2284
Langmuir 2008, 24, 2284-2287
Simultaneous Investigation of the Influence of Topography and Charge on Protein Adsorption Using Artificial Nanopatterns E. Macarena Blanco,† Michael A. Horton,† and Patrick Mesquida*,‡ London Centre for Nanotechnology and Department of Medicine, UniVersity College London, 5 UniVersity Street, London WC1E 6JJ, United Kingdom, and Department of Mechanical Engineering, King’s College London, Strand, London WC2R 2LS, United Kingdom ReceiVed September 24, 2007. In Final Form: NoVember 14, 2007 The combined influence of surface topography and charge of a polymer surface on the adsorption of the protein avidin has been investigated. Atomic force microscopy contact mode imaging and charge writing were used to create defined topographical roughness and electrostatic charge patterns on the surface of polystyrene. Increased avidin adsorption was found on nanometer-size topographical patterns, but the adsorption remained unaffected by electrostatic patterns.
Introduction Understanding the adsorption of proteins on solid surfaces is paramount in the search for biocompatible implant materials, the design of medical devices, in the field of tissue engineering, and in the development of protein-based biosensors.1 Protein adsorption is influenced by the physical and chemical properties of the surface such as roughness, hydrophobicity, surface charge, chemical surface composition, and structure as well as the properties of the protein itself such as size, shape, and charge.1 Hence, a large number of experiments would have to be carried out to quantitatively investigate the influence of all these parameters. Current nanotechnology methods, as applied herein, offer possibilities to create micro- and nanopatterned surfaces whose properties can be tailored on an individual sample basis to perform quantitative adsorption studies simultaneously and with a high degree of accuracy and repeatability.2-5 Two important parameters which affect protein adsorption are surface roughness and charge. A greater surface roughness increases the effective surface area. Thus, more surface is available for protein attachment through nonspecific interactions such as van der Waals forces whereas the overall number of specific interaction sites such as polar or charged groups remains constant. Furthermore, topographical surface features whose size and shape correspond to the size and shape of the protein can also lead to increased adsorption as the protein finds suitable “dock-in” sites. If the surface features are smaller than the protein, however, these features (e.g., small holes) might be inaccessible to the protein and thus not contribute to increased adsorption. The surface charge can lead to attraction or repulsion of proteins depending on their isoelectric point and charge. The ability to adjust these two parameters, surface roughness and surface charge, independently of each other on the same sample allows the effect of these parameters upon protein adsorption to be investigated directly. * To whom correspondence should be addressed. E-mail: patrick.
[email protected]. † University College London. ‡ King’s College London. (1) Castner, D. G.; Ratner, B. D. Surf. Sci. 2002, 500, 28. (2) Galli, C.; Collaud Coen, M.; Hauert, R.; Katanaev, V. L.; Gro¨ning, P.; Schlapbach, L. Colloids Surf., B 2002, 26, 255. (3) Lubarsky, G. V.; Browne, M. M.; Mitchell, S. A.; Davidson, M. R.; Bradley, R. H. Colloids Surf., B 2005, 44, 56. (4) Shi, H. Q.; Tsai, W. B.; Garrison, M. D.; Ferrari, S.; Ratner, B. D. Nature 1999, 398, 593. (5) Christman, K. L.; Requa, M. V.; Enriquez-Rios, V. D.; Ward, S. C.; Bradley, K. A.; Turner, K. L.; Maynard, H. D. Langmuir 2006, 22, 7444.
In the present work, we demonstrate the use of atomic force microscopy-based charge writing (AFM-CW)6 to investigate, simultaneously, the influence of topography and surface charge on protein adsorption. As a model system we have chosen polystyrene (PS) as the surface because biochemical assay plates (microwell plates) usually consist of this material and because topographical modification by AFM imaging has already been demonstrated with this polymer.7,8 Avidin, conjugated with a fluorophore, was chosen as a well-characterized protein, and we investigated its adsorption by semiquantitative fluorescence microscopy. AFM-CW is a technique in which conductive tips are used to inject positive or negative electrical charges into an insulating material, such as a polymer, that can trap charges for a sufficient amount of time so that the surface exhibits an electrostatic field.6,9 The charges are injected by applying a bias voltage to the tip while scanning it over the surface. Besides electrostatic patterning, AFM can also be used to create permanent topographical patterns on a sufficiently soft surface by scanning the tip in contact mode at relatively high force and thereby “scratching” the surface.7,8,10 In contrast, scanning the tip in tapping mode is much gentler to the surface and does normally not produce topographical modifications even on soft polymer surfaces. Both patterning techniques are combined here by applying a bias voltage while scanning in contact mode to produce a charge as well as a topographical pattern. Materials and Methods Sample Preparation. Flat polymer surfaces were prepared by spin coating polystyrene (PS) on polished silicon wafers. The wafers had a specific resistivity of 14.0-20.0 Ω‚cm and were purchased from the Scottish Microelectronics Centre (University of Edinburgh, Edinburgh, UK). Before spin-coating, the wafers were cut into pieces of ca. 5 mm × 5 mm and cleaned by ultrasonication in ethanol and in deionized water (18.2 MΩ‚cm, Elga Labwater, Bucks, U.K.) for 10 min each. The Si pieces were dried in a stream of nitrogen. PS powder (Mw ) 200 000, Sigma-Aldrich Chemical Co., Poole, U.K.) was dissolved in xylene analar (BDH Ltd., Poole, U.K.) at varying concentrations between 0.05 and 0.07 g/mL. Spin coating for 45 s (6) Stern, J. E.; Terris, B. D.; Mamin, H. J.; Rugar, D. Appl. Phys. Lett. 1988, 53, 2717. (7) Aoike, T.; Yamamoto, T.; Uehara, H.; Yamanobe, T.; Komoto, T. Langmuir 2001, 17, 5688. (8) Leung, O. M.; Goh, M. C. Science 1992, 255, 64. (9) Wright, W. M. D.; Chetwynd, D. G. Nanotechnology 1998, 9, 133. (10) Kim, Y.; Lieber, C. M. Science 1992, 257, 375.
10.1021/la702957f CCC: $40.75 © 2008 American Chemical Society Published on Web 02/16/2008
Letters
Langmuir, Vol. 24, No. 6, 2008 2285
at 2000 rpm and baking the film on a hot plate at 60 °C for 20 min provided film thicknesses of ca. 300-600 nm. Sample Characterization. The topography of the samples was determined by atomic force microscopy (AFM) using a Digital Instruments Multimode AFM (Veeco Metrology LLC, Santa Barbara, CA) with Nanoscope V612 software. The local surface potential was simultaneously determined by Kelvin-probe force microscopy (KFM)11 with the same instrument. The AFM tips used were conductive, doped, plain silicon tips (MikroMasch Eesti OU, Tallinn, Estonia). The roughness, effective surface area and projected surface area were analyzed with the function “roughness analysis” of the program WSxM (Nanotec Electronica S.L., Madrid, Spain). Optical fluorescence microscopy has been used to estimate protein adsorption of fluorescently labeled avidin. The fluorescence images were made using a cooled, digital Qimaging 3.3 Micropublisher camera (Digital Pixel, Brighton, U.K.) installed in an Axioplan universal microscope for transmitted light and incident-light fluorescence (Zeiss, Go¨ttingen, Germany). Charge Writing. Localized surface charges were produced by applying a positive or negative bias voltage to the conductive tips with an external, home-built, voltage pulse generator while scanning the tip in contact or tapping mode.12 The pulse generator output was connected to an analog input of the AFM’s signal access module (SAM, Veeco Metrology LLC, Santa Barbara, CA), which provided a direct electrical connection to the AFM tip. The “nanolithography” function of the AFM operating software Nanoscope V612 was used to position the tip in a defined manner in horizontal direction on the sample surface and to trigger the voltage pulses of the pulse generator via the SAM. Protein Solution Preparation and Adsorption. ImmunoPure Avidin, Fluorescein (FITC conjugated) 5 mg/mL in a buffer of 10 mM HEPES, 0.15 M NaCl at pH 8.5, and 0.08% sodium azide (Perbio Science UK Ltd., Tottenhall, U.K.) was used as test protein for adsorption. Avidin is a highly glycosylated, globular tetramer of eight-stranded, antiparallel β-barrels13 with a total molecular mass of approximately 67 kDa and a size of 4 to 5 nm.14 The protein was diluted in 10 mM HEPES (Sigma-Aldrich Chemical Co., Poole, U.K.) at pH 7 to a protein concentration of 0.1 mg/mL. The chargeand topography-patterned samples were immersed for 3 min in the solution and thereafter rinsed in clear buffer and deionized water and then dried in a gentle stream of nitrogen for a few seconds.
Results and Discussion Although there are a number of reports of AFM-CW on polymeric substrates in the literature,6,15,16 PS has not been used as material for charge writing so far. Therefore, it was first checked whether positive as well as negative charge patterns can be created on PS and if they are stable for a sufficient time to allow protein adsorption. Figure 1 confirms that AFM-CW on PS can be performed, with KFM surface potential images of positive (bright) and negative (dark) charge patterns evident on the same individual PS sample. Four rows of eight 1 µm × 1 µm charge patterns were written on PS in air under ambient conditions: positive bias voltage in tapping mode (Figure 1a, top), negative bias voltage in tapping mode (Figure 1a, bottom), positive bias voltage in contact mode (Figure 1b, top), and negative bias voltage in contact mode (Figure 1b, bottom). The observed surface potentials in air directly after (11) Nonnenmacher, M.; O’Boyle, M. P.; Wickramasinghe, H. K. Appl. Phys. Lett. 1991, 58, 2921. (12) Mesquida, P. Ph.D. Thesis No. 14854 (Appendices B.1 and B.2), ETHZurich, Switzerland, 2002, (available free of charge at http://e-collection.ethbib.ethz.ch/show?type)diss&nr)14854). (13) Livnah, O.; Bayer, E. A.; Wilchek, M.; Sussman, J. L. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5076. (14) PDB Protein Data Bank (available online at www.pdb.org); Rutgers, the State University of New Jersey, Piscataway NJ. (15) Scho¨nenberger, C. Phys. ReV. B 1992, 45, 3861. (16) Mesquida, P.; Knapp, H.F.; Stemmer, A. Surf. Interface Anal. 2002, 33, 159.
Figure 1. Charge patterns on PS. KFM image of positive (bright) and negative (dark) patterns written in tapping mode (a) and contact mode (b) on the same PS film (thickness ) 350 nm). AFM-CW was performed by applying +70 V (-70 V) tip bias voltage pulses for the positive (negative) charges, pulse length ) 0.5 ms. Each individual spot was written by scanning the tip on a 1 µm × 1 µm area consisting of ten 1-µm-long lines separated by 0.1 µm. The tip speed was 10 µm/s, and the pulse frequency was 50 Hz. Grey scale ) 10 V, scale bars ) 6 µm.
performing AFM-CW were approximately +2.2 V (Figure 1a, top), -2.8 V (Figure 1a, bottom), +4.0 V (Figure 1b, top), and -5.1 V (Figure 1b, bottom). The patterns in Figure 1 appear larger than the written 1 µm × 1 µm patterns because of the broadening effect of the tip in KFM.17 For the same bias voltage magnitude, the resulting surface potential is higher for negative than for positive polarity, which indicates a higher capacity of PS to trap negative than positive charges. For the same bias voltage polarity, the resulting surface potential is higher when writing in contact mode than when writing using tapping mode. This is probably due to the fact that the overall contact time of the AFM tip with the sample surface is longer in contact mode than in tapping mode. To test protein adsorption on charge patterns the surface charges must remain stable in water for a sufficient time. The effect of water immersion on the stability of the charge patterns is shown in Figure 2. A PS sample with the same pattern as in Figure 1 was imaged by KFM, immersed in deionized water (specific resistivity ) 18.2 MΩ‚cm) for 20 s, dried, and subsequently imaged by KFM again. The charge pattern was still visible after immersion with the surface potential peak reduced to ca. 80% for both polarities (Figure 2), and the horizontal, spatial extent of the charge patterns was not significantly affected. This indicates that the charge carriers are injected into the material sufficiently deep enough to prevent immediate neutralization at the surface and that the conductivity of PS is sufficiently low to prevent charge spreading. It was observed that scanning PS in contact mode resulted in topographical modification of the surfacesno matter whether a bias voltage was applied or notswhereas scanning in tapping mode left the surface topographically unchanged. This fact enables topographical and charge patterns to be produced as independent variables. Figure 3 shows the typical topographic modification created mechanically by scanning the AFM tip in contact mode. In Figure 3a no bias voltage was applied; in Figure 3b,c bias voltages of +80 V and -80 V, respectively, were applied. (17) Jacobs, H.O.; Leuchtmann, P.; Homan, O.J.; Stemmer, A. J. Appl. Phys. 1998, 84, 1168.
2286 Langmuir, Vol. 24, No. 6, 2008
Figure 2. Charge pattern stability in water. Average horizontal surface potential profiles of two rows of eight 1 µm × 1 µm charge patterns, written in contact mode, for (a) positive charges and (b) negative charges. (-) Before immersion in deionized water; (---) after 20 s of immersion in deionized water and drying in air. Surface potential scales are normalized to the values before immersion.
Regular patterns of protrusions, typically of 50-100 nm in size and 5-10 nm in height, were observed. The surface rms roughness of the patterns was 2.1 (a), 2.2 (b), and 3.8 nm (c), compared to the unmodified PS rms roughness of 0.2 nm. The increase of the effective surface area of the patterns compared to the unmodified PS was 3 (a), 4 (b), and 6% (c). Because the occurrence of topographical pattern was independent of the bias voltage the topography and the surface charge could be modified independently of each other. This flexibility is exploited in the experiment shown in Figure 4. Five patterns, each consisting of a rectangular area of approximately 40 µm × 4 µm, were created on the same individual PS sample, three in contact mode and two in tapping mode. The spatial arrangement and the parameters are shown in Figure 4a. The patterns were created at a sufficiently large distance from each other to prevent any interference between them while maintaining them within the field of view of the optical microscope. The different parameter combinations led to the following results (numbering and conditions as per Figure 4a): (1) rough, negative pattern; (2) rough, neutral pattern; (3) rough, positive pattern; (4) smooth, negative pattern; and (5) smooth, positive pattern. Here, rough refers to an rms roughness >2 nm
Letters
Figure 3. Topographical surface modification of PS created by scanning in contact mode. All patterns were produced with the same AFM tip, plain doped silicon, with a spring constant of 3.5-12.5 N/m, a tip speed of 10 µm/s, 21 vertical lines scanned per µm traveled in the slow (horizontal) scan axis, and a PS film thickness of 570 nm with (a) no bias voltage, (b) bias voltage ) +80 V, and (c) bias voltage ) -80 V. Images were taken in tapping mode; scale bars ) 200 nm and z scale ) 30 nm.
whereas smooth refers to the original roughness of unmodified PS of 0.2 nm. The combination bias voltage ) 0 and tapping mode was not performed as this corresponds to a smooth, neutral pattern, which is equivalent to the lack of any pattern. After AFM-CW, the sample was incubated for 3 min in FITClabeled avidin solution (concentration ) 0.01 mg/mL, 10 mM HEPES buffer, pH 7). The sample was then sequentially rinsed in pure HEPES buffer and deionized water, dried and imaged by fluorescence microscopy. Figure 4b shows that an increased fluorescent signal is obtained only on the patterns written in contact mode (1-3) (i.e., the ones that exhibit a roughness >2 nm). The patterns written in tapping mode (4 and 5) both showed the same fluorescence intensity as the surrounding background. It can therefore be concluded that protein adsorption is increased by a greater roughness of the PS surface but not by the surface charge. The influence of roughness on protein adsorption depends on the mutual relation of protein size and size of the roughness features such as protrusions or indentations. Indentations smaller than the protein are unlikely to result in an overall increase in
Letters
Langmuir, Vol. 24, No. 6, 2008 2287
not affect the protein adsorption. This is further supported by the observation that the smooth electrical patterns 4 and 5 did not exhibit any increase in fluorescence. Because the isoelectric point of avidin is 10 the protein carries a positive charge at pH 7. Therefore, one could expect higher adsorption on the oppositely charged pattern and lower adsorption on the equally charged pattern. However, no influence of surface charges was observed at all. This could be due to several factors. First, the magnitude of the surface charges created in our experiments could be too small to have any significant effect on avidin adsorption on PS, which could be dominated by other surface properties such as hydrophobicity, topography, etc. Increasing the surface charge further was not possible by AFMCW. Second, although protein adsorption should take place within a few seconds one cannot completely exclude the decay of any surface charge produced by AFM-CW in the buffer solution that is too rapid. However, the results in Figure 2 show that the lifetime of the surface charges should be long enough to allow adsorption. A more important factor preventing increased adsorption could be the surrounding of the protein molecules with negative chlorine ions from the buffer effectively screening the positive charge of the protein. Third, it is possible that avidin adsorption on PS is reversible if the adsorption was governed solely by weak electrostatic forces and, last, the protein could change conformation upon adsorption on the surface, for example, such that more hydrophobic parts of the protein are exposed to the hydrophobic PS and thus making hydrophobic forces become the dominant protein-surface interaction independent of the surface charge.
Conclusions Figure 4. Protein adsorption. (a) Schematic showing the spatial arrangement of the patterned areas on the PS sample (500 nm thickness); the square in the middle is a manually produced marker to locate the patterns in the steps following immersion. (b) Fluorescent optical microscopy image of the patterned areas after immersion and adsorption of FITC-labeled avidin. (c-e) enlarged areas of image b.
adsorption as the protein cannot penetrate into them without unfolding. However, Figure 3 shows that the roughness features are much larger than the protein. As mentioned above, the features have a typical lateral size of 50-100 nm, which is more than 10 times larger than for avidin. Consequently, the protein molecules experience a largely smooth surface whose effective surface area is simply increased by a few percent. Thus, the most likely cause for the observed increase in protein adsorption is the increase of the effective surface area. The fluorescence increase of patterns 1-3 is approximately 30% compared to their immediate surroundings. This signal increase is only an indirect and qualitative indicator of increased protein adsorption because the fluorescence signal depends on the optical and technical properties of the digital camera used to acquire this image. However, the fact that the fluorescence signal increase is similar on patterns 1-3, which have different electrical charges, indicates that the electrostatic properties do
We have demonstrated that AFM can be used to investigate roughness- and charge-influenced protein adsorption by producing rough patterns of different electrical polarity on PS. The advantage of such micropatterns is that a direct comparison of the adsorption can be made using the same individual sample, thereby cancelling out any variations of the protein solution or between different samples. In the present work it was observed that avidin adsorption on PS increases with roughness but is not affected by surface charges. Our results are in contrast to experiments reported in the literature where negative surface charges produced by electronbeam writing led to increased adsorption on PS.18 We do not know the physical basis for this discrepancy. One possible reason could be that the amount of charge and thereby the strength of the surface potential generated by electron-beam writing are much higher than by AFM-CW. Further investigations would be required to elucidate the contribution of electrostatic charges on protein adsorption on solid surfaces. Acknowledgment. This research was funded by the Interdisciplinary Research Collaboration (IRC) in Nanotechnology and was further supported by the London Centre for Nanotechnology (LCN). M.A.H. is funded by the Wellcome Trust, U.K. LA702957F (18) Wybourne, M. N.; Yan, M.; Keana, J. F. W.; Wu, J. C. Nanotechnology 1996, 7, 302.
