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Fabrication of silica hollow particles using Escherichia coli as a template
Materials Letters 62 (2008) 3727–3729
Contents lists available at ScienceDirect
Materials Letters j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m a t l e t
Fabrication of silica hollow particles using Escherichia coli as a template T. Nomura ⁎, Y. Morimoto, H. Tokumoto, Y. Konishi Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
A R T I C L E
I N F O
Article history: Received 5 March 2008 Accepted 10 April 2008 Available online 16 April 2008 Keywords: Hollow particle Escherichia coli coli Escherichia Template Silica
A B S T R A C T Inorganic hollow particles have attracted great interest in recent years due to their unique physico-chemical properties, as well as their potential use in various applications. In this study, we attempted to fabricate hollow particles using a gram-negative bacterium, Escherichia coli, as a biological template. Silica was synthesized by the hydrolysis of tetraethyl orthosilicate (TEOS) in the presence of ammonia following the Stöber process. Silica was smoothly coated to the surface of the bacterial cells. The bacterial template could be removed by calcining at 600 °C. Interestingly, the calcinated particles retained the morphology of the biological cells even though the template was removed. This result indicates that this bacteria template technique enables the synthesis of hollow spheres, rods, wires, and other three-dimensional structures which retain the morphology of the original bacterium. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Inorganic hollow particles have attracted considerable attention because of their conspicuous properties of density, specific surface area, thermal insulation, and optical activity that differ significantly from those of dense particles. These hollow particles are used in a wide number of applications, such as drug delivery systems, bioencapsulations, and catalysts [1–3]. Various methods have been employed to prepare hollow particles such as a polymer bead template method [4], an inorganic template method [5], a sol–gel method used for surfactant stabilized emulsions [6], spray drying [7], a spray precipitation method [8], and spray pyrolysis [9]. Recently, bacterial cells have been used as a template to fabricate hollow structures [10–12]. The morphology of bacteria can be spherical (cocci), rod-shape (bacilli), curved rod-shape (vibrio), spiral-shaped (spirilla), or tightly coiled (spirochetes). They can exist as single, chained or clumped cells. This wide variety of shapes can be exploited for use as a template for the synthesis of hollow particles or for the construction of three-dimensional hollow structures. Bacteria can be divided into gram-positive and gramnegative bacteria based on the chemical and physical properties of their cell walls. To date, bacteria used as templates have been mainly gram-positive bacteria because the structure of their solid cell walls lends itself to rigid structure preparation. If gram-negative bacteria can be easily used as templates, the applicability of the technique for employing the well-defined morphology of bacteria will become much greater. In this study, the potential of a gram-negative bacterium, Escherichia coli, as a template for silica hollow microparticles was examined. In addition, the synthetic conditions for fabricating the complete ⁎ Corresponding author. Tel.: +81 72 254 9300; fax: +81 72 254 9911. E-mail address: [email protected] (T. Nomura). 0167-577X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2008.04.035
hollow structure were investigated by varying both the concentrations of TEOS as the silica source and biological cells as the template. 2. Experimental 2.1. Materials The analytical grade of tetraethyl orthosilicate (TEOS), 2 mol/L ammonia ethanol solution, and ethanol were obtained from Wako Co. Ltd., Japan. All the chemicals were used without further purification. 2.2. Preparation E. coli (KP7600) was kindly provided by the National BioResource Project (NIG, Japan): E. coli. E. coli was grown at 37 °C in LB medium with shaking. Cells were harvested in late exponential growth phase by centrifugation and washed in triplicate using a sterile NaCl aqueous solution. Silica was directly coated onto the surface of bacterial cells to form core shell particles by the Stöber method. Bacterial cells, ammonia ethanol solution and deionized water were sonicated for 1 min to ensure complete cell dispersion. Then a desired amount of TEOS dissolved in ethanol was added to the previous cell dispersion solution to carry out the silica growth reaction in a conical flask at room temperature under continuous stirring for 72 h. Particles were then collected by centrifugation, and were washed with deionized water and ethanol. The collected particles were dried in a desiccator containing silica gel at room temperature. Finally, the dried particles were calcinated at the desired temperature to remove the biological cells. The standard experimental conditions were [TEOS] = 0.1 M, [NH3] = 0.8 M, [H2O] = 1.0 M, [Cell] = 1.0 g/L ( = 8.2 × 1014 cells/m3), and the total amount of solution was 100 mL. The number of cells in the solution was counted using a Petroff–Hausser counting chamber.
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neous TG/DTA instrument (DTG-60, Shimadzu) to characterize the thermal evolution of the synthesized particles. The heating rate was 10 °C/min under an air atmosphere. 3. Results and discussion Fig. 1 shows the FESEM images of the biological cells and silica coated core shell particles. Cells were fixed with glutaraldehyde to observe their morphology by FESEM according to our previous work [13]. The synthesized particles have retained the morphology of the biological cells. Silica was smoothly coated to the surface of the bacterial cells. The particle size was ca 1.7 × 0.8 μm. This result indicates that the formation of a silica shell on the surface of the bacterial cells was clearly defined. The majority of bacterial cells are generally negatively charged because of the dissociations of the carboxyl group and phosphate group at the bacterial surface. Since the silicate species are negatively charged under this synthetic condition, the electrostatic repulsive force acts between bacteria and silica species. However, since the ammonia added as a catalyst is positively charged, ammonia catalyst localizes near the surface of negatively charged bacteria. It has been assumed that heterogeneous nucleation occurred on the bacterial surface and resulted in the formation of the smoothed surface of silica shell. TG/DTA analysis was carried out to determine the calcining temperature to remove the biological cells. Fig. 2 shows the TG/DTA curves of the synthesized particles. The TG/DTA result of the synthesized particles reveals that three different peaks were detected in the DTA curve. An endothermic peak was observed at 60 °C because of the desorption of adsorbed moisture and the evaporation of the moisture remaining inside the biological cells. This result was supported by the TG curve which shows a weight loss of ca 6%. Two exothermic peaks at 367 °C and 539 °C are because of the decomposition of organic matter composing the body of bacteria. A consecutive weight loss of ca 49% from 200 °C to 600 °C was accompanied by the previous two exothermic peaks corresponding to the burning of organic matter. No reduction in weight was observed in the TG curve above 600 °C. Therefore, heat treatment at 600 °C is required to remove the templates completely. Fig. 3 shows the FESEM images of the calcined particles and their cross-sectional view. The calcined particles removed the template were prepared by calcining the synthesized particles at 600 °C. The calcined particles have retained the morphology of the biological cells even though the template was removed. It was confirmed that the
Fig. 1. FESEM images of (a) the biological cells (E. coli) used as a template, and (b) the synthesized particles prepared by the standard experimental conditions.
2.3. Characterization The morphology of the obtained particles was observed using a field emission scanning electron microscope (FESEM) (JSM-6700F, JEOL) in high-vacuum mode at 10 kV. The differential thermal analysis (DTA) and thermogravimetry (TG) were performed using a simulta-
Fig. 2. TG/DTA curves of the synthesized particles prepared by the standard experimental conditions.
Fig. 3. FESEM images of (a) the calcined particles, and (b) the cross-sectional view of the calcined particle. The calcined particles were heated at 600 °C for 3 h under an air atmosphere.
T. Nomura et al. / Materials Letters 62 (2008) 3727–3729
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crinched particles were observed when a vacuum drying pretreatment was used. In order to fabricate the hollow structure completely, the pretreatment to remove the template must be taken into consideration. Fig. 4 shows the FESEM images of the calcined particles when the concentrations of TEOS or biological cells were decreased. In Fig. 4a, a part of the hollow structure of the calcined particles has been collapsed. When the concentration of TEOS as a source of silica further decreased, many collapsed particles were observed in Fig. 4b. The majority of the calcined particles have not kept the shape of the biological cells. The reasons for this may be because the thickness of the silica shell became gradually thinner with a decrease in TEOS. As a result, the silica did not completely coat onto the bacterial surface due to the lack of a silica source. By contrast, the new silica nanoparticles were generated when the concentration of biological cells decreases (Fig. 4c). Silicic acid monomers generated in the liquid phase by the hydrolysis of TEOS cannot be adequately consumed by the heterogeneous nucleation on the bacterial surface because of the lack of surface area on the templates. This result leads to the generation of silica nanoparticles by homogeneous nucleation in the liquid phase. Therefore, we have to consider the relative importance of homogeneous and heterogeneous nucleation to suppress the formation of silica nanoparticles [14,15]. To fabricate the hollow structure appropriately it is necessary to enlarge the surface area of the bacterial template to act as a reaction field due to the high concentration of the bacterial cells. However, if the surface area is too large, the hollow structure of the particle will collapse due to the thinness of the silica shell.
4. Conclusions Silica hollow particles were prepared using a gram-negative bacterium, E. coli, as a biological template. Silica hollow particles were synthesized by the hydrolysis of TEOS in the presence of ammonia following the Stöber process. The generated particles were calcined at 600 °C to remove the template and then were observed by FESEM. The calcinated particles have retained the morphology of the biological cells. In order to fabricate the hollow structure appropriately, the concentration of TEOS or bacterial cells must be taken into consideration. References [1] Mathiowitz E, Jacob JS, Jong YS, Carino GP, Chickering DE, Chaturvedi P, et al. Nature 1997;386:410–4. [2] Marinakos SM, Novak JP, Brousseau III LC, House AB, Edeki EM, Feldhaus JC, et al. J Am Chem Soc 1999;121:8518–24. [3] Kim SW, Kim M, Lee WY, Hyeon T. J Am Chem Soc 2002;124:7642–3. [4] Im SH, Jeong U, Xia Y. Nat Mater 2005;4:671–5. [5] Fuji M, Takai C, Tarutani Y, Takei T, Takahashi M. Adv Powder Technol 2007;18:81–91. [6] Fujiwara M, Shiokawa K, Tanaka Y, Nakahara Y. Chem Mater 2004;16:5420–6. [7] Roy P, Bertrand G, Coddet C. Powder Technol 2005;157:20–6. [8] Nagamine S, Sugioka A, Konishi Y. Mater Lett 2007;61:444–7. [9] Chung YS, Lim JS, Park SB, Okuyama K. J Chem Eng Jpn 2004;37:1099–104. [10] Zhou H, Fan T, Zhang D, Guo Q, Ogawa H. Chem Mater 2007;19:2144–6. [11] Zhou H, Fan T, Zhang D. Microporous Mesoporous Mater 2007;100:322–7. [12] Zhang Y, Shi EW, Chen ZZ, Xiao B. Mater Lett 2008;62:1435–7. [13] Nomura T, Yoshihara A, Nagao T, Tokumoto H, Konishi Y. Adv Powder Technol 2007;18:489–501. [14] Nomura T, Alonso M, Kousaka Y, Tanaka K. J Colloid Interface Sci 1998;203:170–6. [15] Kousaka Y, Nomura T, Alonso M. Adv Powder Technol 2001;12:291–309.
Fig. 4. FESEM images of the calcined particles prepared by (a) [TEOS] = 10 mM, (b) [TEOS] = 2 mM, and (c) [Cell] = 0.2 g/L. The calcined particles were heated at 600 °C for 3 h under an air atmosphere. coating of silica onto the cells had succeeded. The thickness of the silica hollow particles was ca 70 nm as shown in Fig. 3b. In addition, when no drying pretreatment in the desiccator was employed, the punctured particles were observed. By contrast, the
Contents lists available at ScienceDirect
Materials Letters j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m a t l e t
Fabrication of silica hollow particles using Escherichia coli as a template T. Nomura ⁎, Y. Morimoto, H. Tokumoto, Y. Konishi Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
A R T I C L E
I N F O
Article history: Received 5 March 2008 Accepted 10 April 2008 Available online 16 April 2008 Keywords: Hollow particle Escherichia coli coli Escherichia Template Silica
A B S T R A C T Inorganic hollow particles have attracted great interest in recent years due to their unique physico-chemical properties, as well as their potential use in various applications. In this study, we attempted to fabricate hollow particles using a gram-negative bacterium, Escherichia coli, as a biological template. Silica was synthesized by the hydrolysis of tetraethyl orthosilicate (TEOS) in the presence of ammonia following the Stöber process. Silica was smoothly coated to the surface of the bacterial cells. The bacterial template could be removed by calcining at 600 °C. Interestingly, the calcinated particles retained the morphology of the biological cells even though the template was removed. This result indicates that this bacteria template technique enables the synthesis of hollow spheres, rods, wires, and other three-dimensional structures which retain the morphology of the original bacterium. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Inorganic hollow particles have attracted considerable attention because of their conspicuous properties of density, specific surface area, thermal insulation, and optical activity that differ significantly from those of dense particles. These hollow particles are used in a wide number of applications, such as drug delivery systems, bioencapsulations, and catalysts [1–3]. Various methods have been employed to prepare hollow particles such as a polymer bead template method [4], an inorganic template method [5], a sol–gel method used for surfactant stabilized emulsions [6], spray drying [7], a spray precipitation method [8], and spray pyrolysis [9]. Recently, bacterial cells have been used as a template to fabricate hollow structures [10–12]. The morphology of bacteria can be spherical (cocci), rod-shape (bacilli), curved rod-shape (vibrio), spiral-shaped (spirilla), or tightly coiled (spirochetes). They can exist as single, chained or clumped cells. This wide variety of shapes can be exploited for use as a template for the synthesis of hollow particles or for the construction of three-dimensional hollow structures. Bacteria can be divided into gram-positive and gramnegative bacteria based on the chemical and physical properties of their cell walls. To date, bacteria used as templates have been mainly gram-positive bacteria because the structure of their solid cell walls lends itself to rigid structure preparation. If gram-negative bacteria can be easily used as templates, the applicability of the technique for employing the well-defined morphology of bacteria will become much greater. In this study, the potential of a gram-negative bacterium, Escherichia coli, as a template for silica hollow microparticles was examined. In addition, the synthetic conditions for fabricating the complete ⁎ Corresponding author. Tel.: +81 72 254 9300; fax: +81 72 254 9911. E-mail address: [email protected] (T. Nomura). 0167-577X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2008.04.035
hollow structure were investigated by varying both the concentrations of TEOS as the silica source and biological cells as the template. 2. Experimental 2.1. Materials The analytical grade of tetraethyl orthosilicate (TEOS), 2 mol/L ammonia ethanol solution, and ethanol were obtained from Wako Co. Ltd., Japan. All the chemicals were used without further purification. 2.2. Preparation E. coli (KP7600) was kindly provided by the National BioResource Project (NIG, Japan): E. coli. E. coli was grown at 37 °C in LB medium with shaking. Cells were harvested in late exponential growth phase by centrifugation and washed in triplicate using a sterile NaCl aqueous solution. Silica was directly coated onto the surface of bacterial cells to form core shell particles by the Stöber method. Bacterial cells, ammonia ethanol solution and deionized water were sonicated for 1 min to ensure complete cell dispersion. Then a desired amount of TEOS dissolved in ethanol was added to the previous cell dispersion solution to carry out the silica growth reaction in a conical flask at room temperature under continuous stirring for 72 h. Particles were then collected by centrifugation, and were washed with deionized water and ethanol. The collected particles were dried in a desiccator containing silica gel at room temperature. Finally, the dried particles were calcinated at the desired temperature to remove the biological cells. The standard experimental conditions were [TEOS] = 0.1 M, [NH3] = 0.8 M, [H2O] = 1.0 M, [Cell] = 1.0 g/L ( = 8.2 × 1014 cells/m3), and the total amount of solution was 100 mL. The number of cells in the solution was counted using a Petroff–Hausser counting chamber.
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neous TG/DTA instrument (DTG-60, Shimadzu) to characterize the thermal evolution of the synthesized particles. The heating rate was 10 °C/min under an air atmosphere. 3. Results and discussion Fig. 1 shows the FESEM images of the biological cells and silica coated core shell particles. Cells were fixed with glutaraldehyde to observe their morphology by FESEM according to our previous work [13]. The synthesized particles have retained the morphology of the biological cells. Silica was smoothly coated to the surface of the bacterial cells. The particle size was ca 1.7 × 0.8 μm. This result indicates that the formation of a silica shell on the surface of the bacterial cells was clearly defined. The majority of bacterial cells are generally negatively charged because of the dissociations of the carboxyl group and phosphate group at the bacterial surface. Since the silicate species are negatively charged under this synthetic condition, the electrostatic repulsive force acts between bacteria and silica species. However, since the ammonia added as a catalyst is positively charged, ammonia catalyst localizes near the surface of negatively charged bacteria. It has been assumed that heterogeneous nucleation occurred on the bacterial surface and resulted in the formation of the smoothed surface of silica shell. TG/DTA analysis was carried out to determine the calcining temperature to remove the biological cells. Fig. 2 shows the TG/DTA curves of the synthesized particles. The TG/DTA result of the synthesized particles reveals that three different peaks were detected in the DTA curve. An endothermic peak was observed at 60 °C because of the desorption of adsorbed moisture and the evaporation of the moisture remaining inside the biological cells. This result was supported by the TG curve which shows a weight loss of ca 6%. Two exothermic peaks at 367 °C and 539 °C are because of the decomposition of organic matter composing the body of bacteria. A consecutive weight loss of ca 49% from 200 °C to 600 °C was accompanied by the previous two exothermic peaks corresponding to the burning of organic matter. No reduction in weight was observed in the TG curve above 600 °C. Therefore, heat treatment at 600 °C is required to remove the templates completely. Fig. 3 shows the FESEM images of the calcined particles and their cross-sectional view. The calcined particles removed the template were prepared by calcining the synthesized particles at 600 °C. The calcined particles have retained the morphology of the biological cells even though the template was removed. It was confirmed that the
Fig. 1. FESEM images of (a) the biological cells (E. coli) used as a template, and (b) the synthesized particles prepared by the standard experimental conditions.
2.3. Characterization The morphology of the obtained particles was observed using a field emission scanning electron microscope (FESEM) (JSM-6700F, JEOL) in high-vacuum mode at 10 kV. The differential thermal analysis (DTA) and thermogravimetry (TG) were performed using a simulta-
Fig. 2. TG/DTA curves of the synthesized particles prepared by the standard experimental conditions.
Fig. 3. FESEM images of (a) the calcined particles, and (b) the cross-sectional view of the calcined particle. The calcined particles were heated at 600 °C for 3 h under an air atmosphere.
T. Nomura et al. / Materials Letters 62 (2008) 3727–3729
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crinched particles were observed when a vacuum drying pretreatment was used. In order to fabricate the hollow structure completely, the pretreatment to remove the template must be taken into consideration. Fig. 4 shows the FESEM images of the calcined particles when the concentrations of TEOS or biological cells were decreased. In Fig. 4a, a part of the hollow structure of the calcined particles has been collapsed. When the concentration of TEOS as a source of silica further decreased, many collapsed particles were observed in Fig. 4b. The majority of the calcined particles have not kept the shape of the biological cells. The reasons for this may be because the thickness of the silica shell became gradually thinner with a decrease in TEOS. As a result, the silica did not completely coat onto the bacterial surface due to the lack of a silica source. By contrast, the new silica nanoparticles were generated when the concentration of biological cells decreases (Fig. 4c). Silicic acid monomers generated in the liquid phase by the hydrolysis of TEOS cannot be adequately consumed by the heterogeneous nucleation on the bacterial surface because of the lack of surface area on the templates. This result leads to the generation of silica nanoparticles by homogeneous nucleation in the liquid phase. Therefore, we have to consider the relative importance of homogeneous and heterogeneous nucleation to suppress the formation of silica nanoparticles [14,15]. To fabricate the hollow structure appropriately it is necessary to enlarge the surface area of the bacterial template to act as a reaction field due to the high concentration of the bacterial cells. However, if the surface area is too large, the hollow structure of the particle will collapse due to the thinness of the silica shell.
4. Conclusions Silica hollow particles were prepared using a gram-negative bacterium, E. coli, as a biological template. Silica hollow particles were synthesized by the hydrolysis of TEOS in the presence of ammonia following the Stöber process. The generated particles were calcined at 600 °C to remove the template and then were observed by FESEM. The calcinated particles have retained the morphology of the biological cells. In order to fabricate the hollow structure appropriately, the concentration of TEOS or bacterial cells must be taken into consideration. References [1] Mathiowitz E, Jacob JS, Jong YS, Carino GP, Chickering DE, Chaturvedi P, et al. Nature 1997;386:410–4. [2] Marinakos SM, Novak JP, Brousseau III LC, House AB, Edeki EM, Feldhaus JC, et al. J Am Chem Soc 1999;121:8518–24. [3] Kim SW, Kim M, Lee WY, Hyeon T. J Am Chem Soc 2002;124:7642–3. [4] Im SH, Jeong U, Xia Y. Nat Mater 2005;4:671–5. [5] Fuji M, Takai C, Tarutani Y, Takei T, Takahashi M. Adv Powder Technol 2007;18:81–91. [6] Fujiwara M, Shiokawa K, Tanaka Y, Nakahara Y. Chem Mater 2004;16:5420–6. [7] Roy P, Bertrand G, Coddet C. Powder Technol 2005;157:20–6. [8] Nagamine S, Sugioka A, Konishi Y. Mater Lett 2007;61:444–7. [9] Chung YS, Lim JS, Park SB, Okuyama K. J Chem Eng Jpn 2004;37:1099–104. [10] Zhou H, Fan T, Zhang D, Guo Q, Ogawa H. Chem Mater 2007;19:2144–6. [11] Zhou H, Fan T, Zhang D. Microporous Mesoporous Mater 2007;100:322–7. [12] Zhang Y, Shi EW, Chen ZZ, Xiao B. Mater Lett 2008;62:1435–7. [13] Nomura T, Yoshihara A, Nagao T, Tokumoto H, Konishi Y. Adv Powder Technol 2007;18:489–501. [14] Nomura T, Alonso M, Kousaka Y, Tanaka K. J Colloid Interface Sci 1998;203:170–6. [15] Kousaka Y, Nomura T, Alonso M. Adv Powder Technol 2001;12:291–309.
Fig. 4. FESEM images of the calcined particles prepared by (a) [TEOS] = 10 mM, (b) [TEOS] = 2 mM, and (c) [Cell] = 0.2 g/L. The calcined particles were heated at 600 °C for 3 h under an air atmosphere. coating of silica onto the cells had succeeded. The thickness of the silica hollow particles was ca 70 nm as shown in Fig. 3b. In addition, when no drying pretreatment in the desiccator was employed, the punctured particles were observed. By contrast, the