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Journal of Colloid and Interface Science 278 (2004) 63– www.elsevier.com/locate/jcis
Nanostructured collagen layers obtained by adsorption and drying
I. Jacquemart, E. Pamuła 1 , V.M. De Cupere, P.G. Rouxhet, Ch.C. Dupont-Gillain ∗
Unité de chimie des interfaces, Université catholique de Louvain, Croix du Sud 2/18, 1348 Louvain-la-Neuve, Belgium Received 7 November 2003; accepted 24 May 2004 Available online 2 July 2004
Abstract
The supramolecular organization of collagen adsorbed from a 7 μg/ml solution on polystyrene was investigated as a function of the ad- sorption duration (from 1 min to 24 h) and of the drying conditions (fast drying under a nitrogen flow, slow drying in a water-saturated atmosphere). The morphology of the created surfaces was examined by atomic force microscopy (AFM), while complementary information regarding the adsorbed amount and the organization of the adsorbed layers was obtained using radioassays, X-ray photoelectron spec- troscopy (XPS), and wetting measurements. The collagen adsorbed amount increased up to an adsorption duration of 5 h and then leveled off at a value of 0.9 μg/cm^2. For samples obtained by fast drying, modeling of the N/C ratios obtained by XPS in terms of thickness and surface coverage, in combination with the adsorbed amount, water contact angle measurements and AFM images, indicated that the adsorbed layer formed a felt starting from 30 min of adsorption, the density and/or the thickness of which increased with the adsorption time. Upon slow drying, the collagen layers formed after adsorption times up to about 2 h underwent a strong reorganization. The obtained nanopatterns were attributed to dewetting, the liquid film being ruptured and adsorbed collagen being displaced by the water meniscus. At higher adsorption times, the organization of the collagen layer was similar to that obtained after fast drying, because the onset of dewetting and/or collagen displacement were prevented by the high density of the collagen felt. 2004 Elsevier Inc. All rights reserved.
Keywords: Collagen; Protein adsorption; Adsorption kinetics; Dewetting; XPS; AFM; Radiolabeling; Water contact angle
1. Introduction
Understanding and controlling the spatial organization of adsorbed protein layers on the nanometer scale is expected to bring significant progress in several fields (biosensors, immobilized enzymes, biomaterials, etc.). Organized lay- ers of biological molecules may also serve as templates or as inspiring models to create other nanostructured materials [1–3]. Lithographic methods, which were first used in the mi- crometer range, have been adapted to the creation of protein patterns down to the nanometer scale [4–6]. However, these methods are not suitable for the modification of large ar- eas. Methods based on the deposition of colloidal particles, which create excluded areas for the proteins and then are
- (^) Corresponding author. Fax: 32-10-472005. E-mail address: [email protected] (Ch.C. Dupont-Gillain). (^1) Present address: Department of Biomaterials, Faculty of Materials Science and Ceramics, AGH—University of Science and Technology, 30 Mickiewicza Ave., 30-059 Kraków, Poland.
removed from the surface, have also been proposed [7,8]. These offer the advantage of a higher throughput. Another possible approach relies on the self-organizing properties of molecules, a feature which is commonly encountered among biological molecules [9]. For example, proteins orig- inating from the so-called S-layer of bacteria form ordered monolayers, which can be used as nanotemplates or nanore- sists [10]. Dewetting of thin liquid films on solid substrata may also produce patterns in the nanometer range. Dewet- ting has been investigated mainly for pure water [11] or polymer films [12,13], but it has also been applied to col- lagen solutions [14,15]. In comparison to other methods al- lowing surfaces to be nanostructured (nanolithography, self- assembly), dewetting offers different possibilities in terms of characteristic sizes and order and presents the advantage of a high throughput, which is important in view of industrial applications. Collagen is an extracellular matrix protein (molecular mass ∼300 kg/mol) found in a wide range of vertebrate and invertebrate species. The type I collagen molecule (length ∼300 nm, diameter = 1.4 nm) is made of three helical
0021-9797/$ – see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2004.05.
polypeptides wrapped together in a helix. Nonhelical por- tions, named telopeptides, are found at each end of the mole- cule. In vivo, collagen assembles into fibrils with a periodic structure. It can also be precipitated in vitro into several fib- rillar forms [16]. Collagen offers promising perspectives for creating nanostructured protein layers, due to its shape and dimensions and to its self-assembling properties. The base- ment membrane of corneal epithelium was shown to exhibit a complex network of pores and fibers, with dimensions in the range of 30–400 nm. This was thought to modulate cell behavior [17]. Recently, it was also shown that the mor- phology of smooth muscle cells was affected by the density of collagen fibrils present on the substratum [18]. Control- ling the nanometer-scale organization of adsorbed collagen layers could thus lead to a better control of cell–material in- teractions. The patterns formed from collagen films prepared by spin-coating were found to depend on drying conditions and were interpreted by the occurrence of two distinct dewetting mechanisms: heterogeneous pore nucleation and spinodal dewetting [19,20]. Collagen layers adsorbed on hydrophobic substrates may rearrange to form a net-like structure upon slow drying [15,21]. Such nets were used as templates to de- sign nanostructured polymer surfaces [2]. The aim of the present study was to explore further the influence of experimental conditions on the formation of nanostructures from adsorbed collagen layers, in order to get deeper insight into the nanostructure formation process and to create a variety of nanostructures. Polystyrene, a low- cost polymer frequently used as a support for cell culture, was chosen as a substratum for adsorption. Two parame- ters able to affect the organization of the adsorbed layers were examined: the adsorbed amount (through the varia- tion of the adsorption duration) and the rate of drying (with the aim of forming dewetting patterns). While the morphol- ogy of the surfaces was examined using atomic force mi- croscopy (AFM), complementary information regarding the adsorbed amount and the organization of the adsorbed layers was obtained using radioassays, X-ray photoelectron spec- troscopy (XPS), and wetting measurements.
2. Materials and methods
2.1. Sample preparation
Polystyrene (PS) (PS05232 from BP, Zwijndrecht, Bel- gium) was spincoated (speed 5000 rpm, acceleration 20,000 rpm/s, time 60 s, volume 30 μl) on 12-mm-diameter glass coverslips (Menzel–Gläzer, Germany) from a 10.4% (w/w) solution in toluene (analytical grade; VEL, Leuven, Belgium). The PS substrates obtained in these conditions were shown to be smooth (root-mean-square roughness mea- sured on 5 × 5 μm 2 AFM images equal to 0.2 nm) and featureless [22].
Collagen (type I from calf skin; Roche Diagnostics, Mannheim, Germany) was received as an aqueous solu- tion (3 mg/ml at pH 3.0). It was diluted to a concen- tration of 7 μg/ml in phosphate-buffered saline (PBS) (pH 7.2; 137 mM NaCl (Merck, Leuven, Belgium), 6.44 mM KH 2 PO 4 (VEL), 2.7 mM KCl (Merck), 8 mM Na 2 HPO (^4) (VEL)). At this pH, collagen aggregation could occur, as in- dicated in the literature [16]; we found that the aggregation in 150 μg/ml solutions, monitored using the absorbance at 400 nm, reached a maximum at pH 5.0 and was not detected at pH 7.2 (Dupont-Gillain et al., to be published). Adsorption of collagen on PS substrates was performed as follows. Substrates were deposited in the wells of a tissue culture plate (Falcon 3043 from Becton Dickinson, Franklin Lakes, NJ, USA). Two milliliters of the collagen solution was added to each well and the samples were incubated at 37 ◦C for the desired period of time. When the adsorption was completed, the samples were rinsed without being ex- posed to air by adding 2 ml of deionized water (produced by a Milli-Q plus system from Millipore, Molsheim, France), stirring gently, pumping 3 ml of the solution, adding 3 ml of water, and repeating these two last steps four times. Two different drying modes were then applied to the sam- ples. A fast drying procedure was performed by flushing the samples with a nitrogen flow and storing them in a desicca- tor containing P 2 O 5. A slow drying procedure consisted in placing the samples on a metal grid in a vessel containing a saturated solution of Na 2 CO 3 (VEL), which ensured a rel- ative humidity of about 95%. Before the vessel was closed a 200-μl drop of deionized water was placed on top of the samples and gentle agitation produced a uniform film of wa- ter on the sample surface. The samples were left to dry in the vessel for at least 24 h at a constant temperature of 30 ◦C.
2.2. Surface characterization
2.2.1. Atomic force microscopy AFM images were obtained in air in the contact mode, us- ing a commercial microscope (Nanoscope III, Digital Instru- ments, Santa Barbara, CA, USA) equipped with Si 3 N 4 tri- angular levers (ThermoMicroscopes, Sunnyvale, CA, USA; nominal radius of curvature = 20 nm). Images were flattened with a third-order polynomial, using the image analysis soft- ware provided with the instrument.
2.2.2. X-ray photoelectron spectroscopy (XPS) XPS spectra were recorded using an SSX-100 spectrome- ter (Model 206 from Surface Science Instruments, Mountain View, CA, USA) equipped with a monochromatized and mi- crofocused X-ray source (aluminum anode; 10 kV, 12 mA). The angle between the normal to the sample and the direc- tion of photoelectron collection was 55◦. The pressure in the analysis chamber was about 10−^7 Pa. Charge stabilization was achieved using an electron flood gun set at 6–8 eV and placing a grounded nickel grid 3 mm above the sample sur- face. The analyzed area was approximately 1.4 mm 2 and the
Fig. 1. AFM images (5 × 5 μm 2 ; z = 10 nm) of PS surfaces after collagen adsorption from a 7 μg/ml solution for different periods of time (from top to bottom: 1 min, 30 min, 2 h, 5 h, 24 h) followed by rinsing and fast (left) or slow (right) drying. The two half-images presented for 2 h of adsorp- tion and slow drying illustrate the low reproducibility obtained under these conditions.
up to an adsorption duration of 30 min. Low reproducibility is observed at 2 h of adsorption, with either a low (0.05) or a much higher (0.17) N/C value. Beyond 2 h of adsorption, the N/C value levels off at a value similar to that observed after fast drying. On pure PS, the water contact angle θ is equal to 90◦ (Fig. 2b). θ measured on adsorbed layers obtained after fast drying decreases progressively from 90◦^ to about 40◦^ when the adsorption duration is increased up to 2 h. Beyond 2 h, θ remains close to 40◦. On the collagen layers obtained after slow drying, θ remains close to 83◦^ up to an adsorption dura- tion of 30 min. After 2 h of adsorption, low reproducibility is found again, with either a high (81◦) or a much lower (57◦)
(a)
(b)
Fig. 2. N/C ratios obtained by XPS (a) and water contact angle θ measured by the sessile drop method (b) on PS surfaces after collagen adsorption from a 7 μg/ml solution for different periods of time followed by rinsing and fast (") or slow (!) drying. The broken and dotted lines highlight the different trends found at short adsorption times for fast and slow drying, respectively; the plain line shows that similar behavior is then observed at long adsorption times.
Fig. 3. Collagen adsorbed amount (left y-axis, Q) determined using radioas- says on PS samples treated for different periods of time with a 7 μg/ml collagen solution. The data are compared to the N/C ratios obtained by XPS on fast-dried samples (right y-axis, "; see Fig. 2).
value of θ. Beyond 2 h of adsorption, θ remains close to the value observed after fast drying for the same adsorption du- ration. The amount of collagen found on PS substrata following adsorption for different periods of time was quantified using radioassays. The results are presented in Fig. 3, together with
the N/C ratios obtained by XPS on the fast-dried samples (from Fig. 2a). The shape of the two curves is similar, with a progressive increase up to 5 h and a leveling off beyond 5 h. A value of 0.9 μg/cm 2 is reached after 24 h of adsorption.
4. Discussion
4.1. Collagen layers obtained after fast drying
The amount of collagen adsorbed on PS from a 7 μg/ml solution after different periods of time, measured using ra- dioassays (Fig. 3), is in good agreement with values re- ported in previous studies. Using a collagen concentration of 5 μg/ml and glass as a substratum, Penners et al. [27] found that a plateau was reached after about 2 h of adsorption, at a level of 0.75 μg/cm 2. Deyme et al. [28] measured an ad- sorbed amount of about 0.3 μg/cm 2 after 5 h of adsorption on polyethylene, starting from a 10 μg/ml collagen solu- tion. More recently, Dewez et al. [26] reported a collagen adsorbed amount of about 0.4 μg/cm 2 after 2 h of adsorp- tion on PS, for a residual collagen concentration of 7 μg/ml. To give a point of comparison, monolayers of closely packed molecules lying or standing on the surface would give ad- sorbed amounts of 0.1 or 25.4 μg/cm 2 , respectively. Note that due to its structure, collagen is not expected to undergo conformation changes upon adsorption. The driving force for adsorption is thus expected to be a gain of entropy due to the liberation of water molecules, the role of electrostatic interactions having been ruled out [26]. The nitrogen signal detected by XPS depends on both the adsorbed amount and the spatial organization of the ad- sorbed protein layer. The N/C molar concentration ratio to be expected from XPS analysis was computed using a model of the adsorbed layer defined by a degree of surface cov- erage (f ) and a constant layer thickness (t) in the covered zones [22]. On the other hand, the adsorbed amount (Q) may also be related to t–f pairs using the relationship
Q = f tρ,
where ρ is the density of collagen (ρ = 1 .4 g/cm 3 ) [29]. A chart of the variation of the computed adsorbed amount as a function of computed N/C is presented in Fig. 4a, consid- ering f constant and t variable or t constant and f vari- able. The experimental data obtained by XPS (N/C ratio) and radioassays (adsorbed amount) are plotted in Fig. 4b. A comparison with the chart shows that, for the fast-dried samples, the progressive increase of N/C and of the adsorbed amount with the adsorption duration is mainly due to a pro- gressive increase of the surface coverage. The thickness of about 8 nm for the adsorbed layer must be taken with cau- tion due to simplifications inherent in the model. It is an apparent value, while the reality must be a distribution of different thicknesses. It is not the thickness of the layer but a cumulated thickness of the collagen molecules forming the
(a)
(b) Fig. 4. Variation of the amount of collagen adsorbed on PS as a function of the N/C ratio given by XPS: (a) chart of the computed variations at the indicated constant surface coverage f (—) and at the indicated con- stant thickness t (- - -); (b) experimental data for collagen adsorption from a 7 μg/ml solution for different periods of time followed by rinsing and fast (") or slow (!) drying. Mean values are presented for the sake of clar- ity, except for the results obtained after 2 h of adsorption, which showed low reproducibility. The broken and the dotted lines in (b) highlight the different trends found at low adsorbed amount for fast and slow drying, respectively; the plain line shows that similar behavior is then observed at high adsorbed amount.
layer. However, it is in the same range as the total thick- ness of the collagen layer obtained after 2 h of adsorption from a 30 μg/ml solution on CH 3 -terminated self-assembled monolayers and fast drying, found to be about 7.5 nm from AFM images [30]. Fig. 4 indicates that adsorption does not proceed through the accumulation of layers (t increase) of closely-packed molecules (f = 1). A direct visualization of the organization of the adsorbed collagen layers is provided by AFM. After 1 or 10 min of adsorption, the layer is so loose that it is perturbed by the scanning AFM probe. After 30 min, it is dense enough to resist damage while scanning. The AFM images suggest
odal dewetting. The polygonal network was attributed to the growth of heterogeneously nucleated holes which oc- cupied the whole area before the spinodal dewetting could take place. These authors considered the process as involv- ing dewetting of a liquid film, the collagen molecules being only a marker of the dewetting of the solvent. The results presented here show that similar patterns can be obtained by slowly drying a wet layer of adsorbed col- lagen. This process could be viewed as the dewetting of a liquid (water) on a solid (PS), the liquid phase being modi- fied by the presence of collagen. In this case, decreasing the adsorbed amount would be equivalent to decreasing the con- centration of the dewetting solution. This would be similar to the process described by Stange et al. [13], where more developed PS patterns were obtained by spin-coating less concentrated PS solutions on silicon. However, as collagen is adsorbed on the PS substrate, its interaction with the sub- strate must certainly be taken into account. Alternatively, the process could be viewed as the dewet- ting of a liquid (water) on a solid (PS) modified by the adsorbed collagen layer, which results in decreasing hy- drophobicity of the surface. The increase of the amount of adsorbed collagen would then shift the interfacial potential curve and stabilize the thinning film. However, the situation is more complex here, due to the characteristics of the ad- sorbed layer and its displacement by the receding film. It has been observed that thin films of styrene oligomers on sil- icon were stabilized by the combination of long polystyrene chains added in solution and end-functionalized polystyrene molecules forming a brush at the silicon surface. Film stabi- lization was shown to depend on the entanglement between this brush and free polymer molecules [37]. At this stage, it is difficult to speculate about the mechanisms by which adsorbed collagen influences the stability of the film and the respective role of spinodal dewetting with respect to nucleation-controlled dewetting. Above 2 h of adsorption, the collagen molecules are sub- ject to self-association in the adsorbed phase, as revealed by the observation of fibrillar structures on dried specimens (Fig. 1) as well as on specimens left in water (Gurdak et al., to be published). In these conditions, the collagen felt may hinder the oscillations responsible for spinodal dewetting or be strong enough to reduce the mobility of collagen mole- cules and their displacement by the water meniscus. In any case, while collagen reveals a dewetting process, it may not be considered simply as a witness of the dewetting process. It may influence this process by affecting the liquid film viscosity and thereby spinodal dewetting, by screening the hydrophobicity of the PS substrate, and/or by serving as nu- cleation points. In contrast with common observations, there is no for- mation of droplets at the latest stage of dewetting. This last step, attributed to Rayleigh instabilities [33,38], is probably prevented by the anisotropy of the collagen molecule, which is a rather rigid filament compared to PS coils, and to self- aggregation of collagen molecules.
5. Conclusion
Adsorbed collagen layers presenting a variety of nano- morphologies have been obtained at the PS surface by vary- ing the adsorption duration and the drying rate. Radioassays show that the collagen adsorbed amount increases up to 5 h of adsorption and then levels off. If fast drying is applied to the samples, combination of XPS and radioassays reveals a progressive increase of the surface coverage with the ad- sorption duration, indicating the formation of a collagen felt, the density and/or the thickness of which increase with the adsorbed amount. This is further supported by the wettabil- ity data, which show that cos θ increases progressively with the adsorbed amount. A continuous layer is observed on the AFM images starting from 30 min of adsorption. This again may be explained by the formation of a felt presenting in- terstices which are small compared to the size of the AFM probe. At that stage, the adsorbed layer is more resistant to scanning. Discontinuous structures are formed by collagen adsorp- tion on PS for short periods of time and slow drying. The observed morphologies are similar to those obtained by a dewetting process. The wet adsorbed collagen film is rup- tured and collagen is then displaced by the progression of the contact line formed by dewetting, and geometric patterns are created, with characteristics which are influenced by the ad- sorbed amount. This no longer occurs when the adsorption duration is longer (2 h) and the adsorbed amount is higher ( 0 .6 μg/cm 2 ), due to hindering of the onset of dewetting and/or to the increased collagen–collagen intermolecular in- teractions, preventing collagen displacement.
Acknowledgments
The authors thank P. Bertrand and the late P. Grange for the use of the spin-coater and of the atomic force mi- croscope, respectively. Ch. Dupont-Gillain is a postdoctoral researcher of the Belgian National Foundation for Scien- tific Research (F.N.R.S.). P. Rouxhet is a member of the Research Center in Micro- and Nanoscopic Materials and Electronic Devices (CERMIN). The support of the F.N.R.S., of the Federal Office for Scientific, Technical and Cultural Affairs (Interuniversity Poles of Attraction Program) and of the Research Department of the Communauté Française de Belgique (Concerted Research Action) is gratefully ac- knowledged.
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