Formation of Holoferritin Hexagonal Arrays in Secondary Films Due to

Apr 3, 1996 - Such a secondary film can provide a two-dimensional container for nanometer-sized particles, such as proteins, and bring about an Alder-...
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Langmuir 1996, 12, 1836-1839

Formation of Holoferritin Hexagonal Arrays in Secondary Films Due to Alder-Type Transition Eiki Adachi† Nagayama Protein Array Project, ERATO, JRDC, 5-9-1 Tokodai, Tsukuba, Ibaraki 300-26, Japan

Kuniaki Nagayama* Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Kamaba, Meguro-ku, Tokyo 153, Japan Received September 12, 1995. In Final Form: December 5, 1995X Hexagonally packed holoferritin arrays were produced on a silicon wafer by mechanically expanding (using a scratching method) the holoferritin solution to form a wetting film on the surface. This film is then dried, during which the protein array forms. The packing state of holoferritin depended on the surface charge of the molecules in the wetting film and on the drying rate of the film (i.e., evaporation rate of the water from the film). We found that a secondary minimum was created and then annihilated near the air/water interface and had a potential profile during the evaporation of the water from the wetting film. Consequently, we introduced a concept called the lifetime of a secondary minimum. Since this minimum formed in two-dimensions in the wetting film (primary film), we called it a secondary film in contrast to the primary film that was primarily created in two-dimensions. Such a secondary film can provide a two-dimensional container for nanometer-sized particles, such as proteins, and bring about an Alder-type transition. This new formation technique has widespread application in industry, such as mass production of high-sensitivity bio-sensors.

1. Introduction Protein arrays interest many researchers and engineers as a novel type of condensed material for structural analysis1,2 and industrial applications.3 Protein arrays have been formed on liquid surfaces, for example, on mercury2,4,5 and on water.6-8 However, the arrays formed on liquid surfaces are not suitable for industrial applications because the arrays must be transferred to a solid surface.9 This transfer process inevitably causes deterioration in the alignment of the array. Therefore, direct formation of two-dimensional protein arrays on a solid surface is necessary. To survey this possibility, experiments have been done using polystyrene (PS) spherical particles10-14 ranging in diameter from several hundred nanometers to several * To whom correspondence should be addressed. Phone: +813-5454-6739. Fax: +81-3-5454-4332. † Phone: +81-298-47-9017. Fax: +81-298-47-3599. E-mail: [email protected]/[email protected]. X Abstract published in Advance ACS Abstracts, March 1, 1996. (1) Jap, B. K.; Zulauf, M.; Scheybani, T.; Hefti, A.; Baumeister, W.; Aebi, U.; Engel, A. Ultramicroscopy 1992, 46, 45. (2) Yoshimura, H.; Matsumoto, M.; Endo, S.; Nagayama, K. Ultramicroscopy 1990, 32, 265. (3) Pum, D.; Sa`ra, M.; Messner, P.; Sleytr, U. B. Nanotechnology 1991, 1, 5. (4) Ishii, N.; Taguchi, H.; Matsumoto, M.; Endo, S.; Nagayama, K. Ultramicroscopy 1990, 32, 265. (5) Ishii, N.; Taguchi, H.; Yoshida, M.; Yoshimura, H.; Nagayama, K. J. Biochem. 1991, 110, 905. (6) Fromherz, P. Nature 1971, 231, 267. (7) Uzgiris, E. E.; Kornberg, R. D. Nature 1983, 301, 125. (8) Yoshimura, H.; Scheybani, T.; Baumeister, W.; Nagayama, K. Langmuir 1994, 10, 3290. (9) Furumo, T.; Sasabe, H.; Ulmer, K. M. Thin Solid Films 1989, 180, 23. (10) Dushkin, C. D.; Yoshimura, H.; Nagayama, K. Chem. Phys. Lett. 1993, 294, 455. (11) Denkov, N. D.; Velev, O. D.; Kralchevsky, P. A.; Ivanov, I. B.; Yoshimura, H.; Nagayama, K. Langmuir 1992, 8, 3183. (12) Dimitrov, A. S.; Dushkin, C. D.; Yoshimura, H.; Nagayama, K. Langmuir 1993, 10, 432. (13) Adachi, E.; Dimitrov, A. S.; Nagayama, K. Langmuir 1995, 11, 1060.

microns. The particles are assembled on a glass surface by a convective self-assembly process. Recently such assembling is done simply by slowly pulling up a glass plate that had been submerged in a reservoir (e.g., a beaker) of a solution that contains PS particles and water.15 The convective assembly of particles that occurs in the wetting process is stimulated by water evaporation, which induces a particle flow from the solution reservoir to the boundary of the wetting film. When the thickness of the wetting film approaches the diameter of the particles, a particle monolayer is formed. The packing of this monolayer is governed by a lateral immersion force16 originating from the surface tension of water. This results in a hexagonally packed polystyrene monolayer, which size of domain is approximately 1 mm2, on the glass surface. It was established that direct formation of arrays of submicron particles on a solid surface in the wetting process is achieved by the combination of the convective selfassembly and the lateral immersion force. Unfortunately, unlike arrays of micron-sized particles, it is difficult to apply this combination to form arrays of nanometer-sized particles (such as protein arrays) on solid surfaces. This is due to the lateral immersion force that appears between particles when the film thickness is comparable to the particle diameter. It is very difficult to form stable thin films that have a thickness less than 10 nm, which is a typical size of proteins. Thus, a new formation method is needed for nanometer-thick films, which can act as two-dimensional containers for protein molecules. Historically, protein arrays have been produced at air/ water interfaces8 although they are prepared on bulk liquids. Even if the bulky phases are replaced by a wetting film of a protein solution introduced onto a solid surface, (14) Dushkin, C. D.; Nagayama, K.; Miwa, T.; Kralchevsky, P. A. Langmuir 1993, 9, 3695. (15) Dimitrov, A. S.; Nagayama, K. Chem. Phys. Lett. 1995, 243, 462. (16) Kralchevsky, P. A.; Nagayama, K. Langmuir 1994, 10, 23.

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Formation of Holoferritin Hexagonal Arrays

Figure 1. Schematic representation of the scratching method used in our array formation. The silicon wafer is fixed (by adhesive tape) on the movable stage (S). The platinum plate (P) is stationary during film formation. The stage is moved in the horizontal direction at a constant velocity by a stepping motor (M). As the stage moves, a protein solution introduced at the contact area between the silicon wafer and the plate expands and forms a wetting film. This film is then dried, during which protein arrays form.

protein arrays can be formed at the air/water interface of the film. The formed array is then attached to the solid surface after the water in the film has evaporated, although the array has multilayers. Such a process is the basis of our method for direct formation of protein arrays on solid surfaces. We formed protein arrays on solid surfaces by mechanically expanding a protein solution on a surface to form the wetting film. Although this array formation resembles that at the surface of a bulk liquid, it differs in that the formation occurs in an electrolytic thin wetting film (it includes charged protein particles) as the water evaporates. In the electrolytic film, a characteristic ordering mechanism is expected as reported in this paper. To clarify the details of this ordering mechanism, we evaluated the quality of the ordering of protein molecules at the top layer of array films by varying the surface charge of the protein molecules and the evaporation rate of water during film formation.

Langmuir, Vol. 12, No. 7, 1996 1837 liquid nitrogen cooling system to obtain microscopic images from different positions of 5-6 points on each protein array sample. We then used the Fourier transformation of these FE-SEM images to evaluate the array quality. (This transformation of the images was done using EMIDO, an image-processing software developed by our ERATO project team.) We determined the average distances between molecules by measuring distances between the first diffraction spots in Fourier space. 2.3. Controlling Surface Charge of Proteins. To control the surface charge of the protein molecules, we varied the pH of the protein solution. Each solution had a pH of 5.0, 7.0, or 9.0. The pH was varied by adding GTA buffer containing 3,3dimethylglutaric acid, Tris (2-amino-2-(hydroxymethyl)-1,3propanediol), and 2-amino-2-methyl-1,3-propanediol at a molar ratio of 1:1:1. 2.4. Controlling Drying Rate of Wetting Films. To control the drying rate of the film (i.e., the evaporation rate of the water), the atmosphere around the wetting film during protein film formation was kept at constant humidity by introducing dry N2 gas (0% RH), room humidity (35% RH), and humid N2 gas (50% RH) that had passed through a water reservoir. Furthermore, the movable stage with a silicon wafer (see Figure 1) was sealed by a plastic cover. We determined the evaporation rate of water from droplets of pure water on a glass plate at 20% RH and 40% RH by measuring the quantity of water that evaporates during the initial 2 min using an electronic balance (AND, ER-182A) for evaluation of the drying rate of the film.

3. Results Figure 2 shows the quality of array ordering of holoferritin films formed on silicon wafers which depends on the pH of the protein solution. The films were formed at 35% RH and at 23 °C. The film thickness was about 100 nm. The bright spots show iron cores of the holoferritins. Holoferritins formed a hexagonal lattice at the top layer of the dry film, with an average distance of about 11 nm between iron cores. Fourier-transformed images (lower left of each image) demonstrate that the array quality is improved by an increase in the pH of the protein solution. Table 1 shows this dependence. Figure 3 shows the dependence of array quality on the evaporation rate of water, which was controlled by varying humidity around the scratching setup, for a protein solution at pH 7.0 (also shown in Table 1). The evaporation rate was 0.16 g/m2 s for 20% RH and 0.06 g/m2 s for 40% RH.

2. Experiments 2.1. Protein and Substrate. We used holoferritin (Sigma Co., Ltd.) to produce array films on solid surfaces. Ferritin is spherical (13 nm in diameter) and has a cavity in its center that holds an iron core. The ferritin solution was purified using gel filtration and then diluted with 10 mM NaCl to a concentration of 20 mg/mL. For film formation, we prepared a protein solution at a concentration of 2 mg/mL by diluting the original solution with 10 mM NaCl and 10 mM buffer (GTA). As a solid substrate, we used a silicon wafer (N-type, Shinetsu Co., Ltd.) whose surface had been purified in a 5% HF solution for 1 min and then washed with a chromic acid mixture to make the surface hydrophilic. 2.2. Scratching Method and Evaluation of Arrays. Figure 1 shows a schematic drawing of a scratching method used to form the protein array films on a silicon wafer. A clean silicon wafer (about 20 mm × 20 mm × 1 mm) was fixed to a movable stage (S) and set against one edge of the platinum (P) plate (about 20 mm × 20 mm × 2 mm). Then 10 µL of protein solution was introduced at the edge of the plate. The solution formed a meniscus between the silicon wafer and the plate. The stage was then moved horizontally at a constant velocity (2 µm/s) by a stepping motor (M), which movement formed a wetting film on the wafer. As the water evaporated from the film, a dry protein film started to form at the boundary of the meniscus. After evaporation was completed, we cut the protein array formed on the silicon wafer into small pieces (about 3 mm × 6 mm × 1 mm) for use in a field emission scanning electron microscope (FESEM, HITACHI S5000-H). We used the FE-SEM at an acceleration voltage of 5 kV with a heat stage (∼100 °C) and a

4. Discussions 4.1. Array Quality Depending on Surface Charges of Proteins. Alignment of the array was found to strongly depend on the solution pH and on the evaporation rate of the water. Hexagonally packed arrays were always at the top layer of holoferritin films for pH 7.0 and 9.0, but not for pH 5.0. Although the surface charge of holoferritin17 was calculated to be +1.9 × 10-19 C for pH 5.0, -45.4 × 10-19 C for pH 7.0, and -104.0 × 10-19 C for pH 9.0 (all for an ionic strength I ) 0.01), the negative charge seen for the pH 5.0 solution was expected because the reported isoelectric point of holoferritin is pH 4.8.18 Because the surface charge of holoferritin appears to correlate with the quality of ordering, we created a model that explains the pH dependency of the array ordering. 4.2. A Model for Interpretation of Experimental Results. In our formation technique, the protein array forms during the drying of the electrolytic wetting film. Consequently, the electrostatic condition in the wetting (17) Personal communication with Dr. T. Takahashi. His correspondence address is Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153, Japan. (18) Urushizaki, I.; Niitsu, Y.; Ishitani, K.; Matsuda, M.; Fukuda, M. Biochim. Biophys. Acta 1971, 243, 187.

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Adachi and Nagayama

Figure 2. Dependence of array quality on the pH of the protein solution. The scale bar corresponds to 100 nm. The images were taken using a FE-SEM (HITACHI S5000-H) at an acceleration voltage of 5 kV with a heat stage and liquid nitrogen cooling system. The array quality improved as the pH of the solution was increased.

Figure 3. Dependence of array quality on the evaporation rate of water for a pH 7.0 protein solution for different humidity conditions. The array quality improved as the humidity was increased. Table 1. Dependence of the Quality of Array Ordering on the pH of the Protein Solution and the Humiditya humidity

pH 5.0 (nm)

pH 7.0 (nm)

pH 9.0 (nm)

0% RH 35% RH 50% RH

- (10.4 ( 0.5) - (11.3 ( 0.7) - (11.0 ( 0.4)

+ (10.6 ( 0.5) ++ (11.1 ( 0.8) ++ (11.0 ( 0.3)

++ (11.6 ( 0.3) ++ (11.1 ( 0.6) ++ (11.3 ( 0.4)

a Five to six points were observed for each sample by changing the position for evaluation of array ordering. The symbol - is used when the sample was amorphous in all area. The symbols + and ++ were used when we found ordered arrays at 2-3 points, at least, and at all points for the evaluation, respectively. In parentheses, average distances are shown between iron cores of holoferritins. These were obtained from distances between first diffraction spots in Fourier spaces, which were obtained from original images by using EMIDO.

films changes with time. According to DLVO (DerjaguinLandau, Verwey-Overbeek) theory, a potential (both electrostatic and van der Waals) to the charged particles from interfaces is created19 inside an electrolytic thin wetting film in the vertical direction (see Figure 4). The potential, often characterized by a secondary minimum, always exists at the air/water interface of a thin film (and also at the air/water interface of a bulk liquid). However, since in our formation technique the array forms during the evaporation of the water, the potential profile also changes as the concentration of electrolyte in the film increases. This means the secondary minimum appears at the beginning of the drying process and disappears at the end of the process (note that the macroscopic instability of wetting films is neglected). This creation and annihilation of the secondary minimum in the film is (19) Israelachivili, J. N. Intermolecular & Surface Forces, 2nd ed., Academic Press Inc.: New York, 1991.

Figure 4. Schematic representation of an electrolytic thin film. The parameter σi is the charge density per unit area and Ai (i ) 1, 2) is the Hamaker constant at the interface. The particle has charge q and diameter R. The film thickness is h. We assumed a total potential to the particle as V(x) ) qφ(x) + W(x), where φ(x) ) [1/(0rκ(eκh - e-κh))][(σ1eκh - σ2)e-κx + (σ1e-κh σ2)eκx] and W(x) ) -(R/6)([(A1/x) + (A2/(h - x))]) and κ ) ((2nz2e2)/ 0rkBT))1/2 is a Debye shielding parameter. Here, n, z, e, 0, r, kB, and T are the number density of ions far from interfaces, ionic charge number, elementary electric charge, dielectric constant of vacuum, relative dielectric constant of water, Boltyzmann constant, and temperature, respectively. This potential has a secondary minimum under certain conditions. Since the secondary minimum is formed in two-dimensions in the film, it is called a secondary film.

characteristic to our array formation technique. Thus, we introduce a concept called the lifetime of a secondary minimum, which we defined as the time interval between the creation and annihilation of this minimum. Figure 5 shows the relation between the lifetime of the

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Figure 5. Dependence of the lifetime of a secondary minimum on the surface charge of a particle, q, of film thickness h (see Figure 4). We calculated the zero positions of dV(x)/dx with changing ionic strength (with an initial strength value of 0.01). The lifetime is proportional to the interval between two ionic strengths at which the zero point appears and disappears. Parameters for the calculation were σ1 ) σ2 ) -0.16 C/m2, A1 ) A2 ) 10-19 J, R ) 6.5 nm, r ) 80, 0 ) 8.85 × 10-12 Fm-1, z ) 1, e ) 1.6 × 10-19 C, n ) 6 × 1026 I, and kBT ) 4.1 × 10-21 J. Surface charges of holoferritin at pH 5.0, 7.0, and 9.0 are pointed by arrows. The energy of the minimum is larger than kBT in this case (∼3kBT).

secondary minimum and the surface charge of a charged particle (calculated using DLVO theory) when the concentration of electrolyte constantly increases with the evaporation of water. This figure shows that the lifetime is increased by an increase in the surface charge of the particle. This effect is due to the large surface charge, which then makes the electrostatic energy comparable to the van der Waals energy. Since the secondary minimum is formed in two-dimensions in the electrolytic thin wetting film, the minimum can be thought of as a kind of film. We call it a secondary film (see Figure 4) in contrast to the electrolytic film (primary film) that must be primarily formed in two-dimensions. The ordering process of the protein molecules in the horizontal direction appears to occur in the secondary film during the lifetime of the secondary minimum by the electrostatic repulsive interaction of these molecules. If this is the case, the longer lifetime (produced by using a protein solution of higher pH) gives better ordering of arrays. If we consider holo-

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ferritin as colloidal particles that have the same net charge, then the two-dimensional ordering in the secondary film can be considered as an Alder transition,20 which occurs in a system of particles that interact repulsively. Alder transition is a liquid-solid transition of hard spheres, which repulsively interact with each other, in a twodimensional container. Our system, proteins and the secondary film (container), resembles the system of the transition. 4.3. Array Quality Depending on Drying Rate of Wetting Films. Our results showed that the increase in lifetime caused by the decrease in the evaporation rate of water improves the quality of array ordering. Actually, the quality of the array film for pH 7.0 was improved when the humidity was increased from 0% to 50% RH, which caused a 26-fold increase in the lifetime of the secondary film (it was calculated from evaporation rates of water at 20% and 40% RH). However, change in the lifetime did not affect the array ordering for protein solutions at either pH 5.0 or 9.0. The reason is that the increase in the lifetime for pH 5.0 was not sufficient for ordering because the original lifetime for pH 5.0 was too short (Figure 5). On the contrary, the original lifetime for pH 9.0 was sufficient for ordering (Figure 5) even at low humidity. 5. Conclusions We succeeded in developing a technique to form protein arrays directly on solid surfaces. This successful array formation revealed two new concepts of the ordering mechanism: the lifetime of a secondary minimum and secondary films. These concepts are not limited to our film formation technique. Any electrolytic thin film on a solid surfaces or any free film may contain secondary films. If the secondary film can be retained in an electrolytic thin film for a sufficient time, a large single domain of small colloidal particles, such as proteins, can be produced due to an Alder-type transition. Acknowledgment. We are grateful to Dr. Michael Seul (AT&T Bell Laboratory) for his illuminating discussions on trapping of particles in the secondary minimum. LA950761T (20) Alder, B. J.; Hoover, W. G.; Young, D. A. J. Chem. Phys. 1968, 49, 3688.