent fields of tissue engineering and microfluidics. We place into perspective the tools and capabilities brought into the OOAC field by early tissue engineers and microfluidics experts. Liver-on-a-chip and heart-on-a-chip are used as two case studies of systems that heavily relied on tissue engineering techniques and that were amongst the first OOAC models to be implemented, motivated by the need to better assess toxicity to human tissues in preclinical drug development. We review current challenges in OOAC that often stem from the same challenges in the parent fields, such as stable vascularization and drug absorption.

Rapid prototyping of polydimethylsiloxane (PDMS) microchips using electrohydrodynamic jet printing: Application to electrokinetic assays.

Polydimethylsiloxane (PDMS) based microfluidic devices have found increasing utility for electrophoretic and electrokinetic assays because of their ease of fabrication using replica molding. However, the fabrication of high-resolution molds for replica molding still requires the resource-intensive and time-consuming photolithography process, which precludes quick design iterations and device optimization. We here demonstrate a low-cost, rapid microfabrication process, based on electrohydrodynamic jet printing (EJP), for fabricating non-sacrificial master molds for replica molding of PDMS microfluidic devices. The method is based on the precise deposition of an electrically stretched polymeric solution of polycaprolactone in acetic acid on a silicon wafer placed on a computer-controlled motion stage. This process offers the high-resolution (order 10  µ $\umu$ m) capability of photolithography and rapid prototyping capability of inkjet printing to print high-resolution templates for elastomeric microfluidic devices within a few minutes. Through proper selection of the operating parameters such as solution flow rate, applied electric field, and stage speed, we demonstrate microfabrication of intricate master molds and corresponding PDMS microfluidic devices for electrokinetic applications. We demonstrate the utility of the fabricated PDMS microchips for nonlinear electrokinetic processes such as electrokinetic instability and controlled sample splitting in ITP. The ability to rapid prototype customized reusable master molds with order 10  µ $\umu$ m resolution within a few minutes can help in designing and optimizing microfluidic devices for various electrokinetic applications.

Microfluidic Organ-on-A-chip: A Guide to Biomaterial Choice and Fabrication.

Organ-on-A-chip (OoAC) devices are miniaturized, functional, in vitro constructs that aim to recapitulate the in vivo physiology of an organ using different cell types and extracellular matrix, while maintaining the chemical and mechanical properties of the surrounding microenvironments. From an end-point perspective, the success of a microfluidic OoAC relies mainly on the type of biomaterial and the fabrication strategy employed. Certain biomaterials, such as PDMS (polydimethylsiloxane), are preferred over others due to their ease of fabrication and proven success in modelling complex organ systems. However, the inherent nature of human microtissues to respond differently to surrounding stimulations has led to the combination of biomaterials ranging from simple PDMS chips to 3D-printed polymers coated with natural and synthetic materials, including hydrogels. In addition, recent advances in 3D printing and bioprinting techniques have led to the powerful combination of utilizing these materials to develop microfluidic OoAC devices. In this narrative review, we evaluate the different materials used to fabricate microfluidic OoAC devices while outlining their pros and cons in different organ systems. A note on combining the advances made in additive manufacturing (AM) techniques for the microfabrication of these complex systems is also discussed.

Progress of Microfluidic Hydrogel-Based Scaffolds and Organ-on-Chips for the Cartilage Tissue Engineering.

Cartilage degeneration is among the fundamental reasons behind disability and pain across the globe. Numerous approaches have been employed to treat cartilage diseases. Nevertheless, none have shown acceptable outcomes in the long run. In this regard, the convergence of tissue engineering and microfabrication principles can allow developing more advanced microfluidic technologies, thus offering attractive alternatives to current treatments and traditional constructs used in tissue engineering applications. Herein, the current developments involving microfluidic hydrogel-based scaffolds, promising structures for cartilage regeneration, ranging from hydrogels with microfluidic channels to hydrogels prepared by the microfluidic devices, that enable therapeutic delivery of cells, drugs, and growth factors, as well as cartilage-related organ-on-chips are reviewed. Thereafter, cartilage anatomy and types of damages, and present treatment options are briefly overviewed. Various hydrogels are introduced, and the advantages of microfluidic hydrogel-based scaffolds over traditional hydrogels are thoroughly discussed. Furthermore, available technologies for fabricating microfluidic hydrogel-based scaffolds and microfluidic chips are presented. The preclinical and clinical applications of microfluidic hydrogel-based scaffolds in cartilage regeneration and the development of cartilage-related microfluidic chips over time are further explained. The current developments, recent key challenges, and attractive prospects that should be considered so as to develop microfluidic systems in cartilage repair are highlighted.

Laser Micromachining of Bone as a Tool for Studying Bone Marrow Biology.

The bone marrow (BM) has traditionally been a difficult tissue to access because it is embedded deep within the bone matrix. It is home to the hematopoietic stem cells (HSCs) that give rise to all blood cells in the body. It is also the site of origin for malignant blood cells such as leukemia and multiple myeloma, as well as a frequent site of metastasis for many solid tumors including prostate and breast cancer. The following chapter describes how laser micromachining of bone can be used to improve both optical and physical access to the BM. For example, laser thinning of the overlying bone can improve optical access, enabling deeper imaging into the BM as well as enhancing optical resolution by reducing scattering and aberration. Laser micromachining can also be used to provide physical access into the BM by creating access ports for micropipette insertion and delivery of cells to precise locations in the BM, as well as for the extraction of BM cells and interstitial fluid, all under image guidance. This chapter provides a detailed protocol for installing a laser-micromachining capability for users with an existing multiphoton microscope. Additionally, we briefly outline how such a system improves the optical resolution during imaging as well as its potential use to study injury response.

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 microfabrication by applying the laser-induced bubble method to the thermoset polymer PDMS using a conventional nanosecond laser.

We recently developed a microfabrication technique [microfabrication using laser-induced bubble (microFLIB)] and applied it to polydimethylsiloxane (PDMS), a thermoset polymer. The technique enabled the rapid fabrication of a microchannel on a PDMS substrate and selective metallization of the channel via subsequent plating; however, the technique was limited to surface microfabrication. Therefore, we explored the feasibility of three-dimensional (3D) microFLIB of PDMS using a nanosecond laser. In the experiment, a laser beam was focused inside pre-curing liquid PDMS and was scanned both perpendicular and parallel to the laser-beam axis to generate a 3D line of laser-induced bubbles. In the microFLIB processing, the shape of the created bubbles was retained in the pre-curing PDMS for more than 24 h; thus, the line of bubbles generated by the perpendicular laser scanning successfully produced a 3D hollow transverse microchannel inside the PDMS substrate after subsequent thermal curing. In addition, a through-hole with an aspect ratio greater than ∼200 was easily fabricated in the PDMS substrate by parallel laser scanning. The fabrication of a 3D microfluidic device comprising two open reservoirs in a PDMS substrate was also demonstrated for biochip applications.

Miniaturized systems for gas chromatography: Developments in sample preparation and instrumentation.

An important field of research is the miniaturization of analytical systems for laboratory applications and on-field analysis. In particular, gas chromatography (GC) has benefited from the recent advances in enabling technologies like photolithography, micromachining, hot embossing, and 3D-printing to improve sampling and sample preparation, microcolumn technologies, and detection. In this article, the developments and applications reported since 2015 were reviewed and summarized. Important applications using benchtop instruments, portable GCs, and micro-GCs (µGCs) were showcased to illustrate the current challenges associated with each miniaturized interfaces and systems. For instance, portable instruments need to be energy-efficient and ideally depend on renewable sources for carrier gas generation. Lastly, multidimensional separations were addressed using miniaturized systems to effectively improve the peak capacity of portable systems.

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.