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Fabrication of monodisperse silica microspheres using synechocystis cell as a template
Materials Chemistry and Physics 138 (2013) 762e766
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Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Fabrication of monodisperse silica microspheres using synechocystis cell as a template Bo Zhang a, Tian-Rui Ren a, b, *, Bao-An Song a, Quan-Xi Wang b a
State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, PR China b The Key Laboratory of Resource Chemistry of Ministry of Education, Development Center of Plant Germplasm, College of Life and Environmental Science, Shanghai Normal University, 100 Guilin Road, Shanghai 200234, PR China
h i g h l i g h t s < Monodisperse silica microspheres were prepared using Synechocystis as biotemplate. < The silica microspheres retained the original morphology of the template. < The mean diameter of the particles is uniform and coincides with the template. < The silica particles modified with agarose are effective in separating protein.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 March 2012 Received in revised form 1 December 2012 Accepted 18 December 2012
A novel technique for synthesis of monodisperse silica microparticles (MSMs), using spherical synechocystis sp. PCC 6803 as bio-templates, has been successfully developed. Various approaches including FESEM, TEM, FTIR and Small-angle X-ray scattering were used to characterize the intermediate and the final products. The results showed that MSMs retained the original morphology and uniformity of the PCC 6803 cells, and the average diameter of MSMs is almost the same with the cells. The possible mechanism for the formation of MSMs is proposed. With the surface modification by Sepharose CL-6B, the resultant MSMs were demonstrated as the good chromatography packings for protein separation. Therefore, this convenient, low cost and reproducible synthesis process may be used to prepare the MSMs with various sizes and shapes by the proper selection of original bio-templates. Ó 2013 Elsevier B.V. All rights reserved.
Keywords: Oxides Solegel growth Chemical synthesis Inorganic compounds
1. Introduction Monodisperse silica particles with uniform size, shape and composition have attracted great attention due to their many scientific and industrial applications. In particular, the monodisperse silica microspheres (MSMs) have wide applications not only in the field of fundamental physical chemistry processes such as light scattering and colloidal stability, but also in important industries including ceramics, catalysts and chromatography [1e5]. Achieving the control of the particle size and uniformity of MSMs is critical for many of these applications. For example, the non-porous silica
* Corresponding author. State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, PR China. Tel./fax: þ86 21 64328850. E-mail addresses: [email protected], [email protected] (T.-R. Ren). 0254-0584/$ e see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matchemphys.2012.12.054
particles of 1e2 mm is recommended for improving the separation performance in chromatography of proteins. The binding capacity of 1.5 mm monodisperse silica beads as packings for affinity chromatography of proteins is comparable to those of 100- to 200-nm pore size silicas [5]. The synthesis of monodisperse silica particles is generally performed with the hydrolysis and condensation of alkoxysilanes (often Tetraethyl Orthosilicate (TEOS)) in the presence of the polar alcohol/water medium and using ammonia a catalyst. The maximum particle size of MSMs produced via the well-known Stöber synthesis is less than 5 mm in diameter [6]. To overcome this size limitation, a variety of techniques have been developed, including aerospray, micrometer-sized injection nozzles, and ultrasonic oscillation [7e9]. However, the produced silica microparticles by all these methods have a high degree of polydispersity and poor colloidal stability. Additionally, the template method has been widely employed for the exquisite fabrication of particles with fine structures. Comparing with the artificial templates such as polymer bead and inorganic template, the
B. Zhang et al. / Materials Chemistry and Physics 138 (2013) 762e766
uniform particles with good dispersion, different morphology and tuning sizes are more easily synthesized by using the biological templates including bacterium [10], DNA [11], virus particles [12] and fungi [13]. Recently, Nomura et al. [14,15] reported the fabrication of the silica hollow particles using Escherichia coli as a template. To the best of our knowledge, there are few studies of the preparation of MSMs using a bio-template method. Here we describe a novel strategy to fabricate MSMs with tunable size through a combination of the Stöber synthesis with the biotemplate method. With the goal of applications for protein chromatography, we attempted to fabricate MSMs using synechocystis cell as a biological template. Owing to the uniform property of PCC 6803 synechocystis cells, the MSMs were successfully synthesized by the hydrolysis of TEOS in the presence of ammonia following the Stöber process. This process was characterized using optical microscopy, SEM, TEM, FTIR and Small-angle X-ray scattering. Correspondingly, the mechanism of the formation of MSMs was also proposed. To demonstrate their excellent chromatographic properties, the resultant MSMs were further surface modified with Sepharose CL-6B as packings to separate the binary protein mixture of bovine serum albumin (BSA) and lysozyme (Lys). 2. Experimental 2.1. Materials Tetraethoxysilane (TEOS, 99.9%), absolute ethanol (99.9%), ammonia solution (NH4OH, 25e28%), formaldehyde (37%), sodium hydroxide (NaOH, 96%) and sodium borohydride (NaBH4, 96%) were purchased from Sinopharm Chemical Regent Co. (China). 1,4-Butanedioldiglycidylether, bovine serum albumin (BSA), lysozyme (Lys), g-aminobutyric acid (GABA) and Sepharose CL-6B were provided by SigmaeAldrich (St. Louis, MO, USA). PCC 6803 cells were obtained from the Department of Biology, Shanghai Normal University (China). Double distilled water was used in the experiment. All chemicals were analytic grade and were used without further purification. 2.2. Synthesis of MSMs using PCC 6803 as a bio-template PCC 6803 cells were fixed in 4% formaldehyde solution for 2 h at room temperature. The fixed cells were centrifuged and washed
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with distilled water for several times. Then the cells were dehydrated in a gradient series of ethanol (50%, 70%, 80%, 90%, and 100%) and collected by centrifugation. The dehydrated cells, ammonia solution and double distilled water were sonicated for 1 min to ensure complete cell dispersion. Then, the mixture was harvested under shaking condition for 36 h at room temperature. The cells were separated by centrifugation and washed with double distilled water. Afterward, a desired amount of cell dispersion solution was added to the TEOS ethanol solution to carry out silica growth reaction at room temperature under continuous shaking for 36 h. The resulting particles were collected by centrifugation, and were washed with double distilled water and ethanol for several times. The collected particles were dried at 70 C. Finally, the dried particles were calcined at 550 C for 6 h to remove the biological cells. The standard experimental conditions were TEOS ¼ 10 ml, NH3 ¼ 3 ml, H2O ¼ 10 ml, ethanol ¼ 10 ml, cell ¼ 1.5 g L1 (7.1 1010 cells m3). The number of cells in the solution was counted using a PetroffeHausser counting chamber. 2.3. Surface modification of MSMs with Sepharose CL-6B As previously reported [16,17], the surface modification of MSMs with Sepharose CL-6B is illustrated in Fig. S1 of Supporting information. In a typical procedure, 5 g suction-dried Sepharose CL-6B was washed on a glass filter-funnel with water and then mixed with 5 ml 1,4-butanedioldiglycidylether and 5 ml 0.6 M NaOH solution containing 10 mg NaBH4. The mixture was rotated mechanically at 25 C for 8 h (Supporting information Fig. S1①). The epoxide-activated Sepharose CL-6B was subsequently collected on a sintered glass funnel and was washed with a large excess of double distilled water. Then 1.0 g GABA in 20 ml of 2.0 M of sodium carbonate buffer (pH 10.5) was added to 5 g suction-dried epoxideactivated Sepharose CL-6B and the mixture was rotated mechanically at 65 C for 24 h. The resulting solid was filtered out and washed with double distilled water to obtain the coupling-agarose. (Supporting information Fig. S1②). Finally, 3 g coupling-agarose was dissolved in 30 ml H2O at 90 C. Next, 1.0 g MSMs was added to the above solution under vigorous stirring for 10 min. MSMs suspension was transferred to a 500 ml glass reactor containing a mixture of 300 ml soybean oil and 10 ml sorbitan monooleate (Span 80) (Supporting information Fig. S1③). Then, the mixture was stirred with a speed of 400 rpm at 90 C for 10 min and cooled
Fig. 1. Morphology characterizations of the formation of MSMs. (a) SEM micrographs of PCC 6803; (b and c) FESEM images of PCC 6803/silica particles complex observed under different magnifications; (d, e and f) FESEM images of solid silica after removal of PCC 6803 templates under different magnifications.
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Fig. 2. TEM images of (a, b) a non-dividing cell and a cell in early division the PCC 6803. Scale bar corresponds to 200 nm (c) PCC 6803/silica particles complex observed (inset: high-resolution TEM images) and (d, e, f) solid silica removed bio-template. (Insets: high-resolution TEM images).
2.4. Chromatography of the protein mixture The chromatographic experiments for the protein mixture of BSA and Lys were carried out at room temperature. The MSMs of surface modification with Sepharose CL-6B were slurry packed into a column (15 1 cm I.D.) and equilibrated with a sodium phosphate buffer (20 mM, pH 7.1) prior to sample application. Following the injection of the protein mixture, the column was eluted with a sodium phosphate buffer (20 mM, pH 5.0) containing NaC1 (0.5 M), at a flow-rate of 1 ml min1. The absorbance of the eluate was continuously measured at 280 nm. 2.5. Characterization Fourier transform infrared (FT-IR) spectra were collected on a FTIR EQUINO 55 apparatus using KBr pellets. Field emission scanning microscopy (FESEM) was taken with Hitachi S-4800 operating at 1.5 kV. A thin gold film was sprayed on the sample before the characterization. Transmission electron microscopy (TEM) images were conducted on a Hitachi H-600 microscope (Japan) operated at 200 kV. The sample for TEM measurements was suspended in ethanol and supported on a holey carbon film on a copper grid. Particle size distribution of the samples was determined using ZetasizerNano-ZS90 (Malvern Instruments). The analysis was performed at a scattering angle of 90 at a temperature of 25 C using samples diluted to different intensity concentration with de-ionized distilled water.
continues to furrow inwards, until the cytoplasm is divided producing two daughter cells. The mean diameter of PCC 6803 cells is 1.5 0.1 mm (Supporting information Fig. S2a). The uniform property and the optimal particle size of PCC 6803 cells enable them as a bio-template for the application of MSMs in chromatography. The typical FESEM images of PCC 6803/silica particles complex (Fig. 1b and c) reveal that the PCC 6803 cells were fully permeated and the complexes possess a roughly spherical structure with a diameter range of 1.5 0.1 mm. As seen in FESEM images of Fig. 1def, the calcinated particles retained the original morphology of the PCC 6803 cells even though the template was removed. The resultant MSMs show the spherical shape and the average diameter of MSMs is relatively uniform (1.5 0.1 mm, Supporting information Fig. S2b), which is almost the same with the initial PCC 6803 cells. It is especially noteworthy that the MSMs (Fig. 1f) faithfully retain the original morphologies of the cells at the various division phases. The morphology and structure of the as-obtained samples were further investigated by TEM micrograph (Fig. 2). The representative TEM images of the isolated and proliferated PCC 6803 cells are shown in Fig. 2a and b, respectively. It further suggests that the
110
b 105
100
T (% )
down to 10 C. The as-made products were separated from the solvent by filtration and washed with acetone, double diluted water and ethanolewater (50:50, v/v). Thus, the MSMs of surface modification with Sepharose CL-6B were obtained.
a
95
90
3. Results and discussion
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To fabricate MSMs, the Stöber synthesis is designed to occur inside the biological templates of micrometer dimensions and finally the bio-templates are removed by calcining. As shown in Fig. 1a, Cells of PCC 6803 are essentially spherical immediately after cell division. Some are at the various stages of cell division (the inset of Fig. 1a). During growth, the cells elongate and an incipient septum invaginates into the cytoplasm. The septum
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75 4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750
500 250
-1
v (c m ) Fig. 3. FTIR spectrum of (a) as-synthesized and (b) calcined samples.
B. Zhang et al. / Materials Chemistry and Physics 138 (2013) 762e766
Fixed Cell
Washing by water
NH3.H2O, H2O C2H5OH
Drying and calcination
TEOS,C 2H 5OH
765
SiO2
Hydrolysis and Condensation of TEOS
NH3.H2O
H 2O
C2H5OH TEOS
Scheme 1. Schematic illustration of the synthetic procedures of solid silica particles synthesized using PCC 6803.
original morphology of PCC 6803 cell is approximately spherical. The TEM images in Fig. 2c reveal the morphology of as-obtained precursor of PCC 6803/silica particles. Obviously, PCC 6803 cells, as templates, were well permeated. The TEM images in Fig. 2cee reveal the morphology of monodisperse silica particles with the removal of the PCC 6803 template under different magnifications. It is apparent that the some relatively irregular micropores in channel arrangements, correlating well with the SAXS result (Supporting information Fig. S3b). Furthermore, it is noteworthy to mention that the final product could well mimic the internal structure of PCC 6803 and some MSMs still retained the state of cell division, as illustrated in Fig. 2d and f. Overall, these results suggest that this synechocystis cell template technique enables the synthesis of spherical silica microparticles. Our synthesis of MSMs is low cost and reproducible. FTIR measurements were employed to analyze various vibration modes of different groups in the silica products. The respective FTIR spectrum of the as-synthesized samples and calcined samples are demonstrated in Fig. 3. The patterns of two samples appear to consist of peaks around 1700 and 3430 cm1 corresponding to the carboxyl and hydroxyl groups [18], respectively. The adsorption peak attributing to the SieO stretching vibration of SieOH bond appears at 960 cm1 [19]. The weak peaks at 2855 and 2920 cm1 belong to the stretching vibrations of CeH bonds, indicating a few
organic groups adsorbed on the spheres. The strong peaks near 1100, 802 and 467 cm1 agree to the SieOeSi bonds which imply the condensation of silicon alkoxide [20]. The peak at 802 cm1 is related to the symmetric vibration mode of the SieOeSi siloxane bridges, and the 467 cm1 peak corresponds to the vibrational adsorption peak of the SieOeSi siloxane bridges, which is the characteristic peak of silica. According to the FTIR analysis, the locations of the characteristic peaks of as-synthesized and calcined samples remain unchanged, indicating that the skeleton structure of SiO2 was not damaged during heat treatment. The above results correlate well with the normal FTIR spectra of SiO2. The clear FTIR spectra also suggest that there are no other impurities adsorbed onto the surface of the silica particles, which is confirmed by energy-dispersive X-ray (EDAX) analysis (Supporting information Fig. S4). Based on the above analysis, a possible synthesis mechanism of the MSMs was proposed in Scheme 1. The PCC 6803 cell templates offer a multitude of nucleation sites for target materials that induce the nucleation and grow of the MSMs. The whole process mainly includes two steps. In the first step, the extracellular ammonia, across a cell membrane against the concentration gradient, permeates into the entire cells when the ammonia solution and the PCC 6803 are mixed and shaked at room temperature. The PCC 6803 cells are filled with ammonia based on infiltration and diffusion
Fig. 4. FESEM images of (a) solid silica microspheres; (b) solid silica modified with agarose.
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synechocystis sp. PCC 6803 cells. The entire synthesis process of MSMs is safe, convenient and low cost. The final products have a well-defined spherical morphology and uniform diameters of approximately 1.5 mm, which retain the morphology of the initial PCC 6803 cells. This result indicates that this technique enables the synthesis of solid silica spherical particles with various sizes and other types such as rods and wires by selecting the suitable biotemplates. The two-step formation mechanism of MSMs was discussed based on the theory of diffusion and permeation. Finally, after surface modification with Sepharose CL-6B, the good chromatographic property of the resultant MSMs was demonstrated in the protein separation chromatography.
Lys 0.12
BSA
Absorbance, 280 nm
0.10 0.08 0.06 0.04 0.02
Acknowledgments 0.00 5
10
15
Time (h) Fig. 5. Chromatograms of Lys and BSA separated by self-made column.
under a concentration gradient. Then the cells were washed quickly with double distilled water to remove extracellular ammonia. In the second step, the cells treated with ammonia were added to the TEOS ethanol solution. Consequently, TEOS may enter the cell and react to form the cell/silica spheres under the catalysis of ammonia. This phenomenon has been proved by the FT-IR spectrum of cell/silica spheres as shown in Fig. 3a. Finally, when the biotemplate was removed by heat treatment, the resultant spheres faithfully retained the original morphology of the PCC 6803 (Fig. 2b and e), and the average diameter of the spheres was similar with that of biotemplates, showing the reaction of TEOS was inside the cells. From this proposed mechanism, we can see that the PCC 6803 cells not only serve as nucleation sites of silica microparticles but also act as hard templates to direct the morphology of the products. To explore the potential application of these MSMs as protein separation matrix, we first decorated the surface of silica with Sepharose CL-6B (Supporting information Fig. S1) and selected a mixture of binary proteins with different isoelectric point (pI), Lys (pI ¼ 11.0) and BSA (pI ¼ 4.9), as model proteins. In comparison to the original silica particles, the cloud-like dots occurred on the surface of silica modified with agarose (Fig. 4), which is in agreement with the IR data (Supporting information Fig. S5). These results demonstrated that the SiO2 substrate coated with agarose were fabricated successfully. The chromatogram of the protein mixture of Lys and BSA using self-made Sepharose CL-6B-silica composite particles column is depicted in Fig. 5. The mixture of Lys and BSA was eluted into two split peaks. BSA that has no affinity to ligand was eluted at 3 h. The results suggest that BSA is the non-retained component with respect to this chromatographic column. The MSMs of surface modification with Sepharose CL-6B can be used for protein separation. Due to the small diameter of column packing and use of open column chromatography, the separation efficiency of current column is not very high. The separation efficiency of chromatography is to be further enhanced. 4. Conclusions In summary, a novel method for the preparation of MSMs was developed by control of the Stöber synthesis inside the template of
The work was financially supported by the National Natural Science Foundation of China (21172147), Innovation Program of Shanghai Municipal Education Commission (11ZZ122), the National High Technology Research and Development Program of China (863: 2011AA100503), National Program on Key Basic Research Project (973: 2010CB126106), the Opening Foundation of the Key Laboratory of Green Pesticide and Agricultural Bioengineering (2011GDGP0101).
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.matchemphys.2012.12.054.
References [1] K. Nozawa, H. Gailhanou, L. Raison, P. Panizza, H. Ushiki, E. Sellier, J.P. Delville, M.H. Delville, Langmuir 21 (2005) 1516. [2] S.-Y. Kim, E.-H. Kim, S.-S. Kim, W. Kim, J. Colloid Interface Sci. 292 (2005) 93. [3] C.R. Miller, R. Vogel, P.P.T. Surawski, K.S. Jack, S.R. Corrie, M. Trau, Langmuir 21 (2005) 9733. [4] M. Marquez, B.P. Grady, I. Robb, Colloids Surf. A 266 (2005) 18. [5] G. Jilge, K.K. Unger, U. Esser, H.J. Schäfer, G. Rathgeber, W. Müller, J. Chromatogr. A 476 (1989) 37. [6] W. Stober, A. Fink, E. Bohn, J. Colloid Interface Sci. 26 (1968) 62. [7] P. Kortesuo, M. Ahola, M. Kangas, A. Yli-Urpo, J. Kiesvaara, M. Marvola, Int. J. Pharm. 221 (2001) 107. [8] R. Masuda, W. Takahashi, M. Ishii, J. Non-Cryst. Solids 121 (1990) 389. [9] H. Isobe, K. Kaneko, J. Colloid Interface Sci. 212 (1999) 234. [10] (a) M. Mertig, R. Kirsch, W. Pompe, Appl. Phys. A 66 (1998) 723; (b) R. Mogul, J.J.G. Kelly, M.L. Cable, A.F. Hebard, Mater. Lett. 60 (2006) 19; (c) R. Kirsch, M. Mertig, W. Pompe, R. Wahl, G. Sadowski, K.J. Bohm, E. Unger, Thin Solid Films 305 (1997) 248; (d) K. Pollmann, J. Raff, M. Merroun, K. Fahmy, S. Selenska-Pobell, Biotechnol. Adv. 24 (2006) 58.(e) H. Zhou, T.X. Fan, D. Zhang, Q.X. Guo, H. Ogawa, Chem. Mater. 19 (2007) 2144; (f) T. Nomura, Y. Morimoto, M. Ishikawa, H. Tokumoto, Y. Konishi, Adv. Powder Technol. 21 (2010) 218. [11] S. Hou, J. Wang, C.R. Maetin, J. Am. Chem. Soc. 127 (2005) 8586. [12] C.E. Flynn, S.W. Lee, B.R. Peelle, A.M. Belcher, Acta Mater. 51 (2003) 5867. [13] B. Bai, P.P. Wang, L. Wu, L. Yang, Z.H. Chen, Mater. Chem. Phys. 114 (2009) 26. [14] T. Nomura, Y. Morimoto, H. Tokumoto, Y. Konishi, Mater. Lett. 62 (2008) 3727. [15] T. Nomura, Y. Morimoto, M. Ishikawa, H. Tokumoto, Y. Konishi, Adv. Powder Technol. 21 (2010) 8. [16] L. Sundberg, J. Porath, J.Chromatogr. 90 (1974) 87. [17] X. Zhou, Q.H. Shi, S. Bai, Y. Sun, Biochem. Eng. J. 18 (2004) 81. [18] Y. Li, C. Xu, B. Wei, X. Zhang, M. Zheng, D. Wu, P.M. Ajayan, Chem. Mater. 14 (2002) 483. [19] Y. Shan, L. Gao, S. Zheng, Mater. Chem. Phys. 88 (2004) 192. [20] J.R. Agger, M.W. Anderson, M.E. Pemble, O. Terasaki, Y. Nozue, J. Phys. Chem. B 102 (1998) 3345.
Contents lists available at SciVerse ScienceDirect
Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Fabrication of monodisperse silica microspheres using synechocystis cell as a template Bo Zhang a, Tian-Rui Ren a, b, *, Bao-An Song a, Quan-Xi Wang b a
State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, PR China b The Key Laboratory of Resource Chemistry of Ministry of Education, Development Center of Plant Germplasm, College of Life and Environmental Science, Shanghai Normal University, 100 Guilin Road, Shanghai 200234, PR China
h i g h l i g h t s < Monodisperse silica microspheres were prepared using Synechocystis as biotemplate. < The silica microspheres retained the original morphology of the template. < The mean diameter of the particles is uniform and coincides with the template. < The silica particles modified with agarose are effective in separating protein.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 March 2012 Received in revised form 1 December 2012 Accepted 18 December 2012
A novel technique for synthesis of monodisperse silica microparticles (MSMs), using spherical synechocystis sp. PCC 6803 as bio-templates, has been successfully developed. Various approaches including FESEM, TEM, FTIR and Small-angle X-ray scattering were used to characterize the intermediate and the final products. The results showed that MSMs retained the original morphology and uniformity of the PCC 6803 cells, and the average diameter of MSMs is almost the same with the cells. The possible mechanism for the formation of MSMs is proposed. With the surface modification by Sepharose CL-6B, the resultant MSMs were demonstrated as the good chromatography packings for protein separation. Therefore, this convenient, low cost and reproducible synthesis process may be used to prepare the MSMs with various sizes and shapes by the proper selection of original bio-templates. Ó 2013 Elsevier B.V. All rights reserved.
Keywords: Oxides Solegel growth Chemical synthesis Inorganic compounds
1. Introduction Monodisperse silica particles with uniform size, shape and composition have attracted great attention due to their many scientific and industrial applications. In particular, the monodisperse silica microspheres (MSMs) have wide applications not only in the field of fundamental physical chemistry processes such as light scattering and colloidal stability, but also in important industries including ceramics, catalysts and chromatography [1e5]. Achieving the control of the particle size and uniformity of MSMs is critical for many of these applications. For example, the non-porous silica
* Corresponding author. State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, PR China. Tel./fax: þ86 21 64328850. E-mail addresses: [email protected], [email protected] (T.-R. Ren). 0254-0584/$ e see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matchemphys.2012.12.054
particles of 1e2 mm is recommended for improving the separation performance in chromatography of proteins. The binding capacity of 1.5 mm monodisperse silica beads as packings for affinity chromatography of proteins is comparable to those of 100- to 200-nm pore size silicas [5]. The synthesis of monodisperse silica particles is generally performed with the hydrolysis and condensation of alkoxysilanes (often Tetraethyl Orthosilicate (TEOS)) in the presence of the polar alcohol/water medium and using ammonia a catalyst. The maximum particle size of MSMs produced via the well-known Stöber synthesis is less than 5 mm in diameter [6]. To overcome this size limitation, a variety of techniques have been developed, including aerospray, micrometer-sized injection nozzles, and ultrasonic oscillation [7e9]. However, the produced silica microparticles by all these methods have a high degree of polydispersity and poor colloidal stability. Additionally, the template method has been widely employed for the exquisite fabrication of particles with fine structures. Comparing with the artificial templates such as polymer bead and inorganic template, the
B. Zhang et al. / Materials Chemistry and Physics 138 (2013) 762e766
uniform particles with good dispersion, different morphology and tuning sizes are more easily synthesized by using the biological templates including bacterium [10], DNA [11], virus particles [12] and fungi [13]. Recently, Nomura et al. [14,15] reported the fabrication of the silica hollow particles using Escherichia coli as a template. To the best of our knowledge, there are few studies of the preparation of MSMs using a bio-template method. Here we describe a novel strategy to fabricate MSMs with tunable size through a combination of the Stöber synthesis with the biotemplate method. With the goal of applications for protein chromatography, we attempted to fabricate MSMs using synechocystis cell as a biological template. Owing to the uniform property of PCC 6803 synechocystis cells, the MSMs were successfully synthesized by the hydrolysis of TEOS in the presence of ammonia following the Stöber process. This process was characterized using optical microscopy, SEM, TEM, FTIR and Small-angle X-ray scattering. Correspondingly, the mechanism of the formation of MSMs was also proposed. To demonstrate their excellent chromatographic properties, the resultant MSMs were further surface modified with Sepharose CL-6B as packings to separate the binary protein mixture of bovine serum albumin (BSA) and lysozyme (Lys). 2. Experimental 2.1. Materials Tetraethoxysilane (TEOS, 99.9%), absolute ethanol (99.9%), ammonia solution (NH4OH, 25e28%), formaldehyde (37%), sodium hydroxide (NaOH, 96%) and sodium borohydride (NaBH4, 96%) were purchased from Sinopharm Chemical Regent Co. (China). 1,4-Butanedioldiglycidylether, bovine serum albumin (BSA), lysozyme (Lys), g-aminobutyric acid (GABA) and Sepharose CL-6B were provided by SigmaeAldrich (St. Louis, MO, USA). PCC 6803 cells were obtained from the Department of Biology, Shanghai Normal University (China). Double distilled water was used in the experiment. All chemicals were analytic grade and were used without further purification. 2.2. Synthesis of MSMs using PCC 6803 as a bio-template PCC 6803 cells were fixed in 4% formaldehyde solution for 2 h at room temperature. The fixed cells were centrifuged and washed
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with distilled water for several times. Then the cells were dehydrated in a gradient series of ethanol (50%, 70%, 80%, 90%, and 100%) and collected by centrifugation. The dehydrated cells, ammonia solution and double distilled water were sonicated for 1 min to ensure complete cell dispersion. Then, the mixture was harvested under shaking condition for 36 h at room temperature. The cells were separated by centrifugation and washed with double distilled water. Afterward, a desired amount of cell dispersion solution was added to the TEOS ethanol solution to carry out silica growth reaction at room temperature under continuous shaking for 36 h. The resulting particles were collected by centrifugation, and were washed with double distilled water and ethanol for several times. The collected particles were dried at 70 C. Finally, the dried particles were calcined at 550 C for 6 h to remove the biological cells. The standard experimental conditions were TEOS ¼ 10 ml, NH3 ¼ 3 ml, H2O ¼ 10 ml, ethanol ¼ 10 ml, cell ¼ 1.5 g L1 (7.1 1010 cells m3). The number of cells in the solution was counted using a PetroffeHausser counting chamber. 2.3. Surface modification of MSMs with Sepharose CL-6B As previously reported [16,17], the surface modification of MSMs with Sepharose CL-6B is illustrated in Fig. S1 of Supporting information. In a typical procedure, 5 g suction-dried Sepharose CL-6B was washed on a glass filter-funnel with water and then mixed with 5 ml 1,4-butanedioldiglycidylether and 5 ml 0.6 M NaOH solution containing 10 mg NaBH4. The mixture was rotated mechanically at 25 C for 8 h (Supporting information Fig. S1①). The epoxide-activated Sepharose CL-6B was subsequently collected on a sintered glass funnel and was washed with a large excess of double distilled water. Then 1.0 g GABA in 20 ml of 2.0 M of sodium carbonate buffer (pH 10.5) was added to 5 g suction-dried epoxideactivated Sepharose CL-6B and the mixture was rotated mechanically at 65 C for 24 h. The resulting solid was filtered out and washed with double distilled water to obtain the coupling-agarose. (Supporting information Fig. S1②). Finally, 3 g coupling-agarose was dissolved in 30 ml H2O at 90 C. Next, 1.0 g MSMs was added to the above solution under vigorous stirring for 10 min. MSMs suspension was transferred to a 500 ml glass reactor containing a mixture of 300 ml soybean oil and 10 ml sorbitan monooleate (Span 80) (Supporting information Fig. S1③). Then, the mixture was stirred with a speed of 400 rpm at 90 C for 10 min and cooled
Fig. 1. Morphology characterizations of the formation of MSMs. (a) SEM micrographs of PCC 6803; (b and c) FESEM images of PCC 6803/silica particles complex observed under different magnifications; (d, e and f) FESEM images of solid silica after removal of PCC 6803 templates under different magnifications.
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Fig. 2. TEM images of (a, b) a non-dividing cell and a cell in early division the PCC 6803. Scale bar corresponds to 200 nm (c) PCC 6803/silica particles complex observed (inset: high-resolution TEM images) and (d, e, f) solid silica removed bio-template. (Insets: high-resolution TEM images).
2.4. Chromatography of the protein mixture The chromatographic experiments for the protein mixture of BSA and Lys were carried out at room temperature. The MSMs of surface modification with Sepharose CL-6B were slurry packed into a column (15 1 cm I.D.) and equilibrated with a sodium phosphate buffer (20 mM, pH 7.1) prior to sample application. Following the injection of the protein mixture, the column was eluted with a sodium phosphate buffer (20 mM, pH 5.0) containing NaC1 (0.5 M), at a flow-rate of 1 ml min1. The absorbance of the eluate was continuously measured at 280 nm. 2.5. Characterization Fourier transform infrared (FT-IR) spectra were collected on a FTIR EQUINO 55 apparatus using KBr pellets. Field emission scanning microscopy (FESEM) was taken with Hitachi S-4800 operating at 1.5 kV. A thin gold film was sprayed on the sample before the characterization. Transmission electron microscopy (TEM) images were conducted on a Hitachi H-600 microscope (Japan) operated at 200 kV. The sample for TEM measurements was suspended in ethanol and supported on a holey carbon film on a copper grid. Particle size distribution of the samples was determined using ZetasizerNano-ZS90 (Malvern Instruments). The analysis was performed at a scattering angle of 90 at a temperature of 25 C using samples diluted to different intensity concentration with de-ionized distilled water.
continues to furrow inwards, until the cytoplasm is divided producing two daughter cells. The mean diameter of PCC 6803 cells is 1.5 0.1 mm (Supporting information Fig. S2a). The uniform property and the optimal particle size of PCC 6803 cells enable them as a bio-template for the application of MSMs in chromatography. The typical FESEM images of PCC 6803/silica particles complex (Fig. 1b and c) reveal that the PCC 6803 cells were fully permeated and the complexes possess a roughly spherical structure with a diameter range of 1.5 0.1 mm. As seen in FESEM images of Fig. 1def, the calcinated particles retained the original morphology of the PCC 6803 cells even though the template was removed. The resultant MSMs show the spherical shape and the average diameter of MSMs is relatively uniform (1.5 0.1 mm, Supporting information Fig. S2b), which is almost the same with the initial PCC 6803 cells. It is especially noteworthy that the MSMs (Fig. 1f) faithfully retain the original morphologies of the cells at the various division phases. The morphology and structure of the as-obtained samples were further investigated by TEM micrograph (Fig. 2). The representative TEM images of the isolated and proliferated PCC 6803 cells are shown in Fig. 2a and b, respectively. It further suggests that the
110
b 105
100
T (% )
down to 10 C. The as-made products were separated from the solvent by filtration and washed with acetone, double diluted water and ethanolewater (50:50, v/v). Thus, the MSMs of surface modification with Sepharose CL-6B were obtained.
a
95
90
3. Results and discussion
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To fabricate MSMs, the Stöber synthesis is designed to occur inside the biological templates of micrometer dimensions and finally the bio-templates are removed by calcining. As shown in Fig. 1a, Cells of PCC 6803 are essentially spherical immediately after cell division. Some are at the various stages of cell division (the inset of Fig. 1a). During growth, the cells elongate and an incipient septum invaginates into the cytoplasm. The septum
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75 4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750
500 250
-1
v (c m ) Fig. 3. FTIR spectrum of (a) as-synthesized and (b) calcined samples.
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Fixed Cell
Washing by water
NH3.H2O, H2O C2H5OH
Drying and calcination
TEOS,C 2H 5OH
765
SiO2
Hydrolysis and Condensation of TEOS
NH3.H2O
H 2O
C2H5OH TEOS
Scheme 1. Schematic illustration of the synthetic procedures of solid silica particles synthesized using PCC 6803.
original morphology of PCC 6803 cell is approximately spherical. The TEM images in Fig. 2c reveal the morphology of as-obtained precursor of PCC 6803/silica particles. Obviously, PCC 6803 cells, as templates, were well permeated. The TEM images in Fig. 2cee reveal the morphology of monodisperse silica particles with the removal of the PCC 6803 template under different magnifications. It is apparent that the some relatively irregular micropores in channel arrangements, correlating well with the SAXS result (Supporting information Fig. S3b). Furthermore, it is noteworthy to mention that the final product could well mimic the internal structure of PCC 6803 and some MSMs still retained the state of cell division, as illustrated in Fig. 2d and f. Overall, these results suggest that this synechocystis cell template technique enables the synthesis of spherical silica microparticles. Our synthesis of MSMs is low cost and reproducible. FTIR measurements were employed to analyze various vibration modes of different groups in the silica products. The respective FTIR spectrum of the as-synthesized samples and calcined samples are demonstrated in Fig. 3. The patterns of two samples appear to consist of peaks around 1700 and 3430 cm1 corresponding to the carboxyl and hydroxyl groups [18], respectively. The adsorption peak attributing to the SieO stretching vibration of SieOH bond appears at 960 cm1 [19]. The weak peaks at 2855 and 2920 cm1 belong to the stretching vibrations of CeH bonds, indicating a few
organic groups adsorbed on the spheres. The strong peaks near 1100, 802 and 467 cm1 agree to the SieOeSi bonds which imply the condensation of silicon alkoxide [20]. The peak at 802 cm1 is related to the symmetric vibration mode of the SieOeSi siloxane bridges, and the 467 cm1 peak corresponds to the vibrational adsorption peak of the SieOeSi siloxane bridges, which is the characteristic peak of silica. According to the FTIR analysis, the locations of the characteristic peaks of as-synthesized and calcined samples remain unchanged, indicating that the skeleton structure of SiO2 was not damaged during heat treatment. The above results correlate well with the normal FTIR spectra of SiO2. The clear FTIR spectra also suggest that there are no other impurities adsorbed onto the surface of the silica particles, which is confirmed by energy-dispersive X-ray (EDAX) analysis (Supporting information Fig. S4). Based on the above analysis, a possible synthesis mechanism of the MSMs was proposed in Scheme 1. The PCC 6803 cell templates offer a multitude of nucleation sites for target materials that induce the nucleation and grow of the MSMs. The whole process mainly includes two steps. In the first step, the extracellular ammonia, across a cell membrane against the concentration gradient, permeates into the entire cells when the ammonia solution and the PCC 6803 are mixed and shaked at room temperature. The PCC 6803 cells are filled with ammonia based on infiltration and diffusion
Fig. 4. FESEM images of (a) solid silica microspheres; (b) solid silica modified with agarose.
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synechocystis sp. PCC 6803 cells. The entire synthesis process of MSMs is safe, convenient and low cost. The final products have a well-defined spherical morphology and uniform diameters of approximately 1.5 mm, which retain the morphology of the initial PCC 6803 cells. This result indicates that this technique enables the synthesis of solid silica spherical particles with various sizes and other types such as rods and wires by selecting the suitable biotemplates. The two-step formation mechanism of MSMs was discussed based on the theory of diffusion and permeation. Finally, after surface modification with Sepharose CL-6B, the good chromatographic property of the resultant MSMs was demonstrated in the protein separation chromatography.
Lys 0.12
BSA
Absorbance, 280 nm
0.10 0.08 0.06 0.04 0.02
Acknowledgments 0.00 5
10
15
Time (h) Fig. 5. Chromatograms of Lys and BSA separated by self-made column.
under a concentration gradient. Then the cells were washed quickly with double distilled water to remove extracellular ammonia. In the second step, the cells treated with ammonia were added to the TEOS ethanol solution. Consequently, TEOS may enter the cell and react to form the cell/silica spheres under the catalysis of ammonia. This phenomenon has been proved by the FT-IR spectrum of cell/silica spheres as shown in Fig. 3a. Finally, when the biotemplate was removed by heat treatment, the resultant spheres faithfully retained the original morphology of the PCC 6803 (Fig. 2b and e), and the average diameter of the spheres was similar with that of biotemplates, showing the reaction of TEOS was inside the cells. From this proposed mechanism, we can see that the PCC 6803 cells not only serve as nucleation sites of silica microparticles but also act as hard templates to direct the morphology of the products. To explore the potential application of these MSMs as protein separation matrix, we first decorated the surface of silica with Sepharose CL-6B (Supporting information Fig. S1) and selected a mixture of binary proteins with different isoelectric point (pI), Lys (pI ¼ 11.0) and BSA (pI ¼ 4.9), as model proteins. In comparison to the original silica particles, the cloud-like dots occurred on the surface of silica modified with agarose (Fig. 4), which is in agreement with the IR data (Supporting information Fig. S5). These results demonstrated that the SiO2 substrate coated with agarose were fabricated successfully. The chromatogram of the protein mixture of Lys and BSA using self-made Sepharose CL-6B-silica composite particles column is depicted in Fig. 5. The mixture of Lys and BSA was eluted into two split peaks. BSA that has no affinity to ligand was eluted at 3 h. The results suggest that BSA is the non-retained component with respect to this chromatographic column. The MSMs of surface modification with Sepharose CL-6B can be used for protein separation. Due to the small diameter of column packing and use of open column chromatography, the separation efficiency of current column is not very high. The separation efficiency of chromatography is to be further enhanced. 4. Conclusions In summary, a novel method for the preparation of MSMs was developed by control of the Stöber synthesis inside the template of
The work was financially supported by the National Natural Science Foundation of China (21172147), Innovation Program of Shanghai Municipal Education Commission (11ZZ122), the National High Technology Research and Development Program of China (863: 2011AA100503), National Program on Key Basic Research Project (973: 2010CB126106), the Opening Foundation of the Key Laboratory of Green Pesticide and Agricultural Bioengineering (2011GDGP0101).
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.matchemphys.2012.12.054.
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