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Preparation of Calcium Hydroxyapatite Nanoparticles Using Microreactor and Their Characteristics of Protein Adsorption Kazuhiko Kandori,*,† Tomohiko Kuroda,† Shigenori Togashi,‡ and Erika Katayama‡ School of Chemistry, Osaka UniVersity of Education, 4-698-1Asahigaoka, Kashiwara-shi, Osaka 582-8582, Japan, and Mechanical Engineering Research Laboratory, Hitachi, Ltd., 832-2, Horiguchi, Hitachinaka, Ibaraki 312-0034, Japan ReceiVed: NoVember 1, 2010; ReVised Manuscript ReceiVed: NoVember 30, 2010
The calcium hydroxyapatite Ca10(PO4)6(OH)2 (Hap) nanoparticles were prepared by using microreactor and employed these Hap nanopartices to clarify the adsorption behavior of proteins. The size of Hap particles produced by the microreactor reduced in the order of a hardness of the reaction conditions for mixing Ca(OH)2 and H3PO4 aqueous solutions, such as flow rates of both solutions and temperature. Finally, the size of the smallest Hap nanoparticle became 2 × 15 nm2, similar to that of BSA molecule (4 × 14 nm2). It is noteworthy that the smallest Hap nanoparticles still possesses rodlike shape, suggesting that particles are grown along c-axis even though the reactants mixed very rapidly in narrow channels of the microreactors. The X-ray diffraction patterns of the Hap nanoparticles revealed that the crystallinity of the materials produced by the microreactor is low. The FTIR measurement indicated that the Hap nanoparticles produced by microreactor were carbonate-substituted type B Hap, where the carbonate ions replace the phosphate ions in the crystal lattice. All the adsorption isotherms of acidic bovine serum albumin (BSA), neutral myoglobin (MGB), and basic lysozyme (LSZ) onto Hap nanoparticles from 1 × 10-4 mol/dm3 KCl solution were the Langmuirian type. The saturated amounts of adsorbed BSA (nSBSA) for the Hap nanoparticles produced by microreactor were decreased with decrease in the mean particle length, and finally it reduced to zero for the smallest Hap nanoparticles. Similar results were observed for the adsorption of LSZ; the saturated amounts of adsorbed LSZ (nLSZ S ) also reduced to zero for the smallest Hap nanoparticles. However, in the case of MGB, the saturated mounts of adsorbed MGB (nSMGB) are also depressed with decreased in their particle size, but about half of MGB molecules still adsorbed onto the smallest Hap nanoparticles. This difference in the protein adsorption behavior was explained by the difference in the size and flexibility of three kinds of proteins. The reduction of nSBSA is due to the decrease in the fraction of C sites on the side face of each Hap nanoparticle; i.e., there is not enough area left on the nanoparticle surface to adsorb large BSA molecules even though the BSA molecules are soft and their conformations are alterable. The reduction of nLSZ was explained by the reduction S of P sites. Further, rigidity of the LSZ molecules was given another possibility of the depression of nSLSZ for the Hap nanoparticles. However, MGB molecules with small and soft structure were adsorbed on the Hap nanaoparticle surface by changing their conformation. We could control the amounts of adsorbed proteins by changing the particle size of Hap in the nanometer range and kinds of proteins. These obtained results may be useful for developing biomimetic materials for bone grafts and successful surgical devices in the biochemical field. Introduction It is well-known that a synthetic calcium hydroxyapatite Ca10(PO4)6(OH)2, designated as Hap, shows high affinity for proteins, and much attention has been focused on Hap and substituted Hap nanocrystals as sorbents to remove pathogenic proteins from blood in blood purification therapy1,2 or to act as a carrier for protein delivery.3,4 Hap is in the space group P63/ m; its unit cell parameters are a ) b ) 0.943 nm and c ) 0.688 nm, and it possesses two different binding sites (C and P sites) on the particle surface as is depicted in the crystal structure of Hap in Figure 1. Thus, it contains a multiple-site binding character for proteins.5-7 After dispersing Hap particles in aqueous media, calcium atoms (Ca(II) atoms in Figure 1a) are exposed on the Hap surface by dissolution of OH- ions at the * To whom all correspondence should be addressed. E-mail: kandori@ cc.osaka-kyoiku.ac.jp. † Osaka University of Education. ‡ Hitachi, Ltd.
particle surface to produce rich in calcium ions or positively charged sites to bind to acidic groups of proteins, so-called C sites. These C sites are arranged on ac or bc particle face in a rectangular manner with the interdistances of 0.943 and 0.344 nm (c/2) for the a (or b) and c directions, respectively (Figure 1a). The P sites, negatively charged adsorbing sites, each formed by six oxygen atoms belonging to three crystal phosphate ions, are arranged hexagonally on the ab particle face with a minimal interdistance in both a and b directions equal to |a| () |b|) ) 0.943 nm (Figure 1b). In addition, Hap is the most stable calcium phosphate under physiological conditions. Hence, Hap is widely applied for separating various proteins using as a column for a high-performance liquid chromatograph (HPLC) apparatus, and many essential studies have been reported.6,8,9 In the past decade, the authors’ group has been conducted fundamental studies on the adsorption of acidic bovine serum albumin (BSA), neutral myoglobin (MGB), and basic lysozyme (LSZ) onto various kinds of synthetic Hap particles.10-19 In these
10.1021/jp110441e 2011 American Chemical Society Published on Web 12/16/2010
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Figure 1. (a) Projection of unit cell of Hap crystal viewed along the [110] face and (b) surface structure of the c crystal surface viewed along the [001] face.
studies, we investigated by changing many factors of Hap particles, such as calcium-to-phosphorus (Ca/P) atomic ratio, kinds of divalent cations (Ca, Sr, and Ca-Sr solid solution), mean particle length, contents of carbonate ions incorporated, and heat treatment temperature. From these investigations, we concluded that the mean particle length is a main factor for determining the amounts of adsorbed proteins because the number of C sites, i.e., the fraction of ac or bc particle face, is a dominant factor especially for the adsorption of acidic BSA molecules.12,15 On the other hand, calcium phosphate including Hap is inorganic component of many biological hard tissues, namely bone and teeth. In these tissues, the biomineral consists of platelike Hap 10 nm thick and tens of nanometers long.20 Such nanoparticles have been studied in vitro for two main purposes: (i) learning more about the biological processes responsible for the formation of this mineral phase in vivo21 and (ii) preparing biomimetic materials for bone grafts closely resembling the natural ones.22 To our best knowledge, however, no study has been reported on the protein adsorption behavior onto nanometers size Hap particles. One of the reasons will be attributed to the difficulty of the preparation of Hap nanopartices with a same property not only their size but also surface properties. The sol-gel method,23 the hydrothermal method,24 the microemulsion route,25 the triblock copolymer method,26 hard templating method,27 mechanochemical treatment,28 and the surfactant-assisted methods29 have been reported to prepare Hap nanoparticles. However, no matter how the particle preparation conditions are controlled, the particle properties cannot be free from difference in particle properties. Recently, micromachining technologies have been applied to design miniaturized devices for synthetic applications, i.e., microreactors.30,31 A microreactor is a device that has microchannels on the order of micrometers and that enables chemical reactants to be performed in reaction space several orders of magnitude smaller than conventional batch reactors.32 The potential advantages of using a microreactor, rather than a conventional batch reactor, include high-speed mixing, better control of reaction conditions, improved safety, and improved yield. Many papers have been reported fine nanoparticles such
as Au,33 Ag,34 AgCl,35 and Hap36 being generated using a microreactor. Yang et al. prepared Hap nanoparticles using tubein-tube microchannel reactor.36 They reported that the particle size, and their size distribution depended on the flow rate at the mixing distance. However, the Hap nanoparticles they prepared possess rodlike shape with mean particle length of 58 nm that are of similar size as Hap particles prepared by a batch method and used for protein adsorption experiments reported by the authors.10-14,16-19 To disclose the protein adsorption behavior onto Hap nanoparticles, Hap particles with much smaller size are desired. One of the authors developed microelectromechanical systems (MEMS) technology to miniaturize channels for the combined flow of liquids,37 which enables uniform mixing of liquids on the micrometer order (as will be shown in Figure 3). Since the MEMS technology is performed by using fluid dynamics simulation technique to figure out the best fluid channel structure, such as channel width and channel length for the best mixture,38 it can be expected that this technology can produce Hap nanoparticles less than 50 nm in mean particle length. Accordingly, the objectives on the present study are to confirm the formation of Hap nanoparticles by using our microreactor and also to disclose the adsorption behaviors of proteins onto the Hap nanopartices. The results obtained in the present study must serve to elucidate not only the interaction of proteins to Hap nanoparticles but also the researchers in the fields of biomaterials, biomineralization, and biosensors. Experimental Section Materials and Methods. Figure 2 shows a diagram of the microreactor system used in this study. The microreactor was placed in a temperature-controlled bath. Reactants A and B were introduced into the microreactor through the introduction part (polytetrafluoroethylene (PTFE) tube) and mixed in equal quantity. The reaction progressed in the reaction part, and we produced a product solution. Figure 3 shows the micro-process server, a laboratory microreactor system produced by Hitachi Plant Technologies, Ltd.38 The micro-process server consists of the flow control part, the temperature control part, and a microreactor. The microreactor consists of a housing and a chip
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Figure 2. Laboratory experimental setup using a microreactor unit.
Figure 3. Micro-process server (Hitachi Plant Technologies, Ltd.) and microreactors.
TABLE 1: Reaction Conditions and Properties of Hap Particles Produced by a Batch and Microreactor Methods
particles
flow rates of reactants A and B (mL/min)
reaction temperature (°C)
size (width × length) (nm × nm)
SN (m2/g)
SW (m2/g)
SW/SN
Ca/P atomic ratioa
batch HAP-0 HAP-1 HAP-2 HAP-3 HAP-4
5 50 50 75 75
25 20 20 60 60 70
12 × 75 4 × 37 4 × 32 3 × 30 2 × 17 2 × 15
84 264 321 271 264 229
78 243 229 220 231 243
0.92 0.92 0.71 0.81 0.86 1.06
1.57 1.57 1.69 1.56 1.64 1.64
a
Assayed by ICP-AES measurement.
with multilayer flows. In this study, we used a microreactor in which the channel width of 1 mm and the number of flows of 48, as shown in Figure 3. Furthermore, 1 dm3 of 0.02 M Ca(OH)2 and 0.012 M H3PO4 aqueous solutions were used as reactants A and B, respectively. The reaction conditions of flow rates of reactants A and B and reaction temperature were varied from 5-75 mL/min and 20-70 °C, respectively. We prepared five Hap nanoparticles under various reaction conditions as shown in Table 1. To compare the adsorption behavior of proteins, we also employed rodlike Hap particles (abbreviated as batch). These Hap particles were prepared by a usual batch method13-16 as follows: 0.40 mol of Ca(OH)2 was dissolved into 20 dm3 of deionized-distilled water free of CO2 in a sealed Teflon vessel. After being stirred for 24 h at room temperature, 0.24 mol of H3PO4 was added into the solution and the suspension was stirred for a further 24 h at room temperature. This suspension was aged in an air oven at 100 °C for 48 h. All the Hap particles generated from both the microreactor and batch methods were filtered off, thoroughly washed with distilled water, and finally dried at 70 °C in an air oven for 24 h. All chemicals were reagent grade supplied from Wako Chemical Co. and were used without further purification. Characterization. The shape, specific surface area, crystal phase, and Ca2+ and PO43- contents of Hap particles were
determined by a transmission electron microscope (TEM; JEOL JEM-2100), N2 and H2O adsorption measurements, X-ray diffraction (XRD; Rigaku Rad-RC, Ni-filtered Cu KR radiation, 40 kV, 120 mA), Fourier-transform infrared spectrometer (FTIR; Nicolet Prote´ge´ 460), and inductively coupled plasma atomic emission spectroscopy (ICP-AES; SII SPS 3520UV-2). The adsorption isotherm of N2 was measured at the boiling point of liquid nitrogen with the use of a computerized automatic volumetric apparatus built in-house. Adsorption isotherms of H2O were also determined at 25 °C by a gravimetric apparatus built in-house. Specific surface areas were obtained by fitting the BET equation to these N2 and H2O adsorption isotherms and were abbreviated as SN and SW, respectively. Prior to these gas adsorption measurements, the samples were evacuated at 100 °C for 2 h. Protein Adsorption Measurement. The amounts of proteins adsorbed on the Hap particles were measured by a batch method as following the method employed in our previous papers.16-19 This measurement was conducted at 15 °C employing a 1 × 10-4 mol/dm3 KCl solution of the protein in 10 cm3 Nalgen polypropylene centrifugation tubes. The centrifugation tubes were gently rotated end-over-end at 15 °C for 48 h in a thermostat. The concentrations of proteins were measured by the microbiuret method using an UV absorption band at 310
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TABLE 2: Properties of Proteins numbers of functional groups per molecule proteins
isoelectric point
molecular weight (Da)
size (nm)
-NH2
-COOH
BSA LSZ MGB
4.4 11.1 7.0
67 200 14 600 17 800
4 × 14 3 × 3.5 3.5 × 4.5
680 155 34
680 32 36
nm after centrifuging the dispersions. Most of the UV experiments were triplicated and reproducible within 2%, indicating an uncertainty of 2 × 10-2 mg/m2 for the amounts of protein adsorbed. All proteins were purchased from Sigma Co. (BSA: A-7030, MGB: M-0630 and LSZ: L-6876). The properties of proteins used in this study are listed in Table 2. Results and Discussion Properties of Hap Particles. The TEM pictures of Hap particles prepared in this study are shown in Figure 4. The particles precipitated by a batch method are rodlike with 12 × 75 nm2 in size. The Ca/P atomic ratio of the sample was 1.57, lower than the stoichiometric value of 1.67 (Table 1), suggesting that the particles are calcium-deficient. As seen in Figure 4, the size of Hap particles produced by the microreactor reduced in the order of Hap-0 < 1 < 2 < 3 < 4, coinciding with that of the hardness of the reaction conditions for mixing Ca(OH)2 in water and H3PO4 aqueous solutions, because the mixing performance of the microreactor will be improved by increase in the flow rate of the reactant solutions and also the solubility of Ca(OH)2 is decreased with increase in the solution temperature. Finally, the size of Hap-4 became 2 × 15 nm2, similar to that of BSA molecule (4 × 14 nm2). It is noteworthy that Hap-4 produced by the microreactor with the smallest size still possesses rodlike shape, suggesting that particles are grown along c-axis even though the reactants mixed very rapidly in narrow channels. This result is due to the neutralization reaction of Ca(OH)2 and H3PO4. Indeed, the yields of the particles produced from the microreactor were ca. 100%. The Ca/P atomic ratios of the Hap nanoparticles produced by microreactor were ranging from 1.57 to 1.64, indicating that the particles are calcium-deficient as well as the Hap particles produced by the batch method except for Hap-1, of which Ca/P atomic ratio is 1.69. The X-ray diffraction patterns of the Hap nanoparticles are shown in Figure 5. The diffraction patterns showed characteristic peaks of Hap (JCPDS card: 9-432), though the crystallinity of the materials produced by the microreactor is lower than the particle produced by the batch method. This depression of the
particle crystallinity strongly suggests that the crystallinity of these Hap nanoparticles is low. The diffraction intensity of (002) face at 2θ ) 25.9° becomes small by increase in the hardness of the reaction conditions though that of (300) face at 2θ ) 32.9° is not changed. This fact indicates that the reduction of the particle length but not of particle width. Since broad hump can be seen in 2θ from 15° to 35°, the produced particles include amorphous calcium phosphate particle. However, this possibility is low because of the following two reasons: (i) higher Ca/P atomic ratio of these particles rather than that of batch method (1.57) and (ii) relatively sharp peaks in FTIR spectra as will be shown in Figure 6. Even though their low crystallinity, since the adsorption behavior is only governed by the surface structure of adsorbent but not by whole crystal structure, the change in the crystallinity of the Hap nanoparticles was neglected for discussing the protein adsorption behavior in a later section. The SW/SN ratios in Table 1, representing the surface hydrophilicity of the Hap nanoparticles, ranged between 0.71 and 1.06. This result indicates that the surface hydrophilicity of Hap nanoparticles is equivalent. This result signifies that there is no need to consider the difference in the surface hydrophilicity on the protein adsorption behavior. The t-plot analysis for N2 adsorption isotherms also revealed that there is no micropore in these Hap nanoparticles. FTIR spectra of the particles are given in Figure 6. The FTIR spectra show all the stretching and bending vibrations of the phosphate modes and the OH functional groups. The strong peaks present at 567 and 604 cm-1 in all the spectra indicate the presence of a ν4 phosphate bending mode. The strong broad band around 1045 cm-1 confirms the presence of the ν3 PO4 mode. The broad peak around 3440 cm-1 is from the lattices of water molecules. The sharp peaks at 631 and 3569 cm-1 for the particles produced from the batch method belong to the stretching vibrations of the OH- ions and are considered the characteristic peaks of crystallized rodlike Hap.39 The small band at 875 cm-1 and the doublet at 1419 and 1480 cm-1 of the Hap prepared by microreactor exhibit the CO32- carbonate mode of vibration.40 The presence of these bands at 1419 and 1480 cm-1
Figure 4. TEM pictures of the Hap particles produced by batch and microreactor methods.
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Ca10-x(HPO4)x(PO4)6-x(OH)2-x(H2O)x · nH2O f Ca10-x(P2O7)x(PO4)6-2x(OH)2(H2O)x + nH2O (1) Ca10-x(P2O7)x(PO4)6-2x(OH)2(H2O)x f (1 - x)Ca10(PO4)6(OH)2 + 3xCa3(PO4)2 + 2xH2O (2)
Figure 5. XRD patterns of the Hap particles produced by batch and microreactor methods.
Figure 6. FTIR spectra of the Hap particles produced by batch and microreactor methods.
Figure 7. TG curves of the Hap particles produced by batch and microreactor methods.
is consistent with a carbonate substituted type-B Hap, where the carbonate ions replace the phosphate ions in the crystal lattice.41 Figure 7 shows TG curves of the Hap particles. The weight loss in all the materials occurs in a temperature range of 25-1000 °C. The weight loss accompanies a small step at ∼800 °C except for Hap-1. Berry42 and Monma et al.43 have reported for nonstoichiometric HAP that the large weight loss up to ca. 700 °C is caused by the release of adsorbed and bound H2O molecules and evolve a small amount of H2O molecules by dehydration of HPO42- ions while the smaller weight loss above 700 °C resulted from a reaction of the P2O74- ions formed by the reaction up to 700 °C with OH- ions. Applying the proposed mechanisms to the present materials with H2O molecules in the lattice, the corresponding reactions can be expressed by eqs 1 and 2.
This result strongly suggests that Hap particles are Ca2+deficient, corresponding to the Ca/P molar ratio assayed by the ICP-AES measurement in Table 1, except Hap-1 of which Ca/P molar ratio is 1.69. Indeed, the weight loss step is absent at ∼800 °C for Hap-1. It is well-known that nonstoichiometry occurs not only in biological Haps but also in synthetic Hap (both precipitated and high temperature preparation) by formation of vacant lattice sites and complicates their crystal chemistry. Adsorption Behavior of Proteins onto Hap Particles. Adsorption isotherms of BSA onto the Hap particles are shown in Figure 8 along with the zeta potential (zp). All the adsorption isotherms of BSA from 1 × 10-4 mol/dm3 KCl solution are the Langmuirian type. The saturated amount of adsorbed BSA (nBSA S ) for Hap (batch) (O) was 0.62 mg/m2. The adsorption coverage of BSA (θBSA) on this system, defined as the ratio of the experimental amounts of adsorbed BSA (nBSA S ) to the theoretical value, is 0.25. The latter value was estimated as 2.52 mg/m2 by assuming a side-on adsorption of globular BSA molecules, which are prolate ellipsoids of 14 × 4 nm2.44 Since the solution pH of the system was ca. 8, BSA molecules are negatively charged. Therefore, the negative values of zp of this system were increased with an increase in the amount of adsorbed BSA. As is seen in Figure 8, however, the nSBSA values for the Hap nanoparticles produced by microreactor are clearly decreased in the order of Hap-0 < 1 < 2 < 3 < 4, coinciding with the order of the hardness of the reaction conditions as described before. That is to say, the nSBSA values are strongly decreased with decreased in the particles size. It should be mentioned here that the nSBSA value reduces to zero for Hap-4 (b) with a particle size similar to the BSA molecule. Similar results can be seen in the adsorption of LSZ as shown in Figure 9; the saturated amounts of adsorbed LSZ (nSLSZ) also reduced to zero for Hap4. In this system, the LSZ molecules are positively charged under the experimental condition, and the zp values are revised from negative values to positive ones. However, a slightly different result can be seen for the system of MGB adsorption as is displayed in Figure 10. In this case, the saturated amounts of adsorbed MGB (nSMGB) are also depressed with decrease in value diminished, about the particle size. Even though the nMGB S half of MGB molecules still adsorbed onto Hap nanoparticles, completely different from the results on the BSA and LSZ systems. Comparison with the Protein Adsorption Behavior onto Hap Nanoparticles. To describe the dependence of adsorption of proteins onto particle size of Hap, the nSBSA, nSLSZ, and nSMGB values are plotted as a function of mean particle length (Lp) of Hap in Figure 11. Clearly, the nSBSA (O) and nSLSZ (4) values reached zero at Lp ) 15 nm, though the nSMGB one (0) shows 0.29 mg/m2. As already suggested in the Introduction, BSA molecules strongly bind to C sites on ac or bc particle surface. Therefore, the reduction of nSBSA is due to the decrease in the fraction of C sites on the side face of each particle. Finally, there is no enough area left on the Hap-4 particle surface to adsorb large BSA molecules even though the BSA molecules are soft and their conformations are alterable. In the case of LSZ, it can be regarded that P sites on ab face are adsorption
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Figure 8. (a) Adsorption isotherms of BSA and (b) their zeta potential for Hap particles produced by batch and microreactor methods: (O) batch, (3) Hap-0, (4) Hap-1, (0) Hap-2, (]) Hap-3, and (b) Hap-4.
Figure 9. (a) Adsorption isotherms of LSZ and (b) their zeta potential for Hap particles produced by batch and microreactor methods: (O) batch, (3) Hap-0, (4) Hap-1, (0) Hap-2, (]) Hap-3, and (b) Hap-4.
Figure 10. (a) Adsorption isotherms of MGB and (b) their zeta potential for Hap particles produced by batch and microreactor methods: (O) batch, (3) Hap-0, (4) Hap-1, (0) Hap-2, (]) Hap-3, and (b) Hap-4.
sites for LSZ. Since the mean particle width are also decreased in the order of Hap-0 < 1 < 2 < 3 < 4, nSLSZ may be reduced by the reduction of the number of P sites. Further, the LSZ molecules possess rigid structure even though its small size; LSZ molecules are hard to vary their conformation. This is another possibility of the depression of nSLSZ values for the Hap nanoparticles. On the contrary, nSMGB values only decreased to half value even if the Lp decreased to 15 nm. Since MGB molecules are neutral and soft structure as we already reported,15 phosphate groups existing between C sites are regarded as adsorption sites for MGB through the van der Waals attractive force. Therefore, small MGB molecules are adsorbed on Hap nanaoparticle by changing their conformation. We could control the amounts of adsorbed proteins by altering the particle size in nanometer range
Figure 11. Plots of saturated amounts of adsorbed BSA, LSZ, and MGB as a function of mean particle length of Hap particles produced by batch and microreactor methods.
through microreactor method and kinds of proteins. These obtained results may be useful for developing biomimetic materials for bone grafts and successful surgical devices in the biochemical field. Conclusion The Hap nanoparticles were prepared by using microreactor and clarified the adsorption behavior of proteins onto these Hap nanopartices. The size of Hap particles produced by the microreactor reduced in the order of the hardness of the reaction conditions. Finally, the size of Hap nanoparticle became 2 ×
Calcium Hydroxyapatite Nanoparticles 15 nm2, similar to that of BSA molecule (4 × 14 nm2). The smallest Hap nanoparticles still possesses rodlike shape even though the reactants mixed very rapidly in narrow channels. The X-ray diffraction patterns and FTIR spectra revealed that the crystallinity of the materials produced by the microreactor is low and they were carbonate substituted type-B Hap, where the carbonate ions replace the phosphate ions in the crystal lattice. All the adsorption isotherms of BSA, MGB, and LSZ onto Hap nanoparticles from 1 × 10-4 mol/dm3 KCl solution were the Langmuirian type. The nSBSA and nSLSZ values for the Hap nanoparticles produced by microreactor were decreased with decrease in the size of the particles, and finally it reduced values to zero for the smallest Hap nanoparticles. However, nMGB S were also depressed with decreased in the particle size, but about half of MGB molecules still adsorbed onto the smallest Hap nanoparticles. This difference in the protein adsorption behavior among these proteins was explained by the difference in the size and flexibility of three kinds of proteins. We could control the amounts of adsorbed proteins by changing the particle size in nanometer range and kinds of proteins. These results may be useful to the researchers in the fields of biomaterials, biomineralization, and biosensors. References and Notes (1) Fujimori, E.; Ohkubo, M.; Tsuru, K.; Hayakawa, S.; Osaka, A.; Kawabata, K.; Bonhomme, C.; Babonneau, F. Acta Biomater. 2006, 2, 69– 74. (2) Takemoto, S.; Kusudo, Y.; Tsuru, K.; Hayakawa, S.; Osaka, A.; Takashima, S. J. Biomed. Mater. Res. 2009, 69, 544–551. (3) Paul, W.; Sharma, C. P. J. Mater. Sci.: Mater. Med. 1999, 10, 383– 388. (4) Liu, T. Y.; Liu, S. Y.; Liu, D. M.; Liou, S. C. J. Controlled Release 2005, 107, 112–121. (5) Kawasaki, T.; Takahashi, S.; Ikeda, K. Eur. J. Biochem. 1985, 152, 361–371. (6) Kawasaki, T.; Niikura, M.; Takahashi, S.; Kobayashi, W. Biochem. Int. 1986, 13, 969–982. (7) Kawasaki, T.; Ikeda, K.; Takahashi, S.; Kuboki, Y. Eur. J. Biochem. 1986, 155, 249–257. ¨ . Arch. Biochem. Phys. 1956, (8) Tiselius, A.; Hjerte´n, S.; Levin, O 65, 132–155. (9) Thomann, J. M.; Mura, M. J.; Behr, M. S.; Aptel, J. D.; Schmitt, A.; Bres, E. F.; Voegel, J. C. Colloids Surf. 1989, 40, 293–305. (10) Kandori, K.; Sawai, S.; Yamamoto, Y.; Saito, H.; Ishikawa, T. Colloids Surf. 1992, 68, 283–289. (11) Kandori, K.; Yamamoto, Y.; Saito, H.; Ishikawa, T. Colloids Surf., A 1993, 80, 287–291. (12) Kandori, K.; Saito, M.; Saito, H.; Yasukawa, A.; Ishikawa, T. Colloids Surf., A 1995, 94, 225–230. (13) Kandori, K.; Saito, M.; Takebe, T.; Yasukawa, A.; Ishikawa, T. J. Colloid Interface Sci. 1995, 174, 124–129.
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