Substrate chemistry-dependent conformations of single laminin molecules on polymer surfaces are revealed by the phase signal of atomic force microscopy

Abstract

The conformation of single laminin molecules adsorbed on synthetic substrates is directly observed making use of the phase magnitude in tapping mode atomic force microscopy (AFM). With AFM, it is not possible to differentiate the proteins on the substrate if use is made of the height signal, since the roughness of the material becomes of the same order of magnitude as the adsorbed protein, typically 10 nm height. This work shows how AFM can be exploited to reveal protein conformation on polymer materials. Different laminin morphologies are observed on a series of different copolymers based on ethyl acrylate and hydroxyethyl acrylate as a function of the surface density of -OH groups: from globular to completely extended morphologies of the protein molecules are obtained, and the onset of laminin network formation on some substrates can be clearly identified. The results stress the importance of the underlying synthetic substrate's surface chemistry for the biofunctional conformation of adsorbed proteins.

FIGURE 1

AFM images for the P(EA-co-HEA) 50:50 copolymer scanned on a very smooth area of the sample (R_max = 2.8 nm, RMS = 0.3 nm). The three characteristic magnitudes of the tapping mode were taken simultaneously: height (a), phase (b), and amplitude (c). The image shows that the phase mode is able to reveal the conformation of the protein on the substrate as compared both with the height and amplitude magnitudes.

FIGURE 2

AFM images for the P(EA-co-HEA) 70:30 (a,b) and the 50:50 copolymers (c,d). The height image (left) shows a uniform surface in the 15 nm scale (a) and some evidence of the adsorbed protein (c), note the small arrows. The phase image (right) shows laminin adsorbed on the substrate. The maximum height scale is 15 nm, the scanned area is 1 × 1 μm.

FIGURE 3

AFM phase images of laminin on the different copolymers at different magnifications. (a–c) PHEA homopolymer; (d–f) P(EA-co-HEA) 30:70; (g–i) P(EA-co-HEA) 50:50; (j–l) P(EA-co-HEA) 30:70; and (m–o) PEA homopolymer. The first column shows 1 × 1 μm, the second column shows 500 × 500 nm and the third one 200 × 200 nm. The vertical scale is the same for all images.

FIGURE 4

Fraction of the substrate surface coated with laminin (•) and average end-to-end distance (○) of the protein as a function of the molar fraction of −OH groups in the system as calculated from the AFM phase images. The error bars represent the mean ± SD after measuring at least 20 molecules on three different pictures. The line between points is only a guide to the eye.