Biological systems often outperform artificial ones in ordering, assembly, and diversity of structure at the nanoscale. Proteins are particularly remarkable in this context. Through oligomerization, protein monomers assemble on multiple length scales, into larger and more complex structures such as viral capsids, filaments, and regulatory complexes. It is this structural diversity that makes proteins attractive candidates for use as functionalizable scaffolds. Well-established protein structure databases such as the protein data bank (PDB) allow researchers to search for a structure that fits their requirements, allowing them access to shapes and assembly mechanisms that would otherwise be difficult to achieve. Then, by employing functionalization techniques to conjugate artificial or biological molecules to their protein scaffold of choice, researchers can access chemistries beyond the limits of the 20 commonly occurring natural amino acids. Additionally, proteins, with a few exceptions, operate at physiological pH and temperature, making them ideal for medical applications and/or low-cost manufacture. Additionally, proteins sourced from extremophiles such as Thermus aquaticus (a bacterial species sourced from hot springs) display stability across a wide range of temperatures, expanding the scope for scaffold selection. This chapter will cover some of the common methods of protein functionalization as well as some specific examples of popular functionalization methods reported in the literature. It will then present three case studies showing examples of how functionalization and imaging of proteins and protein-based structures can be achieved.

Hypothesis: Our ability to dictate the colloid geometry is intimately related to self-assembly. The synthesis of anisotropic colloidal particles is currently dominated by wet chemistry and lithographic techniques. The wet chemical synthesis offers limited particle geometries at bulk quantities. Lithographic techniques, on the other hand, provide precise control over the particle shape, although at lower yields. In this respect, two-photon polymerization (2PP)¹ has attracted growing attention due to its ability to automatically fabricate complex micro/nano structures with high resolution. Experiments: We manufacture precisely designed colloids with sizes ranging from 1 µm to 10 µm with 2PP and optimize the process parameters for each dimension. Moreover, we study the shape dependent Brownian motion of these particles with video microscopy and estimate their diffusion coefficients. Findings: We observe that increasing the geometrical anisotropy leads to a pronounced deviation from the analytically predicted diffusion coefficient for disks with a given aspect ratio. The deviation is attributed to stronger hydrodynamic coupling with increasing anisotropy. We demonstrate, for the first time, 2PP manufacturing of colloids with tailored geometry. This study opens synthesis of colloidal building blocks to a broader audience with limited access to cleanrooms or wet-chemistry know-how.

Micro-patterned diamond has been investigated for numerous applications, such as biomimetic surfaces, electrodes for cell stimulation and energy storage, photonic structures, imprint lithography, and others. Controlled patterning of diamond substrates and moulds typically requires lithography-based top-down processing, which is costly and complex. In this work, we introduce an alternative, cleanroom-free approach consisting of the bottom-up growth of nanocrystalline diamond (NCD) micropillar arrays by chemical vapour deposition (CVD) using a commercial porous Si membrane as a template. Conformal pillars of ~4.7 μm in height and ~2.2 μm in width were achieved after a maximum growth time of 9 h by hot-filament CVD (2% CH ₄ in H ₂ , 725 °C at 10 mbar). In order to demonstrate one of many possible applications, micropillar arrays grown for 6 h, with ~2 μm in height, were evaluated as moulds for imprint lithography by replication onto hard cyclic olefin copolymer (COC) and onto soft polydimethylsiloxane (PDMS) elastomer. The results showed preserved mechanical integrity of the diamond moulds after replication, as well as full pattern transfer onto the two polymers, with matching dimensions between the grown pillars and the replicated holes. Prior surface treatment of the diamond mould was not required for releasing the PDMS replica, whereas the functionalisation of the diamond surface with a perfluorododecyltrichlorosilane (FDDTS) anti-stiction layer was necessary for the successful release of the COC replica from the mould. In summary, this paper presents an alternative and facile route for the fabrication of diamond micropillar arrays and functional micro-textured surfaces.

The concept and synthesis approach for planar Compliant Fluidic Control Structures (CFCSs), monolithic flexible continua with embedded functional pores, is presented in this manuscript. Such structures are envisioned to find application in biomedicine as tunable microfluidic devices for drug/nutrient delivery. The functional pores enlarge and/or contract upon deformation of the compliant structure in response to external stimuli, facilitating the regulated control of fluid/nutrient/drug transport. A thickness design variable based topology optimization problem is formulated to generate effective designs of these structures. An objective based on hydraulic diameter(s) is conceptualized, and it is extremized using a gradient based optimizer. Both geometrical and material nonlinearities are considered. The nonlinear behaviour of employed hyperelastic material is modeled via the Arruda-Boyce constitutive material model. Large-displacement finite element analysis is performed using the updated Lagrangian formulation in plane-stress setting. The proposed synthesis approach is applied to various CFCSs for a variety of fluidic control functionalities. The optimized designs of various CFCSs with single and/or multiple functional pores are fabricated via a Polydimethylsiloxane (PDMS) soft lithography process, using a high precision 3D printed mold and their performances are compared with the numerical predictions.

Manufacturing high throughput in vitro models resembling the tissue microenvironment is highly demanded for studying bone regeneration. Tissues such as bone have complex multiscale architectures inside which cells reside. To this end, engineering a microfluidic platform incorporated with three-dimensional (3D) microscaffolds and submicron/nanoscale topographies can provide a promising model for 3D cell cultures. There are, however, certain challenges associated with this goal, such as the need to decorate large surfaces area with high-fidelity 3D submicron structures. Here, we succeeded in fabricating a microfluidic platform embedded with a large area (mm range) of reproducible submicron pillar-based topographies. Using the two-photon polymerization (2PP) as a 3D printing technique based on direct laser writing, uniform submicron patterns were created through optimization of the process parameters and writing strategy. To demonstrate the multiscale fabrication capabilities of this approach, submicron pillars of various heights were integrated onto the surfaces of a 3D microscaffold in a single-step 2PP process. The created submicron topography was also found to improve the hydrophilicity of the surface while being able to withstand flow rates of up to 8 mL/min. The material (IP-Dip resin) used for patterning did not have cytotoxic effects against human mesenchymal stromal cells after 3 days of dynamic culture in the microfluidic device. This proof-of-principle study, therefore, marks a significant step forward in manufacturing submicron structure-on-a-chip models for bone regeneration studies.

The controlled patterning of polymeric surfaces at the micro- and nanoscale offers potential in the technological development of small-scale devices, particularly within the fields of photovoltaics, micro-optics and lab- and organ-on-chip, where the topological arrangement of the surface can influence a system's power generation, optical properties or biological function - such as, in the latter case, biomimicking surfaces or topological control of cellular differentiation.One of the most promising approaches in reducing manufacturing costs and complexity is by exploitation of the self-assembling properties of colloidal particles. Self-assembly techniques can be used to produce colloidal crystals onto surfaces, which can act as replicative masks, as has previously been demonstrated with colloidal lithography, or templates in mold-replication methods with resolutions dependent on particle size. Within this context, a particular emerging interest is focused on the use of self-assembled colloidal crystal surfaces in polymer replication methods such as soft lithography, hot and soft embossing and nano-imprint lithography, offering low-cost and high-resolution alternatives to conventional lithographic techniques.However, there are still challenges to overcome for this surface patterning approach to reach a manufacturing reliability and process robustness comparable to competitive technologies already available in the market, as self-assembly processes are not always 100% effective in organizing colloids within a structural pattern onto the surface. Defects often occur during template fabrication. Furthermore, issues often arise mainly at the interface between colloidal crystals and other surfaces and substrates. Particularly when utilized in high-temperature pattern replication processes, poor adhesion of colloidal particles onto the substrate results in degradation of the patterning template. These effects can render difficulties in creating stable structures with little defect that are well controlled such that a large variety of shapes can be reproduced reliably.This review presents an overview of available self-assembly methods for the creation of colloidal crystals, organized by the type of forces governing the self-assembly process: fluidic, physical, external fields, and chemical. The main focus lies on the use of spherical particles, which are favorable due to their high commercial availability and ease of synthesis. However, also shape-anisotropic particle self-assembly will be introduced, since it has recently been gaining research momentum, offering a greater flexibility in terms of patterning. Finally, an overview is provided of recent research on the fabrication of polymer nano- and microstructures by making use of colloidal self-assembled templates.

The patterning of conductive polymers is a major challenge in the implementation of these materials in several research and industrial applications, spanning from photovoltaics to biosensors. Within this context, we have developed a reliable technique to pattern a thin layer of the conductive polymer poly(3,4-ethylenedioxythiophene) (PEDOT) by means of a low cost and high throughput soft embossing process. We were able to reproduce a functional conductive pattern with a minimum dimension of 1 μm and to fabricate electrically decoupled electrodes. Moreover, the conductivity of the PEDOT films has been characterized, finding that a post-processing treatment with Ethylene Glycol allows an increase in conductivity and a decrease in water solubility of the PEDOT film. Finally, cyclic voltammetry demonstrates that the post-treatment also ensures the electrochemical activity of the film. Our technology offers a facile solution for the patterning of organic conductors with resolution in the micro scale, and can be the basis for the realization and development of polymeric microdevices with electrical and electrochemical functionalities.