NANOSTRUCTURED SURFACES

Introduction
A nanostructure is an object of intermediate size between microscopic and molecular structures. In describing nanostructures it is necessary to differentiate between the numbers of dimensions on the nanoscale. Nanotextured surfaces have one dimension on the nanoscale, i.e., only the thickness of the surface of an object is between 0.1 and 100 nm. Nanotubes have two dimensions on the nanoscale, i.e., the diameter of the tube is between 0.1 and 100 nm; its length could be much greater. Finally, spherical nanoparticles have three dimensions on the nanoscale, i.e., the particle is between 0.1 and 100 nm in each spatial dimension. The terms nanoparticles and ultrafine particles (UFP) often are used synonymously although UFP can reach into the micrometre range. The term 'nanostructure' is often used when referring to magnetic technology.

Figure 1:- The DNA structure at left (schematic shown) will self-assemble into the structure visualized by atomic force microscopy at right.

Nanostructured surfaces can be broadly defined as substrates in which the typical features have dimensions in the range 1100 nm (although the upper limit of 100 nm may be relaxed to greater sizes in some cases, depending on the material and the specific property being investigated). The recent surge of interest in these systems stems from the remarkable effects that may arise from the critical size reduction. Interesting novel properties (catalytic, magnetic, ferroelectric, mechanical, optical and electronic) occur as we reduce the dimensions from a practically infinite (and periodic) solid crystal to a

system composed of a relatively small number of atoms. So far, nanostructured materials or nanomaterials are perhaps the only sub-field of nanoscience that has made the transition from fundamental science to real world applications, thus becoming a technology (a good example of this are nanostructured surface coatings).

Fabrication of Nanostructured Surfaces

Living creatures make extensive use of nanostructured surfaces to conduct their relations with their environment. A wide assortment of unique topographies keep water from soaking leaves and insects, enable deep-sea sponges to collect light, allow beetles to adjust their adhesion to surfaces, and generate the collective motion that prevents microorganisms from settling on echinoderms skin and facilitates bacterial swimming. This strategy offers enticing possibilities for designing multifunctional, dynamic materials, but creating nanoarrays with comparable mechanical flexibility and feature complexity is beyond the capacity of previously existing fabrication techniques. We have devised a two-step lithography technique that meets this challenge and provides almost unlimited design versatility: we can create arrays of nanostructures from a wide range of materials with arbitrary stiffness and built-in environmental sensitivities and can specify any nanostructure shape, geometry, orientation, and spatial organization.

Figure 2:-Two step sot lithography

Each array can be further modified after fabrication by controlled selfassembly and/or environmentally stimulated reconfiguration. On top of this technique, we have also discovered that deposition of conductive polymers transforms each array into a variety of highly complex hierarchical structures comparable to those used in nature but previously unattainable synthetically. By simply varying the deposition conditions, we can now generate entire libraries of re-shaped, mechanically reinforced structures. The unique wetting properties of the arrays additionally confer the capacity for selective functionalization of the nanostructures. These surfaces provide a tremendously versatile foundation for deciphering wetting, crystal growth, self-assembly, optical and biological processes at biomimetic interfaces, and for designing a wide range of multiscale, multifunctional, and multisensitive adaptive materials.

Figure 3:- STEPS: Structural Transformation by Electrodeposition on Patterned Substrates

Method for Making Nanostructured Surfaces

In view of the foregoing, we recognize that there is a need for alternative approaches for providing subwavelength surfaces in order to reduce the

reflection of surfaces. Furthermore, we recognize that in order for such an approach to be commercially viable, it needs to be relatively simple and inexpensive. Briefly, in one aspect, the present invention provides a continuous method for making a nanostructured surface. The method comprises placing a substrate comprising a nanoscale mask on a cylindrical electrode in a vacuum vessel introducing etchant gas to the vessel at a predetermined pressure generating plasma between the cylindrical electrode and a counterelectrode rotating the cylindrical electrode to translate the substrate anisotropically etching a surface of the substrate to provide anisotropic nanoscale features on the surface. As used herein, nanoscale means submicron (for example, between about 1 nm and about 500 nm); nanostructured means having one dimension on the nanoscale; and anisotropic means having a height to width (that is, average width) ratio of about 1.5:1 or greater (preferably, 2:1 or greater; more preferably, 5:1 or greater); and plasma means a partially ionized gaseous or fluid state of matter containing electrons, ions, neutral molecules, and free radicals. The nanostructured surfaces prepared by the method of the invention exhibit a significant reduction in reflectance compared to an unstructured surface comprising the same materials. In addition, the nanostructured articles can be durable and possess scratch resistance. The methods of the invention can be carried out at moderate vacuum conditions (for example, between about 5 mTorr and about 10 mTorr). They can also be carried out as a roll-to-roll (that is, continuous) process. The present invention therefore meets the need in the art for a method of making AR surfaces that is relatively simple and inexpensive. Mechanical methods Mechanical methods of surface structuring encompass methods such as nanomanipulation, nanoindentation and nanoshaving. Nanografting, is an extension of nanoshaving. Based on AFM and STM tools, these patterning

techniques enable the manipulation, damage or removal of structures at the atomic level. Mechanical methods, such as nanoshaving, closely mimic methods currently employed for fabrication of structures and devices on the macroscale, such as the use of a lathe to shave down a bulk material into the desired shape. Not only nanoshaving, but also nanoindentation and nanoscratching have originated from nanotribology studies of friction and adhesion of materials to surfaces. Nanomanipulation Nanomanipulation experiments can represent the absolute limit of fabrication, where individual atoms are placed into the desired pattern or structure on a material surface. Nanomanipulation operations usually are one of two types. The first type involves deposition of a nanoscale species or atom on a surface, and then the use of either an AFM or STM to manoeuvre/push the species into position. The second type of operation involves deposition of a species on a temporary location using standard deposition techniques and then picking up of the species and moving it to its new location. This is usually accomplished by means of manipulating the tip/sample bias of a STM. Structure formation by nanomanipulation processes is at present very time consuming, with no equipment specifically designed for the purpose, hence such experiments are at present mostly confined to research and prototyping. Species and the resolution of assembled species can range from nanoparticles right through to individual atoms. The iconic experiment in the development of such techniques was carried out in 1990 by Eigler and Schweizer180 where xenon atoms were manipulated on a Ni(110) surface in a manner such as to write IBM with the xenon atoms. Also, stadium-type quantum corrals have been assembled by depositing a close line of Fe atoms on a Cu(111) surface. Nanomanipulation techniques are continually being developed and they provide a route to the ultimate limit of fabrication. Nanografting The technique of nanografting is usually (but not exclusively) utilized on surfaces modified with SAMs (figure 4.a) and is achieved by nanoshaving in the presence of a second replacement surfactant molecule with a greater affinity for the surface than the molecule being removed by the AFM tip (figure 4.b).182 Therefore, once the mature SAM is removed from the desired area by the AFM tip it will be replaced with the new incoming SAM (figure 4.c). Nanografting allows for the formation of positive and negative patterns

via tailoring of the incoming surfactant molecule lengths. In order to successfully perform a nanografting operation, there are certain requirements that SAMs should meet.

Figure 4: Diagram showing the process of nanografting; a) a complete SAM is b) shaved away in the presence of a secondary SAM molecule, which c) selectively replaces the original SAM in the regions which were shaved.

The SAMs must be readily removable with the force generated by the AFM tip, but more importantly, the SAMs must form on the surface of the substrate being used in a quick and precise manner. It is for these reasons that thiol SAMs on gold are usually the system of choice for nanografting experiments, due to the way in which thiols rapidly form homogenous monolayers on exposed gold surfaces and formation of the Au-S bond is reversible in solution. Nanoindentation Nanoindentation is a method that was first employed to probe the deformation characteristics of a surface or material using the tip of an AFM. More recently, this technique has been used for fabrication of nanosize wells or indentations on the surface of a material (figure 5.a). The basic principle of nanoindentation fabrication involves: bringing the tip of an AFM in close proximity with a substrate; using the controls of the AFM to lower the tip into

contact with the substrate surface; controlling the force of the lowering operation and hence the depth up to which the tip penetrates into the material surface. The AFM tip is subsequently lifted from the surface and then the substrate or tip is moved to a new position for the repetition of the process.

Figure 5: Diagrammatic representation of nanoindentation, nanoscratching, and nanoshaving fabrication processes from side views and top views: a) nanoindentation is the local deformation of a material formed by indenting the surface; b) nanoscratching is the extended formation of an abrasion on the surface of a material; and c) nanoshaving is the removal of material from the surface of a material.

Nanoscratching Nanoscratching is a further development of the nanoindentation process (figure 5.b). The nanoscratching process involves bringing the AFM tip in contact with a surface and then moving either the substrate or tip ensuring the tip is still in contact with the surface, allowing formation of trench like structures on the substrate surface. Two types of nanoscratching operation can be performed, namely, static and dynamic scratching. The dynamic process involves the operation of AFM tip in an oscillating mode, conversely no oscillation is applied during a static

process. Substrates such as silicon wafers coated with soft materials such as polyimide layers several micrometers thick have been patterned using AFM scratching (figure 6).

Figure 6: AFM image of a furrow scratched into polyimide; inset, cross-section of feature.

Grooves were made by single passes across the surface at loads of up to 490 nN on polyimide. The narrowest achievable grooves using this method and polymer are 20 nm wide and 2 nm deep, fabricated at a force of 100 nN using Kapton-H as the commercial polyimide. Nanoscratched patterns in soft polyimide layers can be transferred through an underlying germanium layer by dry etching. The dry etching removes any remaining polyimide and etches through a small portion of germanium, followed by the removal of underlying PMMA-MAA to form a suspended germanium mask above a lower gold substrate. These mask structures can then be further employed to pattern evaporated metallic layers. The AFM scratching operations are carried out

using sharp silicon tips at speeds in a range of 0.2-2 m s1 and at applied vertical forces in the range 1.5-3 N. These conditions produce furrows diameter of approximately 50 nm. With scratching of PMMA resist films, trenches of up to 50 m in length and widths of approximately 40 nm have also been formed. It has been observed that material can accumulate on either or both sides of the scratch depending on the fabrication direction relative to the AFM tip. Also, feature depth is dependent on the setpoint amplitude of the tip. Nanoscratching can further be employed to pattern much more robust materials than just organic molecules and polymers. Nanoscratching allows alteration of the conduction behaviour of AlGaAs layered heterostructures. Features roughly 4 nm in depth with widths of approximately 120 nm can be fabricated in such materials using standard AFM tips at a force of approximately 50 N. Advances in tip design, such as being able to modify tips with electron beam deposition, allow the user to plough scratches into superconducting materials. Scratches in superconducting materials are used to define a weak link (also known as a Josephson junction) in the superconducting material. More conventional microelectronic materials (e.g. silicon) have also been modified using AFM nanoscratching. It is possible to scratch through the native oxide layer (approximately 2.0 nm in thickness) of silicon (100) wafers using a diamond coated tip at loads in excess of 13 N. Grooves show a uniform V-shape and sizes range from 8 nm in depth at a force of 14 N to 25 nm in depth at a force of 23 N. Widths range from 90 to 240 nm for forces of 14 and 23 N, respectively. As expected, feature size increases linearly with applied force. To further modify the substrate surface, features can be coated in Cu via electrochemical deposition. Nanoscratching fabrication has also been exploited to create nanostructures on aluminium surfaces. Pyramidal diamond tips with a radius of approximately 15 nm have been employed to create square features of approximately 600 nm 600 nm with depth ranges of 1-10 nm. Nanoshaving The Oxford English dictionary definition of shaving is: to scrape away the surface of, to cut down or pare away with a sharp tool, thereby removing very thin portions of the surface. If we transfer this definition into the context of nanostructure formation, we can define nanoshaving as the process of dragging a sharp object, usually an AFM tip over a material to remove selected resist nanomaterial for creating nanometre scale patterns on surfaces (figure 5.c). From this definition, the major difference between the processes of

scratching and shaving is the fate of the displaced material during and after the fabrication process. Shaving is a process, which involves the removal of material, whereas scratching purely involves marking the surface or displacing material from one location to another on the surface. Overall, nanoscratching is a highly useful technique for defining structures in bulk materials, especially soft materials where material can be easily displaced.

Design of Nanostructured Surfaces for Manipulating Cells

Due to current limitations in experimental models, we still lack a general understanding of how cells integrate simultaneous chemical and physical stimuli into a specific output. In addition to the well-studied effect of growth factors, signaling- and adhesion-molecules, over the last decade it has been recognized that physical properties of the extracellular environment also have a profound influence on cell behavior and differentiation. These physical stimuli include mechanical stiffness and topography of the cellular environment, as well as spatial patterns of ligand presentation. The overall aim of this article is to manipulate cell behavior and differentiation via biochemically and mechanically tailored nanostructured two-dimensional (2D) substrates and three-dimensional (3D) scaffolds.

Surface Bio-Functionalization
The geometry of ligand presentation spans different length scales, ranging from nanoscale distributions at the cell membrane to several micrometers for the arrangement of single sites of adhesion at the cellular level. In addition, graded distributions of cell-adhesion molecules and signaling factors play major roles during development and regeneration of multicellular organisms. Microcontact printing (CP) or microfluidic networks (FN) are employed to reliably produce regular patterns or gradients of substrate-bound biomolecules. For membrane bound proteins, a prerequisite for achieving the desired biological effects of a surface-tethered ligand is its correct orientation as well as a suitable surface-ligand separation. Suitable methods to biofunctionalize surfaces with proteins in a specific orientation and conformation therefore need to be worked out. A future challenge in this field is the reliable production of cellular growth-substrates with multiple bio-functionalities that can be spatially controlled.

Forces and Elasticity

In addition to the geometry of ligand presentation, the stiffness of the environment has emerged as a major and formerly overlooked regulator of cell behavior and fate. Cells do not only apply forces to their growth substrate, they also measure elastic modes in their environment and react to them. As a consequence, cell behavior, including stem-cell differentiation, changes dramatically as cells are grown on increasingly softer substrates. Hence, a profound understanding on the influence of elasticity on cell behavior not only depends on the fabrication and calibration of flexible growth substrates but also on precise measurements of cell adhesion forces on biofunctionalized surfaces.

Three-Dimensional Growth Substrates

Cells grown on 2D tissue culture substrates often differ considerably from those grown in more physiological 3D environments regarding their morphology, cell-cell and cell-matrix interactions, and cell differentiation. In vitro 3D models currently used include scaffolds fabricated from purified ECM molecules and synthetic biomaterials. However, the geometry and physical properties of these scaffolds are difficult to control. In a collaborative approach, direct laser writing has successfully been applied to realize threedimensional designer Petri-dishes for the study of cell growth. The longterm goal of this project is to realize flexible 3D templates with multiple biofunctionalities, e.g., adhesive RGD-peptides or ECM-proteins in combination with signaling molecules like cadherins or ephrins. For that purpose, flexible monomeric as well as polymeric materials decorated with wavelength specific photo-click functionalities will be developed in the group of Christopher Barner-Kowollik, which can react rapidly in a spatially highly resolved fashion with suitably functionalized biomarkers as counterparts in the photo-induced ligation chemistries. These novel materials will allow for systematic studies of the effects of spatial ligand distributions and mechanical scaffold stiffness on cell behavior and stem cell differentiation in 3D environments.

Nanostructured surfaces from size-selected clusters

The deposition of ionized beams of size-selected atomic clusters onto welldefined substrates represents a new method of preparing nanostructured surfaces, with lateral feature sizes in the range 110 nm. 'Pinning' of the

incident clusters prevents the diffusion of the clusters on the surface, and thus preserves the gas-phase cluster size, even at room temperature and above. At the same time, advances in diblock copolymer techniques allow the preparation of ordered two-dimensional arrays of clusters. Here we discuss the creation and applications of these nanostructured surfaces, ranging from the fabrication of semiconductor nanostructures to the immobilization of protein molecules.

Nanostructured Surfaces for Bone Biotemplating Applications

A major goal of orthopedic biomaterials research is to design better surface chemistries and configurations to control behavior of bone cells such as osteoblasts. Nanostructured architecture significantly affects the response of several cell lines. In this work, nanostructured surfaces were prepared by vapor liquid solid growth of silicon nanowires from size-controlled gold colloid catalysts deposited on fused silica substrates. The lengths and surface densities of the nanowires were varied to assess the effect of these parameters on bone cell response. Osteoblasts were seeded on nanowire surfaces to investigate both short-term adhesion and proliferation and longterm functionality and matrix production. Cell adhesion and proliferation were characterized using a standard 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay and cell counting for up to 4 days of culture. The total protein content, alkaline phosphatase activity, and matrix production were quantified using standard colorimetric assays for up to 4 weeks of culture. Matrix production was also characterized by measuring surface concentrations of calcium and phosphorus using X-ray photoelectron spectroscopy. Further, scanning electron microscopy was used to investigate osteoblast morphology on nanostructured surfaces. Over the 4-week study, the nanostructured surfaces demonstrated improved osteoblast adhesion and proliferation and increased alkaline phosphatase activity and matrix production compared to non-nanostructured control surfaces.