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Synthesis of positively charged calcium hydroxyapatite nano-crystals and their adsorption behavior of proteins
Colloids and Surfaces B: Biointerfaces 73 (2009) 140–145
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Synthesis of positively charged calcium hydroxyapatite nano-crystals and their adsorption behavior of proteins Kazuhiko Kandori a,∗ , Shohei Oda a , Masao Fukusumi b , Yoshiaki Morisada b a b
School of Chemistry, Osaka University of Education, Asahigaoka 4-698-1, Kashiwara-shi, Osaka 582-8582, Japan Department of Processing Technology, Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka 536-8553, Japan
a r t i c l e
i n f o
Article history: Received 16 March 2009 Received in revised form 7 May 2009 Accepted 12 May 2009 Available online 19 May 2009 Keywords: Calcium hydroxyapatite Nano-crystals Positive charge Protein adsorption
a b s t r a c t Positively charged Hap nano-crystals were prepared by using -alanine and clarified the adsorption affinity of these surface amide functionalized Hap nano-crystals to proteins. Colloidal surface amide functionalized Hap nano-crystals were prepared by wet method in the presence of various amounts of -alanine by changing molar ratio of -alanine/Ca (/Ca ratio) in the solution. The rod-like nano-crystals were lengthened with addition of -alanine though their width did not vary; carboxyl groups of -alanine are strongly coordinated to Ca2+ ions exposed on ac and/or bc faces to inhibit particle growth to a- and/or b-axis directions and enhance the particle growth along to the c-axis. No difference can be recognized on the crystal structure among the synthesized Hap nano-crystals by XRD measurements. However, the large difference was recognized by TG-DTA and FTIR measurements. Those measurements revealed that -alanine is incorporated on the Hap nano-crystal surface up to the /Ca ratio of 1.0, though they are absent in the nano-crystals synthesized at /Ca ratio ≥ 2.0. The zeta potential (zp) of -alanine-Hap nano-crystals prepared at /Ca = 0.4 and 1.0 of those incorporating -alanine exhibited positive charge at pH ≤ 5.9. The saturated amounts of adsorbed BSA for the positively charged -alanine-Hap nano-crystals were increased 2.3–2.4-fold by their electrostatic attraction force between positively charged -alanineHap nano-crystals and negatively charged BSA molecules. We were able to control the adsorption affinity of Hap nano-crystal by changing their surface charge. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The interaction of protein molecules with inorganic materials has received much interest in many fields, such as biomineralization, biomaterials, biochemistry, biosensors, and industry [1–3]. It is well known that calcium hydroxyapatite [Ca10 (PO4 )6 (OH)2 , Hap] is the major inorganic component of mammalian bones and teeth, and it possesses high affinity to the proteins. 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 nano-crystal surface. Thus, Hap contains a multiple-site binding character for proteins [4–6]. After dispersing Hap nano-crystals in aqueous media, calcium atoms (C sites) are exposed on the Hap surface by dissolution of OH− ions at the nano-crystal surface. Therefore, the C sites, rich in calcium ions or positive charge to bind to acidic groups of proteins, 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. Indeed, Chen et al. reported that the –COO− claw of protein grasps the calcium atoms
∗ Corresponding author. E-mail address: [email protected] (K. Kandori). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.05.011
of Hap surface with its two oxygen atoms in a triangle form [7]. The solid state NMR study also revealed that the –COO− terminus of amelogenin is orientated to the Hap surface [8]. The P sites, lack calcium ions or positive charge attach to basic groups of proteins, are arranged hexagonally on the ab particle face with a minimal distance of 0.943 nm. In addition, Hap is the most stable calcium phosphate under physiological conditions. Hence, Hap is widely applied for separating various proteins in a high-performance liquid chromatography (HPLC) system. Many essential studies therefore have been reported [5,9,10]. Even though there are many C sites exposed on ac and/or bc faces, the C sites are localized between orthophosphate ions. Ishikawa et al. have been revealed that a part of surface orthophosphate ions are protonated to maintain charge balance due to the cation-deficiency and produce surface P–OH groups [11]. The surface P–OH groups are dissociative and produce negative charge. Therefore, Hap nanocrystals tend to possess total negative charge in aqueous media even at pH value around neutrality. However, nevertheless of its total negative surface charge, Hap has a high adsorption affinity to negatively charged bovine serum albumin (BSA) due to the presence of localized C sites. If we can provide Hap nano-crystals with positive charge in these neutral solutions, the saturated amounts of adsorbed BSA will be increased in cooperation with the function
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RU-200) and inductively coupled plasma atomic emission spectroscopy (ICP-AES; Seiko SPS1200VR). The adsorption isotherm of N2 was measured at the boiling point of liquid nitrogen with the use of a computerized automatic volumetric apparatus built inhouse. Prior to the measurement, the samples were evacuated at 100 ◦ C for 2 h. The XRD patterns were taken with Ni-filtered Cu K␣ radiation (40 kV, 120 A). The transmission IR spectra of the selfsupporting sample disk were recorded in situ using Nicolet Protégé 460 FTIR interferometer using a specially designed vacuum cell that was capable of heating the samples. The number of scans was 200 and the resolution was 4 cm−1 . Samples (30 mg) pressed to a disk of 10 mm diameter were pretreated under 10−2 Pa at 300 ◦ C. The zeta potential (zp) of the nano-crystals was also measured by an electrophoresis apparatus (PEN KEM 500). 2.2. Adsorption measurement of BSA Scheme 1. Illustration of surface amide functionalized Hap nano-crystal.
of C sites. In practice, Yin et al. reported that domination interaction was attributed to the electrostatic interaction between protein and Hap surface [12]. Therefore, the positively charged Hap nanocrystals are desired to produce a high-quality column for separating acidic and basic proteins in HPLC apparatus. Recently, Lee et al. reported on the synthesis of reactive Hap nano-crystals [13]. The surface modification of Hap nanocrystals was described based on in situ synthesis of surface thiol-functionalized Hap and subsequent grafting polymerization of ethylene glycol methacylate phosphate. They used 3-mercaptopropionic acid (MAP: HS–CH2 –CH2 –COOH) as thiol source. They explained that MAP are incorporated onto the Hap surface directing thiol groups to outside due to the strong ionic bonding (electrostatic attraction) between the –COO− groups of MPA and Ca2+ at the surface layer of Hap. By using -alanine (H2 N–CH2 –CH2 –COOH) instead of MAP, it can be expected that similar reaction takes place and amide groups will be directing to outside. If this surface structure is completed, Hap surface will possess positive charge in acidic solution by protonation of amide groups (Scheme 1). Hence, the aim of this study was to elucidate the preparation of positively charged Hap nano-crystals by using alanine and further clarify the adsorption affinity of these surface amide functionalized Hap nano-crystals to proteins. 2. Experimental 2.1. Materials and methods Colloidal Hap nano-crystals incorporating -alanine onto the surface (-alanine-Hap) were prepared by the following wet method [14–18]: 0.040 mol of Ca(OH)2 was dissolved into 2 dm3 of deionized-distilled water free of CO2 in a sealed Teflon vessel. After being stirred for 24 h at room temperature, 0.024 mol of H3 PO4 with various amounts of -alanine was added into the solution and the suspension was stirred for further 24 h at room temperature. This suspension was aged in an air oven at 100 ◦ C for 48 h. The concentration of -alanine was changed from 0 to 0.4 mol, corresponding to the molar ratio of -alanine/Ca = 0–10. Hereafter, the molar ratio of -alanine/Ca is abbreviated as /Ca. The Hap nano-crystals generated 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. The shape, specific surface area, crystal phase, and Ca2+ and PO4 3− contents of Hap nano-crystals were determined by a transmission electron microscope (TEM; JEOL JEM2100), N2 adsorption measurements, X-ray diffraction (XRD; Rigaku
The amounts of BSA adsorbed on the -alanine-Hap nanocrystals were measured by a batch method as following the method employed in our previous papers [19–22]. This measurement was conducted at 15 ◦ C and pH 5.9 because -alanine-Hap nanocrystals showed positive charge at this pH. The solution of pH 5.9 was developed by using acetic acid (HAc )–sodium acetate (NaAc ) buffer solution system. The concentration of HAc and NaAc were 5.71 × 10−2 and 1.0 M, respectively. The adsorption experiment was performed in the buffer solution of BSA 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 concentration of BSA was measured by the microbiuret method using an UV absorption band at 310 nm after centrifuging the dispersions. Most of the UV experiments were triplicated and reproducible within 2%, indicating an uncertainty of 2 × 10−2 mg m−2 for the amounts of protein adsorbed. BSA was purchased from Sigma Co. (A-7030, 67,200 Da, isoelectric point = 4.7). 3. Results and discussion 3.1. Properties of ˇ-alanine-Hap nano-crystals Table 1 summarized the preparation conditions and properties of -alanine-Hap nano-crystals. The contents of -alanine measured by CHN elemental analysis and number of -alanine on Hap surface estimated by using specific surface area of each particle are also listed in Table 1. The TEM images of the nano-crystals synthesized in this study are shown in Fig. 1. The nano-crystals produced without (/Ca = 0) are rod-like and 15 nm × 60 nm in size. The rod-like nano-crystals are lengthened with addition of -alanine though their width does not vary. Fig. 2 shows changes of short and long axis lengths of the nano-crystals together with specific surface area of these nano-crystals (䊉) as a function of the /Ca molar ratio in a starting solution. Clearly, the long axis lengths of the nano-crystals are increased with increase in the /Ca ratio up to 1, exhibiting a maximum at this point, and they are held almost constant ca. 80 nm. The specific surface area is increased with the /Ca ratio from ca. 90 to 125 m2 /g in spite of increase in their size. Since the t-plot analysis revealed that these nano-crystals are nonporous, the increase in the specific surface area with increase in the /Ca molar ratio may be due to the growth the surface roughness of the particles with -alanine in the solution. The growth along c-axis can be explained as follows: carboxyl groups of alanine are strongly coordinated to Ca2+ ions exposed on ac and/or bc faces to inhibit particle growth to a- and/or b-axis directions and enhance the particle growth along to the c-axis (Scheme 1). Similar effect has been reported on the formation of whisker type Hap nano-crystals from amorphous calcium phosphate in the presence
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Table 1 Preparation conditions and properties of -alanine-Hap nano-crystals. /Ca ratio
0 0.4 1.0 2.0 4.0 10.0 a b
Ca/P atomic ratio
1.63 1.63 1.64 1.61 1.58 1.58
Specific surface area (m2 /g)
95.2 82.4 91.3 105.5 106.5 124.2
Crystallite size L (nm)a
65 80 95 78 84 83
Amounts of -alanine in particles (wt.%)b
(molecules/nm2 )
0 0.98 0.91 0 0 0
0 0.80 0.75 0 0 0
From (0 0 2) face. Measured by CHN elemental analysis.
of acetic acids on the hydrothermal reaction [23]. No difference can be also recognized on the crystal structure among the nano-crystals as is displayed XRD patterns in Fig. 3. The crystallite size of the particles (L) from the (0 0 2) face calculated by using Scherrer’s equation coincided to the particle length as is shown in Table 1, indicating that all the Hap particles prepared are single crystal. However, the large difference was recognized by TG-DTA measurement as is shown in Fig. 4.
The DTA curves of the nano-crystals produced at /Ca = 0.4 and 1.0 possess exothermic peaks at ca. 150–350 ◦ C, indicating that -alanine is present on the nano-crystal surface. However, these peaks disappear at /Ca ≥ 2.0, suggesting -alanine molecules are absent in these nano-crystals irrespective of their high /Ca ratio. To elucidate the state of -alanine, in situ FTIR spectra of the nanocrystals were measured. Fig. 5 depicts the FTIR spectra recorded in vacuo. The absorption bands at 3600–3700 cm−1 is due to the
Fig. 1. TEM pictures of Hap nano-crystals produced at various /Ca ratios.
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Fig. 2. Plots of short () and long () particle lengths and their specific surface area (䊉) as a function of /Ca ratio.
surface P–OH groups constituting surface acidic phosphate ions, HPO4 2− . These ions would have been converted from PO4 3− ions in order to maintain the overall charge balance of calcium-deficient Hap [11]. The symmetry of distorted tetrahedral PO4 3− ions or their sp3 orbital might have been changed in this surface structure. Ishikawa et al. mentioned that these groups may be free or isolated OH groups of similar bond nature and possess ion-exchange properties [11]. Besides these bands, absorption bands newly appear at 2800–3000 cm−1 by increase in the /Ca ratio up to 1.0. These bands are assigned to the stretch mode of C–H groups of -alanine. However, these bands suddenly disappear at the /Ca ratio over 2.0. This result of FTIR coincides with that of TG-DTA in Fig. 4, indicating that -alanine is incorporated on the -alanine-Hap nano-crystal surface up to the /Ca ratio of 1.0. It is noteworthy in Fig. 5 that
Fig. 4. TG-DTA curves of Hap nano-crystals produced at various /Ca ratios.
the absorption band of surface P–OH groups was not altered by -alanine incorporation. This fact strongly implies that carboxyl groups of -alanine molecules are binding to Ca2+ ions (C sites) exposed on the nano-crystal surface but not to surface P–OH groups. Now we should discuss the optimum point for incorporation of -alanine at /Ca = 1.0. Since -alanine is dissolved in acidic H3 PO4 solution, -alanine is positively charged by protonation as is expressed as equation (1). NH3 + CH2 CH2 –COOH
(1)
After poured this acidic H3 PO4 solution into Ca(OH)2 one, Hap nano-crystals are immediately formed and solution pH is decreased. In order to produce dissociated form of two -alanine molecules (Eq. (2)) to bind Ca2+ ions such as depicted in Scheme 1, the concentration of Ca(OH)2 should be the same as that of alanine because Ca(OH)2 promotes two OH− ions by dissociation. NH2 CH2 CH2 –COO−
Fig. 3. XRD patterns of Hap nano-crystals produced at various /Ca ratios.
Fig. 5. FTIR spectra of Hap nano-crystals produced at various /Ca ratios.
(2)
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Fig. 6. Plots of Ca/P atomic ratio () and solution pH (䊉) after the reaction as a function of /Ca ratio.
Therefore, the optimum concentration for incorporation of alanine was observed at the /Ca ratio of 1.0. Above this molar ratio, the number of OH− ions to dissociate carboxyl groups of -alanine is insufficient and reduce their binding ability to Ca2+ ions. Fig. 6 plots the solution pH after the reaction. The solution pH is decreased from 9.7 to 6.9 with increase in the /Ca ratio up to 2.0 and shows almost constant value of 6.9–7.4 at above this /Ca ratio. Since the constant pH value of solution is close to the isoelectric point (iep) of -alanine (6.9), it can be presumed that -alanine is uncharged in this /Ca region. This result strongly supports the discussion of -alanine molecules described above.
Fig. 8. Adsorption isotherms of BSA onto Hap nano-crystals produced at /Ca ratios. /Ca ratios: () 0; () 0.4; (䊉) 1.0; () 2.0; (×) 4.0; () 10.0.
3.3. Adsorption behavior of BSA onto ˇ-alanine-Hap nano-crystals
Fig. 7 displays the changes of zp of the -alanine-Hap nanocrystals prepared at different /Ca ratios as a function of solution pH. The untreated Hap particle exhibits negative value over all pH range measured (: 5.5 ≤ pH ≤ 7.9). However, the zp of -alanineHap nano-crystals prepared at /Ca = 0.4 () and 1.0 (䊉) moves to positive side, especially the latter ones possess definitive positive zp (,, ) at pH ≤ 5.9. However, zp of the nano-crystals prepared at the /Ca ratio over 2.0 exhibits again negative values even at pH ≤ 5.9. This result is compatible with the results of TG-DTA (Fig. 4) and FTIR (Fig. 5); only -alanine molecules incorporated onto Hap surface produced at the /Ca ratios of 0.4 and 1.0 attain positive charge. This fact further substantiated by the fact that the zp only vary at pH < 7, coinciding with the iep of -alanine (6.9).
Adsorption isotherms of BSA onto -alanine-Hap nano-crystals were measured in HAc –NaAc buffer solution of pH 5.9, where the -alanine-Hap produced at /Ca = 1.0 exhibits positive charge. The obtained isotherms are shown in Fig. 8. The adsorption isotherms of BSA are the Langmuirian type. The adsorption coverage of BSA, defined as the ratio of the experimental saturated amounts of adsorbed BSA (nBSA s ) to the theoretical value, is 0.24. The latter value was estimated as 2.52 mg m−2 by assuming side-on adsorption of globular BSA molecules, which are prolate ellipsoids of 14 nm × 4 nm. Since the solution pH of the system is 5.9, BSA molecules are negatively charged. The nBSA values were determined s by Fig. 8 and plotted in Fig. 9 as a function of the /Ca ratio. As is increases rapidly at the /Ca ratio of 0.4 and 1.0 and expected, nBSA s exhibits a maximum at 1.0, though they decrease again over the /Ca ratio of 2.0. The nBSA value for the nano-crystals produced s at /Ca = 0.4 and 1.0 are 2.3–2.4-fold than the untreated original ones. Clearly this enhancement of BSA adsorption is attributed to the electrostatic attractive force between positively charged Hap nano-crystals and negatively charged BSA molecules, comparable with the results of Yin et al. [12]. Of course, the conformation of BSA will be changed after attached BSA molecules to -alanine-Hap nano-crystals because secondary and tertiary structures of protein
Fig. 7. Plots of zeta potential of Hap nano-crystals produced at various /Ca ratios as a function of pH.
Fig. 9. Plots of saturated amounts of adsorbed BSA as a function of /Ca ratio.
3.2. Surface charges of ˇ-alanine-Hap nano-crystals
K. Kandori et al. / Colloids and Surfaces B: Biointerfaces 73 (2009) 140–145
molecules would be affected easily by the biological materials contacted with [24–26]. Though this conformational rearrangement is another possibility of the increase of nBSA s , we cannot discuss this subject from our results at the moment. Even though this important subject, BSA molecules are attracted to the -alanine-Hap nanocrystal surface by electrostatic attractive force before attached to the surface. Therefore, we believe the electrostatic interaction is the dominating factor in the present study. The results described on the -alanine-Hap nano-crystals could be expected to give valuable information for the development of a high-quality HPLC column and may be useful to the researchers in the fields of biomaterials, biomineralization, biosensors and protein separation. 4. Conclusion Colloidal surface amide functionalized Hap nano-crystals were prepared by wet method in the presence of various amounts of alanine. The rod-like nano-crystals were lengthened with addition of -alanine though their width did not vary. No difference was recognized on the crystal structure among the synthesized Hap nano-crystals by XRD measurements. However, TG-DTA and FTIR measurements revealed that -alanine is incorporated on the alanine-Hap nano-crystal surface up to the /Ca ratio of 1.0, though they are absent in these nano-crystals synthesized over the /Ca ratio of 2.0. The zeta potential (zp) of -alanine-Hap nano-crystals prepared at /Ca ≤ 1.0 exhibited positive charge at pH < 5.9. The saturated amounts of adsorbed BSA value for the positively charged -alanine-Hap nano-crystals were increased 2.3–2.4-fold by their electrostatic attraction force between positively charged -alanineHap nano-crystals and negatively charged BSA molecules. Acknowledgment The authors wish to express their deepest gratitude to Hosokawa Powder Technology Foundation for its support in their present research.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Synthesis of positively charged calcium hydroxyapatite nano-crystals and their adsorption behavior of proteins Kazuhiko Kandori a,∗ , Shohei Oda a , Masao Fukusumi b , Yoshiaki Morisada b a b
School of Chemistry, Osaka University of Education, Asahigaoka 4-698-1, Kashiwara-shi, Osaka 582-8582, Japan Department of Processing Technology, Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka 536-8553, Japan
a r t i c l e
i n f o
Article history: Received 16 March 2009 Received in revised form 7 May 2009 Accepted 12 May 2009 Available online 19 May 2009 Keywords: Calcium hydroxyapatite Nano-crystals Positive charge Protein adsorption
a b s t r a c t Positively charged Hap nano-crystals were prepared by using -alanine and clarified the adsorption affinity of these surface amide functionalized Hap nano-crystals to proteins. Colloidal surface amide functionalized Hap nano-crystals were prepared by wet method in the presence of various amounts of -alanine by changing molar ratio of -alanine/Ca (/Ca ratio) in the solution. The rod-like nano-crystals were lengthened with addition of -alanine though their width did not vary; carboxyl groups of -alanine are strongly coordinated to Ca2+ ions exposed on ac and/or bc faces to inhibit particle growth to a- and/or b-axis directions and enhance the particle growth along to the c-axis. No difference can be recognized on the crystal structure among the synthesized Hap nano-crystals by XRD measurements. However, the large difference was recognized by TG-DTA and FTIR measurements. Those measurements revealed that -alanine is incorporated on the Hap nano-crystal surface up to the /Ca ratio of 1.0, though they are absent in the nano-crystals synthesized at /Ca ratio ≥ 2.0. The zeta potential (zp) of -alanine-Hap nano-crystals prepared at /Ca = 0.4 and 1.0 of those incorporating -alanine exhibited positive charge at pH ≤ 5.9. The saturated amounts of adsorbed BSA for the positively charged -alanine-Hap nano-crystals were increased 2.3–2.4-fold by their electrostatic attraction force between positively charged -alanineHap nano-crystals and negatively charged BSA molecules. We were able to control the adsorption affinity of Hap nano-crystal by changing their surface charge. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The interaction of protein molecules with inorganic materials has received much interest in many fields, such as biomineralization, biomaterials, biochemistry, biosensors, and industry [1–3]. It is well known that calcium hydroxyapatite [Ca10 (PO4 )6 (OH)2 , Hap] is the major inorganic component of mammalian bones and teeth, and it possesses high affinity to the proteins. 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 nano-crystal surface. Thus, Hap contains a multiple-site binding character for proteins [4–6]. After dispersing Hap nano-crystals in aqueous media, calcium atoms (C sites) are exposed on the Hap surface by dissolution of OH− ions at the nano-crystal surface. Therefore, the C sites, rich in calcium ions or positive charge to bind to acidic groups of proteins, 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. Indeed, Chen et al. reported that the –COO− claw of protein grasps the calcium atoms
∗ Corresponding author. E-mail address: [email protected] (K. Kandori). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.05.011
of Hap surface with its two oxygen atoms in a triangle form [7]. The solid state NMR study also revealed that the –COO− terminus of amelogenin is orientated to the Hap surface [8]. The P sites, lack calcium ions or positive charge attach to basic groups of proteins, are arranged hexagonally on the ab particle face with a minimal distance of 0.943 nm. In addition, Hap is the most stable calcium phosphate under physiological conditions. Hence, Hap is widely applied for separating various proteins in a high-performance liquid chromatography (HPLC) system. Many essential studies therefore have been reported [5,9,10]. Even though there are many C sites exposed on ac and/or bc faces, the C sites are localized between orthophosphate ions. Ishikawa et al. have been revealed that a part of surface orthophosphate ions are protonated to maintain charge balance due to the cation-deficiency and produce surface P–OH groups [11]. The surface P–OH groups are dissociative and produce negative charge. Therefore, Hap nanocrystals tend to possess total negative charge in aqueous media even at pH value around neutrality. However, nevertheless of its total negative surface charge, Hap has a high adsorption affinity to negatively charged bovine serum albumin (BSA) due to the presence of localized C sites. If we can provide Hap nano-crystals with positive charge in these neutral solutions, the saturated amounts of adsorbed BSA will be increased in cooperation with the function
K. Kandori et al. / Colloids and Surfaces B: Biointerfaces 73 (2009) 140–145
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RU-200) and inductively coupled plasma atomic emission spectroscopy (ICP-AES; Seiko SPS1200VR). The adsorption isotherm of N2 was measured at the boiling point of liquid nitrogen with the use of a computerized automatic volumetric apparatus built inhouse. Prior to the measurement, the samples were evacuated at 100 ◦ C for 2 h. The XRD patterns were taken with Ni-filtered Cu K␣ radiation (40 kV, 120 A). The transmission IR spectra of the selfsupporting sample disk were recorded in situ using Nicolet Protégé 460 FTIR interferometer using a specially designed vacuum cell that was capable of heating the samples. The number of scans was 200 and the resolution was 4 cm−1 . Samples (30 mg) pressed to a disk of 10 mm diameter were pretreated under 10−2 Pa at 300 ◦ C. The zeta potential (zp) of the nano-crystals was also measured by an electrophoresis apparatus (PEN KEM 500). 2.2. Adsorption measurement of BSA Scheme 1. Illustration of surface amide functionalized Hap nano-crystal.
of C sites. In practice, Yin et al. reported that domination interaction was attributed to the electrostatic interaction between protein and Hap surface [12]. Therefore, the positively charged Hap nanocrystals are desired to produce a high-quality column for separating acidic and basic proteins in HPLC apparatus. Recently, Lee et al. reported on the synthesis of reactive Hap nano-crystals [13]. The surface modification of Hap nanocrystals was described based on in situ synthesis of surface thiol-functionalized Hap and subsequent grafting polymerization of ethylene glycol methacylate phosphate. They used 3-mercaptopropionic acid (MAP: HS–CH2 –CH2 –COOH) as thiol source. They explained that MAP are incorporated onto the Hap surface directing thiol groups to outside due to the strong ionic bonding (electrostatic attraction) between the –COO− groups of MPA and Ca2+ at the surface layer of Hap. By using -alanine (H2 N–CH2 –CH2 –COOH) instead of MAP, it can be expected that similar reaction takes place and amide groups will be directing to outside. If this surface structure is completed, Hap surface will possess positive charge in acidic solution by protonation of amide groups (Scheme 1). Hence, the aim of this study was to elucidate the preparation of positively charged Hap nano-crystals by using alanine and further clarify the adsorption affinity of these surface amide functionalized Hap nano-crystals to proteins. 2. Experimental 2.1. Materials and methods Colloidal Hap nano-crystals incorporating -alanine onto the surface (-alanine-Hap) were prepared by the following wet method [14–18]: 0.040 mol of Ca(OH)2 was dissolved into 2 dm3 of deionized-distilled water free of CO2 in a sealed Teflon vessel. After being stirred for 24 h at room temperature, 0.024 mol of H3 PO4 with various amounts of -alanine was added into the solution and the suspension was stirred for further 24 h at room temperature. This suspension was aged in an air oven at 100 ◦ C for 48 h. The concentration of -alanine was changed from 0 to 0.4 mol, corresponding to the molar ratio of -alanine/Ca = 0–10. Hereafter, the molar ratio of -alanine/Ca is abbreviated as /Ca. The Hap nano-crystals generated 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. The shape, specific surface area, crystal phase, and Ca2+ and PO4 3− contents of Hap nano-crystals were determined by a transmission electron microscope (TEM; JEOL JEM2100), N2 adsorption measurements, X-ray diffraction (XRD; Rigaku
The amounts of BSA adsorbed on the -alanine-Hap nanocrystals were measured by a batch method as following the method employed in our previous papers [19–22]. This measurement was conducted at 15 ◦ C and pH 5.9 because -alanine-Hap nanocrystals showed positive charge at this pH. The solution of pH 5.9 was developed by using acetic acid (HAc )–sodium acetate (NaAc ) buffer solution system. The concentration of HAc and NaAc were 5.71 × 10−2 and 1.0 M, respectively. The adsorption experiment was performed in the buffer solution of BSA 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 concentration of BSA was measured by the microbiuret method using an UV absorption band at 310 nm after centrifuging the dispersions. Most of the UV experiments were triplicated and reproducible within 2%, indicating an uncertainty of 2 × 10−2 mg m−2 for the amounts of protein adsorbed. BSA was purchased from Sigma Co. (A-7030, 67,200 Da, isoelectric point = 4.7). 3. Results and discussion 3.1. Properties of ˇ-alanine-Hap nano-crystals Table 1 summarized the preparation conditions and properties of -alanine-Hap nano-crystals. The contents of -alanine measured by CHN elemental analysis and number of -alanine on Hap surface estimated by using specific surface area of each particle are also listed in Table 1. The TEM images of the nano-crystals synthesized in this study are shown in Fig. 1. The nano-crystals produced without (/Ca = 0) are rod-like and 15 nm × 60 nm in size. The rod-like nano-crystals are lengthened with addition of -alanine though their width does not vary. Fig. 2 shows changes of short and long axis lengths of the nano-crystals together with specific surface area of these nano-crystals (䊉) as a function of the /Ca molar ratio in a starting solution. Clearly, the long axis lengths of the nano-crystals are increased with increase in the /Ca ratio up to 1, exhibiting a maximum at this point, and they are held almost constant ca. 80 nm. The specific surface area is increased with the /Ca ratio from ca. 90 to 125 m2 /g in spite of increase in their size. Since the t-plot analysis revealed that these nano-crystals are nonporous, the increase in the specific surface area with increase in the /Ca molar ratio may be due to the growth the surface roughness of the particles with -alanine in the solution. The growth along c-axis can be explained as follows: carboxyl groups of alanine are strongly coordinated to Ca2+ ions exposed on ac and/or bc faces to inhibit particle growth to a- and/or b-axis directions and enhance the particle growth along to the c-axis (Scheme 1). Similar effect has been reported on the formation of whisker type Hap nano-crystals from amorphous calcium phosphate in the presence
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Table 1 Preparation conditions and properties of -alanine-Hap nano-crystals. /Ca ratio
0 0.4 1.0 2.0 4.0 10.0 a b
Ca/P atomic ratio
1.63 1.63 1.64 1.61 1.58 1.58
Specific surface area (m2 /g)
95.2 82.4 91.3 105.5 106.5 124.2
Crystallite size L (nm)a
65 80 95 78 84 83
Amounts of -alanine in particles (wt.%)b
(molecules/nm2 )
0 0.98 0.91 0 0 0
0 0.80 0.75 0 0 0
From (0 0 2) face. Measured by CHN elemental analysis.
of acetic acids on the hydrothermal reaction [23]. No difference can be also recognized on the crystal structure among the nano-crystals as is displayed XRD patterns in Fig. 3. The crystallite size of the particles (L) from the (0 0 2) face calculated by using Scherrer’s equation coincided to the particle length as is shown in Table 1, indicating that all the Hap particles prepared are single crystal. However, the large difference was recognized by TG-DTA measurement as is shown in Fig. 4.
The DTA curves of the nano-crystals produced at /Ca = 0.4 and 1.0 possess exothermic peaks at ca. 150–350 ◦ C, indicating that -alanine is present on the nano-crystal surface. However, these peaks disappear at /Ca ≥ 2.0, suggesting -alanine molecules are absent in these nano-crystals irrespective of their high /Ca ratio. To elucidate the state of -alanine, in situ FTIR spectra of the nanocrystals were measured. Fig. 5 depicts the FTIR spectra recorded in vacuo. The absorption bands at 3600–3700 cm−1 is due to the
Fig. 1. TEM pictures of Hap nano-crystals produced at various /Ca ratios.
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Fig. 2. Plots of short () and long () particle lengths and their specific surface area (䊉) as a function of /Ca ratio.
surface P–OH groups constituting surface acidic phosphate ions, HPO4 2− . These ions would have been converted from PO4 3− ions in order to maintain the overall charge balance of calcium-deficient Hap [11]. The symmetry of distorted tetrahedral PO4 3− ions or their sp3 orbital might have been changed in this surface structure. Ishikawa et al. mentioned that these groups may be free or isolated OH groups of similar bond nature and possess ion-exchange properties [11]. Besides these bands, absorption bands newly appear at 2800–3000 cm−1 by increase in the /Ca ratio up to 1.0. These bands are assigned to the stretch mode of C–H groups of -alanine. However, these bands suddenly disappear at the /Ca ratio over 2.0. This result of FTIR coincides with that of TG-DTA in Fig. 4, indicating that -alanine is incorporated on the -alanine-Hap nano-crystal surface up to the /Ca ratio of 1.0. It is noteworthy in Fig. 5 that
Fig. 4. TG-DTA curves of Hap nano-crystals produced at various /Ca ratios.
the absorption band of surface P–OH groups was not altered by -alanine incorporation. This fact strongly implies that carboxyl groups of -alanine molecules are binding to Ca2+ ions (C sites) exposed on the nano-crystal surface but not to surface P–OH groups. Now we should discuss the optimum point for incorporation of -alanine at /Ca = 1.0. Since -alanine is dissolved in acidic H3 PO4 solution, -alanine is positively charged by protonation as is expressed as equation (1). NH3 + CH2 CH2 –COOH
(1)
After poured this acidic H3 PO4 solution into Ca(OH)2 one, Hap nano-crystals are immediately formed and solution pH is decreased. In order to produce dissociated form of two -alanine molecules (Eq. (2)) to bind Ca2+ ions such as depicted in Scheme 1, the concentration of Ca(OH)2 should be the same as that of alanine because Ca(OH)2 promotes two OH− ions by dissociation. NH2 CH2 CH2 –COO−
Fig. 3. XRD patterns of Hap nano-crystals produced at various /Ca ratios.
Fig. 5. FTIR spectra of Hap nano-crystals produced at various /Ca ratios.
(2)
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Fig. 6. Plots of Ca/P atomic ratio () and solution pH (䊉) after the reaction as a function of /Ca ratio.
Therefore, the optimum concentration for incorporation of alanine was observed at the /Ca ratio of 1.0. Above this molar ratio, the number of OH− ions to dissociate carboxyl groups of -alanine is insufficient and reduce their binding ability to Ca2+ ions. Fig. 6 plots the solution pH after the reaction. The solution pH is decreased from 9.7 to 6.9 with increase in the /Ca ratio up to 2.0 and shows almost constant value of 6.9–7.4 at above this /Ca ratio. Since the constant pH value of solution is close to the isoelectric point (iep) of -alanine (6.9), it can be presumed that -alanine is uncharged in this /Ca region. This result strongly supports the discussion of -alanine molecules described above.
Fig. 8. Adsorption isotherms of BSA onto Hap nano-crystals produced at /Ca ratios. /Ca ratios: () 0; () 0.4; (䊉) 1.0; () 2.0; (×) 4.0; () 10.0.
3.3. Adsorption behavior of BSA onto ˇ-alanine-Hap nano-crystals
Fig. 7 displays the changes of zp of the -alanine-Hap nanocrystals prepared at different /Ca ratios as a function of solution pH. The untreated Hap particle exhibits negative value over all pH range measured (: 5.5 ≤ pH ≤ 7.9). However, the zp of -alanineHap nano-crystals prepared at /Ca = 0.4 () and 1.0 (䊉) moves to positive side, especially the latter ones possess definitive positive zp (,, ) at pH ≤ 5.9. However, zp of the nano-crystals prepared at the /Ca ratio over 2.0 exhibits again negative values even at pH ≤ 5.9. This result is compatible with the results of TG-DTA (Fig. 4) and FTIR (Fig. 5); only -alanine molecules incorporated onto Hap surface produced at the /Ca ratios of 0.4 and 1.0 attain positive charge. This fact further substantiated by the fact that the zp only vary at pH < 7, coinciding with the iep of -alanine (6.9).
Adsorption isotherms of BSA onto -alanine-Hap nano-crystals were measured in HAc –NaAc buffer solution of pH 5.9, where the -alanine-Hap produced at /Ca = 1.0 exhibits positive charge. The obtained isotherms are shown in Fig. 8. The adsorption isotherms of BSA are the Langmuirian type. The adsorption coverage of BSA, defined as the ratio of the experimental saturated amounts of adsorbed BSA (nBSA s ) to the theoretical value, is 0.24. The latter value was estimated as 2.52 mg m−2 by assuming side-on adsorption of globular BSA molecules, which are prolate ellipsoids of 14 nm × 4 nm. Since the solution pH of the system is 5.9, BSA molecules are negatively charged. The nBSA values were determined s by Fig. 8 and plotted in Fig. 9 as a function of the /Ca ratio. As is increases rapidly at the /Ca ratio of 0.4 and 1.0 and expected, nBSA s exhibits a maximum at 1.0, though they decrease again over the /Ca ratio of 2.0. The nBSA value for the nano-crystals produced s at /Ca = 0.4 and 1.0 are 2.3–2.4-fold than the untreated original ones. Clearly this enhancement of BSA adsorption is attributed to the electrostatic attractive force between positively charged Hap nano-crystals and negatively charged BSA molecules, comparable with the results of Yin et al. [12]. Of course, the conformation of BSA will be changed after attached BSA molecules to -alanine-Hap nano-crystals because secondary and tertiary structures of protein
Fig. 7. Plots of zeta potential of Hap nano-crystals produced at various /Ca ratios as a function of pH.
Fig. 9. Plots of saturated amounts of adsorbed BSA as a function of /Ca ratio.
3.2. Surface charges of ˇ-alanine-Hap nano-crystals
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molecules would be affected easily by the biological materials contacted with [24–26]. Though this conformational rearrangement is another possibility of the increase of nBSA s , we cannot discuss this subject from our results at the moment. Even though this important subject, BSA molecules are attracted to the -alanine-Hap nanocrystal surface by electrostatic attractive force before attached to the surface. Therefore, we believe the electrostatic interaction is the dominating factor in the present study. The results described on the -alanine-Hap nano-crystals could be expected to give valuable information for the development of a high-quality HPLC column and may be useful to the researchers in the fields of biomaterials, biomineralization, biosensors and protein separation. 4. Conclusion Colloidal surface amide functionalized Hap nano-crystals were prepared by wet method in the presence of various amounts of alanine. The rod-like nano-crystals were lengthened with addition of -alanine though their width did not vary. No difference was recognized on the crystal structure among the synthesized Hap nano-crystals by XRD measurements. However, TG-DTA and FTIR measurements revealed that -alanine is incorporated on the alanine-Hap nano-crystal surface up to the /Ca ratio of 1.0, though they are absent in these nano-crystals synthesized over the /Ca ratio of 2.0. The zeta potential (zp) of -alanine-Hap nano-crystals prepared at /Ca ≤ 1.0 exhibited positive charge at pH < 5.9. The saturated amounts of adsorbed BSA value for the positively charged -alanine-Hap nano-crystals were increased 2.3–2.4-fold by their electrostatic attraction force between positively charged -alanineHap nano-crystals and negatively charged BSA molecules. Acknowledgment The authors wish to express their deepest gratitude to Hosokawa Powder Technology Foundation for its support in their present research.
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