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Immobilization of protein molecules on step-controlled sapphire surfaces
Available online at www.sciencedirect.com
Surface Science 601 (2007) 4915–4921 www.elsevier.com/locate/susc
Immobilization of protein molecules on step-controlled sapphire surfaces R. Aoki a, T. Arakawa a, N. Misawa b, R. Tero c, T. Urisu c, A. Takeuchi d, T. Ogino
a,e,*
a
Yokohama National University, Japan b The University of Tokyo, Japan c Institute of Molecular Science, Japan d Namiki Precision Jewel Co., Ltd, Japan e CREST/JST, Japan Received 6 May 2007; accepted for publication 10 August 2007 Available online 31 August 2007
Abstract We have studied effects of surface morphology on immobilization of protein molecules using step-controlled sapphire surfaces. Preferential adsorption of avidin molecules on the step edges was observed on the single-stepped sapphire surface. A randomly-stepped sapphire surface was found to be suitable for high-density immobilization of protein molecules. These results indicate atomic scale structures of the substrate surface influence the adsorption efficiency of the proteins. By using an atomic force microscopy (AFM) equipped with a biotin-modified cantilever, we have confirmed that the immobilized avidin molecules on the substrates keep their biological activity. This means that the ligand–receptor interaction can be detected using the phase image mode of a standard AFM. 2007 Elsevier B.V. All rights reserved. Keywords: Atomic force microscopy; Adhesion; Biological molecules – proteins; Aluminum oxide; Steps
1. Introduction In recent biotechnology such as biosensors, sensing techniques using solid surfaces attract much attention because electronic functions based on semiconductor device technology can be integrated with bio-sensing units. In those devices, immobilization of bio-materials on solid surfaces is one of the most critical issues because the biological activities are often inhibited through the immobilization process. For example, when a protein molecule is deposited on a solid surface, denaturation takes place and its activity often disappears [1–6]. In the strategy of the bio-sensing on solid surfaces, therefore, properties of the solid surfaces should play crucial roles. Avidin is a protein derived from egg-white and the molecule interacts specifically with *
Corresponding author. Address: Yokohama National University, Japan. E-mail address: [email protected] (T. Ogino). 0039-6028/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.susc.2007.08.018
biotin, a kind of vitamins. The avidin–biotin interaction is well-known as one of the strongest binding among the ligand–receptor interactions and often utilized to bind bio-molecules to other bio-molecules, organic molecules, or inorganic materials [7–10]. In order to detect the interaction between an avidin molecule and a biotin molecule, an atomic force microscopy (AFM) can be used because the tip can be modified with the biotin molecules and the binding process changes the tip-surface force [11,12]. In this paper, we describe avidin molecule immobilization on sapphire surfaces and show that a sapphire substrate is suitable for arrangement control of the bio-molecules. Sapphire surfaces are chemically stable and optically transparent. Moreover, it has been shown that step arrangement can be controlled by high temperature annealing. The chemical properties specific to the step edge are preserved in air and liquid in the case of sapphire surfaces because a further oxidation does not occur. Atomic structures on Si surfaces, on the other hand, can be precisely controlled,
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R. Aoki et al. / Surface Science 601 (2007) 4915–4921
but the selectivity in the chemical reactions at the step edges is not preserved in air or in liquid because the Si surfaces are quickly oxidized in such environments. Therefore, step-controlled sapphire surfaces including step-free areas are very useful to examine the surface effects on bio-material immobilization where liquid environment is required. Since atomic steps are often preferential adsorption and reaction sites in the surface processes, step-controlled sapphire substrates are expected to be powerful to obtain well-ordered immobilized biomaterial arrays. 2. Experiment We used two kinds of sapphire C surfaces. One is a surface with randomly-distributed atomic steps (randomlystepped surface) and the other a single-stepped substrate whose step height is about 0.2 nm that is equal to an atomic monolayer of the sapphire single crystal (single-stepped surface). Sapphire substrates with a misorientation angle of 0.15 were annealed in air at 1000 C for single-stepped
surfaces and at 1300 C for randomly-stepped surfaces, respectively. Fig. 1 shows a chemical process of immobilization of avidin molecules onto the surfaces. A sapphire surface was treated with a H2SO4 and H2O2 mixture. After this treatment, the surface is terminated with –OH groups (Fig. 1a). We used 2-(carbomethoxy)ethyltrichlorosilane (CMETS) as a coupling molecule between the sapphire surface and the avidin molecules. A CMETS layer was deposited by dipping the substrate in a 0.5 mM ([M] = [mol/l]) CMETS/toluene solution at 10 C for 1 h (Fig. 1b). Then the substrate was immersed in the concentrated HCl at room temperature (RT) for 24 h to carboxylate the surface (Fig. 1c). N-hydroxysuccinimide (NHS, 3.0 mM) was reacted with the COOH groups in a buffer solution (N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HAPES) and NaOH, pH 7.4) at RT for 30 min to form an NHS-terminated surface. In this process, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 1.0 mM) was added to promote the dehydration reaction (Fig. 1d).
Fig. 1. Chemical processes in the immobilization of avidin molecules: (a) surface state of the sapphire substrate after the acid treatment and chemical structure of a CMETS molecule; (b) polymerization and deposition of CMETS molecules to form a self-assembled monolayer on the sapphire surface; (c) hydrolysis and carboxylation of the CMETS layer surface; (d) chemical structure of an NHS molecule and NHS-termination of the surface; (e) immobilization of avidin molecules.
R. Aoki et al. / Surface Science 601 (2007) 4915–4921
Finally the sample was immersed in the avidin solution at RT for 1 h (Fig. 1e). In this step, the density of avidin molecules on the surfaces was controlled by changing the avidin mole fraction from 20 to 160 nM. The avidinimmobilized substrates were observed in air and in liquid by the dynamic force mode (DFM) of an AFM. In order to examine the biological activity of the avidin molecules immobilized on the sapphire substrates, a biotinfunctionalized AFM cantilever was used, and the avidin– biotin interaction was detected in the phase image mode of the AFM. In the phase image mode, the phase delay of the oscillation of the cantilever with respect to the input signal is detected. This shift is caused by the adsorption and the viscoelastic character of the surfaces while the topography is detected by the change in the oscillation amplitude
4917
of the cantilever. The phase image is sensitive to a weak interaction between the tip and the substrate and can be obtained at the same time as the topography. Biotin molecules were covalently tethered to the amino-functionalized tip in a single coupling step using a ‘‘biotin–PEG–NHS’’ system which consists of a PEG chain with a biotin on one end and an amino-reactive N-hydroxysuccinimide ester function on the other. 3. Results and discussion Fig. 2 shows DFM images of the sapphire surfaces corresponding to the chemical steps shown in Fig. 1. All images were taken in air. Fig. 2a shows a typical morphology of a single-stepped surface. Fig. 2b and c shows DFM
Fig. 2. DFM images and height profiles of the sapphire surface in the avidin immobilization process. These images were taken in air: (a) single-stepped sapphire surface; (b) and (c) the CMETS-layer-deposited surface before and after the hydrolyzing process; (d) NHS-terminated surface; (e) and (f) avidinterminated surfaces where avidin densities in the solutions were 20 and 160 nM, respectively.
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Fig. 2 (continued)
images of the CMETS-deposited surface before and after hydrolyzing. Although CMETS basically forms a selfassembled monolayer, excess molecules were deposited before immersing it in the concentrated HCl. After the hydrolyzing process, however, excess CMETS was removed. We believe that the recovery of the surface flatness shows formation of a CMETS monolayer. Fig. 2d was taken after the NHS-termination. In this stage, the surface is atomically flat without any particle because atomic steps are clearly observed. Fig. 2e and f shows the avidin-immobilized surfaces when the avidin densities of the solution were 20 and 160 nM, respectively. Preferential adsorption of avidin molecules on the step edges is observed in Fig. 2e. This suggests the possibility of arrangement control of bio-molecules by surface structure control of the substrate. The height of avidin molecules in air, shown in Fig. 2e, is smal-
ler than the molecule size in aqueous environment (approximately 5 nm) due to denaturation, as Misawa et al. reported [6]. When the avidin density of the solution was higher, avidin molecules were immobilized also on the terrace as well as the step edges, as shown in Fig. 2f. We discuss the origin of the selectivity in the avidin adsorption between the step edges and the terraces. One possibility is the difference in the adsorption probability of CMETS molecules. Since avidin molecules are not directly bound on the sapphire surface, the density of the CMETS influences the density of the immobilized avidin molecules when the avidin density in the solution is low. In other experiments, we have observed that OTS (octadecytrichlorosilane, C18H37SiCl3) monolayer islands are selfassembled along the step edges on a sapphire surface when the averaged coverage was smaller than one monolayer.
R. Aoki et al. / Surface Science 601 (2007) 4915–4921
Since the reaction mechanism of CMETS with the sapphire surfaces is same as that of OTS, higher adsorption probability of CMETS along the step edges cannot be excluded as the origin of the selective adsorption of the avidin molecules. Although the surface before the hydrolyzation process is rough as shown in Fig. 2b, the flatness is recovered during the hydrolyzation as shown in Fig. 2c. The surface morphology taken by the AFM shows that the surface is uniformly covered with a CMETS film in the present experiment. Another explanation for the selective adsorption is a topological effect. The length of the linker molecule (CMETS) is about 0.3 nm which is larger than the single step height, and the sharp step edges of the sapphire surface should become gentle. The difference in the chemical reaction probabilities between the terraces and the step edges may be small if the reacted molecule is small. However, an avidin molecule (5 nm in diameter in liquid) is much larger than the step height. A steric effect on the reac-
4919
tion between avidin molecules and the CMETS film may be caused by the single steps. Further study, however, is necessary to determine the origin of the adsorption selectivity of avidin molecules. Fig. 3a shows a DFM image of a randomly-stepped sapphire surface taken in air. The roughness is about 0.15 nm in RMS (root-mean-square) which is comparable to the commercial Si wafer’s roughness. Fig. 3b and c shows the results of the immobilization of avidin molecules on the randomly-stepped surfaces when the avidin densities of the solutions were 20 and 160 nM, respectively. These AFM images were taken in air. It is obvious from these images that randomly-stepped surfaces are suitable for high-density immobilization of avidin molecules compared to the single-stepped surfaces. The reason for a higher adsorption of the avidin molecules on the randomly-stepped surface is same as that for the preferential adsorption on single-stepped surfaces because the
Fig. 3. DFM images and height profiles of the sapphire surface in the avidin immobilization process. These images were taken in air: (a) randomly-stepped sapphire surface; (b) and (c) avidin-terminated surfaces where avidin densities in the solutions were 20 and 160 nM, respectively.
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Fig. 4. DFM images and phase images of avidin-terminated randomly-stepped surfaces taken in the buffer solution (1lm · 1 lm): (a) topography; (b) phase image observed using a normal AFM tip; (c) topography; (d) phase image observed using a biotinylated tip.
randomly-stepped surface is a high-density-stepped surface. In conclusion, the atomic scale structure of the sapphire surface influences the immobilization efficiency of avidin molecules. Fig. 4a and b shows a topographic image and a phase image of the avidin-terminated surface on the randomlystepped surface, respectively. Both images were simultaneously taken in a buffer solution using a normal cantilever without biotin-modification. In this experiment, the avidin density of the solution was selected to be 10 nM to clearly observe individual molecules. Even if considering this difference, the molecule concentrations in Fig. 4 may be smaller than that expected from Fig. 3. One possible explanation is difference in the measurement environment. The images in Fig. 3 were taken in air after removing the solution, and some physically adsorbed molecules may remain on the surface after the drying process. In our separate experiment using AFM, the physical adsorption force of avidin molecules onto the solid surfaces was found to be much smaller in the aqueous environment than in air (not shown in this paper). In AFM imaging in the aqueous environment, physically adsorbed molecules are not included because they are easily swept out from the scan area by the tip. Therefore, apparent concentrations observed in a solution is smaller than those in air. The height of the particle in the circle in Fig. 4a is about 5 nm, that is the theoretical height of an avidin molecule. The height of an avidin molecule observed in a buffer solution using AFM was
reported to be comparable to the theoretical size [6]. We can conclude that avidin molecules exist on this surface, though the resolution is generally poor in the imaging in a buffer solution even when using the DFM mode. Any particle image, however, is not observed in Fig. 4b. This means that the interaction between the normal cantilever without biotin-modification and the avidin molecules is very weak. Fig. 4c and d shows a topographic image and a phase image of the avidin-terminated surface which were simultaneously taken in a buffer solution using a biotinylated cantilever. The only difference in the experimental conditions between the set of Fig. 4a and b and that of Fig. 4c and d was whether the tip was biotinylated or not. In Fig. 4d, dips can be observed at the same positions as the avidin molecules were observed in the topographic image shown in Fig. 4c. These results show that an interaction between the biotinylated cantilever and the avidin molecules is taking place and observed as the phase shift of the cantilever oscillation. Since the avidin and the biotin molecules have a specific reaction, these results are expected. In fact, the force between avidin and biotin was measured using AFM by Lo et al. [13] and estimated to be 173 ± 19 pN from the force curves. In our experiments, phase-shift images were taken during scans and quantitative estimation of the avidin–biotin interaction force is difficult. Our results, however, suggest that the position of avidin–biotin interaction can be detected using the biotinylated cantilever. This specific interaction appears only when the bio-
R. Aoki et al. / Surface Science 601 (2007) 4915–4921
activity of the avidin molecules are maintained. Another important result of the present experiments is that the immobilized avidin molecules on sapphire substrates preserve the biological activity in a buffer solution.
4. Conclusion We have studied the roles of atomic structures on the immobilization of proteins on solid surfaces. On the randomly-stepped surfaces, avidin molecules are immobilized in a high density. We found that preferential immobilization of avidin molecules takes place at the step edges on the single-stepped sapphire surfaces. This indicates that surface atomic-level structure influences the efficiency of the immobilization. Moreover, a possibility of self-organized protein array formation based on a well-ordered surface is also suggested. Any signal from the avidin molecules was not detected in a phase image of an AFM when we used the normal cantilevers. On the other hand, we succeeded to observe the positions of the avidin molecules in the phase image mode by using the biotinylated cantilevers. This result also suggest that the immobilized avidin molecules on the sapphire surfaces preserve their bio-activity. In general, we can detect the interaction between a protein on a solid surface
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and a molecule that specifically reacts with the protein by using an AFM tip modified with the reacting molecules. Acknowledgements This work was partly supported by CREST/JST and Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology. References [1] F. Caruso, E. Rodda, D.N. Furlong, J. Colloid Interf. Sci. 178 (1996) 104. [2] B. Lu, M.R. Smyth, R. O’Kennedy, Analyst 121 (1996) 29R. [3] M. Minier, M. Salmain, N. Yacoubi, L. Barbes, C. Methivier, S. Zanna, C.-M. Pradier, Langmuir 21 (2005) 5957. [4] T. Cha, A. Guo, X.-Y. Zhu, Proteomics 5 (2005) 416. [5] Y.-Y. Luk, M.L. Tingey, K.A. Dickson, R.T. Raines, N.L. Abbott, J. Am. Chem. Soc. 126 (2004) 9024. [6] N. Misawa, S. Yamamura, K. Yong-Hoon, R. Tero, Y. Nonogaki, T. Urisu, Chem. Phys. Lett. 419 (2006) 86. [7] T.-S. Huang, R.J. DeLange, J. Biol. Chem. 246 (1971) 686. [8] R.J. DeLange, T.-S. Huang, J. Biol. Chem. 246 (1971) 698. [9] G. Gatti, M. Bolognesi, A. Coda, F. Chiolerio, E. Filippini, M. Malcovati, J. Mol. Biol. 178 (1984) 787. [10] M. Wilchek, E.A. Bayer, O. Livnah, Immunol. Lett. 103 (2006) 27. [11] K.L. Brogan, M.H. Schoenfisch, Langmuir 21 (2005) 3054. [12] A. Ebner et al., ChemPhysChem 6 (2005) 897. [13] Y.-S. Lo, N.D. Huefner, W.S. Chan, F. Stevens, J.M. Harris, T.P. Beebe Jr., Langmuir 15 (1999) 1373.
Surface Science 601 (2007) 4915–4921 www.elsevier.com/locate/susc
Immobilization of protein molecules on step-controlled sapphire surfaces R. Aoki a, T. Arakawa a, N. Misawa b, R. Tero c, T. Urisu c, A. Takeuchi d, T. Ogino
a,e,*
a
Yokohama National University, Japan b The University of Tokyo, Japan c Institute of Molecular Science, Japan d Namiki Precision Jewel Co., Ltd, Japan e CREST/JST, Japan Received 6 May 2007; accepted for publication 10 August 2007 Available online 31 August 2007
Abstract We have studied effects of surface morphology on immobilization of protein molecules using step-controlled sapphire surfaces. Preferential adsorption of avidin molecules on the step edges was observed on the single-stepped sapphire surface. A randomly-stepped sapphire surface was found to be suitable for high-density immobilization of protein molecules. These results indicate atomic scale structures of the substrate surface influence the adsorption efficiency of the proteins. By using an atomic force microscopy (AFM) equipped with a biotin-modified cantilever, we have confirmed that the immobilized avidin molecules on the substrates keep their biological activity. This means that the ligand–receptor interaction can be detected using the phase image mode of a standard AFM. 2007 Elsevier B.V. All rights reserved. Keywords: Atomic force microscopy; Adhesion; Biological molecules – proteins; Aluminum oxide; Steps
1. Introduction In recent biotechnology such as biosensors, sensing techniques using solid surfaces attract much attention because electronic functions based on semiconductor device technology can be integrated with bio-sensing units. In those devices, immobilization of bio-materials on solid surfaces is one of the most critical issues because the biological activities are often inhibited through the immobilization process. For example, when a protein molecule is deposited on a solid surface, denaturation takes place and its activity often disappears [1–6]. In the strategy of the bio-sensing on solid surfaces, therefore, properties of the solid surfaces should play crucial roles. Avidin is a protein derived from egg-white and the molecule interacts specifically with *
Corresponding author. Address: Yokohama National University, Japan. E-mail address: [email protected] (T. Ogino). 0039-6028/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.susc.2007.08.018
biotin, a kind of vitamins. The avidin–biotin interaction is well-known as one of the strongest binding among the ligand–receptor interactions and often utilized to bind bio-molecules to other bio-molecules, organic molecules, or inorganic materials [7–10]. In order to detect the interaction between an avidin molecule and a biotin molecule, an atomic force microscopy (AFM) can be used because the tip can be modified with the biotin molecules and the binding process changes the tip-surface force [11,12]. In this paper, we describe avidin molecule immobilization on sapphire surfaces and show that a sapphire substrate is suitable for arrangement control of the bio-molecules. Sapphire surfaces are chemically stable and optically transparent. Moreover, it has been shown that step arrangement can be controlled by high temperature annealing. The chemical properties specific to the step edge are preserved in air and liquid in the case of sapphire surfaces because a further oxidation does not occur. Atomic structures on Si surfaces, on the other hand, can be precisely controlled,
4916
R. Aoki et al. / Surface Science 601 (2007) 4915–4921
but the selectivity in the chemical reactions at the step edges is not preserved in air or in liquid because the Si surfaces are quickly oxidized in such environments. Therefore, step-controlled sapphire surfaces including step-free areas are very useful to examine the surface effects on bio-material immobilization where liquid environment is required. Since atomic steps are often preferential adsorption and reaction sites in the surface processes, step-controlled sapphire substrates are expected to be powerful to obtain well-ordered immobilized biomaterial arrays. 2. Experiment We used two kinds of sapphire C surfaces. One is a surface with randomly-distributed atomic steps (randomlystepped surface) and the other a single-stepped substrate whose step height is about 0.2 nm that is equal to an atomic monolayer of the sapphire single crystal (single-stepped surface). Sapphire substrates with a misorientation angle of 0.15 were annealed in air at 1000 C for single-stepped
surfaces and at 1300 C for randomly-stepped surfaces, respectively. Fig. 1 shows a chemical process of immobilization of avidin molecules onto the surfaces. A sapphire surface was treated with a H2SO4 and H2O2 mixture. After this treatment, the surface is terminated with –OH groups (Fig. 1a). We used 2-(carbomethoxy)ethyltrichlorosilane (CMETS) as a coupling molecule between the sapphire surface and the avidin molecules. A CMETS layer was deposited by dipping the substrate in a 0.5 mM ([M] = [mol/l]) CMETS/toluene solution at 10 C for 1 h (Fig. 1b). Then the substrate was immersed in the concentrated HCl at room temperature (RT) for 24 h to carboxylate the surface (Fig. 1c). N-hydroxysuccinimide (NHS, 3.0 mM) was reacted with the COOH groups in a buffer solution (N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HAPES) and NaOH, pH 7.4) at RT for 30 min to form an NHS-terminated surface. In this process, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 1.0 mM) was added to promote the dehydration reaction (Fig. 1d).
Fig. 1. Chemical processes in the immobilization of avidin molecules: (a) surface state of the sapphire substrate after the acid treatment and chemical structure of a CMETS molecule; (b) polymerization and deposition of CMETS molecules to form a self-assembled monolayer on the sapphire surface; (c) hydrolysis and carboxylation of the CMETS layer surface; (d) chemical structure of an NHS molecule and NHS-termination of the surface; (e) immobilization of avidin molecules.
R. Aoki et al. / Surface Science 601 (2007) 4915–4921
Finally the sample was immersed in the avidin solution at RT for 1 h (Fig. 1e). In this step, the density of avidin molecules on the surfaces was controlled by changing the avidin mole fraction from 20 to 160 nM. The avidinimmobilized substrates were observed in air and in liquid by the dynamic force mode (DFM) of an AFM. In order to examine the biological activity of the avidin molecules immobilized on the sapphire substrates, a biotinfunctionalized AFM cantilever was used, and the avidin– biotin interaction was detected in the phase image mode of the AFM. In the phase image mode, the phase delay of the oscillation of the cantilever with respect to the input signal is detected. This shift is caused by the adsorption and the viscoelastic character of the surfaces while the topography is detected by the change in the oscillation amplitude
4917
of the cantilever. The phase image is sensitive to a weak interaction between the tip and the substrate and can be obtained at the same time as the topography. Biotin molecules were covalently tethered to the amino-functionalized tip in a single coupling step using a ‘‘biotin–PEG–NHS’’ system which consists of a PEG chain with a biotin on one end and an amino-reactive N-hydroxysuccinimide ester function on the other. 3. Results and discussion Fig. 2 shows DFM images of the sapphire surfaces corresponding to the chemical steps shown in Fig. 1. All images were taken in air. Fig. 2a shows a typical morphology of a single-stepped surface. Fig. 2b and c shows DFM
Fig. 2. DFM images and height profiles of the sapphire surface in the avidin immobilization process. These images were taken in air: (a) single-stepped sapphire surface; (b) and (c) the CMETS-layer-deposited surface before and after the hydrolyzing process; (d) NHS-terminated surface; (e) and (f) avidinterminated surfaces where avidin densities in the solutions were 20 and 160 nM, respectively.
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Fig. 2 (continued)
images of the CMETS-deposited surface before and after hydrolyzing. Although CMETS basically forms a selfassembled monolayer, excess molecules were deposited before immersing it in the concentrated HCl. After the hydrolyzing process, however, excess CMETS was removed. We believe that the recovery of the surface flatness shows formation of a CMETS monolayer. Fig. 2d was taken after the NHS-termination. In this stage, the surface is atomically flat without any particle because atomic steps are clearly observed. Fig. 2e and f shows the avidin-immobilized surfaces when the avidin densities of the solution were 20 and 160 nM, respectively. Preferential adsorption of avidin molecules on the step edges is observed in Fig. 2e. This suggests the possibility of arrangement control of bio-molecules by surface structure control of the substrate. The height of avidin molecules in air, shown in Fig. 2e, is smal-
ler than the molecule size in aqueous environment (approximately 5 nm) due to denaturation, as Misawa et al. reported [6]. When the avidin density of the solution was higher, avidin molecules were immobilized also on the terrace as well as the step edges, as shown in Fig. 2f. We discuss the origin of the selectivity in the avidin adsorption between the step edges and the terraces. One possibility is the difference in the adsorption probability of CMETS molecules. Since avidin molecules are not directly bound on the sapphire surface, the density of the CMETS influences the density of the immobilized avidin molecules when the avidin density in the solution is low. In other experiments, we have observed that OTS (octadecytrichlorosilane, C18H37SiCl3) monolayer islands are selfassembled along the step edges on a sapphire surface when the averaged coverage was smaller than one monolayer.
R. Aoki et al. / Surface Science 601 (2007) 4915–4921
Since the reaction mechanism of CMETS with the sapphire surfaces is same as that of OTS, higher adsorption probability of CMETS along the step edges cannot be excluded as the origin of the selective adsorption of the avidin molecules. Although the surface before the hydrolyzation process is rough as shown in Fig. 2b, the flatness is recovered during the hydrolyzation as shown in Fig. 2c. The surface morphology taken by the AFM shows that the surface is uniformly covered with a CMETS film in the present experiment. Another explanation for the selective adsorption is a topological effect. The length of the linker molecule (CMETS) is about 0.3 nm which is larger than the single step height, and the sharp step edges of the sapphire surface should become gentle. The difference in the chemical reaction probabilities between the terraces and the step edges may be small if the reacted molecule is small. However, an avidin molecule (5 nm in diameter in liquid) is much larger than the step height. A steric effect on the reac-
4919
tion between avidin molecules and the CMETS film may be caused by the single steps. Further study, however, is necessary to determine the origin of the adsorption selectivity of avidin molecules. Fig. 3a shows a DFM image of a randomly-stepped sapphire surface taken in air. The roughness is about 0.15 nm in RMS (root-mean-square) which is comparable to the commercial Si wafer’s roughness. Fig. 3b and c shows the results of the immobilization of avidin molecules on the randomly-stepped surfaces when the avidin densities of the solutions were 20 and 160 nM, respectively. These AFM images were taken in air. It is obvious from these images that randomly-stepped surfaces are suitable for high-density immobilization of avidin molecules compared to the single-stepped surfaces. The reason for a higher adsorption of the avidin molecules on the randomly-stepped surface is same as that for the preferential adsorption on single-stepped surfaces because the
Fig. 3. DFM images and height profiles of the sapphire surface in the avidin immobilization process. These images were taken in air: (a) randomly-stepped sapphire surface; (b) and (c) avidin-terminated surfaces where avidin densities in the solutions were 20 and 160 nM, respectively.
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Fig. 4. DFM images and phase images of avidin-terminated randomly-stepped surfaces taken in the buffer solution (1lm · 1 lm): (a) topography; (b) phase image observed using a normal AFM tip; (c) topography; (d) phase image observed using a biotinylated tip.
randomly-stepped surface is a high-density-stepped surface. In conclusion, the atomic scale structure of the sapphire surface influences the immobilization efficiency of avidin molecules. Fig. 4a and b shows a topographic image and a phase image of the avidin-terminated surface on the randomlystepped surface, respectively. Both images were simultaneously taken in a buffer solution using a normal cantilever without biotin-modification. In this experiment, the avidin density of the solution was selected to be 10 nM to clearly observe individual molecules. Even if considering this difference, the molecule concentrations in Fig. 4 may be smaller than that expected from Fig. 3. One possible explanation is difference in the measurement environment. The images in Fig. 3 were taken in air after removing the solution, and some physically adsorbed molecules may remain on the surface after the drying process. In our separate experiment using AFM, the physical adsorption force of avidin molecules onto the solid surfaces was found to be much smaller in the aqueous environment than in air (not shown in this paper). In AFM imaging in the aqueous environment, physically adsorbed molecules are not included because they are easily swept out from the scan area by the tip. Therefore, apparent concentrations observed in a solution is smaller than those in air. The height of the particle in the circle in Fig. 4a is about 5 nm, that is the theoretical height of an avidin molecule. The height of an avidin molecule observed in a buffer solution using AFM was
reported to be comparable to the theoretical size [6]. We can conclude that avidin molecules exist on this surface, though the resolution is generally poor in the imaging in a buffer solution even when using the DFM mode. Any particle image, however, is not observed in Fig. 4b. This means that the interaction between the normal cantilever without biotin-modification and the avidin molecules is very weak. Fig. 4c and d shows a topographic image and a phase image of the avidin-terminated surface which were simultaneously taken in a buffer solution using a biotinylated cantilever. The only difference in the experimental conditions between the set of Fig. 4a and b and that of Fig. 4c and d was whether the tip was biotinylated or not. In Fig. 4d, dips can be observed at the same positions as the avidin molecules were observed in the topographic image shown in Fig. 4c. These results show that an interaction between the biotinylated cantilever and the avidin molecules is taking place and observed as the phase shift of the cantilever oscillation. Since the avidin and the biotin molecules have a specific reaction, these results are expected. In fact, the force between avidin and biotin was measured using AFM by Lo et al. [13] and estimated to be 173 ± 19 pN from the force curves. In our experiments, phase-shift images were taken during scans and quantitative estimation of the avidin–biotin interaction force is difficult. Our results, however, suggest that the position of avidin–biotin interaction can be detected using the biotinylated cantilever. This specific interaction appears only when the bio-
R. Aoki et al. / Surface Science 601 (2007) 4915–4921
activity of the avidin molecules are maintained. Another important result of the present experiments is that the immobilized avidin molecules on sapphire substrates preserve the biological activity in a buffer solution.
4. Conclusion We have studied the roles of atomic structures on the immobilization of proteins on solid surfaces. On the randomly-stepped surfaces, avidin molecules are immobilized in a high density. We found that preferential immobilization of avidin molecules takes place at the step edges on the single-stepped sapphire surfaces. This indicates that surface atomic-level structure influences the efficiency of the immobilization. Moreover, a possibility of self-organized protein array formation based on a well-ordered surface is also suggested. Any signal from the avidin molecules was not detected in a phase image of an AFM when we used the normal cantilevers. On the other hand, we succeeded to observe the positions of the avidin molecules in the phase image mode by using the biotinylated cantilevers. This result also suggest that the immobilized avidin molecules on the sapphire surfaces preserve their bio-activity. In general, we can detect the interaction between a protein on a solid surface
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and a molecule that specifically reacts with the protein by using an AFM tip modified with the reacting molecules. Acknowledgements This work was partly supported by CREST/JST and Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology. References [1] F. Caruso, E. Rodda, D.N. Furlong, J. Colloid Interf. Sci. 178 (1996) 104. [2] B. Lu, M.R. Smyth, R. O’Kennedy, Analyst 121 (1996) 29R. [3] M. Minier, M. Salmain, N. Yacoubi, L. Barbes, C. Methivier, S. Zanna, C.-M. Pradier, Langmuir 21 (2005) 5957. [4] T. Cha, A. Guo, X.-Y. Zhu, Proteomics 5 (2005) 416. [5] Y.-Y. Luk, M.L. Tingey, K.A. Dickson, R.T. Raines, N.L. Abbott, J. Am. Chem. Soc. 126 (2004) 9024. [6] N. Misawa, S. Yamamura, K. Yong-Hoon, R. Tero, Y. Nonogaki, T. Urisu, Chem. Phys. Lett. 419 (2006) 86. [7] T.-S. Huang, R.J. DeLange, J. Biol. Chem. 246 (1971) 686. [8] R.J. DeLange, T.-S. Huang, J. Biol. Chem. 246 (1971) 698. [9] G. Gatti, M. Bolognesi, A. Coda, F. Chiolerio, E. Filippini, M. Malcovati, J. Mol. Biol. 178 (1984) 787. [10] M. Wilchek, E.A. Bayer, O. Livnah, Immunol. Lett. 103 (2006) 27. [11] K.L. Brogan, M.H. Schoenfisch, Langmuir 21 (2005) 3054. [12] A. Ebner et al., ChemPhysChem 6 (2005) 897. [13] Y.-S. Lo, N.D. Huefner, W.S. Chan, F. Stevens, J.M. Harris, T.P. Beebe Jr., Langmuir 15 (1999) 1373.