- Open Access
Protein Adsorption on Nano-scaled, Rippled TiO2 and Si Surfaces
© The Author(s) 2012
- Received: 12 July 2012
- Accepted: 13 August 2012
- Published: 7 September 2012
We synthesized nano-scaled periodic ripple patterns on silicon and titanium dioxide (TiO2) surfaces by xenon ion irradiation, and performed adsorption experiments with human plasma fibrinogen (HPF) on such surfaces as a function of the ripple wavelength. Atomic force microscopy showed the adsorption of HPF in mostly globular conformation on crystalline and amorphous flat Si surfaces as well as on nano-structured Si with long ripple wavelengths. For short ripple wavelengths the proteins seem to adsorb in a stretched formation and align across or along the ripples. In contrast to that, the proteins adsorb in a globular assembly on flat and long-wavelength rippled TiO2, but no adsorbed proteins could be observed on TiO2 with short ripple wavelengths due to a decrease of the adsorption energy caused by surface curvature. Consequently, the adsorption behavior of HPF can be tuned on biomedically interesting materials by introducing a nano-sized morphology while not modifying the stoichiometry/chemistry.
- Contact Angle
- Adsorption Energy
- Adsorption Behavior
- Ripple Structure
The acceptance of artificial replacements is chiefly determined by cell and platelet adsorption out of the surrounding human environment. This in turn is mediated by protein adsorption on the surface of the corresponding device [1–3]. An improvement and acceleration of the healing after surgical intervention would cause a tremendous enhancement of life quality. Therefore, finding optimized functional materials that are able to attract or repel specific molecules is recently in a strong focus of life sciences [4–6]. Thus, the question arises how one can improve and modify the interface between a specific material and its biological surrounding by subsequent physical treatments, e.g. such as ion, laser, or electron irradiation.
A controlled manipulation of the surface morphology can be realized by means of ion beam bombardment, which can be used to cause a periodical structure on a nanometer scale that matches the diameter of proteins. When an energetic ion hits a target, its entire energy is transferred to the target system. If the impact energy is high enough and the energy devolution takes place close to the surface, a fraction of target atoms nearest to the surface will be sputtered off the substrate. A detailed theory of the sputter process itself and the thereby caused roughening of the surface was elaborated by Sigmund . Due to self-diffusion of the surface atoms, a smoothening process can be observed. Finally, the interaction of the described roughening and smoothing causes periodical structures which are called ripples . It was recently shown that osteoblasts show an enhanced response on ion beam irradiated, rippled titanium surfaces pointing to an effect of the ripple pattern on the cell attachment process .
Therefore, introducing morphological changes by means of ion beams offers a new approach to influence the adsorption behavior of proteins on established biomaterials without changing their surface chemistry. In this work, we concentrated on TiO2 that is often used for artificial replacements as this material is well known for its non-toxic, biocompatible character. We investigated the adsorption behavior of HPF on TiO2 as a function of the ripple wavelength and observed a clear influence of the nanostructure on the protein adsorption. Additionally, we performed the same experiments on rippled Si as a reference.
Contact angle measurements (CAM) of water reveal that the contact angle Θ slightly but not significantly increases from 60.9° ± 3.2° to 65.1° ± 12.0° for flat TiO2 after irradiation with xenon ions which had an energy of 20 keV. The fluence was 5 × 1016 cm−2 and the sample was irradiated under an incident ion beam angle of 0° to the surface normal. The same result was also found for flat silicon surfaces: no change of the contact angle after ion irradiation. Note, the latter is accompanied with an amorphization of the surface. However, changes of the hydrophobicity might occur with structural changes. Hence, Fig. 2b shows the obtained contact angles for rippled TiO2 with different reciprocal wavelengths. Within the range of error, we found an increase of the contact angle, about 25–30°, for high wavelengths (small reciprocal wavelength) indicating an influence of the surface curvature on the surface chemistry. However, the contact angles remain unchanged within the range of error for small wavelengths (high reciprocal wavelengths). Therefore, we can exclude an influence of the ripple wavelength on the surface chemistry.
X-ray photoelectron spectroscopy (XPS) was performed on both non- and Xe-irradiated TiO2 and Si surfaces (see supplementary information). The untreated TiO2 samples show an excess of oxygen due to the coverage with adsorbed OH molecules over long periods in atmosphere that is decreased after ion beam irradiation. Nonetheless, the irradiated TiO2 had a perfect stoichiometric surface even after weeks of exposure to air. In contrast, Si surfaces oxidize very fast in air and we observed the common oxygen features of SiO2 , irrespective of performing ion irradiation or not.
Summarizing the part above, Xe ion irradiation induces a clear periodic nano-patterned ripple surface structure for both material systems. The structure can be tuned by the used ion energy. Nonetheless, Xe ion irradiation has only a negligible effect on the surface stoichiometry/chemistry. The latter was detected by both XPS and CAM investigations for flat surfaces. In any case, each set of TiO2 and Si samples exhibits the same surface chemistry before and after the ion irradiation process. However, the ripples have an influence on the hydrophobicity.
We used HPF concentrations (see supplementary information) that were sufficient for a full surface coverage, so that the molecules can connect with each other, as shown for the flat Si or TiO2 substrates. End-to-end interactions of the proteins finally lead to weakly bonded protein networks in the case of the Si substrate. When introducing a nanostructure on a surface, the protein networks cannot muster stable bindings. The adsorption of single proteins on ripple structures with long wavelengths (λ > 100 nm) seems to rely basically on the surface chemistry. In our case, this resulted in a globular conformation of HPF molecules with folded α chains underneath the domains, which is in good agreement with the investigations of Van de Keere et al.  on Ti substrates with TiO2 surfaces and Tunc et al.  on SiO2. Based on the theoretical findings of Melis et al.  for the adsorption of synthetic oligomers, the adhesion of the proteins on our nano-rippled surfaces might be decreased when a curvature appears that is within the size of the protein. Hence, we presume that by increasing the surface curvature (smaller wavelength) for Si, the adsorption energy is decreased. Therefore, it is energetically favorable for the proteins to stretch. By doing this, the contact area between substrate and protein is increased assuring adsorption. This theory accords well with the findings by Rasmusson et al.  and Roach et al.  for the adsorption of HPF on polymer nanostructures and silica nanospheres, for example. The dependence of the orientation of the proteins on the ripple wavelength can be explained as follows. For large wavelengths, the adsorption takes place along the ripple backbones. The situation changes when the wavelength is decreased and thus the curvature is increased. In this case, the required optimization of the adsorption energy is only possible by additional protein–protein interaction. This eventually results in an alignment of the proteins across the ripple backbones, which increases the contact area between neighboring proteins. Protein–protein interaction leading to an increased surface coverage is also described by Roach et al. . Although we observed the described adsorption behavior on short-wavelength ripples only on Si surfaces, we assume a similar effect for TiO2. However, we could not observe any adsorbed proteins on short-wavelength TiO2 ripples, which might be caused by the fact that the adsorption energy of TiO2 is even more decreased than the one of Si. Thus, the adsorption energy was not sufficient for the proteins to attach or to withstand the rinsing procedure. Taking into account that after the adsorption of proteins the samples were rinsed and assuming a small binding energy of short-rippled TiO2, weakly bound proteins on the surface were likely to be removed during the preparation process.
As a consequence, the adsorption behavior of HPF does not seem to rely exclusively on the surface chemistry. A change of the substrate morphology has a major effect on the protein attachment to a substrate. This theory is further supported by the fact that we found an increased water contact angle for TiO2 with short wavelengths, which therefore has a more hydrophobic character. According to the findings by other groups [12, 28], HPF should adsorb more strongly on this hydrophobic surfaces because of the chemistry of the surface. Since this was not the case, we interpret the fact that we did not observe proteins to be a result of the local surface curvature. In order to explain the observation that proteins only adsorbed on the ridges of the ripples, we like to propose the following model: Due to the experimental process, the proteins approach the range of the surface potential of the backbones first and start to adsorb there. According to Siegismund et al. , the migration probability for directions combining HPF molecules is higher than that for isolated molecules. Thus, adjacent proteins adsorb around the backbones as well, which finally leads to the coverage only on top of the ripple backbones.
By preparing nano-sized ripple structures on biomedically relevant materials such as TiO2 and Si via ion beam bombardment, we investigated the influence of the nano-sized morphology on the adsorption behavior of HPF. We found that HPF adsorbs mostly in globular conformation on flat Si and TiO2 surfaces. In the case of Si, some proteins seem also to adsorb in stretched conformation allowing the proteins to interact and cause little network structures. For long ripple wavelengths (λ > 100 nm), the globular adsorption is observed on both materials; whereas, a rather stretched fibrinogen conformation and alignment appeared on short-wavelength (λ ≈ 50 nm) rippled Si. Adsorbed proteins were not found by AFM in the case of short-wavelength rippled TiO2. The observation is mainly explained by a decrease of the adsorption energy due to surface curvature. Concluding, it is possible to tune the adsorption behavior of proteins on biomedical materials just by changing the morphology while not modifying the stoichiometry/chemistry.
We thank Ralf Wagner of the Institute of Materials Science and Technology (University of Jena) for the XPS analysis. Furthermore, we thank Prof. Friedrich Huisken and the Laboratory Astrophysics and Cluster Physics group (University of Jena) for the use of their lab and equipment.
- Doolittle RF (1984) Annu Rev Biochem 53:195–229View ArticleGoogle Scholar
- Lindon J, McManama G, Kushner L, Merrill E, Salzman E (1986) Blood 68:355–362Google Scholar
- Roach P, Farrar D, Perry CC (2005) J Am Chem Soc 127:8168–8173View ArticleGoogle Scholar
- Riedel NA, Williams JD, Popat KC (2011) J Mater Sci 46:6087–6095View ArticleGoogle Scholar
- Lord MS, Foss M, Besenbacher F (2010) Nano Today 5:66–78View ArticleGoogle Scholar
- Stupp SI (2010) Nano Lett 10:4783–4786View ArticleGoogle Scholar
- Cacciafesta P, Humphris LAD, Jandt KD, Miles MJ (2000) Langmuir 16(21):8167–8175View ArticleGoogle Scholar
- Hall CE, Slayter HS (1959) J Biophysic Biochem Cytol 5(1):11–17View ArticleGoogle Scholar
- Feng L, Andrade JD (1995) Proteins at Interfaces II: Fundamentals and Applications; ACS Symposium Series 602; Chapter 5. American Chemical Society, USAGoogle Scholar
- Marchin KL, Berrie CL (2003) Langmuir 19(23):9883–9888View ArticleGoogle Scholar
- Agnihotri A, Siedlecki CA (2004) Langmuir 20:8846–8852View ArticleGoogle Scholar
- Sit PS, Marchant RE (1999) Thromb Haemost 82:1053–1060Google Scholar
- Van De Keere I, Willaert R, Hubin A, Vereecken J (2008) Langmuir 24:1844–1852View ArticleGoogle Scholar
- Vieira EP, Rocha S, Pereira MC, Möhwald H, Coelho MAN (2009) Langmuir 25(17):9879–9886View ArticleGoogle Scholar
- Kim J, Somorjai GA (2003) J Am Chem Soc 125:3150–3158View ArticleGoogle Scholar
- Ta TC, Sykes MT, McDermott MT (1998) Langmuir 14:2435–2443View ArticleGoogle Scholar
- Keller TF, Schönfelder J, Reichert J, Tuccitto N, Licciardello A, Messina GML, Marletta G, Jandt KD (2011) ACS Nano 5(4):3120–3131View ArticleGoogle Scholar
- Ortega-Vinuesa JL, Tengvall P, Lundström I (1998) Thin Solid Films 324:257–273View ArticleGoogle Scholar
- Jung SY, Lim SM, Albertorio F, Kim G, Gurau MC, Yang RD, Holden MA, Cremer PS (2003) J Am Chem Soc 125:12782–12786View ArticleGoogle Scholar
- Cai K, Bossert J, Jandt KD (2006) Colloids Surf B 49:136–144View ArticleGoogle Scholar
- Rasmusson JR, Erlandson E, Salaneck WR, Schott M, Clark DT, Lundström I (1994) Scanning Microsc 8(3):481–490Google Scholar
- Sigmund P (1969) Phys Rev 184(2):383–416View ArticleGoogle Scholar
- Bradley RM, Harper JME (1988) J Vac Sci Technol A 6(4):2390–2395View ArticleGoogle Scholar
- Keller A, Facsko S (2010) Materials 3:4811–4841View ArticleGoogle Scholar
- Yewande EO, Hartmann AK, Kree R (2005) Phys Rev B Condens Matter Mater Phys 71:195405-1–195405-8View ArticleGoogle Scholar
- Chini TK, Datta DP, Bhattacharyya SR (2009) J Phys Condens Matter 21:224004View ArticleGoogle Scholar
- Nefedov VI, Salyn YV, Leonhardt G, Scheibe R (1977) J Electron Spectrosc Relat Phenom 10:121–124View ArticleGoogle Scholar
- Tunc S, Maitz MF, Steiner G, Vázquez L, Pham MT, Salzer R (2005) Colloids Surf B 42:219–225View ArticleGoogle Scholar
- Melis C, Mattoni A, Colombo L (2010) J Phys Chem 114:3401–3406Google Scholar
- Roach P, Farrar D, Perry CC (2006) J Am Chem Soc 128:3939–3945View ArticleGoogle Scholar
- Siegismund D, Keller TF, Jandt KD, Rettenmayr M (2010) Macromol Biosci 10:1216–1223View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited.