Collagen Surface Functionalization
A New Strategy for Molecular Recognition Force Spectroscopy
- Collagen Surface Functionalization - A New Strategy for Molecular Recognition Force Spectroscopy
- Fig. 1: Comparison of the BP of different coating strategies. Red bar: collagen was adhered to a silicon support and fixed. Green bar: collagen was covalently bound to an APTES coated mica sheet using a short EGS linke. Blue bar: covalent immobilization of collagen using GPS chemistry.
- Fig. 2: Stepwise imaging and scratching of different GPS sample layers. (A) Glass slide with GPS coating, (B) Glass slide with GPS coating and a dense layer of PEG-diamine linker molecules, (C) Glass slide with GPS coating, a dense layer of PEG-diamine, actal-NHS linker and collagen.
- Fig. 3: Two specificity proofs. (A) The cantilever was functionalized with the specific collagen ligand VWF A1-A2-A3. MRFS measurements were carried out on samples with and without collagen using the same tip each. (B) Interactions were studied between collagen and VWF A1-A2-A3. Then the VWF A1-A2-A3 construct was blocked with free collagen.
We tested several sample preparation methods for collagen surfaces, suitable for Single Molecule Force Spectroscopy (SMFS). When collagen was adhered to silicon surfaces or bound via the short EGS-linker, it showed a high adhesive behavior and was therefore not apt for SMFS experiments. In contrast, with a sample preparation procedure using substrates with a dense layer of poly-(ethylene glycol) chains and terminal benzaldehyde functions, unspecific adhesion between tip and sample was low.
We aim to study interactions between collagen and von Willebrand Factor (VWF) on a single molecule level using Atomic Force Microscopy (AFM) based SMFS. This interaction is an initial and important event of hemostasis. A requirement for this study is a uniform collagen matrix with low adhesive behavior. In the following, the development and the validation of such a collagen surface preparation method is described.
Materials and Methods
Commercial MSCT cantilevers were functionalized according to  with either an unspecific protein or the cognate VWF A1-A2-A3 domain construct carrying a 6xHis-tag.
Fibrillar collagen type III was used (Southern Biotech, Alabama, USA). For adhering collagen onto silicon we followed , with an additional fixation step using 4% paraformaldehyde (PFA) for 1 hour at room temperature. Covalent immobilization of collagen using an EGS linker (16.1 Å) was done according to . Furthermore, we used a dense layer of poly-(ethylene glycol) chains and terminal benzaldehyde functions for covalent immobilization of collagen (further termed as GPS sample). Here a small amount of PEG800-diamine was pipetted on a GPS (Glycidoxypropyltrimethoxysilane) coated epoxy slides from Schott Nexterion before they were placed in a drying stove for 4 hours at 70°C. Subsequently the slide was rinsed with a warm mixture of chloroform/ methanol/ acetic acid (v/v/v=70/30/4) and then two times with the same mixture at room temperature. Then the slides were dried with nitrogen .
The following coupling of the connecting acetal-NHS linker and the collagen was performed as described in . For control samples without collagen, 100 µl of ethanolamine (1M, in milliQ H2O, pH 8.0 adjusted with NaOH) were pipetted onto the slide instead of the collagen solution to saturate the reactive groups.
SMFS and Mac-mode Imaging
Measurements were performed using a Pico SPM Plus setup (Agilent, USA) at room temperature. For imaging type VII Mac-levers with a nominal spring constant of 0.14 Nm-1 (Agilent Technologies, Santa Clara, USA) were used. In SMFS experiments, the position of the tip relative to the surface was changed every 200 FDCs to statistically avoid position dependent artefacts. At least 1000 force distance cycles (FDCs) were recorded for each tip at a pulling speed of 600 nm/s. For SMFS non-conductive Silicon Nitride MSCT tips (Brucker, MA, USA, C-cantilever, 0.010 Nm-1 nominal spring constant) were used.
Results and Discussion
Firstly, collagen was purely adhered to silicon. The quality of the sample functionalization was verified by mac-mode imaging (see half page illustration). In order to test the adhesive behavior of this sample, we functionalized the tip with an unspecific protein, performed SMFS measurements and compared the binding probability (BP). The BP is the ratio between FDCs showing an unbinding event relative to the total number of FDCs. The red bar in figure 1 shows the unspecific binding probability (BP=52.2±11.78%) of this system. The very high number of unspecific adhesion made it unfeasible for further SMFS measurements. In a next step, we covalently immobilized collagen with a short EGS linker and repeated the SMFS measurements, which revealed lower unspecific adhesion (BP=31.9±5.80%, fig. 1, green bar). Nevertheless, the reduction in unspecific adhesion was still not optimal for SMFS. As a next attempt, we used a GPS chemistry for covalent immobilization of collagen. It was shown previously  that, compared to conventional amino functionalization, a ten times higher density of reactive binding sites was achieved when melting PEG-diamine on the epoxy surface. Subsequently we used an acetal-NHS linker as a connection between the dense brush of PEG-diamine linker molecules and the collagen layer. SMFS experiments showed low unspecific adhesion (BP=6.1±1.82%, fig. 1, blue bar), probably due to steric interchain PEG repulsion  (PEG linker coupled to the tip and the sample surface) and therefore, this procedure seemed to be the best option for further SMFS experiments. No distinct collagen strands could be resolved by mac-mode imaging, probably due to the mobility of the different linker layers (fig. 2C).
So as to characterize the sample functionalization procedure, we prepared three samples and added the different linker layers stepwise. For the first sample, only the GPS coated glass slide was imaged and scratched. The depth of the hole was about 2 nm (fig. 2A). This is in good correlation with the predicted height of GPS molecules . In a next step, the PEG-diamine linker was added. The depth of the hole was determined to be 2.5 nm (fig. 2B). In a fully stretched conformation the PEG-diamine linker is about 4nm in length. However, incubated on the GPS slide, the linker might be folded and compressed, which would result in the decreasing height of 2.5 nm. Subsequently, the connecting acetal-NHS linker and the collagen molecules were incubated. The acetal-NHS linker is about 1nm in length and the collagen molecules about 1.5 nm in height. AFM imaging and scratching revealed a reasonable height of 4 nm of all layers (fig 2C).
So as to proof the quality of the sample for SMFS, we performed two further specific tests. First, we prepared two samples - one with and one lacking collagen. The AFM tips were functionalized with a VWF A1-A2-A3 construct, which specifically binds to collagen. Three different tips were used on both samples and the BP was compared. Figure 3A shows a specific decrease of BP on the sample without collagen. In addition we performed tip block experiments, where free collagen was allowed to bind to the VWF constructs on the tip for two hours. The free collagen saturated the VWF construct on the tip and blocked the interaction with the collagen on the sample surface. The decrease of the BP after the tip block is shown in figure 3B. These experiments suggest a large number of specific interactions between the VWF construct and collagen.
Here, we report about a novel collagen coating method, especially suited for SMFS measurements, due to a low unspecific adhesion behavior. This sample preparation method is based on a dense layer of poly-(ethylene glycol) chains and the terminal benzaldehyde functions will allow the study of the interactions between different types of collagen and specific collagen binding proteins, such as VWF on a single molecule level.
This study was supported by the DFG within the Research group FOR1543: "Shear flow regulation of hemostasis - bridging the gap between nanomechanics and clinical presentation (SHENC) / FWF (Project I 767-B11)" (TO, MAB, RS, UK). We thank Christian Rankl (Agilent, Linz) for his support in data evaluation, Hermann Gruber (JKU, Linz) and Robert Tampé (Goethe-University Frankfurt) for providing heterobifunctional crosslinker, and Peter Hinterdorfer for supervision of this work.
 www.jku.at/biophysics/content/e201852, (23.07.14)
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DI Sandra Posch
Johannes Kepler University Linz
Institute of Biophysics
Department of Applied Experimental Biophysics