F Rossi

P Colpo

Applications and challenges of plasma processes in NanoBiotechnology

Applications and challenges of plasma processes in nanobiotechnology

Introduction

Since many years, the fabrication of surfaces presenting micron and submicron patterns with a welldefined geometry and chemistry has been the object of many research and publications in NanoBiotechnology [START_REF]Nanobiotechnology Concepts, Applications and Perspectives[END_REF][START_REF] Walter | [END_REF]3,4]. For instance micro and nano-patterned surfaces with contrasted physico-chemical functionalities -e.g. protein adhesive/non-adhesive, hydrophilic/hydrophobic, acid/base…-provide unique tools in a large field of applications such as tissue engineering [START_REF] Khademhosseini | Micro and Nanoengineering of the Cellular Microenvironment: Technologies and Applications[END_REF][START_REF] Curtis | [END_REF]7], cell behavior investigations [4,[START_REF] Khademhosseini | Micro and Nanoengineering of the Cellular Microenvironment: Technologies and Applications[END_REF]8,9,10,11,12,13], or microfluidics [14,15]. Nanostructured surfaces have properties leading to very special effects related either to the kinetics of biochemical reactions at the interface [16,17,18,19], or to their interaction with liquid (superhyhophobic or hydrophilic surfaces) [20,21,22].

For a routine and cost-effective use of such micro-nanostructures, the fabrication method must rely on a well chosen combination of surface functionalisation and patterning techniques. Numerous methods have been developed for the production of such surfaces and are based essentially on the use of surface functionalisation techniques e.g. by Self Assembled Monolayers (SAMs) [23,24,25] or electrodeposition of functional polymers [26,27], combined with patterning techniques such as microcontact printing [28,29,31] or classical or advanced photolithography [30]. SAMs provide extremely well controlled surface chemistries but have several limitations particularly in relation to the substrate that has to be used -Gold or Silicon for thiol based or silane based chemistry respectively.

Besides, microcontact printing is the most widely used techniques for proteins and SAMs patterning but has some inherent limitation in terms of resolution due to stamp roof collapsing when high aspect ration structure are fabricated [31]. This is why other possibilities have been investigated, in particular those based on functional plasma polymers that can be deposited on virtually any substrate and at low temperatures. The plasma processes allow, by the choice of the right precursors, to deposit materials a very large range of properties and chemical functionalities, such as -COOH [32], -NH 2 [33,34] and their combination [35] or anti fouling polymers based for instance on polyethylene oxide (PEO) [36,37]. An excellent review of the different functionalities produced up to now can be found in [38].

Due to their variable degree of cross-linking linked to the discharge conditions, these coatings are resistant to classical polar solvents, making them compatible with almost all standard patterning techniques including lift-off processes [39,40,41], but can also be selectively dissolved upon request. Plasma polymers can be then assembled in structures with sub-micron dimensions by combination of colloidal lithography [39,44], deposition and etching, or as well as by direct modifications by electron beam lithography. Reactive etching is also a straighforward method to nanostructure polymer layers and then tune their wettability from superhydrophilicity to superhydrophobicity. We develop here a summary of the work we performed, based on the use of plasma assisted deposition and plasma etching of functional polymers to produce patterns by a combination of techniques, namely colloidal and electron beam lithography and microcontact printing to address several issues in Nanobiotechnology: protein adsorption, high-sensitivity biodetection, control of stem cell maintenance and differentiation. We show thereby that plasma techniques provide a large range of interesting possibilities that offer many unique opportunities that cannot be easily obtained by other techniques, however at the expense of some degree of compromise for the control of surface chemistry. We provide general experimental details and present the combination of processes that have been developed for protein adsorption studies and cell cultures.

Plasma based techniques.

Plasma polymerisation of functional films

In the work summarized below, two different types of plasma polymers were used, namely plasma polymer of acrylic acid (pAA) containing COOH moieties and a protein and cell repellent plasma polymerized Polyethylene Oxide (PEO)-like surface. The choice of the acrylic acid is motivated by the fact that the carboxylic functions are able to promote a large amount of chemical reactions with different biomolecules (enzymes, antibodies, DNA…). However, other types of plasma polymers could be used as well (e.g. providing amine, epoxy moieties), to change the properties of the active layer and its interaction with adsorbed proteins (conformation, bioactivity, packing, denaturation etc…). The PEO-like polymer is resistant to adsorption of proteins in liquid solution but we will see that proteins are readily attached to the PEO-like surface in dry conditions, as the ones used in micro contact printing: this property offers a decisive advantages in terms of process simplicity and robustness [42,43].

The deposition process of the plasma polymerized PEO-like and PAA films are described in details elsewhere [32,44,45,39]. Briefly, the systems used are capacitively coupled reactors with two symmetrical parallel-plate electrodes. For PEO-like coating, plasma polymerization is carried out by using a radio frequency generator (13.56 MHz) in pulsed mode (time on = 10 ms, time off = 100 ms, nominal power = 5 watts) of pure Diethylene Glycol Dimethyl Ether (Di-glyme), ((CH 3 OCH 2 CH 2 ) 2 O) vapors (Sigma Aldrich, used as received). For PAA, acrylic acid vapors are mixed with Ar in pulsed RF mode (13.56 MHz; pulsed plasma: time on = 4 ms; time off =36 ms). The films obtained are uniform and their thickness can be adjusted between 20 and 200 nm. Characterisation of the PAA and PEO films can be found in [32,36]. Typically, the PAA films contain varying degree of carboxylic functions, depending on the power applied during the discharge. As discharge power is increased, the O/C ration as measured by XPS decreases from 50% down to a few percent, increasing the stability of the films, which become insoluble in water for powers larger than 30W. For PEO films, similar results are obtained and the antifouling properties of the films are directly related to the importance of the C-O-C peak in the C1S XPS spectrum, which decreases as the discharge power increases [36,46]. So the composition of the films, as well as the degree of cross-linking, vary continuously as the discharge power is increased, which make these films less well-defined as compared to SAMs for instance.

Surface patterning using colloidal lithography

The patterning of a surface by colloidal lithography consists in creating a 2-D crystalline structure of polystyrene (PS) beads on the PAA, those beads being used as masks during subsequent etching and deposition operation [47,48]. The mask self-assembly is performed by evaporation of solvent containing the PS beads in suspension. During the de-wetting and evaporation, the colloidal particles can be arranged in different geometrical configurations in order to minimize the free energy of the system [39,44,45].

First, the PAA layer (typically thickness from 10 to 200 nm) is deposited on a Si substrate as described above (figure 1a). Then, a micro-drop (Drop Volume = 5 μl) of PS beads (typically of 200 to 500 nm diameter) suspension is deposited on the 1x1 cm 2 samples surface. The bulk volume of the drop is removed by a micro-pipette, in order to obtain a very thin layer of PS beads suspension on the surface. The sample is then spun to produce a slow evaporation of the liquid. It is thus possible to create macroscopic homogeneous areas covered by mono-layered nano-beads of few mm 2 with a surface coverage ranging typically of 90% (figure 1b). The nano-mask pattern is then transferred to the PAA layer by O 2 plasma etching (Figure 1c). The etching operation has to be accurately controlled in order to create the nano-structures on the PAA layer and avoid over-etching. Once the polymeric nano-structures are created on the surface, the chemical contrast is formed by the plasma deposition of the PEO-like coating. The anti-fouling layer is deposited on the surface through the nano-masks (Figure 1d). The deposition is isotropic and the regions unprotected by the masks (both the partially and the totally uncovered regions) are coated. The residual nano-sphere mask is then lifted-off by an ultrasonic treatment of the surfaces in ultra-pure water. The surface finally obtained is composed of areas of carboxylic functionalities of a few hundreds of nm surrounded by the anti-fouling layer (Figure 1e). The SEM and AFM images of Figure 2 show the typical 'nanodome' structure with an hexagonal distribution, the dimensions of which (typically a few hundreds of nm in diameter and few tenth of nm in height) depend on the initial size of the beads (here 500nm), the PAA layer thickness and the etching time. In particular, the distance between active areas can be adjusted by the duration of etching, which reduces the dimension of the mask and leaves the PAA domains farther apart. Structures of 200 nm up to 1000 nm and more can be obtained with this process, with coverage typically of 90% on a few mm 2 . Details of the experiments are given in [67,68]. The shape, aspect ratio of the nanostructures can be controlled as well by tuning the plasma parameters [56]. For instance, reducing the working pressure leads to high aspect ratio structure thanks to the anisotropic character of the plasma etching [56]. Numerical simulations have been performed to predict the nanostructure shape as a function of plasma parameters [49]. This study shows that parameters such as substrate bias, electron temperature, and plasma density influence the ratio of ion flux hitting the nanoparticle interstice and the nanoparticles themselves. Fine-tuning of these plasma parameters can lead to a very accurate control of this plasma aided nanofabrication process. Pulsed bias can as well be used to finely tune the shapes of nanostructures in template-assisted nanoarray fabrication [50].

Direct surface patterning using electron-beam lithography

However, colloidal lithography is limited in the geometry of the patterns that can be produced (shape of the patterns, pitch, size, and distribution). This is why more conventional lithography techniques offer a large advantage, at the expense of time and cost. It has been shown that plasma polymers can be patterned by direct photolithography [51] but also with conventional processes using photoresists [36]. Nanopatterned surfaces can be as well fabricated by E-beam lithography using PMMA as resist [52,53,54]. Nanopatterns of typically 100nm can be obtained by this approach. Lower dimensions can be obtained by optimizing the sequence of developing. E-beam lithography using PMMA as resist has been combined with plasma polymers enabling chemically nano contrasted surface fabrication [55]. In our work, we used the possibility of cross-linking water soluble PAA directly by the electron beam. The process of fabrication is described in Figure 3. First, a 20 nm layer of PEO-like polymer is plasma deposited on a Si substrate in order to form the non-adhesive matrix [28]. Then a second water-soluble layer is deposited by plasma polymerization of acrylic acid in the monomer rich region. The conditions of deposition of this layer are similar to the one of PAA previously described but with a lower power of deposition (nominal power of 10 watts, operating in pulsed mode with a t on of 4 ms and a duty cycle of 10 %). The film thus obtained is not cross-linked and dissolves readily in water. After deposition of the water-soluble layer, the surface is exposed to the electron-beam with an energy of 20 keV and incident dose of 1600 mC/cm 2 , then immersed for 5 minutes in water and dried under nitrogen flow. Local cross-linking of the polymer is thus used to produce the stable domains of PAA. The chemistry of the stabilized PAA layer has been determined by µ-spot XPS on a micro patterned surface (100 x 100 mm 2 ). XPS analysis reveals that the chemistry of the stabilized layer is modified as compared to the initial film, because of the cross-linking occurring during electron beam irradiation. The chemistry of the film thus produced is similar to plasma polymerized acrylic acid layer produced at optimum plasma density and used for bio specific applications with a percentage of retention of COOH groups around 7-10 % [56]. Figure 4 shows AFM and SEM images of functional nano-structures with different heights and shapes created with the patterning process described in this study. Well-defined features sizes down to 200 nm in lateral resolution can be fabricated with several heights (from 80 nm to 500 nm). It must be noted that the resolution limit obtained (or the order of 100nm) depends on the e-beam energy and can be much improved by working at lower energies (typically down to 5keV) provided by a dedicated instrument. These images proves the feasibility of combining electron-beam lithography and plasma processes for direct patterning of surfaces with well defined nano-scale dimensions. More experimental details can be found in [69].

Plasma polymer PEO-like films as platform for proteins and cell patterning.

Protein and cell patterning plays an important role for biological and biochemical applications [30]. Challenges reside in developing of surfaces that can support for long time period well-defined biomolecule patterns in a non-adhesive matrix. A difficulty is that protein adsorption at the interface, which results from a complex interplay of electrostatic, hydrophobic and chemical forces [58] renders the results hard to control. An improvement in the spatial distribution of protein adsorption onto surfaces can be obtained by using microcontact printing. The advantage of the approach is the transfer directly from the stamp to the surface the chemicals, ligands or proteins of interest [59,60,61]. This technique has previously been successfully applied in producing highly contrasted chemical functionalities with sharp contours [62] and to pattern proteins for the study of bio/non-bio interactions and for cell attachment [43,63,64,65]. The main difficulties to design these surfaces concerns le anti fouling properties of the background to keep the proteins or cell microarrays stable in time. In our work, we have optimised a platform based on plasma polymerized PEO like-films for the patterning of human umbilical cord blood -derived neural stem cells. The goal is to study cellular maintenance and differentiation when interacting with polypeptide micropatterns on a biologically non-adhesive surface [49][50]. To produce these surfaces, microcontact printing has been used as a mean to deposit controlled, geometric patterns of Poly-L-lysine (PLL) and fibronectin (FN) on glass substrates previously coated with a protein resistant, cell repellent plasma polymerized PEO film. The PEO layer has the important characteristic of being cell repellent in solution but protein adhesive under the dry conditions used during the microcontact printing operation [42,66]. The composition of the PLL or FN inking solution and the pH were critical to the quality of the pattern, which under optimised conditions could be effectively transferred to the substrate where it was stable for more than four weeks [43,65]. Details of the experiments are reported in [43]. Well-defined microstructures of PLL over a protein free background have been fabricated, as can be seen in figure 5, which shows fluorescence microscopy (Figure 5 A-C) and Time of Flight -Secondary Ion Mass Spectroscopy (ToF-SIMS) images (Figure 5 D-G) of the polypeptide pattern. The geometry of the PLL pattern has been chosen to suit the desired cell response required for the study. Long inter-patch distances (squares 165µm, spaced 400µm) are preferred for cell migration studies, while short distances (squares 100µm, spaced 100µm) are better for cell differentiation and outgrowth projection studies. The extension of cell protrusions was also monitored on interconnected islands by bridging the squares with 15µm width lines (Figure 5C). The PLL ToF-SIMS positive imaging, ascribed to C-N bonding (Figure 5D,5F) clearly shows a large difference in intensity of this ion between the printed area and surrounding PEO background. In comparison, the images derived from peaks associated with PEO, given by C 3 -H 3 -O bond mapping (Figure 5E, 5G), show a much less 200 nm 400 nm 400 nm distinct contrast between the printed areas and the surrounding background PEO. From these results it can be concluded that the PLL is only delivered in the squares while the surrounding background is totally PLL free and that the PLL layer is sufficiently thin or has incomplete coverage (at a sub-micron level) that a significant contribution to the PEO ion image comes from the underlying layer. Fluorescence imaging of the samples after immersion in water for one month revealed a good stability of the PLL attachment to the PEO substrate.

Application to bio interfaces

Protein adsorption on nanopatterns

The nanostructures produced by colloidal and electron-beam lithography have been extensively studied with different biological tests and protein adsorption experiments [17,67,68,69,70,73]. We present below two significant examples indicating the particular behaviour of nanostructures as compared to non-patterned surfaces. In order to illustrate that the potential of these surfaces as biosensors platforms, nanostructured surfaces produced by e-beam stabilization of acrylic acid on a PEO background have been exposed to a fluorescent Poly-L-Lysine (PLL) solution. The fluorescent contrast observed in figure 6 after the incubation shows that PLL is adsorbed selectively on the PAA domains and that the PEO-like has kept its anti adhesive properties. These results show that plasma polymers combined with E-Beam lithography are a very promising technique to produce nanostructured surfaces with chemical nano contrast for protein nanopatterning in sensing and biology applications. Quartz crystal microbalance with dissipation (QCM-D) (Qsense, Sweden) has been used for quantitatively compare the mass of proteins absorbed on uniformly functionalized and nano-structured surfaces fabricated by colloidal lithography. The mass of proteins absorbed on the crystals was estimated by the Sauerbrey equation, corrected for the water coupled to the proteins. For the determination of variation of frequency (i.e. mass absorption) due to protein adsoption, 25% of the measured mass, considered as the contribution of the water in the measurement was subtracted: this estimate is consistent with the typical average retention of water in proteins [22]. The number of absorbed molecules was then calculated by dividing the estimated mass by the molecular weight of the molecule and by multiplying it by the Avogadro's number. Both the probe (human IgG) immobilisation and the antibody/antigen (human IgG/Anti-human IgG) recognition have been monitored on-line. The nano-patterned quartz resonator was held in the measurement cell and the baseline was recorded when the carrier buffer (PBS: 10 mM PBS, pH 7.4) was flowing in the reaction cell. For the probe immobilisation, Human IgGs with concentrations ranging from 1 to 15 µg/ml in PBS solution were injected in the liquid cell. The results in Figure 7 are expressed as the difference between the values measured after the rinsing step and before the sample injection. The same buffer solution was used for all the experiments. In these conditions, the variation is related only to compounds fixed on the surface during the reaction [71,72]. In the case of nano-structures, the areas covered by PEO (anti fouling matrix between the nanostructures and the areas non-structured areas) are considered totally protein repellent since the measurement by QCM did not show detectable protein adsorption on PEO layers (data not shown). Figure 7 shows the result of the immuno-reaction as a function of primary antibody concentration. It can be seen that limit of detection is shifted toward lower concentration, with best results obtained at smaller nanopattern dimensions (200 nm nanostructure). Similar enhancement has been observed with different techniques (SPRi, QCM, ELISA test) [67,68,73,74]. The level of amplification depends on the size of the nanostructure and increases as the size of the patterns decreases. A possible explanation is linked to a better orientation of the antibodies on a nanostructured surfaces and a reduction of the steric hindrance linked to the antibodies adsorption on the edges of the structures, making binding reactions more efficient on the nanostructures as compared to uniform surfaces [75].

Cell culture experiments

Fibronectin and PLL microcontact printed surfaces were used to study the patterned growth of neural stem cells and as a model to study different processes regarding stem cell neural lineage commitment. Human umbilical cord blood derived neural stem cells (HUCB-NSC) [76] have been incubated on the polypeptide functionalized surfaces. Previous studies in non-patterned surfaces [77] showed that the density of plating and the type of polypeptide used for attachment can change the HUCB-NSC commitment to a specific lineage (neuronal, astrocytic, olegodendroglial). In this work, the cells attached to the FN and PLL domains grow following the pattern but show a behaviour that is sensitive to both the geometry and the cell density. It has been reported that different cell morphologies can influence stem cell commitment [78]. In this study, it was noticed that cells on densely crowded islands stayed in non-differentiated mode longer than low populated samples where cell commitment to a differentiated mode is favoured. By varying the conditions of culture (with and without serum, with and without growth factor, on FN and PLL patches), we found that the differentiation of HUCB-NSC could be modulated to a large extent, the population of neurons, astrocytes and oligodendrocytes varying between 10% and 80% depending on the conditions (see Figure 9) [11,76,77]. Reproduced with permission from [11]

Conclusions and perspectives

There is a constant need of new technologies for research innovation in healthcare, environment monitoring and toxicology studies. For these applications, there is a recurrent requirement for the development of new devices that can be used to detect disease marker, pollutant or to improve the reliability of in vitro testing. Bio/non bio interface is the common cornerstone for the fabrication of such a device. Proteins and cells must be immobilised on surfaces in a manner that all their functionality are kept intact. In this domain, surface functionalisation and patterning plays a pivotal role. Many articles have been published on this topics using a variety of different methodologies. Nevertheless, the proposed techniques are often difficult to implement routinely and suffer from reproducibility issues. Most of these methods are based on non-trivial wet chemistry (SAMs, hydrogels…). This paper shows that plasma technology represents an interesting alternative. Derived from plasma processes using in semi conductor industry, the processes of deposition or etching operation quality can be highly controlled by mass or optical spectrometry and process fully automated. In this article, we have shown that plasma technologies, and in particular deposition and etching can be combined with colloidal and electron-beam lithography as well as microcontact printing for production of high quality micrometric and sub-micrometric patterned surfaces with arbitrary geometries and controlled chemistry. The resolutions that can be obtained vary with the techniques used from the micrometer scale down to a few tens of nm. We have seen that the major advantage of plasma polymers is related to the large range of properties that can be obtained, and their relative stability with time and against different solvents. Moreover, due to the versatility of the plasma processes, these techniques can be easily extended to a large panel of surface chemistries without constraints in terms of substrates, and many studies are currently carried out on the use of alternative chemistries, which could benefit from the same approach and extend its application. This is however done at the expense of control of chemistry of the surface, the plasma polymers chemical properties being intrinsically more ill defined as compared to SAMs for instance. We also feel that the combination of conventional biochemistry/chemistry and plasma processing techniques opens a large range of possibilities and offer very interesting opportunities for cell cultures as well as bioanalytics. The approach can also be extended by using for instance other combinations of techniques (ion beam, UV treatments) or stimuli responsive materials [79,80] for e.g. modulating the reactivity of the films and developing new structures. A major challenge of these processes will be optimization of the robustness and control of surface properties of the platforms obtained to make them applicable at a large scale at low cost. Plasma technology has all the necessary requisites to penetrate successfully all life sciences related applications.

Figure 1 .

 1 Figure 1. Experimental procedure for colloidal lithography: a. Plasma polymer deposition of PAA; b. Transfer of PS nanoparticle beads on the substrate; c. O 2 plasma etching; d. Plasma deposition of PEO-like plasma polymer; e. Nanoparticles lift-off by ultrasonication treatment. (AA : acrylic acid, PEO: Poly-ethylene oxide).

Figure 2 .

 2 Figure 2. SEM and AFM pictures of the structures obtained by colloidal lithography. Reproduced with permission [57]

Figure 3 .

 3 Figure 3. Electron-Beam lithography on plasma polymers: A PEO layer is first deposited on a Si substrate (a). A second layer of unstable acrylic acid plasma polymer is deposited on the PEO (b). ebeam irradiation stabilizes locally the PAA (c). After washing, one obtains a nanostructure of stable PAA domains on a non-adhesive PEO background (d).

Figure 4 .

 4 Figure 4. SEM (left) and AFM (right) pictures of nanostructures obtained by e-beam patterning of an unstable PAA film. Typical dimensions in height can vary from 100 to 500 nm.

Figure 5 .

 5 Figure 5. Fluorescence microscopy images of the PLL patterns. Long inter-patch distances (A) are design to study migration while close islands (B) and islands connected by lines (C) are used for monitoring outgrowth projections. ToF-SIMS mapping showed good chemical contrast between PLL positive areas (D, F) recognize by C-N, and PEO-like positive background (E, G) ascribed to C 3 -H 3 -O.Reproduced with permission from[11]

Figure 6 .

 6 Figure 6. (a): SEM and fluorescent (included inside the SEM image) of nanopillars (h= 100nm, d= 200 nm) of stabilized PAA on PEO-like surface and corresponding relative fluorescent intensity after incubation with PLL for different height and pitch of pillars. Reproduced with permission from [69].

Figure 7 .

 7 Figure 7. Dose-response curves for the flat ppAA and the nanostructured surfaces. The solid lines are the sigmoidal-shaped functions best-fitting the calibration curves.The curve for ppAA is reported in each graph for direct comparison with the nanostructured surfaces. Reproduced with permission[71]

Figure 9 .

 9 Figure 9. Percentage of HUCB stem cells in different states (undifferentiated, astroneurons, astrocytes and neurons) depending on the culture conditions, on FN and PLL microcontact printed surfaces.Reproduced with permission from[11]


Evangelos Gogolides

Angeliki Tserepi

Vassilios Constantoudis

George Kokkoris

Dimitrios Kontziampasis

Katerina Tsougeni

George Boulousis

Marilena Vlachopoulou

Controlling roughness: from etching to nanotexturing and plasma-directed organization on organic and inorganic materials

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Lithography followed by plasma etching is the main top-down approach for micro and nanopatterning. Usually, during plasma etching ions "enhance" the removal of matter by neutrals and induce etch anisotropy (i.e. etching proceeds only towards the direction of impinging ions, which are accelerated perpendicularly to the substrate). However, microelectronic material etching has suffered for years from the so-called "grass" formed on plasma etched surfaces: The existence of any unetchable residues on the surface (or inside the material being etched) and the etch anisotropy lead to columnar nanostructure formation (grass), the column being protected by the unetchable material. Scanning-electronmicroscope (SEM) images of grass among structures are standard textbook material of etching problems in microelectronics fabrication classes.

On the other hand, grass may be seen as simply nanoroughness, or as desired nanotexture of a surface to be exploited for several applications. For example, if such a rough surface is coated with a low-surface-energy-film an increase in hydrophobicity will be observed leading to dramatic increases of the contact angle of water and oils [START_REF] Vourdas | Nanotextured super-hydrophobic transparent poly(methyl methacrylate) surfaces using high-density plasma processing[END_REF] (2), [START_REF] Tserepi | Method for the fabrication of high surface area ratio and high aspect ratio surfaces on substrates International Application No[END_REF], and eventually to a surface on which liquids roll, thus permitting self-cleaning action (4), [START_REF] Crick | Preparation and Characterisation of Super-Hydrophobic Surfaces[END_REF] [START_REF] Bhushan | Natural and biomimetic artificial surfaces for superhydrophobicity, self-cleaning, low adhesion, and drag reduction[END_REF] of the surface. Another example where nanotexture is beneficial is when antireflectivity is desired: Etch-induced nanoroughness is known to reduce reflectivity for both Si (the "black Silicon" being a well known example [START_REF] Jansen | Black silicon method: X. A review on high speed and selective plasma etching of silicon with profile control: an in-depth comparison between Bosch and cryostat DRIE processes as a roadmap to next generation equipment[END_REF]) and polymers for which the so called "nanomotheye" plasma roughening has been proposed [START_REF] Kaless | NANO-motheye antireflection pattern by plasma treatment of polymers[END_REF]. In fact for polymers plasma nanotexturing can simultaneously achieve optical transparency, antireflectivity and superhydrophobicity [START_REF] Vourdas | Nanotextured super-hydrophobic transparent poly(methyl methacrylate) surfaces using high-density plasma processing[END_REF][START_REF] Gogolides | Micro and nano structuring and texturing of polymers using plasma processes: Potential manufacturing applications[END_REF] . We would therefore like to emphasize that contrary to the undesirable effects of "grass" for nanoelectronics, controlled nanotexture formation may be valuable for nanomanufacturing of both large areas as well as devices, when one or more "smart" functionalities may be desired [START_REF] Gogolides | A review of line edge roughness and surface nanotexture resulting from patterning processes[END_REF] . As the Greeks would say "there is no bad thing without a good side-effect", in other words one should not always cut the grass but rather control its growth.

Despite the fact that during plasma nanotexturing one is removing material, the result is the formation of grass-like nanostructures on the etched surface. This implies that plasmas may be used for the assembly of nanostructures even when used in the "subtractive-etching mode". The subtractive mode has been used to create nano-grass on polymers [START_REF] Vourdas | Nanotextured super-hydrophobic transparent poly(methyl methacrylate) surfaces using high-density plasma processing[END_REF] (2), [START_REF] Tserepi | Method for the fabrication of high surface area ratio and high aspect ratio surfaces on substrates International Application No[END_REF], to form nanocolums after random deposition of metallic etch inhibitors on the surface and subsequent etching [START_REF] Seeger | Fabrication of silicon cones and pillars using rough metal films as plasma etching masks[END_REF], and recently to create Silicon nanotips and silicon nano-grass in Hydrogen /Argon mixtures [START_REF] Levchenko | Silicon on silicon: self-organized nanotip arrays formed in reactive Ar+H2plasmas[END_REF][START_REF] Xu | Self-organized vertically aligned singlecrystal silicon nanostructures with controlled shape and aspect ratio by reactive plasma etching[END_REF]. Using the "additive mode" on the other hand, several groups have also demonstrated assembly of random nanostructures from depositing plasmas. In fact silicon and other inorganic material nano-grass growth by plasmas is a very active field as evidenced by several publications [START_REF] Tam | Deterministic shape control in plasma-aided nanotip assembly[END_REF], [START_REF] Levchenko | Nanostructures of various dimensionalities from plasma and neutral fluxes[END_REF], [START_REF] Hsu | Generally applicable self-masked dry etching technique for nanotip array fabrication[END_REF][START_REF] Park | Formation of silicon nitride nanopillars in dualfrequency capacitively coupled plasma and their application to Si nanopillar etching[END_REF][START_REF] Gharghi | Formation of nanoscale columnar structures in silicon by a maskless reactive ion etching process[END_REF] including a recent review [START_REF] Zheng | Plasma-Assisted Approaches in Inorganic Nanostructure Fabrication[END_REF]. Plasma has thus been proven to direct the assembly of both inorganic nanostructures [START_REF] Levchenko | Plasma-driven selforganization of Ni nanodot arrays on Si(100)[END_REF], [START_REF] Zheng | Plasma-Assisted Approaches in Inorganic Nanostructure Fabrication[END_REF], and organic nanostructures (1), [START_REF] Tserepi | Nanotexturing of poly(dimethylsiloxane) in plasmas for creating robust super-hydrophobic surfaces[END_REF], while it has been claimed that plasma nanofabrication mimics nature in the creation of the cosmos [START_REF] Ostrikov | Plasma nanoscience: From nature's mastery to deterministic plasmaaided nanofabrication[END_REF]. Thus, additive or subtractive plasma directed assembly emerges as a new field, under the broad title plasma nanoscience.

However, in all the above plasma directed assembly efforts pattern order and periodicity have not been looked at in detail or are missing as random patterns are formed. An exception to this rule has been the organization of parallel ripples on stressed or lithographed poly(dimethyl)siloxane (PDMS) films ( 22),( 23) upon plasma oxidation. However, the formation of organized structures is often more desirable than random ones. For example, fabrication of organized, periodic nanodot, nanocolumn or nanopore arrays on surfaces is extremely useful in many fields ranging from biology, to hard disk drives, and from catalysis to photonics. Typically such structures are made with: a) either expensive nano-lithography (such as immersion, electron beam, Extreme Ultra Violet, or nanoimprint lithography) followed by smooth anisotropic plasma etching, b) self-assembly processes using block copolymers or colloidal nanospheres [START_REF] Boal | Selfassembly of nanoparticles into structured spherical and network aggregates[END_REF][START_REF] Park | Enabling nanotechnology with self assembled block copolymer patterns[END_REF][START_REF] Hulteen | Nanosphere lithography: A materials general fabrication process for periodic particle array surfaces[END_REF][START_REF] Ting | Plasma etch removal of poly(methyl methacrylate) in block copolymer lithography[END_REF], which define an organized pattern usually on an intermediate polymeric layer, subsequently transferred to the substrate with smooth, anisotropic plasma etching [START_REF] Ostrikov | Plasma nanoscience: From nature's mastery to deterministic plasmaaided nanofabrication[END_REF][START_REF] Ting | Plasma etch removal of poly(methyl methacrylate) in block copolymer lithography[END_REF].

Very recently we have demonstrated that plasma etching alone can be used for plasma directed organization to BOTH create the organized nanodot pattern on any nonspecialty commercial polymer, and then transfer it to the substrate (28) [START_REF] Vourdas | Plasma directed assembly and organization: bottom-up nanopatterning using top-down technology[END_REF] . This finding has promoted plasma etching, an inherently top-down technology also as a bottom-up nanomanufacturing tool. The duality of plasma etching (i.e. create and transfer the pattern) is indeed unique, and may change the way we have been doing nanofabrication. Additionally it has added the dimension of plasma directed organization in the plasma nanoscience toolbox. To our knowledge only ion beam etching has up to now led to ion-beam directed organization, as nicely reviewed in several recent papers [START_REF] Frost | The shape and ordering of selforganized nanostructures by ion sputtering[END_REF][START_REF] Frost | Surface engineering with ion beams: From self-organized nanostructures to ultra-smooth surfaces[END_REF][START_REF] Gago | Self-organized surface nanopatterning by ion beam sputtering in Toward Functional Nanomaterials[END_REF]. It is the authors' opinion that despite the "cleaner" environment of ion beams, plasmas offer more opportunities and mechanisms due to chemical and physical synergies, and are inherently more cost-effective. Thus, plasma directed assembly and plasma directed organization are very promising emerging fields with a wealth of applications, modes (additive or subtractive), and materials to act upon.

It is the purpose of this progress and perspective paper to focus on plasma nanotexturing and plasma directed organization using mainly etching chemistries, i.e. a subtractive approach. This is to be complemented by the approaches of other groups which use mainly additive processes [START_REF] Zheng | Plasma-Assisted Approaches in Inorganic Nanostructure Fabrication[END_REF]. We would like to encourage however our colleagues using depositing plasmas to look into the issue of nanostructure organization, as conditions may exist, where order is achieved. We believe that the phenomena we describe are generic, despite the fact that they are applied mostly for etching plasmas. Our approach in assembling nanostructures is twofold: either to do so, on a surface from which material is removed, thus ensuring that the nanostructures are of the same nature as the surface being etched, or to use a sacrificial soft (usually polymer layer) for the nanostructure assembly, and then transfer the pattern to an underlying substrate. Crucial for all the above is the plasma-wall interaction and this will be the focus of this paper.

Before moving on it is good to clarify some terminology. We have been using the terms plasma nanoroughening, plasma nanotexturing, and plasma directed assembly with no difference to denote random nanostructure creation, although nanotexturing could be used more appropriately for soft materials, and plasma directed assembly is a rather broader term initially used for depositing plasmas. To refer to organized structure formation we will be using the term plasma directed organization or simply plasma organization.

What is different in plasma nanotexturing and plasma organization?

In the subtractive mode of patterning the main action is removal of material using typically plasma etching of selected areas of a substrate. While the main action is etching, a very small amount of co-deposition is also taking place. In general co-deposition is so small that it goes by unnoticed in most plasma etching processes. This co-deposition may typically come from sputtering of unetchable materials from the metallic or dielectric reactor wall (we call these "hard" etch inhibitors [START_REF] Kokkoris | Dual nanoscale roughness on plasma-etched Si surfaces: Role of etch inhibitors[END_REF]), from depositing material from the gas phase species, or from etchable wall materials (we call these "soft" inhibitors(33)) as shown schematically in figure 1. Normally all such co-depositing species would cause some type of "grass" formation after prolonged etching, if some etch anisotropy is present. As we have shown, such codeposition may lead to the so called roughness instability, i.e. almost linear increase of roughness with time [START_REF] Kokkoris | Dual nanoscale roughness on plasma-etched Si surfaces: Role of etch inhibitors[END_REF]. However, the role of the etch inhibitors is not limited to the roughness instability. Experimental results [START_REF] Vourdas | Plasma directed assembly and organization: bottom-up nanopatterning using top-down technology[END_REF] show that ordered periodic morphologies can be induced on plasma etched polymeric surfaces. Thus, what is different in plasma directed assembly and organization is that the control of the ratios of ions/neutrals/inhibitors as well as the selectivities and etching yields allows control of the order, the height, and the spacing of the formed nanostructures.

Several recent studies address the problem of plasma roughening of polymers and its growth kinetics and most of them attempt its minimization in order to produce smooth etching for the nanoscale [START_REF] Engelmann | Dependence of Polymer Surface Roughening Rate on Deposited Energy Density During Plasma Processing[END_REF]. However, rather than trying to suppress the appearance of plasma instability we have been allowing its formation in order to use it for several applications as will be discussed below, especially by tuning the geometry and the order of the assembled nanostructures [START_REF] Gogolides | A review of line edge roughness and surface nanotexture resulting from patterning processes[END_REF]. [START_REF] Kokkoris | Dual nanoscale roughness on plasma-etched Si surfaces: Role of etch inhibitors[END_REF] for plasma nanotexturing and roughness instability (i.e. linear growth of roughness with time). The arrows indicate the angular distribution of impinging species. a) Mechanism with "hard" inhibitors coming from reactor walls or electrode: The ratio of ions to inhibitors is higher in the valleys than on the hills, due to more intense shadowing of inhibitors compared to the ions. b) Mechanism with "soft" inhibitors: The ratio of reactive neutral species to inhibitors is higher in the valleys than on the hills, due to the lower sticking probability of reactive neutral species (S RN < S SINH ).

Plasma Nanotexturing and the role of reactor walls

We begin with a brief description of what plasma nanotexturing actually is. When a polymer surface is etched and a few microns of material have been removed, nanotexture may develop on its surface, and roughness may increase linearly with time. This is shown in figure 2a for two organic polymers Poly(methyl methacrylate) PMMA, and Poly(ether ether ketone) PEEK. Starting from a flat surface, within minutes one can get a rough surface, and finally a porous rough layer. X ray photoelectron Spectroscopy (XPS) analysis shown in figure 2b, reveals relatively large surface concentration of Aluminum present in Oxide form [START_REF] Tsougeni | Mechanisms of Oxygen Plasma Nanotexturing of Organic Polymer Surfaces: From Stable Super Hydrophilic to Super Hydrophobic Surfaces[END_REF] coming from sputtering of the Alumina dielectric dome, and the anodized Aluminum clamping ring of the etch tool. This "hard" etch inhibitor creates micromasking and leads to the development of nanotexture. In nanoscience terminology the plasma directs the assembly of a rough nanotexture on the top surface of the polymer.

Figure 2. (a) Column Height on Micro and Nanotextured PMMA and PEEK plates in Helicon Plasma

Reactor with Oxygen Plasma. The SEM images are shown embedded for selected times to reveal the porous surface formed. (b) Elemental XPS analysis of 0.5mm thick PMMA plate versus etching time. Notice the large surface concentration of Aluminum. The reactor dome was made from alumina [START_REF] Tsougeni | Mechanisms of Oxygen Plasma Nanotexturing of Organic Polymer Surfaces: From Stable Super Hydrophilic to Super Hydrophobic Surfaces[END_REF].

The XPS results suggest that nanotexture is a result of plasma-wall interactions. We thus decided to study how the phenomenon may be controlled by changing reactor wall material, and plasma reactor design. In this section we compare for the first time 7 different reactor wall conditions and their effects on the morphology of 3 representative materials, namely PMMA-a typical organic polymer-, PDMS-a typical inorganic polymer-, and Silicon-a typical inorganic material. Five out of the seven reactor conditions refer to different wall conditions of one reactor type, namely the MET system by Adixen-Alcatel. The two remaining conditions refer to an ICP etcher developed by Oxford Plasma Technology (OIPT).

The Adixen reactor is a helicon type reactor with an antenna of one loop around a dielectric (alumina) dome. The etching conditions in the Adixen tool were: a) PMMA etch: -100 V bias, 10 o C, 0.75 Pa, 1900 W, 100 sccm O 2 , PMMA etch rate 1000 nm/min (for plates), 1659 nm/min (for films). b) PDMS etch: -100 V bias, 15 o C, 1.33 Pa, 1900 W, 200 sccm SF 6 , PDMS etch rate 0.75µm/min c) Si etch: -55 V bias, 15 o C, 5.25 Pa, 1800 W, 172 sccm SF 6 , Silicon etch rate 5µm/min. The conditions were chosen to be identical to the etching step of the gas chopping ("Bosch") process. The OIPT reactor is an ICP with alumina dome having dielectric shielding, and a quartz clamping ring. The etching conditions in the OIPT reactor are described below and were chosen so as to achieve similar etching rates with the Adixen tool: the anodized aluminum ring also with photoresist (see figure 3a). Both surfaces were baked after being painted with photoresist polymer. • Wall condition 3 (Polymer, Anodized Al, Helicon), refers to painting only the dome area and NOT the anodized aluminum ring of the Adixen Helicon System (see figure 3a). • Wall condition 4 (Alumina, Polymer, Helicon), refers to painting only the anodized aluminum ring and not the reactor dome of the Adixen Helicon System (see figure 3a). • Wall condition 5 (Quartz, Al, Helicon), refers to using an identical Adixen Helicon System with quartz dome and aluminum ring (not anodized). • Wall condition 6 (Alumina, Quartz, ICP, Shield), refers to using an ICP reactor from OIPT, with quartz dome, and quartz ring, and having electrostatic shielding of the RF fields to minimize dome material sputtering. • Wall condition 7 (Alumina, Quartz, ICP, No Shield), refers to using an ICP reactor from OIPT without electrostatic shielding of the RF dome.

Figure 3 shows several characteristic results from the 7 conditions used for the etching of PMMA plates. Figure 3b shows an SEM picture of a PMMA film etched for 2 min in the helicon reactor (etch depth 3.4µm). A columnar morphology is clearly seen on the film after etching. Figure 3c shows how the morphology is influenced by changing the dome material from alumina to quartz; Instead of columns nanomounds are now observed with height values less (approximately half) than those of the nanocolumns shown in figure 3b, as AFM measurements reveal (see also Table 1). Similar results are observed not only for films, but also for commercial PMMA plates, as shown in figure 3d,e,f. Indeed, after 1min of etching (more than 2µm of etched depth) severe micro and nanotexture appears on the surface of the plate and the root mean square (rms) roughness jumps to more than 40nm (figure 3d). Plasma etching directs the assembly of "grass" like micro-nanocolumns on PMMA as a result of Alumina sputtering and micromasking. Figure 3d is the AFM version of figure 3b but for a plate rather than a film and for only 1min etching. When painting the dome and ring of the reactor with a photoresist polymer, roughness is halved (figure 3e), and the morphology of the assembled nanostructures changes from grass-like to nanomounds, suggesting a transition from hard inhibitors (sputtered Al compounds from the reactor walls) to soft inhibitors (sputtered/partially etched photoresist polymer from the covered reactor wall). We note that this change is not accompanied by any significant etch rate reduction. In the OIPT reactor, an etching system designed for minimized sputtering and equipped with electrostatic shielding, roughness is only 4.5 nm even after 2min of etching (figure 3f), i.e. is reduced by an order of magnitude, resulting in relatively smooth surfaces. Note: AFM images were processed using the Software WSxM [START_REF] Horcas | WSXM: A software for scanning probe microscopy and a tool for nanotechnology[END_REF] Figure 4 shows characteristic etching results of PDMS (an inorganic polymer) with SF 6 plasma. In a reactor with alumina dome and ring, etching for just two minutes leads to roughness value of approximately 100nm (rms) as shown in figure 4a. After painting both the ring and the dome with an organic polymer roughness drops by more than an order of magnitude to only 8.7nm (figure 4b). When only the dome is covered with a polymer, while the anodized aluminum ring is exposed to the plasma, roughness increases slightly, but still remains at low values of 11.6nm. It is also extremely interesting to see that when changing the dome from Alumina to quartz, which is etched by the SF 6 plasma, roughness remains to low levels. Thus for PDMS etching the main contributor for roughness formation is the sputtering of alumina (in the form of Aluminum oxyfluoride) from the dome to the sample, a result also observed for Silicon etching by others [START_REF] Martin | Surface roughness generated by plasma etching processes of silicon[END_REF]. Finally experiments were done also for an inorganic material (Silicon) which is etched mostly isotropically and for which grass formation is not justified on the grounds of anisotropy. Figure 5a shows that after etching silicon in SF 6 plasma for 4 min (20µm etch depth) rough nanomounds appear [START_REF] Boulousis | Formation and metrology of dual scale nano-morphology on SF6 plasma etched silicon surfaces[END_REF] with rms value of 9.3nm. When the dome and ring are covered with photoresist, roughness values drop by almost 4 times (Figure 5b). When moving to the OIPT system even without an electrostatic shield roughness is further reduced as seen in Figure 5c. The complete set of experiments is summarized in Table I below. Some remarks and observations can be made by looking at figures 3,4,5 and roughness values shown on Table I:

First, one can note that the substrate / etch chemistry combination is crucial for roughness formation: For Silicon despite etching for 20micrometers roughness is below 10nm, while for polymers roughness can be one order of magnitude higher for an etch depth of only 2 micrometers.

Second, one can observe that the reactor design is crucial for roughness / nanotexture formation. The ICP reactor with the larger dome diameter results in smooth surfaces (compare condition 1 to condition 6 and 7). Even the absence of electrostatic shielding of the RF fields does not significantly increase roughness (compare conditions 6 and 7), which is one order of magnitude less compared to the unshielded helicon system.

Third, one may notice that in the helicon reactor covering the wall with polymer eliminates any sputtering of wall material (hard inhibitors) and results in almost one order of magnitude decrease of roughness down to values comparable to those of the OIPT system. The roughness reduction is larger for PDMS compared to Silicon and PMMA. Polymeric walls are of course also sputtered or etched and result in soft inhibitors. These are etched more easily thus reducing or eliminating nanotexture. The intermediate wall conditions of the Helicon reactor (see conditions 3 and 4) result in intermediate roughness, suggesting that both sputtering of the dome and the ring material is responsible for roughness formation, with the dome having a more important role.

Fourth, one understands that the chemical interaction of wall materials with the plasma is also crucial in achieving or eliminating roughness. For example in the Helicon reactor a drastic decrease (by a factor of more than 3) of nanotexture of PDMS surfaces occurs when the Alumina dome is painted with photoresist (condition 2). In addition, changing the dome to quartz, or painting the alumina ring (condition 4) results in significant reduction of nanotexture. In condition 2 the plasma interacts with a "polymeric" walls and any sputtered material is etchable (soft inhibitor). The same is partially true for condition 5, where the dome is made from quart a material etchable (but with smaller etching rate compared to polymer) in the SF6 plasma; As a result both conditions 2 and 5 reduced roughness. Thus, a good method to nanotexture PDMS is by having unetchable sputtered material (e.g. Alumina) in the SF6 plasma. Reversing the argument one could say that the only way to have smooth PDMS etching is by using quartz or carbonaceous walls, and a system designed for minimal sputtering; Indeed, note that the OIPT system which gives the smoothest PDMS surfaces (see Table I) has a quartz (etchable) ring and an antenna shielding minimizing sputtering of the Alumina dome.

Finally, we again stress that appropriate reactor design and wall material selection are the main parameters to reduce roughness if that is desired. On the other hand production of smart superhydrophobic surfaces with random nanotexture necessitates controlled wall material sputtering during etching [START_REF] Ramos | Plasma/reactor walls interactions in advanced gate etching processes[END_REF].

Table I. Effect of reactor wall condition and reactor type on rms roughness of nanotextured polymeric

and Silicon surfaces. The numbers given are the RMS roughness in nm, while the etch rate is indicated in parenthesis in µm/min so that the etch depth can be calculated. (

Plasma Directed Organization and the role of reactor walls

We start this section with a brief description of plasma directed organization on polymers. Under specific etching conditions nanotexturing may take the form of organized nanodots on a polymeric surface as shown in figure 6a. The specific etching conditions used for obtaining the results shown in figure 6a were: PMMA film etched in Oxygen plasma, 0 V bias, 65 o C, 0.75 Pa, 1900 W, O 2 flow 100 sccm, etch rate 1659 nm/min, wall condition 1 described in the previous section. Order and organization is witnessed by a peak in the Power Spectrum of the Surface as shown in figure 6h. The question therefore arises as to what is causing plasma directed organization.

Recent modeling results from our team (29) support the hypothesis that plasma directed organization is also due to fast etching of the polymer with simultaneous deposition of etch inhibitors from the plasma reactor walls. In this section we experimentally verify the role of plasma reactor walls on the formation of order and the assembly of organized nanodots. For these experiments both thin (750nm) and thick (5500nm) PMMA films were used, rather than plates as in the previous section. The etching time in most cases was 40 s and the other conditions were the same described above for figure 6a. The 7 wall conditions were the same as those used for nanotexturing, with the exception that we did not perform experiments at wall condition 6 since the Helicon Reactor with the quartz dome did not support etching at 65 C. In addition, for the ICP reactor from OIPT an 8 th plasma-wall condition was used: Wall condition 8 (Alumina, Quartz, ICP, Shield, photoresist carrier plate), refers to using an ICP reactor from OIPT, with alumina dome, and quartz ring, and having electrostatic shielding of the RF fields to minimize dome material sputtering. In addition the carrier 4 inch wafer was coated with a thick photoresist, and the samples were pieces (rather than whole wafers) covered with PMMA film and glued on the photoresist-coated carrier wafer. Figure 6 shows the AFM images of PMMA films etched with Oxygen plasma for different plasmawall conditions and the effects on plasma organization. 6a) and 8 (figure 6g). A peak indicative of the periodicity and organization is shown at wavenumbers of 0.016 and 0.009nm -1 , corresponding to =63nm and 116nm respectively.

As one can see from figure 6, the nanodots formed on the PMMA film (figure 6a) disappear when the dome and the ring of the reactor are coated with photoresist (figure 6b) at least for the same etching time. This shows that by diminishing sputtering from the reactor one can prevent the creation of the nanodots. When only the dome of the reactor is coated with photoresist, the surface is similarly flat without nanodots (figure 6c). We can thus clearly state that sputtering of the Alumina dome plays a major role in the creation of the nanodots. To strengthen our hypothesis we continued by coating only the ring and observed that we got back the nanodot formation on the PMMA surface even when etching for only 15s (the etch time used in almost all other experiments in the figure is 40s).

The next step was to use a different etching system an ICP etcher by OIPT in which the experimental conditions were altered so as to achieve the same etch rate as in the Adixen system. However, heating was not possible in the OIPT system. Figure 6e,f show characteristic results of these experiments. With or without electrostatic shielding the ICP has small random roughness. The nanodots are not totally absent, but they are diminished in size compared to the previous system (rms is 1.5 nm in OIPT and 6.5 nm in Adixen), while they lack in order, uniformity of shape and organization for the same etching time. However, at wall condition 8 (figure 6g) we used as samples pieces of PMMA coated wafers, and mounted these pieces on a carrier 4 inch Silicon wafer coated with a photoresist layer. To our surprise, we observed formation of ordered nanodots after 84s of etching, which we attributed to etching and sputtering of the photoresist on the carrier wafer. Such sputtering of the organic photoresist is possible even at zero bias with the ions having the energy of the plasma potential. We thus have first evidence that even "soft" inhibitors can lead to plasma directed organization. It is however a puzzle why such "soft" inhibitor assisted organization was not observed in the Adixen system when covering the dome with photoresist. It may be that our etching time was too short to observe it, or that the amount of polymer was very large.

Understanding plasma nanotexturing and plasma directed organization through simulation

(1+1) Dimensional ( 33) and (2+1) Dimensional Monte Carlo simulations have been performed considering that the surface is represented by a 2D or 3D lattice and is subjected to fluxes of neutral etchants, ions, and soft or hard inhibitors (hard are those removed only by ions). The morphology, the roughness parameters, and the time evolution are recorded. Surfaces are analyzed with power spectra (PSD) and height-height correlation functions to derive the exponents of growth of the roughness parameters.

Figure 7 below shows an example of both the time evolution of rms roughness and surface morphology when anisotropic ion etching and isotropic deposition of hard inhibitors are considered. Roughness instability is observed in agreement to the experimental behaviour (see figure2a). Furthermore, such simulation results match very closely the AFM images obtained from anisotropic ion enhanced etching of polymers.

Figure 7. Monte Carlo Simulation of ion and hard inhibitor driven plasma roughening. (a) Evolution of the rms roughness with time for assumed mechanism of hard inhibitors and ion enhanced etching. For the sake of comparison the rms increase due only to ions (without inhibitors) is also depicted. (b) Simulation of the evolution of surface morphology with time (AFM-like images) when one considers anisotropic ion-enhanced etching, and isotropic deposition of hard inhibitors. The etching time

increases from the left to the right, the fraction of depositing hard inhibitors is 0.05 and the selectivity of inhibitors vs. bulk material is 10. Notice the formation of columnar structures which are becoming higher, less dense and wider with time. Compare with AFM images for polymer etching and silicon in figures 3, 4, and 5 above.

Preliminary simulations have been also performed with a close look at the PSD of the surface, in order to see if there are cases where a peak is observed, indicative of order formation. The simulation results showed that the combination of non-reflecting ions, arriving at the surface at almost normal incidence and causing ion-enhanced etching, with a small amount (<10%) of depositing soft inhibitors, may induce order on the etched surface. Figure 8a,b shows a simulated surface in a 3D and 2D top down view which reveals a similarity with the experimental surface of figure 6a. Figure 8b shows embedded the circularly averaged PSD of the simulated surface, where the periodicity of the surface is manifested in the peak. Deposition alone has been reported to produce periodic mounds [START_REF] Pelliccione | Mound formation in surface growth under shadowing[END_REF]. Our case is codeposition during ion-enhanced etching and the mounds are not made by deposited inhibitors; indeed for the surface of figure 6a, the surface coverage by depositing particles is only 10%.

Emerging Applications? 6.1. Control of surface wetting

Control of wetting properties on all surfaces and especially on polymers is important in many technological applications. Some applications require the surface to be completely wetted, others require it to be totally water repellant. Plasma processing and plasma nanotexturing is an ideal tool to this extent since it allows control of both the surface chemistry and of the surface topography / texture. Oxygen plasma treatment of polymers produces superhydrophilic surfaces. While such surfaces usually show hydrophobic recovery, plasma nanotextured surfaces may withstand such recovery for a period of more than a month [START_REF] Tsougeni | Mechanisms of Oxygen Plasma Nanotexturing of Organic Polymer Surfaces: From Stable Super Hydrophilic to Super Hydrophobic Surfaces[END_REF].

On the other hand the demand for self-cleaning, antifogging, and anti-icing behaviour poses a need for superhydrophobic surfaces (1), (2) (3), ( 4), [START_REF] Crick | Preparation and Characterisation of Super-Hydrophobic Surfaces[END_REF] [START_REF] Bhushan | Natural and biomimetic artificial surfaces for superhydrophobicity, self-cleaning, low adhesion, and drag reduction[END_REF]. Plasma nanotexturing combined with plasma deposition of low energy coatings is again an ideal technology. It was as early as 1993 (41) that formation of ultra hydrophobic glass surfaces was demonstrated taking advantage of the roughness formation during plasma processing, while analogous demonstrations have been made for polymers (polypropylene [START_REF] Youngblood | Ultrahydrophobic polymer surfaces prepared by simultaneous ablation of polypropylene and sputtering of poly(tetrafluoroethylene) using radio frequency plasma[END_REF], PDMS (2),(3), PMMA (1),(3) and other polymers [START_REF] Mundo | Fluorocarbon plasmas for nanotexturing of polymers: A route to water-repellent antireflective surfaces[END_REF][START_REF] Milella | Plasma Nanostructuring of Polymers: Different Routes to Superhydrophoblicity[END_REF][START_REF] Mundo | Nanotexturing of polystyrene surface in fluorocarbon plasmas: From sticky to slippery superhydrophobicity[END_REF][START_REF] Favia | Deposition of super-hydrophobic fluorocarbon coatings in modulated RF glow discharges[END_REF]). In addition Si nano-tips with low-energy overcoats [START_REF] Shieh | Robust Airlike Superhydrophobic Surfaces[END_REF] and carbon nanotubes without any additional overcoats [START_REF] Han | Electrowetting control of Cassieto-Wenzel transitions in superhydrophobic carbon nanotube-based nanocomposites[END_REF] have been shown to have excellent superhydrophobic properties.

Figure 9a shows how nanotexturing of a polymer followed by thin fluorocarbon layer plasma deposition makes the polymer superhydrophobic. Examples are shown for PMMA and PDMS, two different polymers, the first being etched in oxygen and the second in sulfur hexafluoride discharges. Furthermore, plasma nanotexturing imposed on microstructured polymer surfaces leads to dual-scale topographies exhibiting robust superhydrophobicity, as has been demonstrated for PDMS [START_REF] Cortese | Superhydrophobicity due to the hierarchical scale roughness of PDMS surfaces[END_REF], parylene [START_REF] Chen | Dual hierarchical biomimic superhydrophobic surface with three energy states[END_REF], SU-8 [START_REF] Marquez-Velasco | Stable superhydrophobic surfaces induced by dual-scale topography on SU-8[END_REF], and Si surfaces [START_REF] Shieh | Robust Airlike Superhydrophobic Surfaces[END_REF]. Recently, there is an emerging demand to manufacture not only water but also oil repellant (i.e. amphiphobic) surfaces. Latest reports show that careful control of the geometry, slope, and undercut of micro and nanostructures on a surface allows a superhydrophobic surface to become oleophobic or superoleophobic (52) (53) [START_REF] Liu | Bioinspired Design of a Superoleophobic and Low Adhesive Water/Solid Interface[END_REF]. We are currently investigating this possibility for plasma nanotextured surfaces with impressive results.

There is a large need for superhydrophobic outdoor surfaces such as panels, windows, photovoltaic cells etc, but there exist numerous methods for manufacturing such surfaces. A cost analysis has to be done for a particular application. Low-pressure plasma technology needs vacuum processing, which increases the cost, and may make the technology less competitive for low-cost products. However, if other properties are desired in addition to superhydrophobicity, such as antireflectivity, then plasma technology can be competitive.

Control of optical properties of surfaces

Reduction of surface reflectivity, with simultaneous preservation of the optical transparency is often a requirement for many surfaces, such as windows and photovoltaic cells. In other cases roughening is required so as to reduce both reflectivity and transparency. Plasmas are ideal for both applications. Several reports have appeared on this subject and patents also exist [START_REF] Mundo | Fluorocarbon plasmas for nanotexturing of polymers: A route to water-repellent antireflective surfaces[END_REF][START_REF] Wang | Biomimetic corrugated silicon nanocone arrays for self-cleaning antireflection coatings[END_REF] [START_REF] Kaless | NANO-motheye antireflection pattern by plasma treatment of polymers[END_REF]. However, the key here is how to control the nanotexture in order to reduce reflectance for specific wavelength range. Figure 9b shows that reflectivity is indeed reduced significantly by plasma nanotexturing an organic polymer (9). . For short etch times the surface remains transparent, while for longer etch times it becomes milky. As an alternative to plasma nanotexturing, either plasma organization followed by plasma etching, or some other self-organization technique combined with plasma etching may allow full control. Indeed, nanosphere lithography followed by plasma etching has been recently proposed for antireflective and superhydrophobic surface fabrication [START_REF] Li | Bioinspired Silica Surfaces with Near-Infrared Improved Transmittance and Superhydrophobicity by Colloidal Lithography[END_REF].

Polymeric and Silicon Microfluidic Fabrication and modification

While for open surfaces plasma technology is in competition with other more "chemical" technologies, it is very appropriate for MEMS fabrication (57-59) and surface modification. For polymeric microfluidics plasma technology is of course in competition with injection molding and hot embossing. Nevertheless, the implementation of plasma treatment of microfluidics presents some unique merits in that:

(i) plasma etching is an inherently mass production technique, (ii) the polymer is treated while being in its solid state, (iii) channel formation is performed close to room temperature (cold-plasma), (iv) within the same plasma reactor several surface physicochemical and mechanical properties (wetability, hardness etc) may be modified, towards the requirements of the specific application, (v) master fabrication is not needed, hence using only one lithography step devices of different depths may be manufactured. (vi)

The high surface area of nanotextured polymers may be used for chromatographic separations, or as reaction sites in a lab-on-a-chip.(58,

59) (vii)

Oxygen nanotextured polymeric microfluidics are stable-in-time hydrophilic microchannels, which show capillary pumping. Fluorocarbon deposition through a stencil mask on such microchannels may create hydrophobic valve areas. Thus, pumping and valving through such microfluidic networks is possible due to plasma technology.

An illustration of the above concepts is shown schematically in figure 10 which shows capillary pumping and hydrophobic valving on chip. 6.4 Protein adsorption control Protein microarrays are used for probing the expression of protein function. Typically polymeric substrates such as Polysterene are used and proteins are deposited with a nanoplotter. There are efforts to increase the amount of protein on each spot, and thus the fluorescence intensity of the spot, as well as reduce the size of the spot and increase the array density. Plasma nanotexturing of polymers has been proven to increase protein adsorption by a factor of 3-6 times compared to a flat substrate, mainly due to the higher surface area (60), [START_REF] Tsougeni | Nano-texturing of poly(methyl methacrylate) polymer using plasma processes and applications in wetting control and protein adsorption[END_REF]. This could lead to a method of texturing surfaces as substrates for high quality, high intensity microarrays. Figure 11 illustrates the concept of high intensity microarray [START_REF] Tsougeni | Plasma Nanotextured PMMA Surfaces for Protein Arrays: Increased Protein Binding and Enhanced Detection Sensitivity[END_REF]. It should be noted that adsorption of protein is selectively taking place only in the nearly hydrophobic (CA~70º) oxygen plasma etched and subsequently aged-in-time polymer, while it is suppressed in the superhydrophobic areas (CA~150º), thus permitting local control of protein adsorption on microchannel walls by local control of wetting properties, as we will soon report. Finally, plasma processing can be used for selective protein adsorption on specific substrates and for protein microarray fabrication [START_REF] Bayiati | Highdensity protein patterning through selective plasma-induced fluorocarbon deposition on Si substrates[END_REF], based on the selective chemical modification of surfaces in plasmas.

It should also be noted that nanotexturing permits control of cell growth on the surface as recently discussed in several reports [START_REF] Gristina | Increasing cell adhesion on plasma deposited fluorocarbon coatings by changing the surface topography[END_REF][START_REF] Sardella | Nano-Structured Cell-Adhesive and Cell-Repulsive Plasma-Deposited Coatings: Chemical and Topographical Effects on Keratinocyte Adhesion[END_REF]

Fabrication of organized nanocolumn arrays and Silicon nanoimprint stamps

Plasma directed organization combined with plasma etching may be used for production of moderately ordered silicon nanocolumns. Today this may be accomplished either by electron beam lithography and plasma etching (but this is a costly procedure), or colloidal nanoparticle lithography [START_REF] Boal | Selfassembly of nanoparticles into structured spherical and network aggregates[END_REF][START_REF] Hulteen | Nanosphere lithography: A materials general fabrication process for periodic particle array surfaces[END_REF][START_REF] Zhang | Colloidal Self-Assembly Meets Nanofabrication: From Two-Dimensional Colloidal Crystals to Nanostructure Arrays[END_REF] and plasma etching (low cost but not uniform on large areas), or block-copolymer self-assembly [START_REF] Park | Enabling nanotechnology with self assembled block copolymer patterns[END_REF][START_REF] Ting | Plasma etch removal of poly(methyl methacrylate) in block copolymer lithography[END_REF] and plasma etching (a time consuming process) . Thus, plasma directed assembly especially if combined with order enhancement techniques (graphoepitaxy) may become a promising alternative. Silicon nanopillar arrays may be used as nanoiprint masks for organic polymers or organic photovoltaics to permit nanostructuring of such materials and thus enhanced efficiency [START_REF] Cheyns | Nanoimprinted semiconducting polymer films with 50 nm features and their application to organic heterojunction solar cells[END_REF] . Work in this direction is already in progress in our team.

Outlook

Perhaps the biggest problem of plasma technology is the variability from one reactor system to another and the need to recalibrate processes, when one changes etching system. Plasma nanotexturing / nanoassembly and plasma directed organization being plasma -wall interaction phenomena add to this variability and may provoke skepticism as to the future of this technology. The answer to this challenge will be the design of systems with controlled and adjustable sputtering / deposition. This may be accomplished by a) eliminating sputtering from walls and introducing additional sputtering targets in the system, b) allowing control of wall sputtering, c) eliminating wall sputtering and allowing for gas-phase deposition during etching. Such solutions demand design of new plasma reactors with embedded or controlled sputtering / deposition sources in close collaboration with equipment manufacturers. The understanding of plasma-surface interactions necessitates also modeling and simulation tools to aid the design of better plasma systems and processes.

Figure 1 .

 1 Figure 1. Two mechanisms(33) for plasma nanotexturing and roughness instability (i.e. linear growth of roughness with time). The arrows indicate the angular distribution of impinging species. a) Mechanism with "hard" inhibitors coming from reactor walls or electrode:The ratio of ions to inhibitors is higher in the valleys than on the hills, due to more intense shadowing of inhibitors compared to the ions. b) Mechanism with "soft" inhibitors: The ratio of reactive neutral species to inhibitors is higher in the valleys than on the hills, due to the lower sticking probability of reactive neutral species (S RN < S SINH ).

  a) PMMA etch: -105 V bias, 5 o C, 0.74 Pa, 1000 W, 50 sccm O 2, PMMA etch rate 1194nm/min for films b) PDMS etch: -100 V bias, 15 o C, , 1000 W, 200 sccm SF 6, PDMS etch rate 0.7µm/min c) Si etch: -55 V bias, 5 o C, 5 Pa, 1800 W, 172 sccm SF 6 , Si etch rate 10µm/min The 7 wall conditions were the following: • Wall condition 1 (Alumina dome, Anodized Al ring, Helicon source) briefly referred to as (Alumina, Anodized Al, Helicon) refers to clean alumina dome and anodized aluminum clamping ring of the Adixen Helicon System. • Wall condition 2 (Polymer, Polymer, Helicon) refers to covering (painting with a brush) the interior of the dome of the Adixen Helicon System with photoresist polymer, (especially in the area of high RF fields close to the antenna), and painting

Figure 3 .

 3 Figure 3. Plasma nanotexturing of PMMA in O 2 and the role of reactor walls. For the complete set of experiments refer to Table I. The z axis shows the range of height values in each image. (a) Schematic of the MET Helicon reactor showing the positions of painting with photoresist polymer the walls in order to reduce wall material sputtering. (b) Wall condition 1 for PMMA film etched for 2min. The SEM image shows the morphology of a polymer film and the roughness formed. (c) Wall condition 5 for PMMA film etched for 2min. Conditions are the same as in figure 3b, the main difference being that the dielectric dome material is quartz rather than Alumina. (d) PMMA plate etching at condition 1: Alumina dome and clamping ring with anodized aluminum. Scan size: 10x10µm, Treatment time: 1 min Rms: 41.8nm. (e) PMMA plate etching at condition 2: Photoresist on the ring and in the dome (only the antenna area). Treatment time: 1min, Scan size: 4x4 µm. Rms: 23.7 nm. (f) PMMA plate etching at condition 7, Without Electrostatic Shielding of the RF field of the antenna, Treatment time: 2min, Scan size: 4x4 µm. Rms: 4.5 nm.Note: AFM images were processed using the Software WSxM[START_REF] Horcas | WSXM: A software for scanning probe microscopy and a tool for nanotechnology[END_REF]

Figure 4 .

 4 Figure 4. Plasma Nanotexturing of PDMS in SF 6 and the role of reactor walls. For the complete set of experiments refer to Table I. The z axis shows the range of height values in each image. (a) PDMS etching in SF 6 for 2min at wall conditions 1, rms=98nm. (b) PDMS etching in SF 6 for 2min at wall condition 2, rms=8.7nm. (c) PDMS etching in SF 6 at wall condition 3, rms=11.6nm. (d) PDMS etching in SF 6 at wall condition 5 rms=11.2nm.

.

Figure. 5 .

 5 Figure. 5. Plasma Nanotexturing of Silicon in SF 6 and the role of reactor walls. For the complete set of experiments refer to Table I . The z axis shows the range of height values in each image. (a) Silicon etching for 4min in SF6 at wall condition 1, rms=9.3nm. (b) Silicon etching for 4min in SF6 at wall condition 2, rms=2.5nm. (c) Silicon etching for 4min in SF6 at wall condition 7 rms=1nm.

Figure

  Figure 6. Plasma directed organization on PMMA film in O 2 plasma and the role of reactor walls. AFM images (2x2µm) are shown.Typical etch rates are 600 nm/min, while nanodot formation is observed only at certain wall conditions. (a) Wall condition 1 (Alumina, Anodized Al, Helicon) (rms=6.5nm, etch time 40s). Nanodot formation is evident. (b) Wall condition 2 (Polymer, Polymer, Helicon) (rms=0.3nm, etch time 40s). No nanodots are observed. (c) Wall condition 3 (Polymer, Anodized Al, Helicon) (rms=0.4nm, etch time 40s). No nanodots are observed. (d) Wall condition 4 (Alumina, Polymer, Helicon) (rms=2.6nm, Etch time 15s only). Nanodots are formed even at 1/3 of the etch time. (e) Wall condition 6 (Alumina, Quartz, ICP, Shield, ) Rms: 1.5 nm, Etch time 42s. Nanodots are not sharp, they are short and less periodic. (f) Wall condition 7 (Alumina, Quartz, ICP, No Shield), Rms: 1.6 nm, etch time: 42 sec, Nanodots are not sharp; they are short and less periodic. (g) Wall condition 8 (Alumina, Quartz, ICP, Shield). Base plate (i.e. carrier of the sample) coated with thick layer of photoresist. Etch time 84s, rms=3.6nm.Nanodots are formed with slightly different morpholoby compared to figure 6a. (h) PSD of the surfaces resulting from wall conditions 1 (figure6a) and 8 (figure6g). A peak indicative of the periodicity and organization is shown at wavenumbers of 0.016 and 0.009nm -1 , corresponding to =63nm and 116nm respectively.

Figure 8 .

 8 Figure 8. Monte Carlo simulation of plasma directed organization from ions and soft inhibitors. (a) 3D top-down view of a surface produced by the Monte Carlo simulator (3600 monolayers have been etched and the fraction of depositing inhibitors is 0.05). (29) (b) Top-down view of the surface shown in figure 8a. Notice the similarity with figure 6a Average PSD of 10 simulated surfaces. The surface coverage by the depositing particles is only 0.1.

Figure 9 .

 9 Figure 9. (a) CA and CA hysteresis versus etching time for PDMS and PMMA. The evolution of superhydrophobicity and the process window for optical transparency are also indicated. (b) Reflection spectra (at 6 o ) of 2mm PMMA substrates before and after 1, 2, 3 min O 2 plasma processing on ONE side of the polymer plate only)(9).

Figure 11 .

 11 Figure 11. Fluorescence image of b-BSA and RgG spots microarray deposited by a nanoplotter on 20min O 2 plasma treated highly porous PMMA surface. The fluorescence from a flat PMMA plate is also shown on the right for comparison.[START_REF] Tsougeni | Plasma Nanotextured PMMA Surfaces for Protein Arrays: Increased Protein Binding and Enhanced Detection Sensitivity[END_REF]

  n/m stands for not measured)

	Wall condition	Alumina	Polymer	Polymer	Alumina	Quartz	Alumina	Alumina
	(dome		anod-Al	Polymer	Anod. Al	Polymer	Al	Quartz	Quartz
	clamping ring	Helicon		Helicon	Helicon	Helicon	Helicon	ICP e-shield	ICP No e-shield
	Reactor type)	1		2		3	4	5	6	7
	Substrate
	PMMA plate	98 (1)		29 (0.8)	61 (1)	64 (1)	41	3.7 (0.75)	4.5 (0.75)
	2min etch (
	some values	40 for 1min n/m
	for 1 and 5	240	for	38	for
	min etch)	5min		5min
	PMMA film	65 (1.6)		n/m		n/m	n/m	13 (1.6)	n/m	n/m
	2min etch
	PDMS	film	100 (0.75)	3.4		11.6 (0.85)	12.4 (0.85)	11.2 (1)	1.6 (0.7)	1.4 (0.7)
	(2min some values etch, for 1min given)	average (1.1) 25 for 1min 0.8 1min	for			4.3 1min	for
	Si wafer 20µm	9.1 (5)		2.4		2.9	n/m	n/m	1.1 (10)	1 (10)
	etch	depth
	(4min etch)

6 .

 6 Plasma directed organization on PMMA film in O 2 plasma and the role of reactor walls.

	AFM images (2x2µm) are shown. Typical etch rates are 600 nm/min, while nanodot formation is
	observed only at certain wall conditions.
	(a) Wall condition 1 (Alumina, Anodized Al, Helicon) (rms=6.5nm, etch time 40s). Nanodot formation
	is evident.
	(b) Wall condition 2 (Polymer, Polymer, Helicon) (rms=0.3nm, etch time 40s). No nanodots are
	observed.
	(c) Wall condition 3 (Polymer, Anodized Al, Helicon) (rms=0.4nm, etch time 40s). No nanodots are
	observed.
	(d) Wall condition 4 (Alumina, Polymer, Helicon) (rms=2.6nm, Etch time 15s only). Nanodots are
	formed even at 1/3 of the etch time.
	(e) Wall condition 6 (Alumina, Quartz, ICP, Shield, ) Rms: 1.5 nm, Etch time 42s. Nanodots are not
	sharp, they are short and less periodic.
	(f) Wall condition 7 (Alumina, Quartz, ICP, No Shield), Rms: 1.6 nm, etch time: 42 sec, Nanodots are
	not sharp; they are short and less periodic.
	(g) Wall condition 8 (Alumina, Quartz, ICP, Shield). Base plate (i.e. carrier of the sample) coated with
	thick layer of photoresist. Etch time 84s, rms=3.6nm.Nanodots are formed with slightly different
	morpholoby compared to figure 6a.
	(h) PSD of the surfaces resulting from wall conditions 1 (figure

Acknowledgments

Financial support through various European and National projects is acknowledged, such as Nanoplasma, PENED 03ED202, MD3, Nanotolife, and others. Dr Arun Kumar is kindly acknowledged for the experiments and SEM images for PMMA films, and Mr Athanasios Smyrnakis for providing sketches of the plasma reactor. Dr Nikos Vourdas, who was the first in the group to discover and study plasma directed organization is kindly acknowledged for his early work. Oxford Instruments Plasma Technology and especially Andy Goodyear and Mike Cooke are kindly acknowledged for the experiments done in OIPT in their new plasma source during the course of the Nanoplasma EU project. Adixen-Alcatel is kindly acknowledged for kind discussions and support for the equipment operation. AFM images were processed using the Software WSxM (36)