Supporting Information. for. Angew. Chem. Int. Ed. Z WILEY-VCH Verlag GmbH & Co. KGaA

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1 Supporting Information for Angew. Chem. Int. Ed. Z WILEY-VCH Verlag GmbH & Co. KGaA Weinheim, Germany

2 Spatially Directed Protein Adsorption By Using a Novel, Nanoscale Surface Template Patricia Moraille and Antonella Badia Domain assignment: Several pieces of evidence have led us to assign the stripes and circular domains in the mixed DPPC/DLPC films to DPPC and the surrounding matrix to DLPC. [1] First, the step-height differences measured for the two LB films (Supplementary Figures 2B and 3B) compare favorably with the 0.85 nm height difference expected from half the bilayer thicknesses measured by X-ray diffraction for the lamellar phases of DPPC and DLPC at 25 C. [2] Second, phase imaging (medium to high oscillation damping) of the LB films (supplementary Figures 2B - 2C and 3B - 3C ) reveals a more positive phase shift at the stripes compared to the surrounding matrix, suggesting that the stripes are stiffer. The step-height differences and mechanical responses observed by AFM are thus consistent with phase separation into condensed DPPC-rich domains and a liquid-like DLPC-rich matrix. Third, the area fraction covered by the stripes and circular domains in the AFM images (50 µm x 50 µm areas) of the 0.25/0.75 DPPC/DLPC film, 28 ± 3%, is in reasonable agreement with the area fraction of DPPC in the film, 21%, estimated for a DPPC molar fraction of 0.25 and molecular areas of 46.5 Å 2 for DPPC and 57.2 Å 2 for DLPC at the deposition pressure of 35 mn/m (Supplementary Table 1). For the 0.50/0.50 DPPC/DLPC monolayer, an area fraction of 42% (calculated using molecular areas of 50.6 Å 2 for DPPC and 70.5 Å 2 for DLPC at 15 mn/m) is expected if the stripes were pure DPPC and the surrounding matrix was pure DLPC. The AFM images (50 µm x 50 µm areas) of the 0.50/0.50 DPPC/DLPC films show that the stripes occupy an area fraction of 42 ± 7%. [1] P. Moraille, A. Badia, Langmuir 2002, 18, [2] D. Marsh, Handbook of Lipid Bilayers, CRC Press, Boca Raton, FL,

3 DPPC 0.50 DPPC 0.25 DPPC DLPC π / mn/m A / Å 2 molecule -1 Supplementary Figure 1. Surface pressure-area (π-a) isotherms at 20 ºC of DPPC, DLPC, 0.50/0.50 (mol/mol) DPPC/DLPC, and 0.25/0.75 (mol/mol) DPPC/DLPC. The plateau observed at π 5 mn/m in the DPPC isotherm corresponds to a liquid-expanded (LE) to liquid-condensed (LC) phase transition. There is a chainlength difference of 4 carbons between DPPC and DLPC. DPPC has a main phase transition temperature of 41 ºC and DLPC has a main transition temperature of 1 ºC. [2] -3-

4 Supplementary Table 1: The areas occupied per phospholipid molecule and per alkyl chain in pure DPPC and DLPC monolayers as a function of the surface pressure based on the π-a isotherms in Supplementary Figure 1. Surface pressure (mn/m) Phospholipid Area occupied per molecule (Å 2 ) Area occupied per alkyl chain (Å 2 ) DPPC DLPC DPPC DLPC DPPC DLPC

5 Tapping mode phase imaging of DPPC/DLPC LB films Phase imaging is an extension of tapping or intermittent-contact mode AFM, and involves the monitoring of the phase lag between the signal that drives the cantilever to oscillate and the cantilever oscillation output signal. Under certain conditions, changes in the phase lag reflect changes in the mechanical properties of the sample. Since the contrast in the height (topography) and phase images depends on several imaging parameters (i.e. the cantilever resonance frequency, the free air oscillation amplitude, and the set-point amplitude), it is necessary to examine height and phase images acquired as a function of the % oscillation damping: (free air amplitude imaging setpoint amplitude) N.B. % oscillation damping = free air amplitude x 100 Height and phase images were acquired in a given area of the 0.25 DPPC/0.75 DLPC and 0.50 DPPC/0.50 DLPC films with increasing oscillation damping from top to bottom (Supplementary Figures 2 and 3). The measured step-height difference in the topography images increases with increasing oscillation damping due to a greater extent of tip penetration into the fluid DLPC phase. As for the phase shift, imaging at low oscillation damping or light tapping (1-5%) results in a negative phase lag over the stripes with respect to the surrounding matrix (top of AFM images in Supplementary Figures 2 and 3). For medium oscillation damping (15-30 %), a positive phase lag is observed at the stripes (middle of AFM images in Supplementary Figures 2 and 3). For high oscillation damping or hard tapping (50 %-85 %), the positive phase lag increases (bottom of AFM images in Supplementary Figures 2 and 3). For the cantilever drive amplitudes used here (60-65 mv), the tip-sample interaction becomes repulsive under conditions of moderate to high oscillation damping, so that a larger stiffness leads to a more positive phase shift, and thus, to a brighter contrast in the phase image. [3] [3] G. Bar, Y. Thomann, M.-H. Whangbo, Langmuir 1998, 14,

6 A) 0.51 nm A ) -1.0 B) 0.83 nm B ) 6.6 C) 1.07 nm C ) 9.4 Supplementary Figure 2. Tapping mode AFM images and cross-sections of the 0.25/0.75 (mol/mol) DPPC/DLPC LB film: Height (A)-(C) and phase (A )-(C ). Drive amplitude = mv. Images were acquired while increasing the oscillation damping from top to bottom: (A) & (A ) = 1 % (low oscillation damping or light tapping), (B) & (B ) = 13% (medium oscillation damping or medium tapping), (C) & (C ) = 77% (high oscillation damping or hard tapping). -6-

7 A) 0.24 nm A ) -3.6 B) 0.85 nm B ) 2.9 C) 2.76 nm C ) 6.9 Supplementary Figure 3. Tapping mode AFM images and cross-sections of the 0.50/0.50 (mol/mol) DPPC/DLPC LB film: Height (A)-(C) and phase (A )-(C ). Drive amplitude = 65 mv. Images were acquired while increasing the oscillation damping from top to bottom: (A) & (A ) = 5.2 % (low oscillation damping or light tapping), (B) & (B ) = 20% (medium oscillation damping or medium tapping), (C) & (C ) = 81% (high oscillation damping or hard tapping). -7-

8 A) B) Supplementary Figure 4. AFM topography images in air of mixed DPPC/DLPC monolayers after exposure (15 min) to water: A) 0.25/0.75 DPPC/DLPC monolayer transferred at 35 mn/m and B) 0.50/0.50 DPPC/DLPC monolayer transferred at 15 mn/m. Supplementary Figure 5. AFM topography image in air of a 0.25/0.75 DPPC/DLPC monolayer (35 mn/m) after adsorption (10 min) of Au nanoparticles (10 nm nanoparticle solution; particle diameter = 11 ± 3 nm). -8-

9 A) B) 1 µm 1 µm 20 h / nm l / µm 20 h / nm l / µm Supplementary Figure 6. AFM topography images in air of a 0.50/0.50 DPPC/DLPC monolayer with: A) adsorbed human γ-globulin and B) adsorbed human γ-globulin and Au nanoparticles (d = 11 nm). The area in (B) is not the same area shown in (A). The arrows in the AFM images and height profiles indicate "grooves" in the patterned surface. Groove depths: (a) 3.4 ± 0.6 nm and (b) 12.6 ± 1.9 nm. -9-

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