pubs.acs.org/Langmuir © 2009 American Chemical Society
Selective Nanopatterning Using Citrate-Stabilized Au Nanoparticles and Cystein-Modified Amphiphilic Protein )
:: ,†,‡ Paivi Laaksonen,* Jani Kivioja,§, Arja Paananen,† Markku Kainlauri,§ :: Kyosti Kontturi,‡ Jouni Ahopelto,§ and Markus B. Linder†
)
† VTT Biotechnology, VTT Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT Espoo, Finland, ‡Laboratory of Physical Chemistry and Electrochemistry, TKK Helsinki University of Technology, P.O. Box 6100, FI-02015 TKK Espoo, Finland and §VTT Micro and Nanoelectronics, VTT Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT Espoo, Finland. Present address: Nokia Research Center Cambridge U.K., 11 JJ Thomson Avenue, Cambridge CB3 0FF, U.K
Received December 4, 2008. Revised Manuscript Received January 30, 2009 We present an approach where biomolecular self-assembly is used in combination with lithography to produce patterns of metallic nanoparticles on a silicon substrate. This is achieved through a two-step method, resulting in attachment of nanoparticles on desired sites on the sample surfaces, which allowed a detailed characterization. First, a genetically modified hydrophobin protein, NCysHFBI, was attached by self-assembly on a hydrophobic surface or a surface patterned with hydrophobic and hydrophilic domains. The next step was to label the protein layers with 17.8 nm gold nanoparticles, to allow microscopic characterization of the films. Kinetics and extent of attachment of nanoparticles were characterized by UV-vis spectroscopy and transmission electron microscopy. It was shown that the attachment of citrate-stabilized gold nanoparticles was strongly dependent on the electrostatic properties of the capping ligand layer and the density of nanoparticles in the monolayer could be controlled via pH. The resulting nanoparticle assemblies followed the original pattern created by optical lithography in high accuracy. We demonstrate that combining bottom-up and top-down nanotechnological approaches in a good balance can provide very effective ways to produce nanoscale components providing a functional interface between electronics and the biological world.
Introduction Controlling the surface binding, distribution, and targeting of nanoparticles is a key element of the technology to use them in applications.1 Utilizing the special properties of nanoparticles such as optical, electronic, or magnetic depends on our means of positioning and organizing them in a functional context. For this, self-assembly is a very attractive technique. Ideally, the components contain in their structures the information needed for finding their correct place and position to build functional assemblies. Self-assembly is ubiquitous in biology, where numerous examples have been studied in great detail. It is a great challenge for technology to make use of selfassembly in the manufacture of materials, electronics, devices, etc. The central importance of self-assembly has been widely recognized and clearly shown to be a route toward better performance.2 Because self-assembly is essential to biology at the molecular scale, it is interesting to turn to biology for understanding and inspiration. There is also a possibility to use biological molecules themselves for nanofabrication in a completely different context than what they have been evolved to do. Advantages of biomolecules include the highly selective and specific interactions that they participate in, for instance, with DNA and protein-receptor recognition.3 Proteins *To whom correspondence should be addressed. Telephone: +358 20722 4611. Fax: +358 20722 7071. E-mail:
[email protected]. (1) Stewart, M. E.; Anderton, C. R.; Thompson, L. B.; Maria, J.; Gray, S. K.; Rogers, J. A.; Nuzzo, R. G. Chem. Rev. 2008, 108, 494. (2) Nam, K. T.; Kim, D.; Yoo, P. J.; Chiang, C.; Meethong, N.; Hammond, P. T.; Chiang, Y.; Belcher, A. M. Science 2006, 312, 885. (3) Keren, K.; Krueger, M.; Gilad, R.; Ben-Yoseph, G.; Sivan, U.; Braun, E. Science 2002, 297, 72.
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additionally have the advantage that their structures are highly defined and that techniques are available for very precise engineering of their structures. In nanofabrication, biomolecules have a wide range of possible applications because of their compatibility with living cells and tissue and their environmentally sustainable production. Although self-assembly is very powerful for organizing individual objects, it is often difficult to control structures having several levels of hierarchy at a larger scale. For largerscale structures, we therefore turn to top-down techniques such as lithography and etching. In order to control structures over orders of magnitude in dimensions and hierarchy, it becomes an interesting aim to combine top-down and self-assembly techniques. This work presents new tools for building devices for nanoelectronics by utilizing a selectively binding protein, hydrophobin. The protein forms semi-insulating surfaces on top of hydrophobic materials that can be manufactured at desired positions by lithographical methods. By choosing a protein that has the ability to bind metallic nanoparticles, we could organize individual nanoparticles on top of the semiconductor surface following lithographically manufactured patterns. Such systems are of interest for building nanoscale devices utilizing the peculiar properties carried by gold nanoparticles.4 For instance, particle-particle separation has a key role when building sensing devices based on interactions between individual nanoparticles,5 and controlling it is another benefit of our method. (4) Daniel, M.; Astruc, D. Chem. Rev. 2004, 104, 293. (5) Wang, L.; Shi, X.; Kariuki, N. N.; Schadt, M.; Wang, G. R.; Rendeng, Q.; Choi, J.; Luo, J.; Lu, S.; Zhong, C. J. Am. Chem. Soc. 2007, 129, 2161.
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From a biofabrication approach, this work was inspired by the interfacial self-assembly exhibited by a group of proteins called hydrophobins. This group of proteins is found in filamentous fungi where they have different roles as adhesives, surfactants, and coatings.6 They have received much attention for their surprising properties in assembling at interfaces.7 The self-assembled surface layers have even been tested as masks in lithographic processes and for passivation of optical devices.8 HFBI9 is a hydrophobin that is produced by the filamentous fungi Trichoderma reesei. It is classified as a class II hydrophobin. HFBI contains about 70 amino acid residues and folds into a compact nearly globular structure with a diameter of about 2 nm. On the surface of the folded molecule, there is a coherent hydrophobic patch which makes the molecule amphiphilic. The amphiphilicity gives the protein the ability to stick particularly to hydrophobic surfaces, which makes HFBI suitable, for example, for surface immobilization.10 A variant of HFBI had been modified from the wild type by genetic engineering to display a single reactive -SH group on its surface which is reactive with gold nanoparticles. By first depositing a monolayer of hydrophobin on a solid surface, gold nanoparticles could be bound to the surface in a controlled way by carefully controlling conditions such as pH. A somewhat similar control of particle density has been shown for the assembly of nanoparticles on S-layers.11 As the amphiphilic nature of HFBI is considered the driving force for its binding and self-assembly, it was of interest to explore possibilities for binding HFBI selectively on patterned surfaces. Silicon patterned with silicon oxide was chosen as the substrate material because of the well-known technology associated with silicon and its well-established status in electronics. When looking for enhancement on building processes of small-scale devices, the combination of self-assembly and top-down methods has proven to be very efficient.12 Our approach shows that biomolecular self-assembly in combination with lithography can be used to create patterns of protein monolayers on which a monolayer of well-dispersed metallic nanoparticles can be bound in a controlled manner. Although nanoparticle patterns on silicon wafers resembling our results have been reported earlier,13 the benefits of our method are evident. When letting the formation of the selective patterns happen by self-assembly, one can avoid laborious steps related to conventional nanopatterning, such as aligning of stamps or serial steps with low throughput surface probe methods. Our method demonstrates that the accuracy of biomolecular self-assembly and recognition should be recognized in silicon technology as a potential tool for building devices in small scale.
(6) Linder, M. B.; Szilvay, G. R.; Nakari-Setaelae, T.; Penttilae, M. E. FEMS Microbiol. Rev. 2005, 29, 877. (7) Szilvay, G. R.; Paananen, A.; Laurikainen, K.; Vuorimaa, E.; Lemmetyinen, H.; Peltonen, J.; Linder, M. B. Biochemistry 2007, 46, 2345. (8) De Stefano, L.; Rea, I.; Giardina, P.; Armenante, A.; Rendina, I. Adv. Mater. 2008, 20, 1529. (9) The protein is named HFBI because it was the first hydrophobin found in this particular fungus. (10) Linder, M.; Szilvay, G. R.; Nakari-Setala, T.; Soderlund, H.; Penttila, M. Protein Sci. 2002, 11, 2257. (11) Bergkvist, M.; Mark, S. S.; Yang, X.; Angert, E. R.; Batt, C. A. J. Phys. Chem. B 2004, 108, 8241. (12) Tamerler, C.; Duman, M.; Oren, E. E.; Gungormus, M.; Xiong, X.; Kacar, T.; Parviz, B. A.; Sarikaya, M. Small 2006, 2, 1372. (13) Chen, C.; Tzeng, S.; Lin, M.; Gwo, S. Langmuir 2006, 22, 7819.
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Results and Discussion In this work, we demonstrated the combination of topdown and bottom-up techniques to achieve a pattern of gold nanoparticles by a bionanofabrication approach. We show that a self-assembling hydrophobin protein forms a functional layer on a surface onto which nanoparticles can be bound. We also show that the binding of the protein is selective to a hydrophobic Si surface in a Si/SiO2 pattern and we can use this property to drive the assembly of surface layers of nanoparticles to selected areas. Synthesis and Characterization of Au Nanoparticles. The nanoparticles were synthesized according to a technique described by Turkevich and Enustun.14 The method is based on simultaneous reduction of aqueous gold salt and stabilization of the formed gold nanocolloid with citrate. According to size analysis from transmission electron microscopy (TEM) micrographs, the resulting particle size was 17.8 nm. The detailed description of the particle synthesis and analysis is in the Experimental Methods section. Since the nanoparticles were capped with acid-containing ligands, characterization of their behavior at different pHs was necessary for knowing the effect of surface charge on surface binding. To obtain the pKa value of the carboxylic groups on the particle surface, the nanoparticle solution was titrated with HCl and NaOH. The experiment is described in the Supporting Information and showed that the pKa3 was 7.8 (see Figure S1). Next, the stability of the nanoparticle solution under different pH conditions was investigated. The changes in ionic strength were minimized by using strong electrolytes in pH adjustment. The stability was studied by UV-vis spectroscopy, ζ-potential measurements, and particle size analysis by dynamic light scattering. The observations are described in the Supporting Information. It was found that the nanoparticles tend to aggregate at pH under 4 and over 11. This could be seen independently from all the different measurements. Protein Self-Assembly and Deposition of Protein Layers on Solid Surfaces. Hydrophobin was deposited on the solid surface by a previously developed method. In this method, the hydrophobin first forms a self-assembled monolayer on the air/water interface of an aqueous solution containing the hydrophobin. When the protein layer has formed, the solid surface is carefully brought in contact with the layer whereby it attaches to the surface. The binding of the protein layer on hydrophobic substrates has been shown earlier and can be verified by, for example, atomic force microscopy (AFM),7 but this is the first time also patterned surfaces are studied. The earlier data on hydrophobin monolayers show that the protein layer binds well to surfaces but the crystallinity of the protein may vary in the layer. The sizes of fully crystalline domains depend on the sample preparation method.7 The method employed in this work is illustrated in Figure 1. The naturally occurring HFBI does not have a chemical functionality that would allow coupling to nanoparticles. We therefore used a variant of HFBI where genetic engineering techniques had been used to introduce a reactive -SH group to the protein. This was done by inserting a Cys amino acid residue to the amino terminal of the protein.7 The variant of HFBI with the additional Cys residue at the amino terminal was called NCysHFBI. It had previously been verified that (14) Enustun, B. V.; Turkevich, J. J. Am. Chem. Soc. 1963, 85, 3317.
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Figure 1. Cartoon describing the attachment of a monolayer of hydrophobin proteins on a hydrophobic substrate. (a) A droplet of hydrophobin solution is pipetted on a hydrophobic substrate. (b) Hydrophobins assemble at the air/water interface on the droplet surface, forming a 2D crystalline lattice. A hydrophobic substrate is brought to contact with the protein monolayer. (c) A monolayer of hydrophobin is attached on a substrate, with the hydrophilic side facing up. this -SH functionality is exposed on the hydrophilic side of the protein layer and therefore could likely be used for linking gold nanoparticles to the protein film. There are numerous examples in the literature of how the applied conditions and functionality of the surface affect the formation of nanoparticle monolayers.15-17 Here, we chose to keep the environment otherwise constant, but to change the pH in order to control the electrostatic properties of the nanoparticles and the protein film. Binding of Nanoparticles to Protein Layers. The fundamental properties of the attachment of nanoparticles to the terminal cysteine group of NCysHFBI film were characterized. Conditions for the formation of the protein film were kept constant in all experiments, whereas the conditions of the nanoparticle attachment were varied systematically. Silanized glass slides, carbon/Formvar TEM grids, and patterned Si/SiO2 wafers were used as the hydrophobic substrates for picking up the protein layers. A series of experiments on attaching nanoparticles on NCysHFBI monolayers on silanized glass were carried out within the pH range of 4-11. UV-vis spectra of NCysHFBI-modified glass slides exposed to the nanoparticle solutions for different times are presented in Figure 2. The appearance of the solution-like absorption spectra of the gold nanoparticles on the glass substrates shows that the nanoparticles attach to the surface without precipitating. The surface coverage of the nanoparticles depends strongly on the solution pH, and very little attachment of particles was observed at pH values above 6 (data not shown). Below pH 6, however, the surface coverage increases as a function of descending pH. Also, the time scale of nanoparticle film formation spans as the pH decreases. At long incubation times, the absorbance of all samples showed a strong increase in intensity throughout the whole spectrum as the amount of nanoparticles at the surface achieved a certain limit. As in the case of colloidal solutions in general, the increasing intensity of the baseline can be associated with scattering of light from larger nanoparticle assemblies. Here, the assemblies are twodimensional nanoparticle islands that form as a result of the interactions between the individual nanoparticles. Also, the effect of tight packing of the particles at long dipping times is observed from the appearance of a shoulder at wavelengths higher than the plasmon band. The new band is due to the interaction between nanoparticles at a close proximity to each other. This, so-called plasmon coupling, is sensitive to the particle-particle separation, and the position of the (15) Grabar, K. C.; Brown, K. R.; Keating, C. D.; Stranick, S. J.; Tang, S. L.; Natan, M. J. Anal. Chem. 1997, 69, 471. (16) Brouwer, E. A. M.; Kooij, E. S.; Wormeester, H.; Poelsema, B. Langmuir 2003, 19, 8102. (17) Zhu, T.; Fu, X. Y.; Mu, T.; Wang, J.; Liu, Z. F. Langmuir 1999, 15, 5197.
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new band gives information about the separation of the particles.18,19 In this case, the intensity of the coupled plasmon stayed below the intensity of the original plasmon band, implying low extent of aggregation on the film.19 The position of the coupled plasmon varied between 590 and 646 nm, which is in agreement with the spectral shift observed earlier for citrate-stabilized gold nanoparticles.19 The intensity of the coupled plasmon evolved as a function of time as presented in the Supporting Information in Figure S4. Formation of the nanoparticle monolayer on NCysHFBI film was also studied with TEM. The protein films were first attached on TEM grids which were then dipped into the nanoparticle solution. Three immersion times, 30 min and 4 and 67 h, for different pHs were used to follow the assembly of the nanoparticles. Particle size and distance to the nearest neighbor and particle density on the grid at each time were analyzed. TEM micrographs and the size/separation analysis from the film at pH 5 are presented in Figure 3. The average size of the nanoparticles was 17.8, and the dispersity 15%. From the upper panel of Figure 3, it can be clearly seen that the nanoparticles do not aggregate on the protein film but they are rather well-distributed on the grid. Well-separated nanoparticles drop cast from aqueous solutions are fairly seldom observed on TEM grids, since water evaporates slowly from the sample droplet and the descending solution drags the nanoparticles, which finally precipitate on the surface (see Figure S5 in the Supporting Information). At long incubation times (67 h), the distribution of nanoparticles on the protein layer is no longer even, but they start to form close packed domains. Hexagonal packing observed at pH 4 (Figure S6 in the Supporting Information) is typical for spherical particles having a reasonably narrow size distribution.4 From the point of view of an individual nanoparticle, formation of closely packed areas means that an increasing number of particles has a neighboring particle at very close proximity. This verifies the appearance of the coupled plasmon band in the UV-vis spectra of the nanoparticle assemblies. The lower panel of Figure 3 illustrates the nanoparticle size and separation distributions on the carbon/Formvar TEM grids. Same analysis for pH 4 and 6 can be found in the Supporting Information in Figures S6 and S7. In all cases, the ratio of the particle center to center separation L and diameter D approaches a value of 1 as the surface coverage of the nanoparticles increases as a function of time. The separation/diameter ratios for all three pHs are presented in Figure 4. In each case, particle separation decreases (18) Rechberger, W.; Hohenau, A.; Leitner, A.; Krenn, J. R.; Lamprecht, B.; Aussenegg, F. R. Opt. Commun. 2003, 220, 137. (19) Sendroiu, I. E.; Mertens, S. F. L.; Schiffrin, D. J. Phys. Chem. Chem. Phys. 2006, 8, 1430.
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Figure 2. UV-vis spectra of NCysHFBI-modified glass substrates show clear plasmon absorption bands of the nanoparticles as they attach to the surface. pH 6: Spectra recorded at 15, 30, 60, 120, 180, and 1320 min. pH 5: Spectra recorded at 15, 30, 60, 90, 150, 180, 240, 1200, and 2700 min. pH 4: Spectra recorded at 30, 45, 60, 90, 120, 180, 240, 1200, 2160, and 3660 min. At long incubation times, a coupled plasmon band due to interactions between close packed nanoparticles appears as a shoulder at wavelength near 650 nm.
Figure 3. Upper panel: TEM micrographs of Au nanoparticles attached to NCysHFBI film on carbon/Formvar grids at pH 5. The micrographs are taken from samples with incubation times of 30 min, and 4 and 67 h. Lower panel: Distributions of the diameter (dark gray) and the nearest neighbor center to center separation (light gray) of the nanoparticles on the TEM grids.
Figure 4. Time dependence of the ratio of particle separation and diameter at pH 4 (b), pH 5 (9), and pH 6 (2). Exponential decay is fitted to the data as guides to the eye. exponentially but at a different rate. It should be noted that the particle separation used here is the distance to the nearest neighbor and not the average distance between nanoparticles; thus, the ratio L/D is not proportional to the surface 5188
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coverage of the nanoparticles. From the data, it can be stated that after a certain time there is another nanoparticle next to each particle but the measured quantity L/D bears no information about the total number of particles in the formed nanoparticle island. Thus, long-time behavior of the nanoparticles appears not to be controlled by the pH of the environment, but by the interaction between the nanoparticles and their tendency to form 2D superlattices. However, control of the nanoparticle separations at shorter time scales (up to hours) gives the possibility to manufacture nanoparticle monolayers with certain particle densities. The surface concentration of the nanoparticles was measured from TEM images. The extinction coefficient ε of the nanoparticles at the maximum wavelength was determined from the measured absorbances and the surface concentrations at incubation time 4 h (except at pH 6 where absorbance at 3 h was used instead). Averaging the values at each pH resulted in a ε value 2.49 108 M-1 cm-1, which is very close to the values obtained for citrate-stabilized gold Langmuir 2009, 25(9), 5185–5192
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nanoparticles in aqueous solutions.19-21 This value was used to convert absorbance at 518 nm to surface concentrations of the nanoparticles. From calculation of the particle densities from the TEM micrographs, it was also noticed that, at pH 4 and 5, the amount of nanoparticles did not change significantly after 4 h, although the distance to the nearest neighbor decreased. Thus, it appears that the increase of absorbance at long times arises from the increased extinction of light by the islandlike nanoparticle superstructures formed on the sample surface. This means that the absorbance at longer incubation times behaves nonlinearly with particle concentration and cannot be included in determination of the kinetics of nanoparticle adsorption on the film. Thus, measured data were studied only up to 240 min incubation time. A simple model, assuming first order kinetics for a Langmuir type adsorption of nanoparticles, was fitted to the measured surface concentration as a function of time. The equilibrium of nanoparticle adsorption and desorption was the following: k1
NP þ S S NPS k2
ð1Þ
In the above chemical equilibrium, NP denotes a free nanoparticle in the solution, S is a vacant surface site, complex NPS is a nanoparticle bound to the surface site, k1 is the rate constant of the adsorption, and k2 is the rate constant of the desorption process. The equilibrium constant K is defined as the ratio of k1 and k2. The rate of the nanoparticle binding to the surface can be written using the surface concentration of the bound nanoparticles ΓNPS:
Figure 5. Surface concentration of the nanoparticles as a function of time at pH 4 (b), pH 5 (9), and pH 6 (2). Solid lines are fits to the measured data. Table 1. Kinetic Parameters Obtained for Nanoparticle Adsorption on Protein Films pH
K (10-7 M-1)
k2 (104 s-1)
Γ¥ (pmol/cm2)
4 5 6
9.5 2.8 0.2
1.1 3.6 6.9
3.4 1.3 0.1
θ¥ (%) 30 11 1.0
The above equation was fitted to the measured data (solid lines in Figure 5) by varying the parameters K and k2, and the obtained values are presented in Table 1. From the data, it is observed that the equilibrium of nanoparticle adsorption
depends strongly on pH. The desorption rate of nanoparticles, k2, increases as pH increases, leading to lower surface concentrations. This can be related to a stronger repulsion between the nanoparticles and the protein layer at high pHs. The value of final surface concentration Γ¥ and the corresponding surface coverage θ¥ are also included in Table 1. Ideally, HFBI settles to a hexagonally packed crystal structure at the air/water interface.7 In a protein lattice, the distance between cysteine groups attached to the N-terminus of the protein is around 2.6 nm.22 Compared to the size of the nanoparticles, the crystal units are much smaller than the particles, and it is very likely that several thiol groups bind to the surface of one nanoparticle simultaneously. Nevertheless, attachment of nanoparticles to the hydrophobin layer appears to be under control of the nanoparticles’ surface charge via pH control. In order to bind to the Cys residue, the surface of the nanoparticle covered with citrate molecules needs to be exposed. The replacement of a noncovalently adsorbed citrate by a thiol having a strong ability to bind to gold can be considered as a favored process, and thus, it is very unlikely to be the limiting step for the adsorption of nanoparticles on the protein surface. In case of thiol molecules carrying charge, the ligand exchange reaction has been shown to be under control of the electrostatic interactions between the nanoparticles and the thiol ligands,23 which is consistent with our results and is discussed below. Another factor hindering ligand exchange would be the presence of steric effects. However, in our case, the Cys group is located at the terminus of a tail consisting of 13 amino acids, which can be assumed to move quite freely. Also, the citrate ligands are quite short and do not provide much steric hindrance.
(20) Haiss, W.; Thanh, N. T. K.; Aveyard, J.; Fernig, D. G. Anal. Chem. 2007, 79, 4215. (21) Demers, L. M.; Mirkin, C. A.; Mucic, R. C.; Reynolds, R. A.; Letsinger, R. L.; Elghanian, R.; Viswanadham, G. Anal. Chem. 2000, 72, 5535.
(22) Kurppa, K.; Jiang, H.; Szilvay, G. R.; Nasibulin, A. G.; Kauppinen, E. L.; Linder, M. B. Angew. Chem., Int. Ed. 2007, 46, 6446. (23) Lim, I. S.; Ip, W.; Crew, E.; Njoki, P. N.; Mott, D.; Zhong, C.; Pan, Y.; Zhou, S. Langmuir 2007, 23, 826.
dΓNPS ¼ k1 ΓS CNP -k2 ΓNPS dt
ð2Þ
where CNP is the solution concentration of the free nanoparticles, 4.5 nM, and ΓS is the surface concentration of the vacant sites. CNP was determined from the absorption of the nanoparticle solution by using the extinction coefficient determined above. The mass balance of the surface sites is the following: Γtot ¼ ΓS þ ΓNPS
ð3Þ
where Γtot is the total amount of surface sites, determined from the theoretical maximum surface coverage of spherical particles (area fraction of close packed circles of the total area, 90.7%). By solving the differential equation assuming concentration of the free nanoparticles as constant, the time dependence of the surface concentration gets the following form: ΓNPS ¼ Γtot
KCNP 1 -e -k2 ð1 þKCNP Þt 1 þ KCNP
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Figure 6. (a) Simulated distribution of different citrate species as a function of pH. The mole fraction of the fully protonated form of citric acid is highlighted. (b) Average charge in terms of unit charge per square nanometer for a protein monolayer (black line) and for a citrate SAM (dashed line). Level of zero charge is indicated with the dotted line. To analyze the effect of pH on the nanoparticle’s surface, distribution of different species of citric acid as a function of pH was simulated and the result compared with the measured data. The simulation was made assuming the same shift (+1.4 units) as observed for pKa3 for each pKa value (see the Supporting Information). As illustrated in Figure 6a, the dominant species of citrate above pH 7 are the divalent and trivalent anions, HA2- and A3-. On the other hand, the population of fully protonated H3A starts to increase below pH 6. When comparing the distribution of ions with different valencies to the measured nanoparticle attachment, it can be deduced that the charge state of the citrate adsorbed on the nanoparticles is a major factor defining the probability of nanoparticle attachment on the protein film. In light of the ion distribution, it can even be stated that the nanoparticles carrying charged citrate ions attach to the protein films to a very small extent and practically only nanoparticles capped with uncharged citrate molecules attach to the surface. This assumption is also consistent with the increased rate of desorption as a function of pH, which is likely to be related to the repulsion between the nanoparticles and the protein. Repulsion between nanoparticles and the protein film can be considered as the main reason for low attachment of nanoparticles above pH 6. The net charge of the amino acid sequence of NCysHFBI changes from positive to negative near pH 6.6 (CLC Main Workbench, CLC bio), which matches rather well with the observed threshold for nanoparticle attachment. Thus, it appears that at certain pHs the attachment of nanoparticles is prevented by the negative charge of both the protein and the nanoparticles. Approximated net charges per square nanometer on a protein monolayer and on a self-assembled monolayer of citrate molecules on a Au surface24 are presented as a function of pH in Figure 6b. Charge of the protein layer was calculated from the amino acid sequence, and the citrate charge is based on the ionic distributions. Note that there is attraction between the two surfaces when their charges have different signs and repulsion when the sign is same. This pH region overlaps with the region where nanoparticle attachment is observed. In addition, pH plays a significant role in the final coverage of nanoparticles also at pH below 6. This implies that the repulsion between nanoparticles also regulates the density of the formed monolayer and prevents aggregation on the film. (24) Kunze, J.; Burgess, I.; Nichols, R.; Buess-Herman, C.; Lipkowski, J. J. Electroanal. Chem. 2007, 599, 147.
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When the measured surface coverage is extrapolated toward larger values, full particle coverage would be achieved at pH value close to 2 matching with the pH where uncharged H3A species achieves its maximum concentration (See Figure S8 in the Supporting Information). This pH is however beyond the limits of colloidal stability of the nanoparticles as demonstrated in Figure S2 in the Supporting Information, meaning that the nanoparticles would be already aggregated in the immersion solution if the experiment was carried out at this pH. Thus, achievement of full coverage with these nanoparticles is not possible in this particular environment. Manufacture of Patterned Surfaces. Patterns of Si/SiO2 were studied because of the well-established techniques in this field and the wide use of the material in electronics and sensor applications. Present semiconductor industry strongly relies on Si and SiO2. Here, the Si/SiO2 pattern is considered from a surface energy point of view as domains of hydrophobic and hydrophilic sites. The pattern was made by a hydrofluoric acid (HF) wet etching utilizing a standard photolithography technique on a silicon wafer with a 20 nm thermal oxide on top. The minimum feature size was of the order of 100 nm, and the patterned structure consists of both positive and negative features (SiO2 features on Si surface or vice versa). Prior to protein transfer, the surface was treated with HF to remove natural oxide from the patterned Si surface, which also reduced the final Si/SiO2 step size to the value of 15 nm as analyzed by AFM (data not shown). Protein transfer was done immediately after etching to prevent the oxidation of the exposed Si surface in air. Making Patterns with Proteins and Nanoparticles. The possibility to control the adhesion of hydrophobin to a surface was studied by transferring the protein films on silicon oxide patterned silicon wafers followed by microscopic observation. After attaching the protein to the surface, the samples were dipped into Au nanoparticle solutions which allowed location of the attached protein film by following the nanoparticle binding. The conditions of nanoparticle dipping were chosen based on the results discussed above. To achieve an array of well-separated and evenly distributed nanoparticles, pH 5 and a dipping time of 45 min were chosen. The resulting nanoparticle patterns are shown in Figure 7, where the dark areas are silicon, the light wide areas are silicon oxide, and the white spots are the gold nanoparticles. It is readily observed that the nanoparticles are selectively attached to the parts where silicon is exposed. Some separate particles and particle aggregates still remain at the oxide parts, but they were shown to be loosely attached and easily removed by more thorough washing of the samples (Figure 7c-e). Similar patterns were obtained by AFM, showing a more densely packed monolayer of nanoparticles on the Si/NCysHFBI surfaces and some nanoparticle aggregates on the oxide (see the Supporting Information). There was no evidence of nanoparticle binding from solution to the surface of bare silicon or silicon oxide in large extent (data not shown). These results clearly indicate that the hydrophobin film has been transferred only on the hydrophobic parts of the wafers. The rinsing protocol of the samples appeared essential for washing out all the nonspecifically bound nanoparticles from the sample surface. In Figure 7c-e, 2 min showering of the protein-nanoparticle composite with distilled water was employed. The sample presented in Figure 7d was also rinsed with acidic (pH = 2.5) solution in prior to the water shower, whereas the sample presented in Figure 7e was Langmuir 2009, 25(9), 5185–5192
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the underlying lithographically realized structure of the substrate, the selective self-assembly of the protein, and finally the conditions during the assembly of a nanoparticle monolayer on the film. Each step could be individually controlled, and the final structure undoubtedly followed the original pattern. Thus, a fully characterized method for a reproducible assembly of gold nanoparticle monolayers on desired positions was presented. The driving forces and reasons to investigate the use of biomolecules in the assembly of devices is that we can make use of self-assembly and specific interactions of biomolecules to be able to miniaturize further and at the same time to make more economic processes. One way of seeing the process is that biomolecules contain information in their structure on where they should position themselves and in which orientation. This reduces the effort that goes into processing and produces new technologies that require less energy. We have shown here a nanomanufacturing process where the functional properties of a protein are used to bridge a top-down lithographic process and a bottom-up self-assembly process.
Experimental Methods
Figure 7. SEM images of Au nanoparticles decorated NCysHFBI films on Si/SiO2 patterned surfaces. (a, b) Samples washed gently with distilled water. (c) Sample showered with distilled water for 2 min. (d) Sample washed first gently with HCl solution (pH = 2.5), (e) NaOH solution (pH = 11.5), and then showered for 2 min with distilled water. rinsed with basic solution (pH = 11.5). The effect of pH of the rinsing solution is clear on both the final density of the particles and the probability of finding aggregates on the oxide surface. Washing with acidic solution clearly increased the amount of aggregates in the sample and also led to a denser nanoparticle monolayer, whereas using basic solution helped to detach the physisorbed particles. Repulsion between the more negatively charged nanoparticles at basic pH appeared to play a role in the assembly of the nanoparticles on the protein layer, but also on the nonspecific binding of the particles on the silicon oxide surface. The above observations support the assumption of direct attachment of the protein to the nanoparticle surface via thiol bonding, since most of the nanoparticles on the surface can take the rather harsh washings.
Conclusions A method for constructing a nanoscale architecture consisting of a protein layer and a monolayer of nanoparticles was demonstrated by utilizing selective properties of the protein. The main property, amphiphilicity, was carried by the employed protein naturally, but the chemical functionality leading to nanoparticle attachment was brought to its structure by genetic engineering. Physisorption of the protein on silicon showed to be sufficiently strong and adjustable for supporting a monolayer of gold nanoparticles, which is not necessarily the case for smaller noncovalently binding molecules. The final structure of the nanofabricated assembly was determined by Langmuir 2009, 25(9), 5185–5192
Materials. All the chemicals were used as purchased. Materials of high purity, HAuCl4 3 3H2O (99.9% Sigma-Aldrich), sodium citrate dihydrate (p.a., Sigma-Aldrich), and dichlorodimethylsilane (99.0%, Sigma-Aldrich) were used in the nanoparticle synthesis and the modification of glass. All the other chemicals were of p.a. quality. Water was distilled and purified with a mQ system (Millipore). In experiments carried out in a clean room, a more thorough cleaning was employed, resulting in ultrapure water (UPW) having resistivity higher than 18 MΩ cm. Synthesis of Fusion Protein NCysHFBI. The NCysHFBI variant of HFBI was produced by growing the VTT D-061175 strain of T. reesei as previously described.25 The purification was performed as previously described.25 Briefly, the supernatant of the medium was extracted with surfactant, and the hydrophobin was recovered from the surfactant by addition of isobutanol. The protein was then purified by reversed phase chromatography and lyophilized. The identity of the protein was verified by matrix assisted laser desorption time-of-flight mass spectroscopy (MALDI TOF MS). Synthesis of Citrate-Stabilized Au Nanoparticles. The synthesis of the nanoparticles was carried out following the protocol adapted from Turkevich and Enustun.14 Briefly, 40 mg of HAuCl4 trihydrate was dissolved in 300 mL of mQ water and heated close to the boiling point. A total of 100 mg of sodium citrate dissolved in 10 mL of mQ water was then added rapidly, and the mixture was refluxed for 20 min. During refluxing, the color of the bright yellow gold salt turned first to dark blue due to the reduction of Au3+ ions to metallic gold, and then gradually to ruby red as the citrate-stabilized nanoparticles were dispersed in the solution. After cooling down, 25 mg of citrate was added to the solution to enhance the stability of the colloidal particles. All the glassware employed in the synthesis was washed with aqua regia (1:3 nitric acid/hydrochloric acid) in prior to use. Note that concentrated acids should be handled with caution. Fabrication of Patterned Si/SiO2 Substrate Wafer. Used patterned silicon/silicon dioxide substrate wafers were fabricated by using common silicon processing techniques in clean room conditions. At first, a 20 nm thick silicon oxide layer was grown on silicon wafers by thermal oxidation. Next, the silicon oxide layer was patterned by using UV-photolithography and (25) Szilvay, G. R.; Nakari-Setaelae, T.; Linder, M. B. Biochemistry 2006, 45, 8590.
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buffered hydrofluoric wet etching (NH4F 35.8%, HF 5.3%). Note that hydrogen fluoride is highly corrosive and toxic and should be handled with caution. Buffered hydrofluoric (BHF) etch was used to reduce resist loss during etching process and hence to enhance the resolution of final structures. The photoresist used was AZ5214 (AZ Electronic Materials). After patterning, the resist layer was stripped out using acetone and isopropyl alcohol. Immediately before protein transfer, the silicon native oxide was removed form the patterned Si surface by using a short (15 s) hydrofluoric etch (1%) dip. This treatment reduces the final step size to 15 nm due to lack of protecting resist layer on top of SiO2 parts.
Measurement of UV-Vis Spectra, ζ-Potential, and Size of the Citrate-Stabilized Au Nanoparticles at Different pH Values. The pH of the solution containing citrate-stabilized nanoparticles was adjusted by adding 0.1 M HCl or 0.1 M NaOH in order to make the solution either acidic or basic, respectively. The pHs of the solutions were measured manually with a videotitrator (Radiometer). The solution resulting from the nanoparticle synthesis was used as-prepared in all experiments. UV-vis absorbance spectra of the nanoparticle solutions after pH adjustment were collected with a spectrophotometer (Varian, Cary 50) from 800 to 300 nm using a glass cuvette. The spectrum of water was subtracted from the measured spectra. The spectra of the nanoparticle films on microscopy glass :: (Menzel-Glaser) were collected by measuring the transmission of the films with the same equipment. In these, the spectrum of an NCysHFBI-modified glass slide was used as the blank sample. ζ-Potential and hydrodynamic radii of the nanoparticles were measured with a particle analyzer (Malvern, Zetasizer Nano ZS). In both ζ-potential and size measurements, disposable capillary cells (DTS1060) were used. Hydrodynamic radii of the nanoparticles were measured by dynamic light scattering (DLS). The number of measurements for both methods was optimized automatically by the software.
Adhesion of NCysHFBI Films on Different Substrates. Formation of the NCysHFBI film on hydrophobic and patterned surfaces and attachment of gold nanoparticles on them were studied by UV-vis spectroscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The protein films were formed from 0.1 mg/mL NCysHFBI water solutions by a drop-surface transfer adapted from Szilvay et al.7 (see Figure 1). Briefly, a droplet of the protein solution is pipetted on a hydrophobic surface and left standing for 1-2 h. During this time, a layer of hydrophobin fusion is formed at the water/air interface due to amphiphilic interactions. After formation of the protein film, a hydrophobic substrate is brought in short contact with the surface of the droplet where the protein film floats, with the hydrophobic side pointing upward. The transfer of the film was observed from the resulting highly hydrophilic spot in the sample at the place the film was transferred. The films were then washed with UPW water and dried with N2 stream. Substrates for the UV-vis measurements were prepared by immersing glass slides in dichlorodimethyl silane for 30 min. The samples were then washed copiously with toluene and dried
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with N2. In prior to silanization, the glass slides were cleaned in hot piranha solution (3:1 concentrated sulfuric acid/hydrogen peroxide) for 30 min and washed with mQ water. Note that piranha solution is highly reactive with organic solvents and should be handled with caution. For TEM, films were transferred on to Formvar carbon grids (Electron Microscopy Sciences). Because the grids are hydrophobic, there was no need for surface modification prior to the film transfer. A similar protocol was used for transferring the protein film on Si/SiO2 patterned surfaces.
Attachment of Au Nanoparticles on the NCysHFBI Films. The attachment of citrate-stabilized Au nanoparticles to the protein films in different pHs was studied by immersing the NCysHFBI-modified substrates in the nanoparticle solutions. In the case of glass slides and TEM grids, the immersion time was varied from 30 min to 67 h in order to characterize the kinetics of the NP monolayer formation on the protein film. On the basis of UV-vis and TEM analysis, suitable immersion time and pH were selected for the decoration of the Si/SiO2 patterned surfaces.
Transmission Electron Microscopy and Scanning Electron Microscopy of the NCysHFBI-Au Nanoparticle Composite Films. The distribution of nanoparticles on Formvar/carbon grids was analyzed by TEM using a Tecnai 12 instrument operating at a 120 kV accelerating voltage. Size analysis of the nanoparticles was carried out using ImageJ software. The directed self-assembly was verified and analyzed by using a LEO1560 Zeiss scanning electron microscope. The accelerating voltage used was 10 kV in all experiments. SEM measurements were carried out in clean room conditions. AFM Measurements. AFM measurements were performed using a Digital Instruments Dimension 3100 atomic force microscope. AFM images were acquired using tapping mode and silicon cantilevers with a nominal spring constant of 42 N/m and a resonant frequency of 330 kHz (Nanosensors NCH). The radius of curvature of the tip was less than 10 nm. Imaging of the patterned silicon wafer samples was performed in clean room conditions. The relative humidity and the temperature were 45 ( 5% and 21.0 ( 0.5 C, respectively. Both the topography and the phase shift images were recorded simultaneously at a scan rate of 0.5 Hz. The images were processed after acquisition by flattening the data and compensating for the tilt.
Acknowledgment. This work was supported by the Academy of Finland (Grant No. 118519) and European Commission under the PF6 programme (Design and functionality of nonlinear electrochemical nanoscale devices, DYNAMO). Riitta Suihkonen is thanked for technical assistance. Supporting Information Available: Characterization of electrostatic properties and stability of the nanoparticles. Coupled plasmon interaction of nanoparticles on NCysHFBI film at pH 4. TEM images and analysis on NCysHFBI-Au nanoparticle films at pH 5 and 6. TEM images of control experiments. AFM image of the NCysHFBI-Au-patterned surface. This material is available free of charge via the Internet at http://pubs.acs.org.
Langmuir 2009, 25(9), 5185–5192
