Allergen arrays for antibody screening and immune cell activation profiling generated by parallel lipid dip-pen nanolithography

Multiple-allergen testing for high throughput and high sensitivity requires the development of miniaturized immunoassays that allow for a large test area and require only a small volume of the test analyte, which is often available only in limited amounts. Developing such miniaturized biochips containing arrays of test allergens needs application of a technique able to deposit molecules at high resolution and speed while preserving its functionality. Lipid dip-pen nanolithography (L-DPN) is an ideal technique to create such biologically active surfaces, and it has already been successfully applied for the direct, nanoscale deposition of functional proteins, as well as for the fabrication of biochemical templates for selective adsorption. The work presented here shows the application of L-DPN for the generation of arrays of the ligand 2,4-dinitrophenyl[1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[6-[(2,4-dinitrophenyl)amino]hexanoyl] (DNP)] onto glass surfaces as a model system for detection of allergen-specific Immunoglobin E (IgE) antibodies and for mast cell activation profiling.     

Complex Microstructured 3D Surfaces Using Chitosan Biopolymer

A technique for producing micrometer‐scale structures over large, nonplanar chitosan surfaces is described. The technique makes use of the rheological characteristics (deformability) of the chitosan to create freestanding, three‐dimensional scaffolds with controlled shapes, incorporating defined microtopography. The results of an investigation into the technical limits of molding different combinations of shapes and microtopographies are presented, highlighting the versatility of the technique when used irrespectively with inorganic or delicate organic moulds. The final, replicated scaffolds presented here are patterned with arrays of one‐micrometer‐tall microstructures over large areas. Structural integrity is characterized by the measurement of structural degradation. Human umbilical vein endothelial cells cultured on a tubular scaffold show that early cell growth is conditioned by the microtopography and indicate possible uses for the structures in biomedical applications. For those applications requiring improved chemical and mechanical resistance, the structures can be replicated in poly(dimethyl siloxane).     

Novel anti-reflection technology for GaAs single-junction solar cells using surface patterning and Au nanoparticles

Single-junction GaAs solar cell structures were grown by low-pressure MOCVD on GaAs (100) substrates. Micro-rod arrays with diameters of 2 microm, 5 microm, and 10 microm were fabricated on the surfaces of the GaAs solar cells via photolithography and wet chemical etching. The patterned surfaces were coated with Au nanoparticles using an Au colloidal solution. Characteristics of the GaAs solar cells with and without the micro-rod arrays and Au nanoparticles were investigated. The short-circuit current density of the GaAs solar cell with 2 microm rod arrays and Au nanoparticles increased up to 34.9% compared to that of the reference cell without micro-rod arrays and Au nanoparticles. The conversion efficiency of the GaAs solar cell that was coated with Au nanoparticles on the patterned surface with micro-rod arrays can be improved from 14.1% to 19.9% under 1 sun AM 1.5G illumination. These results show that micro-rod arrays and Au nanoparticle coating can be applied together in surface patterning to achieve a novel cost-effective anti-reflection technology.     

Cellular Deformations Induced by Conical Silicon Nanowire Arrays Facilitate Gene Delivery

Engineered cell–nanostructured interfaces generated by vertically aligned silicon nanowire (SiNW) arrays have become a promising platform for orchestrating cell behavior, function, and fate. However, the underlying mechanism in SiNW‐mediated intracellular access and delivery is still poorly understood. This study demonstrates the development of a gene delivery platform based on conical SiNW arrays for mechanical cell transfection, assisted by centrifugal force, for both adherent and nonadherent cells in vitro. Cells form focal adhesions on SiNWs within 6 h, and maintain high viability and motility. Such a functional and dynamic cell–SiNW interface features conformational changes in the plasma membrane and in some cases the nucleus, promoting both direct penetration and endocytosis; this synergistically facilitates SiNW‐mediated delivery of nucleic acids into immortalized cell lines, and into difficult‐to‐transfect primary immune T cells without pre‐activation. Moreover, transfected cells retrieved from SiNWs retain the capacity to proliferate—crucial to future biomedical applications. The results indicate that SiNW‐mediated intracellular delivery holds great promise for developing increasingly sophisticated investigative and therapeutic tools.     

Interfacing Inorganic Nanowire Arrays and Living Cells for Cellular Function Analysis

Inorganic nanowires are among the most attractive functional materials, which have emerged in the past two decades. They have demonstrated applications in information technology and energy conversion, but their utility in biological or biomedical research remains relatively under‐explored. Although nanowire‐based sensors have been frequently reported for biomolecular detection, interfacing nanowire arrays and living mammalian cells for the direct analysis of cellular functions is a very recent endeavor. Cell‐penetrating nanowires enabled effective delivery of biomolecules, electrical and optical stimulation and recording of intracellular signals over a long period of time. Non‐penetrating, high‐density nanowire arrays display rich interactions between the nanostructured substrate and the micro/nanoscale features of cell surfaces. Such interactions enable efficient capture of rare cells including circulating tumor cells and trafficking leukocytes from complex biospecimens. It also serves as a platform for probing cell traction force and neuronal guidance. The most recent advances in the field that exploits nanowire arrays (both penetrating and non‐penetrating) to perform rapid analysis of cellular functions potentially for disease diagnosis and monitoring are reviewed.     

Inverse Opal Scaffolds with an Embossed Surface Pattern Prepared from Sticky Golf‐Ball‐Shaped Microparticle Assemblies

Inverse opal scaffolds presenting an embossed‐pattern surface are prepared from colloidal crystal assemblies of uniformly sized golf‐ball‐shaped microparticles. Post‐treatments, such as thermal annealing during the bridging of the microparticles for opal preparation, are avoided to prevent deterioration of surface patterns of the sacrificial template. This presents a new approach to increase the surface‐area‐to‐volume ratio (SAV) by the alteration of morphological features in sophisticated 3D structures that remain largely unexamined owing to difficulties in their preparation. Previous results observed in 2D surfaces that show effective performance improvement through an increase in contact area, especially in biomedical applications, also appear applicable to patterned inverse opal scaffolds based on comparable results obtained from cell cultures. As the field of application of opal and inverse opal structures is expanding due to their unique structural advantages, such as 3D interconnectivity and periodic structures, our strategy opens the door for the use of patterned surfaces on highly sophisticated 3D structures, improving their performance via an increase in SAV.     

Specific Capture of Peptide‐Receptive Major Histocompatibility Complex Class I Molecules by Antibody Micropatterns Allows for a Novel Peptide‐Binding Assay in Live Cells

Binding assays with fluorescently labeled ligands and recombinant receptor proteins are commonly performed in 2D arrays. But many cell surface receptors only function in their native membrane environment and/or in a specific conformation, such as they appear on the surface of live cells. Thus, receptors on live cells should be used for ligand binding assays. Here, it is shown that antibodies preprinted on a glass surface can be used to specifically array a peptide receptor of the immune system, i.e., the major histocompatibility complex class I molecule H‐2K^b, into a defined pattern on the surface of live cells. Monoclonal antibodies make it feasible to capture a distinct subpopulation of H‐2K^b and hold it at the cell surface. This patterned receptor enables a novel peptide‐binding assay, in which the specific binding of a fluorescently labeled index peptide is visualized by microscopy. Measurements of ligand binding to captured cell surface receptors in defined confirmations apply to many problems in cell biology and thus represent a promising tool in the field of biosensors.     

Monoclonal Cell Line Generation and CRISPR/Cas9 Manipulation via Single‐Cell Electroporation

Stably transfected cell lines are widely used in drug discovery and biological research to produce recombinant proteins. Generation of these cell lines requires the isolation of multiple clones, using time‐consuming dilution methods, to evaluate the expression levels of the gene of interest. A new and efficient method is described for the generation of monoclonal cell lines, without the need for dilution cloning. In this new method, arrays of patterned cell colonies and single cell transfection are employed to deliver a plasmid coding for a reporter gene and conferring resistance to an antibiotic. Using a nanofountain probe electroporation system, probe positioning is achieved through a micromanipulator with sub‐micron resolution and resistance‐based feedback control. The array of patterned cell colonies allows for rapid selection of numerous stably transfected clonal cell lines located on the same culture well, conferring a significant advantage over slower and labor‐intensive traditional methods. In addition to plasmid integration, this methodology can be seamlessly combined with CRISPR/Cas9 gene editing, paving the way for advanced cell engineering.     

5 nm Silver Nanoparticles Amplify Clinical Features of Atopic Dermatitis in Mice by Activating Mast Cells

The triggering effect of silver nanoparticles (NPs) on the induction of allergic reactions is evaluated, by studying the activation of mast cells and the clinical features of atopic dermatitis in a mouse model. Granule release is induced in RBL‐2H3 mast cells by 5 nm, but not 100 nm silver NPs. Increases in the levels of reactive oxygen species (hydrogen peroxide and mitochondrial superoxide) and intracellular Ca⁺⁺ in mast cells are induced by 5 nm silver NPs. In a mouse model of atopic dermatitis induced by a mite allergen, the skin lesions are more severe and appear earlier in mice treated simultaneously with 5 nm silver NPs and allergen compared with mice treated with allergen alone or 100 nm silver NPs and allergen. The histological findings reveal that number of tryptase‐positive mast cells and total IgE levels in the serum increase in mice treated with 5 nm silver NPs and allergen. The results in this study indicate that cotreatment with 5 nm silver NPs stimulates mast cell degranulation and induces earlier and more severe clinical alterations in allergy‐prone individuals.     

Unveiling the Dynamical Assembly of Magnetic Nanocrystal Zig‐Zag Chains via In Situ TEM Imaging in Liquid

The controlled assembly of colloidal magnetic nanocrystals is key to many applications such as nanoelectronics, storage memory devices, and nanomedicine. Here, the motion and ordering of ferrimagnetic nanocubes in water via liquid‐cell transmission electron microscopy is directly imaged in situ. Through the experimental analysis, combined with molecular dynamics simulations and theoretical considerations, it is shown that the presence of highly competitive interactions leads to the formation of stable monomers and dimers, acting as nuclei, followed by a dynamic growth of zig‐zag chain‐like assemblies. It is demonstrated that such arrays can be explained by first, a maximization of short‐range electrostatic interactions, which at a later stage become surpassed by magnetic forces acting through the easy magnetic axes of the nanocubes, causing their tilted orientation within the arrays. Moreover, in the confined volume of liquid in the experiments, interactions of the nanocube surfaces with the cell membranes, when irradiated at relatively low electron dose, slow down the kinetics of their self‐assembly, facilitating the identification of different stages in the process. The study provides crucial insights for the formation of unconventional linear arrays made of ferrimagnetic nanocubes that are essential for their further exploitation in, for example, magnetic hyperthermia, magneto‐transport devices, and nanotheranostic tools.     

Controlling Cell Behavior Through the Design of Polymer Surfaces

Polymers have gained a remarkable place in the biomedical field as materials for the fabrication of various devices and for tissue engineering applications. The initial acceptance or rejection of an implantable device is dictated by the crosstalk of the material surface with the bioentities present in the physiological environment. Advances in microfabrication and nanotechnology offer new tools to investigate the complex signaling cascade induced by the components of the extracellular matrix and consequently allow cellular responses to be tailored through the mimicking of some elements of the signaling paths. Patterning methods and selective chemical modification schemes at different length scales can provide biocompatible surfaces that control cellular interactions on the micrometer and sub‐micrometer scales on which cells are organized. In this review, the potential of chemically and topographically structured micro‐ and nanopolymer surfaces are discussed in hopes of a better understanding of cell–biomaterial interactions, including the recent use of biomimetic approaches or stimuli‐responsive macromolecules. Additionally, the focus will be on how the knowledge obtained using these surfaces can be incorporated to design biocompatible materials for various biomedical applications, such as tissue engineering, implants, cell‐based biosensors, diagnostic systems, and basic cell biology. The review focusses on the research carried out during the last decade.     

Anisotropic Crystalline Protein Nanolayers as Multi‐Functional Biointerface for Patterned Co‐Cultures of Adherent and Non‐Adherent Cells in Microfluidic Devices

The spatial arrangement of cells in their microenvironment is known to significantly influence cellular behavior, thus making the control of cellular organization an important parameter of in vitro co‐culture models. However, recent advances in micropatterning co‐culture methods within biochips do not address the simultaneous cultivation of anchorage‐dependent and non‐adherent cells. To address this methodological gap we combine S‐layer technology with microfluidics to pattern co‐cultures to study the cell‐to‐cell and cell‐to‐surface interactions under physiologically relevant conditions. We exploit the unique self‐assembly properties of SbpA and SbsB S‐layers to create an anisotropic protein nanobiointerface on‐chip with spatially‐defined cytophilic (adhesive) and cytophobic (repulsive) properties. While microfluidics control physical parameters such as shear force and flow velocities, our anisotropic protein nanobiointerface regulates the biological aspects of the co‐culture method including biocompatibility, biostability, and affinity to non‐adherent cells. The reliability and reproducibility of our microfluidic co‐culture strategy based on laminar flow patterned protein nanolayers is envisioned to advance in vitro models for biomedical research.     

Bioinspired Patterning with Extreme Wettability Contrast on TiO2 Nanotube Array Surface: A Versatile Platform for Biomedical Applications

Binary wettability patterned surfaces with extremely high wetting contrasts can be found in nature on living creatures. They offer a versatile platform for microfluidic management. In this work, a facile approach to fabricating erasable and rewritable surface patterns with extreme wettability contrasts (superhydrophilic/superhydrophobic) on a TiO₂ nanotube array (TNA) surface through self‐assembly and photocatalytic lithography is reported. The multifunctional micropatterned superhydrophobic TNA surface can act as a 2D scaffold for site‐selective cell immobilization and reversible protein absorption. Most importantly, such a high‐contrast wettability template can be used to construct various well‐defined 3D functional patterns, such as calcium phosphate, silver nanoparticles, drugs, and biomolecules in a highly selective manner. The 3D functional patterns would be a versatile platform in a wide range of applications, especial for biomedical devices (e.g., high‐throughput molecular sensing, targeted antibacterials, and drug delivery). In a proof‐of‐concept study, the surface‐enhanced Raman scattering and antibacterial performance of the fabricated 3D AgNP@TNA pattern, and the targeted drug delivery for site‐specific and high‐sensitivity cancer cell assays was investigated.     

Tunable Assembly of Gold Nanoparticles on Nanopatterned Poly(ethylene glycol) Brushes

The organization of metallic nanoparticles (NPs) into ordered arrays on nanopatterned surfaces is an enabling process to fabricate devices and study the properties of the particles. Tailoring the interaction between NPs and nanopatterns is a necessity to gain a high level of control in this process. Here, nanopatterned poly(ethylene glycol) (PEG) brushes are presented as a platform for the organization of Au NPs on surfaces. The binding of citrate‐stabilized Au NPs to the PEG brushes depends on the size of the particles and molecular weight of the brushes: the density of NPs immobilized on the nanopatterns of PEG brushes increases with decreasing the diameter of the particles and increasing the chain length of the brushes. The key aspect of the process is to pattern PEG brushes with high resolution and chemical contrast to provide controllable and specific interaction between Au NPs and nanopatterns at a single particle resolution. The modulation of the number (0–4) of Au NPs (e.g., 30 nm) per patterned feature with a high level of accuracy and the generation of patterned heterostructures that consist of two different sizes (e.g., 40 and 20 nm) of particles constitute two examples showing the capabilities of the presented platform.     

Degradation and failure of field emitting carbon nanotube arrays

It has been observed experimentally that the collective field emission from an array of Carbon Nanotubes (CNTs) exhibits fluctuation and degradation, and produces thermal spikes, resulting in electro-mechanical fatigue and failure of CNTs. Based on a new coupled multiphysics model incorporating the electron-phonon transport and thermo-electrically activated breakdown, a novel method for estimating accurately the lifetime of CNT arrays has been developed in this paper. The main results are discussed for CNT arrays during the field emission process. It is shown that the time-to-failure of CNT arrays increases with the decrease in the angle of tip orientation. This observation has important ramifications for such areas as biomedical X-ray devices using patterned films of CNTs.     

Mass Cytometry and Single‐Cell RNA‐seq Profiling of the Heterogeneity in Human Peripheral Blood Mononuclear Cells Interacting with Silver Nanoparticles

Understanding the interactions between nanoparticles (NPs) and human immune cells is necessary for justifying their utilization in consumer products and biomedical applications. However, conventional assays may be insufficient in describing the complexity and heterogeneity of cell–NP interactions. Herein, mass cytometry and single‐cell RNA‐sequencing (scRNA‐seq) are complementarily used to investigate the heterogeneous interactions between silver nanoparticles (AgNPs) and primary immune cells. Mass cytometry reveals the heterogeneous biodistribution of the positively charged polyethylenimine‐coated AgNPs in various cell types and finds that monocytes and B cells have higher association with the AgNPs than other populations. scRNA‐seq data of these two cell types demonstrate that each type has distinct responses to AgNP treatment: NRF2‐mediated oxidative stress is confined to B cells, whereas monocytes show Fcγ‐mediated phagocytosis. Besides the between‐population heterogeneity, analysis of single‐cell dose–response relationships further reveals within‐population diversity for the B cells and naïve CD4⁺ T cells. Distinct subsets having different levels of cellular responses with respect to their cellular AgNP doses are found. This study demonstrates that the complementary use of mass cytometry and scRNA‐seq is helpful for gaining in‐depth knowledge on the heterogeneous interactions between immune cells and NPs and can be incorporated into future toxicity assessments of nanomaterials.     

Integration of metal organic chemical vapour deposition and wet chemical techniques to obtain highly ordered porous ZnO nanoplatforms

Large-area, highly ordered ZnO micropores-arrays consisting of ZnO nanotubes delimited by ZnO nanorods have been successfully fabricated and tested for protein sensing applications. ZnO seed layers have been deposited by Metal Organic Chemical Vapour Deposition and readily patterned by Colloidal Lithography to attain ZnO nanorods growth at selective sites by Chemical Bath Deposition. The used synthetic approach has been proven effective for the easy assembly of ZnO nanoplatforms into high-density arrays. Both patterned and unpatterned ZnO nanorods have been morphologically and compositionally characterised and, thus, tested for model studies of protein mobility at the interface. The patterned layers, having a higher contribution of surface polar moieties than the corresponding unpatterned surfaces, exhibit a reduced lateral diffusion of the adsorbed protein. This evidence is related to the intrinsic porous nature of the ZnO hemispherical arrays characterised by a nanotube-nanorod hybrid networks. The present study gives a great impetus to the fabrication of tunable ZnO nanoplatforms having multiple morphologies and exceptionally high surface areas suitable for application in sensing devices.     

When Would Immunologists Consider a Nanomaterial to be Safe? Recommendations for Planning Studies on Nanosafety

The immune system is professional in recognizing and responding to non‐self, including nanomaterials. Immune responses by professional and nonprofessional immune cells are thus nearly inevitable upon exposure of cells and organisms to such materials. The state of research into taking the immune system into account in nanosafety studies is reviewed and three aspects in which further improvements are desirable are identified: 1) Due to technical limitations, more stringent testing for endotoxin contamination should be made. 2) Since under overdose conditions immunity shows unphysiological responses, all doses used should be justified by being equivalent to tissue‐delivered doses. 3) When markers of acute inflammation or cell stress are observed, functional assays are necessary to distinguish between homeostatic fluctuation and genuine defensive or tolerogenic responses. Since immune activation can also indicate that the immune system considers a stimulus to be harmless and induces tolerance, activation markers by themselves do not necessarily imply a danger to the body. Guidelines such as these are necessary to approach the point where specific nanomaterials are classified as safe based on reliable testing strategies.     

Anisotropic Wetting Surfaces with One‐Dimesional and Directional Structures: Fabrication Approaches,Wetting Properties and Potential Applications

This review article provides a brief summary of recent research progress on anisotropic wetting on one‐dimensional (1D) and directionally patterned surfaces, as well as the technical importance in various applications. Inspiration from natural structures exhibiting anisotropic wetting behavior is first discussed. Development of fabrication techniques for topographically and chemically 1D patterned surfaces and directional nanomaterials are then reviewed, with emphasis on anisotropic behavior with topographically (structurally) patterned surfaces. The basic investigation of anisotropic wetting behavior and theoretical simulations for anisotropic wetting are also further reviewed. Perspectives concerning future direction of anisotropic wetting research and its potential applications in microfluidic devices, lab‐on‐a‐chip, sensor, microreactor and self‐cleaning are presented.