**2.2.1 Glass substrate conditioning**

54 Advances in Unconventional Lithography

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.,

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.,

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

Glass substrates were microscope coverslips (12 mm diameter, Menzel-Gläzer, Germany). Polyethyleneimine solution (PEI, Mw = 750 000), polyallylamine hydrochloride (PAH, Mw~70 000), poly-l-lysine hydrobromide (PLL, Mw = 70 000 to 150 000), poly(diallyldimethylammonium chloride) solution (lPDDA, Mw < 100 000; hPPDA, Mw = 400 000 to 500 000) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Negatively charged polystyrene particles with two different sizes (sulfate latex, 65±6nm, 470±12nm) were purchased from Interfacial Dynamic Corporation (IDC, Tualatin, OR, USA). The composition of the phosphate buffer saline solution (PBS) was as follows: 137 mM NaCl, 6.4

Figure 1 presents the different steps of sample preparation, from the glass substrate to

mM KH2PO4, 2.68 mM KCl, 8 mM Na2HPO4; adjusted to pH 7.4 with 1M NaOH.

2003).

2007) and immobilization on a specific substrate.

**2. Materials and methods** 

**2.2 Sample preparation** 

surfaces with bimodal roughness.

**2.1 Materials** 

colloidal particle, and thus distinguished from polyelectrolytes.

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 (Figure 1b).

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