**1. Introduction**

From bioengineering to optics and electronics, a great deal of work has been conducted on the development of new materials with structured surfaces. A large range of methods has been used, such as plasma etching, electron beam and colloidal lithography (Denis et al., 2002), electrical deposition (Yang et al., 2009), phase separation (Dekeyser et al., 2004) and polyelectrolyte assembly in order to produce structured surfaces (Agheli et al., 2006). The combination of different methods is also more and more explored. Schaak et al. (2004) described a simple approach to achieve colloidal assembly on a patterned template obtained by lithography.

Densely packed layers of colloidal particles can be produced by lifting a substrate vertically from a suspension (Fustin et al., 2004; Li J. et al., 2007) or by spin coating (Yang et al., 2009). Colloidal lithography utilizes the ability of particles to adhere on oppositely charged surfaces (Johnson & Lenhoff, 1996; Hanarp et al., 2001, 2003)), possibly using surface modification by inorganic or organic polyelectrolytes. The surface coverage is influenced by several factors: ionic strength, particle size and time. The adhesion of microbial cells on various substrates was also achieved by surface treatments with inorganic or organic polycations (Changui et al., 1987; Van haecht et al., 1985) or with positively charged colloidal particles (Boonaert et al., 2002). A review on colloidal lithography and biological applications was published recently (Wood, 2007).

Adsorption of polyelectrolytes is influenced by ionic strength, pH and the polyelectrolyte characteristics (molecular mass, charge density) (Lindquist & Stratton, 1976; Davies et al., 1989; Choi & Rubner, 2005). At low ionic strength, highly charged polyelectrolytes adopt extended conformations and are fairly rigid due to the strong repulsion between charged units. The maximum adsorbed amount and the adsorbed layer thickness do not vary markedly according to molecular weight. As the salt concentration is increased and the electrostatic intrachain repulsion is decreased, the polyelectrolyte becomes more coiled. In this case, the maximum amount adsorbed (expressed in mass) increases as a function of molecular weight (Roberts, 1996; Lafuma, 1996; Claesson et al., 2005; Boonaert et al., 1999).

Build up of polyelectrolyte films may be achieved using layer-by-layer assembly through alternating adsorption of oppositely charged polyelectrolytes (Decher & Hong, 1991).

Fabrication of Surfaces with Bimodal Roughness Through Polyelectrolyte/Colloid Assembly 55

The glass coverslips were cleaned by overnight immersion in sulfochromic solution and rinsed with water, prior to polycation adsorption (Figure 1a). The pH and ionic strength (I) of the polycation solution were adjusted with NaOH and HCl, and NaCl, respectively. The polycation solution (1 ml) was poured into the wells of a tissue culture plate (Falcon, Becton Dickinson, Belgium, Ref. 353226), where the glass coverslips had been placed earlier, and was left in contact with the substrate for at least 2 h. Unless stated otherwise, the polycation solution was 10-5 M at pH 11 and I 10-2 M. The samples were rinsed by 6 successive dilutions to avoid exposure to air. Each rinsing step was performed by adding 2 ml of deionized water (produced by a Milli-Q plus system from Millipore, Molsheim, France), stirring gently, and removing 2 ml of liquid. They were then dried under a gentle nitrogen flow

Fig. 1. Schematic representation of the steps used for sample fabrication. Glass substrate (a), polycation-conditioned glass substrate (b), substrate with a layer of adhering particles (c),

Polycation-conditioned substrates were placed horizontally into wells of another culture plate. 1 ml of a suspension of the desired concentration, pH and ionic strength was poured into the wells and left in contact with the substrate for at least 3 h (Figure 1c). Unless stated otherwise, the procedure used for rinsing and drying the samples was performed as follows: rinsing 3 times with water and 3 times with isopropanol and drying overnight in air. The samples were rinsed by successive dilutions as detailed in the preceding paragraph. After

1 ml of a 0.1 % suspension of 470 nm particles was poured into the wells containing glass substrates conditioned with PAH (10-5 M, pH=11, I=10-2 M) (Figure 1b), placed in horizontal

surface with bimodal I roughness (d), substrate with a layer of adhering particles conditioned with the polycation (e), surface with bimodal II roughness (f).

the last rinsing, as much as possible of the solution was removed with a pipette.

**2.2.2 Adhesion of a single colloid layer** 

**2.2.3 Surfaces with bimodal I roughness** 

**2.2.1 Glass substrate conditioning** 

(Figure 1b).

Polyethyleneimine (PEI), polyallylamine (PAH), poly-l-lysine (PLL) and polydiallyldimethylammonium chloride (PDDA) are common polycations used for multiple layer formation with polyanions such as polystyrene sulfonate (PSS) (Bertrand et al., 2000). Layer-by-layer assembly of polyelectrolytes has been combined with the use of colloidal particles. For instance, a film was made on silicon wafers precoated with thermally evaporated titanium, by adsorption of PDDA, followed by adsorption of PSS, followed by treatment with an aluminium chloride hydroxide solution (Hanarp et al., 2001, 2003). In another study, chemically patterned surfaces made by self-assembled monolayers (SAMs) were covered with a polyelectrolyte multilayer film, before adhesion of SiO2 silica particles or functionalized polystyrene latex particles (Chen et al., 2000). While a polyelectrolyte layer may provide a strong bond to colloidal particles, the drying process applied after particle adhesion may be crucial to obtain a regular and homogeneous monolayer (Hanarp et al., 2003).

Structured hydrophobic surfaces have gained increasing interest because the roughness amplifies the hydrophobicity (Wenzel, 1936). This is exemplified by the Lotus effect, in which a dual size roughness seems to be important (Barthlott & Neinhuis, 1997; Patankar, 2004). Raspberry-like surface morphologies were created in different ways : styrene polymerization (Perro et al., 2006; Reculusa et al., 2002) or gold sputtering (Xiu et al., 2006) on silica particles , controlled aggregation of different surface-functionalized silica particles (Ming et al., 2005) or direct electrochemical synthesis of gold microaggregates (Li Z. et al., 2007) and immobilization on a specific substrate.

In this paper, we prepare surfaces covered with a homogeneous monolayer of colloidal particles, using adhesion of negatively charged polystyrene latex beads on a polycationprecoated glass substrate. The method is then extended to prepare surfaces presenting a bimodal roughness, by using latex particles of different sizes. The influence of substrate surface roughness on the behavior of mammalian cells has been of great concern in the last years (Nonckreman et al., 2010). Therefore, the stability of fabricated samples is tested in phosphate buffer saline (PBS), which simulates the pH and ionic strength of biological fluids. Note that here, the term "colloid" is used with the restrictive sense of lyophobic colloidal particle, and thus distinguished from polyelectrolytes.
