Publications

To interrogate neural circuits and crack their codes, in vivo brain activity imaging must be combined with spatiotemporally precise stimulation in three dimensions using genetic or pharmacological specificity. This challenge requires deep penetration and focusing as provided by infrared light and multiphoton excitation, and has promoted two-photon photopharmacology and optogenetics. However, three-photon brain stimulation in vivo remains to be demonstrated. We report the regulation of neuronal activity in zebrafish larvae by three-photon excitation of a photoswitchable muscarinic agonist at 50 pM, a billion-fold lower concentration than used for uncaging, and with mid-infrared light of 1560 nm, the longest reported photoswitch wavelength. Robust, physiologically relevant photoresponses allow modulating brain activity in wild-type animals with spatiotemporal and pharmacological precision. Computational calculations predict that azobenzene-based ligands have high three-photon absorption cross-section and can be used directly with pulsed infrared light. The expansion of three-photon pharmacology will deeply impact basic neurobiology and neuromodulation phototherapies.© 2023 Wiley-VCH GmbH.

JTD Keywords: absorption, azobenzene photoswitches, deep, glutamate-receptor, intravital microscopy, multiphoton excitation, muscarinic neuromodulation, photopharmacology, two-photon lithography and polymerization, 2-photon excitation, Azobenzene, Multiphoton excitation, Muscarinic neuromodulation, Photopharmacology, Photopharmacology, azobenzene, muscarinic neuromodulation, multiphoton excitation, two-photon lithography and polymerization, Two-photon lithography and polymerization

The intestine is a complex tissue with a characteristic three-dimensional (3D) crypt-villus architecture, which plays a key role in the intestinal function. This function is also regulated by the intestinal stroma that actively supports the intestinal epithelium, maintaining the homeostasis of the tissue. Efforts to account for the 3D complex structure of the intestinal tissue have been focused mainly in mimicking the epithelial barrier, while solutions to include the stromal compartment are scarce and unpractical to be used in routine experiments. Here we demonstrate that by employing an optimized bioink formulation and the suitable printing parameters it is possible to produce fibroblast-laden crypt-villus structures by means of digital light projection stereolithography (DLP-SLA). This process provides excellent cell viability, accurate spatial resolution, and high printing throughput, resulting in a robust biofabrication approach that yields functional gut mucosa tissues compatible with conventional testing techniques.Copyright © 2023 Elsevier B.V. All rights reserved.

JTD Keywords: 3d microstructure, barrier, cells, epithelial-stromal interactions, gelma-pegda soft hydrogels, growth, hydrogel, intestinal mucosa model, methacrylamide, microfabrication, proliferation, scaffold, stereolithography, 3d bioprinting, 3d microstructure, Epithelial-stromal interactions, Fibroblasts, Gelma-pegda soft hydrogels, Intestinal mucosa model

Research with soft materials, that is, polymeric gels, colloidal suspensions, liquid crystals, and most biomaterials often involves the need for microfabrication of confinement channels, cells, and lab-on-a-chip devices. Photolithography techniques are often chosen, as they offer the combination of versatility, precision, and quick delivery demanded by researchers. Beyond fabrication, stimulus-responsive systems, such as photosensitivity biomaterials, are the object of broad study within a very interdisciplinary community. Here, we show that a standard laboratory microscope can be quickly and economically transformed into a powerful maskless photofabrication/photoexcitation station using off-the-shelf DMD development modules and simple optomechanical components allowing real time observation of the fabrication process.

JTD Keywords: Dmd, Microscopy, Photolithography, Soft matter

Gradients of signaling pathways within the intestinal stem cell (ISC) niche are instrumental for cellular compartmentalization and tissue function, yet how are they sensed by the epithelium is still not fully understood. Here a new in vitro model of the small intestine based on primary epithelial cells (i), apically accessible (ii), with native tissue mechanical properties and controlled mesh size (iii), 3D villus-like architecture (iv), and precisely controlled biomolecular gradients of the ISC niche (v) is presented. Biochemical gradients are formed through hydrogel-based scaffolds by free diffusion from a source to a sink chamber. To confirm the establishment of spatiotemporally controlled gradients, light-sheet fluorescence microscopy and in-silico modeling are employed. The ISC niche biochemical gradients coming from the stroma and applied along the villus axis lead to the in vivo-like compartmentalization of the proliferative and differentiated cells, while changing the composition and concentration of the biochemical factors affects the cellular organization along the villus axis. This novel 3D in vitro intestinal model derived from organoids recapitulates both the villus-like architecture and the gradients of ISC biochemical factors, thus opening the possibility to study in vitro the nature of such gradients and the resulting cellular response.© 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.

JTD Keywords: 3d architectures, biomolecular gradients, colon, crypt, engineering organoids, hydrogels, identification, in silico modeling, intestinal stem cell niches, light sheet fluorescence microscopy, niche, permeability, photolithography, regeneration, villus, wnt, 3d architectures, Biomolecular gradients, Engineering organoids, In silico modeling, Intestinal stem cell niches, Light sheet fluorescence microscopy, Photolithography, Stem-cell

Cells sense their environment through the cell membrane receptors. Interaction with extracellular ligands induces receptor clustering at the nanoscale, assembly of the signaling complexes in the cytosol and activation of downstream signaling pathways, regulating cell response. Nanoclusters of receptors can be further organized hierarchically in the cell membrane at the meso- and micro-levels to exert different biological functions. To study and guide cell response, cell culture substrates have been engineered with features that can interact with the cells at different scales, eliciting controlled cell responses. In particular, nanoscale features of 1-100 nm in size allow direct interaction between the material and single cell receptors and their nanoclusters. Since the first "contact guidance" experiments on parallel microstructures, many other studies followed with increasing feature resolution and biological complexity. Here we present an overview of the advances in the field summarizing the biological scenario, substrate fabrication techniques and applications, highlighting the most recent developments.Copyright © 2022 Casanellas, Samitier and Lagunas.

JTD Keywords: cell response, density, differentiation, lithography, micro, nanofabrication, nanopatterning, nanopatterns, nanoscale, nanotopography, organization, photolithography, Cell response, Nanofabrication, Nanopatterning, Nanotopography, Plasma-membrane, Receptor nanoclustering

The combination of microfluidics and photo-polymerization techniques such as stereolithography (SLA) has emerged as a new field which has a lot of potential to influence in such important areas as biological analysis, and chemical detection among others. However, the integration between them is still at an early stage of development. In this article, after analyzing the resolution of a custom SLA 3D printer with commercial resins, microfluidic devices were manufactured using three different approaches. First, printing a mold with the objective of creating a Polydimethylsiloxane (PDMS) replica with the microfluidic channels; secondly, open channels have been printed and then assembled with a flat cover of the same resin material. Finally, a closed microfluidic device has also been produced in a single process of printing. Important results for 3D printing with commercial resins have been achieved by only printing one layer on top of the channel. All microfluidic devices have been tested successfully for pressure-driven fluid flow.

JTD Keywords: 3d printing, additive manufacturing, microfluidics, photo-curable polymers, 3d printing, Additive manufacturing, Microfluidics, Photo-curable polymers, Stereolithography

Thermal scanning probe lithography (tSPL) is a nanofabrication method for the chemical and physical nanopatterning of a large variety of materials and polymer resists with a lateral resolution of 10 nm and a depth resolution of 1 nm. In this Primer, we describe the working principles of tSPL and highlight the characteristics that make it a powerful tool to locally and directly modify material properties in ambient conditions. We introduce the main features of tSPL, which can pattern surfaces by locally delivering heat using nanosized thermal probes. We define the most critical patterning parameters in tSPL and describe post-patterning analysis of the obtained results. The main sources of reproducibility issues related to the probe and the sample as well as the limitations of the tSPL technique are discussed together with mitigation strategies. The applications of tSPL covered in this Primer include those in biomedicine, nanomagnetism and nanoelectronics; specifically, we cover the fabrication of chemical gradients, tissue-mimetic surfaces, spin wave devices and field-effect transistors based on two-dimensional materials. Finally, we provide an outlook on new strategies that can improve tSPL for future research and the fabrication of next-generation devices.

JTD Keywords: Beam lithography, Design, Feature size, Force microscope cantilevers, Mos2, Polymer, Silicon, Speed, Thermochemical nanolithography, Tip

Three-dimensional bioprinting (3D bioprinting) has been at theforefront of tissue engineering research in the past years, with evermore efficient systems reaching the market(1). While existing 3Dbioprinting techniques are numerous and varied, they are limited bylong printing times when used at high resolution(2). The techniquedescribed in this work aims at enabling fast and accurate productionof monolayered skin constructs.To achieve shorter production times, a digital scanned light sheetis used to produce patterns of polymerized hydrogel, which enablesthe printing of a full three-dimensional plane in a matter of a fewhundred milliseconds. The high resolution resides in the properties ofthe light sheet itself – the width of the light sheet represents the z-axial resolution of the system (as low as 10mm) and the x-axialresolution is determined by the intensity profile of the gaussian beam(around 50mm). In order to fully exploit this system, the hydrogelused to encapsulate the cells must therefore be tailor-made for pho-toactivated cross-linking.As a proof of concept, a light sheet microscope is used as a po-lymerization source for novel photosensitive hydrogels. The up-coming hardware, software, chemical and biological improvementsneeded to reach the full potential of this system are expected toeventually be sufficient to print a complete skin construct, whichcould be used in the drug development industry, or as a graft forregenerative medicine therapy. Additionally, the constructs can beused to reduce and even replace animal testing for drug or cosmetictesting.

JTD Keywords: 3d bioprinting, Light sheet microscopy, Stereolithography

Micro- and nanotexturization of surfaces can give to the parts different advanced functionalities, such as superhydrophobicity, self-cleaning, or antibacterial capabilities. These advanced properties in combination with the biocompatibility of Liquid Silicone Rubber are an interesting approach for obtaining high-performance medical devices. The industrial production of surface textures in polymeric materials is through the replication technique, and the best option to attain a high production rate is injection molding. Moreover, its low viscosity during processing can provide an accurate replication capacity by the easy filling by capillarity of the microtextures. An innovative replicating technique for Liquid Silicone Rubber is presented by studying the replication of different shaped textures within a diameter range of between 2 and 50 mu m. The copying process consists in the overmolding of a textured polymeric inlay obtained by nanoimprint lithography. At the end of the process, a textured part is obtained, while the imprinted film remains in the mold. The injection molding parameters are optimized to increase the replication accuracy, and their effect on texture replicability is analyzed and discussed. Finally, it is shown that the textured surfaces improve their wettability behavior, which is a necessary and important characteristic in the development of biomedical devices.

JTD Keywords: Cross-linking density, Injection molding, Microtextures, Nanoimprint lithography, Polymeric inlays, Silicone rubber, Stamp, Wettability

JTD Keywords: DNA nanotechnology, Gold nanoparticles, Lithography, Metasurfaces, Nanopatterning

Suspended planar-array (SPA) chips embody millions of individual miniaturized arrays to work in extremely small volumes. Here, the basis of a robust methodology for the fabrication of SPA silicon chips with on-demand physical and chemical anisotropies is demonstrated. Specifically, physical traits are defined during the fabrication process with special focus on the aspect ratio, branching, faceting, and size gradient of the final chips. Additionally, the chemical attributes augment the functionality of the chips with the inclusion of complete coverage or patterns of selected biomolecules on the surface of the chips with contact printing techniques, offering an extremely high versatility, not only with the choice of the pattern shape and distribution but also in the choice of biomolecular inks to pattern. This approach increases the miniaturization of printed arrays in 3D structures by two orders of magnitude compared to those previously demonstrated. Finally, functional micrometric and sub-micrometric patterned features are demonstrated with an antibody binding assay with the recognition of the printed spots with labeled antibodies from solution. The selective addition of physical and chemical attributes on the suspended chips represents the basis for future biomedical assays performed within extremely small volumes.

JTD Keywords: Microcontact printing, Microparticles, Molecular multiplexing, Polymer pen lithography, Silicon chip technology

We report the first DNA-based origami technique that can print addressable patterns on surfaces with sub-10 nm resolution. Specifically, we have used a two-dimensional DNA origami as a template (DNA origami stamp) to transfer DNA with pre-programmed patterns (DNA ink) on gold surfaces. The DNA ink is composed of thiol-modified staple strands incorporated at specific positions of the DNA origami stamp to create patterns upon thiol-gold bond formation on the surface (DNA ink). The DNA pattern formed is composed of unique oligonucleotide sequences, each of which is individually addressable. As a proof-of-concept, we created a linear pattern of oligonucleotide-modified gold nanoparticles complementary to the DNA ink pattern. We have developed an in silico model to identify key elements in the formation of our DNA origami-driven lithography and nanoparticle patterning as well as simulate more complex nanoparticle patterns on surfaces.

JTD Keywords: DNA nanotechnology, Lithography, Nanopatterning, Gold nanoparticles, Metasurfaces

A simple method for constructing versatile ordered biotin/avidin arrays on UV-curable perfluoropolyethers (PFPEs) is presented. The goal is the realization of a versatile platform where any biotinylated biological ligands can be further linked to the underlying biotin/avidin array. To this end, microcontact arrayer and microcontact printing technologies were developed for photobiotin direct printing on PFPEs. As attested by fluorescence images, we demonstrate that this photoactive form of biotin is capable of grafting onto PFPEs surfaces during irradiation. Bioaffinity conjugation of the biotin/avidin system was subsequently exploited for further self-assembly avidin family proteins onto photobiotin arrays. The excellent fouling release PFPEs surface properties enable performing avidin assembly step simply by arrays incubation without PFPEs surface passivation or chemical modification to avoid unspecific biomolecule adsorption. Finally, as a proof of principle biotinylated heparin was successfully grafted onto photobiotin/avidin arrays.

JTD Keywords: Antifouling, Heparin, Malaria, Microcontact arrayer, Microcontact printing, Micropatterning, Perfluoropolyether, Photobiotin, Polymers, Soft lithography

Dip-pen nanolithography and microcontact printing were used to fabricate mesopatterned substrates for cell differentiation experiments. A biotin-thiol was patterned on gold substrates and subsequently functionalised with streptavidin and biotinylated bone morphogenetic protein-2 (BMP-2). The feasibility of mesopatterned substrates containing immobilised BMP-2 was proven by obtaining similar differentiation outcomes compared to the growth factor in solution. Therefore, these substrates might be suitable for replacing conventional experiments with BMP-2 in solution.

JTD Keywords: Bone morphogenetic protein-2, C2C12 cells, Dip-pen nanolithography, Micro contact printing

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JTD Keywords: Biotin, Click chemistry, Dip-pen nanolithography, Oligonucleotides, Surfaces

Rapid and inexpensive development of electrochemical sensors with good exploitation potential may be produced using a polymer as a substrate material. However, fabrication of polymer-based sensors is challenging. Using photolithography and etching of gold-coated poly(ethylene-2,6-naphthalate) (PEN), we have succeeded in fabricating disk-shaped and interdigitated microelectrodes (uEs). The electrodes have an excellent adhesion to the polymer and are encapsulated using a novel room-temperature process, applicable for low-cost, high-throughput fabrication. The PEN surface has been characterized in respect of wettability, surface energy and surface roughness. Finally, the electrodes give stable and reproducible electrochemical impedance spectroscopy and cyclic voltammetry responses, using the redox couple ferrocyanide and ruthenium hexamine. The results demonstrate the robustness and functionality of the polymer-based sensor platform with minimum feature sizes of 6 um.

JTD Keywords: Poly(ethylene naphthalate), Photolithography, Microelectrodes, Interdigitated electrodes, Electrochemical characterization, Electrochemical sensor

Micro and nanoscale protein patterning based on microcontact printing technique on large substrates have often resolution problems due to roof collapse of the poly(dimethylsiloxane) (PDMS) stamps used. Here, we describe a technique that overcomes these issues by using instead a stamp made of poly(methyl methacrylate) (PMMA), a much more rigid polymer that do not collapse even using stamps with very high aspect ratios (up to 300:1). Conformal contact between the stamp and the substrate is achieved because of the homogeneous pressure applied via the nanoimprint lithography instrument, and it has allowed us to print lines of protein 150 nm wide, at a 400 nm period. This technique, therefore, provides an excellent method for the direct printing of high-density submicrometer scale patterns, or, alternatively, micro/nanopatterns spaced at large distances.

JTD Keywords: Microcontact printing, Nanoimprint lithography, Poly(methyl methacrylate), Protein

A method for the simultaneous (bio)chemical and topographical patterning of enclosed structures in poly(dimethyl siloxane) (PDMS) is presented. The simultaneous chemical and topography transference uses a water-soluble chitosan sacrificial mold to impart a predefined pattern with micrometric accuracy to a PDMS replica. The method is compared to conventional soft-lithography techniques on planar surfaces. Its functionality is demonstrated by the transference of streptavidin directly to the surface of the three-dimensional PDMS structures as well as indirectly using streptavidin-loaded latex nanoparticles. The streptavidin immobilized on the PDMS is tested for bioactivity by coupling with fluorescently labeled biotin. This proves that the streptavidin is immobilized on the PDMS surface, not in the bulk of the polymer, and is therefore accessible for use as signaling/binding element in micro and bioengineering. The use of a biocompatible polymer and processes enables the technique to be used for the chemical patterning of tissue constructions.

JTD Keywords: Biotechnology, Chitosan, Microfabrication, MEMs, Soft lithography

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JTD Keywords: Soft lithography, Micro contact printing, Self-assembled monolayers, Soft Molding techniques

Biological patterned surfaces having sub-micron scale resolution are of great importance in many fields of life science and biomedicine. Different techniques have been proposed for surface patterning at the nanoscale. However, most of them present some limitations regarding the patterned area size or are time-consuming. Micro/nanocontact printing is the most representative soft lithography-based technique for surface patterning at the nanoscale. Unfortunately, conventional micro/nanocontact printing also suffers from problems such as diffusion and stamp collapsing that limit pattern resolution. To overcome these problems, a simple way of patterning thiols under liquid media using submerged nanocontact printing (SnCP) over large areas (similar to cm(2)) achieving nanosize resolution is presented. The technique is also low cost and any special equipment neither laboratory conditions are required. Nanostructured poly(dimethyl siloxane) stamps are replicated from commercially available digital video disks. SnCP is used to stamp patterns of 200 nm 1-octadecanethiol lines in liquid media, avoiding ink diffusion and stamp collapsing, over large areas on gold substrates compared with conventional procedures. Atomic force microscopy measurements reveal that the patterns have been successfully transferred with high fidelity. This is an easy, direct, effective and low cost methodology for molecule patterning immobilization which is of interest in those areas that require nanoscale structures over large areas, such as tissue engineering or biosensor applications.

JTD Keywords: Submerged Nanocontact Printing, Replica Molding, Nanopatterning, Large Area, Dip-pen nanolithography, High-aspect-ratio, Soft lithography, Submicronscale, Nanoimprint lithography, Thin-film, Surfaces, Fabrication, Proteins, Nanofabrication

A technique for imparting micro- and nano-structured topography into the surface of freestanding thin sheets of chitosan is described. Both micro- and nanometric surface structures have been produced using soft lithography. The soft lithography method, based on solvent evaporation, has allowed structures similar to 60 nm tall and similar to 500 X 500 nm(2) to be produced on freestanding similar to 0.5 mm thick sheets of the polymer when cured at 293 K, and structures similar to 400 nm tall and 5 X 5 mu m(2) to be produced when cured at 283 K. Nonstructured chitosan thin sheets (similar to 200 mu m thick) show excellent optical transmission properties in the visible portion of the electromagnetic spectrum. The structured sheets can be used for applications where optical microscopic analysis is required, such as cell interaction experiments and tissue engineering.

JTD Keywords: Chitin/chitosan, Microstructure, Nanotopography, Polymerization, Soft lithography

Novel medical and biological applications are driving increased interest in the fabrication of micropipette or micro-dispensers. Reduced volume samples and drug dosages are prime motivators in this effort. We have combined microfabrication technology with ion beam milling techniques to successfully produce cantilever-type polysilicon micro-dispensers with 3D enclosed microchannels. The microfabrication technology described here allows for the designing of nozzles with multiple shapes. The contribution of ion beam milling has had a large impact on the fabrication process and on further customizing shapes of nozzles and inlet ports. Functionalization tests were conducted to prove the viability of ion beam-fabricated micro-dispensers. Self-assembled monolayers were successfully formed when a gold surface was patterned with a thiol solution dispensed by the fabricated micro-dispensers.

JTD Keywords: Dip-pen nanolithography, Silicon, Deposition, Microneedles, Delivery, Arrays, Polysilicon, Capillary, Systems, Gene

Polymers with high glass transition temperatures, fluorinated ethylene propylene copolymer (FEP) and poly(ethylene naphthalate) (PEN), have been used in imprint lithography as a protective support layer and as a secondary mould, to imprint superficial structures into a polymer with a lower glass transition temperature, namely poly(methyl methacrylate) (PMMA). As a support layer, FEP replaces fragile silicon based supports for the production of freestanding, structured sheets of PMMA, useful, for example, in biomedical applications where transmittance optical microscopy is required. Secondary PEN moulds, produced by imprinting using silicon-based primary moulds, have been used to transfer sub-micrometer tall structures to a freestanding PMMA sheet. Similarly, hole structures, with different dimensions, have been embossed in both sides of a PMMA sheet simultaneously.

JTD Keywords: Polymer engineering, Embossing, Nanoimprint lithography, Biomedical applications

Micro- and nanoscale protein patterns have been produced via a new contact printing method using a nanoimprint lithography apparatus. The main novelty of the technique is the use of poly(methyl methacrylate) (PMMA) instead of the commonly used poly(dimethylsiloxane) (PDMS) stamps. This avoids printing problems due to roof collapse, which limits the usable aspect ratio in microcontact printing to 10:1. The rigidity of the PMMA allows protein patterning using stamps with very high aspect ratios, up to 300 in this case. Conformal contact between the stamp and the substrate is achieved because of the homogeneous pressure applied via the nanoimprint lithography instrument, and it has allowed us to print lines of protein similar to 150 nm wide, at a 400 nm period. This technique, therefore, provides an excellent method for the direct printing of high-density sub-micrometer scale patterns, or, alternatively, micro-/nanopatterns spaced at large distances. The controlled production of these protein patterns is a key factor in biomedical applications such as cell-surface interaction experiments and tissue engineering.

JTD Keywords: Soft lithography, Cell-adhesion, Microstructures, Fabrication, Stability, Patterns

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JTD Keywords: Self-assembled monolayers, High-aspect-ratio, Dip-pen nanolithography, Langmuir-blodgett-films, Conducting polymer, Enzymes, Microstructures, Immunosensors, Fabrication, Stability