CRYSTAL GROWTH & DESIGN
Hollow SiO2 Microspheres Produced by Coating Yeast Cells Daniel Weinzierl,*,† Anke Lind,† and Werner Kunz‡ Papiertechnische Stiftung Mu¨nchen, Heβstraβe 134, D-80798 Mu¨nchen, Germany, and Institute of Physical and Theoretical Chemistry, UniVersity of Regensburg, D-93040 Regensburg, Germany
2009 VOL. 9, NO. 5 2318–2323
ReceiVed October 14, 2008; ReVised Manuscript ReceiVed February 16, 2009
ABSTRACT: Hollow silica microspheres were produced by coating yeast cells with amorphous SiO2. In the precipitation reaction, yeast cells of Saccharomyces cereVisiae were used as a biological template. The silica shell was synthesized by the hydrolysis of tetraethylorthosilicate (TEOS) in water-alcohol mixtures as solvent using ammonia as a catalyst according to the Stoeber process. The hollow microspheres were characterized by means of scanning electron microscopy (SEM) and thermogravimetric analysis (TGA). Both smooth and textured coatings were prepared. The biological template can be removed by calcining, after which the particle morphology persists. Additionally, density and light scattering coefficients of the pigment were measured. Introduction In recent years the synthesis of hollow microspheres with well-defined structures has attracted interest,1-5 since these materials have potential applications in catalysis, chromatography, controlled release of drugs, dyes, perfumes, etc., development of artificial cells, waste removal, and protection of biologically active agents such as proteins and enzymes.6-14 Furthermore, hollow particles exhibit advantages over their solid counterparts due to lower densities, higher surface area, and unique optical properties15 and are thus used as fillers or pigments. Many methods have been developed for the synthesis of hollow mineral structures, such as nozzle reactor approaches (spray-drying or pyrolysis),16-18 emulsion/phase extraction techniques,19-25 and self-assembly processes including layerby-layer (LBL) approaches.6,26-31 Self-assembly processes, also known as core-shell techniques, are probably the most effective of all of these approaches to produce inorganic hollow spherical particles. In core-shell processes composite materials with core-shell structure are built by the assembly of the shell material onto the surface of the core template, followed by the removal of the templates by selective dissolution of the core using appropriate solvents or via calcination in air at elevated temperatures. Thus hollow structures are produced with inner diameters determined by the size of the template. Typically, template particles are coated in solution either by controlled surface precipitation of inorganic molecule precursors (such as silica and titania etc.) or by direct surface reactions utilizing specific functional groups on the cores to create core-shell composites. The sacrificial template cores used are mainly organic templates including polystyrene latex spheres,32,33 resin spheres,34 liquid droplets,35 vesicles36,37 or gas bubbles.38-40 Recently Nomura et al.41 have reported the synthesis of hollow particles using Escherichia coli as a biological template. In our experiments yeast cells of Saccharomyces cereVisiae were used as biological templates. Hollow microparticles were produced by controlled surface precipitation using tetraethyl orthosilicate (TEOS) and ammonia in alcoholic solvent according to the Stoeber process. In this innovative biomimetic process, inexpensive yeast cells are used as templates for the formation * Corresponding author. E-mail:
[email protected]. † PTS Mu¨nchen. ‡ Regensburg University.
of hollow particles in contrast to the expensive polymer particles usually used as templates. The hollow particles were also tested for light scattering ability since hollow particles theoretically exhibit higher light scattering coefficients than their solid counterparts. Experimental Procedures Materials. TEOS (tetraethylorthosilicate, purity g99.8%, Wacker Semicosil TES), 25% ammonia solution (purity g99.5%, Merck), methanol (purity g99.8%, Merck), ethanol (purity g99.5%, Merck), and n-propanol (purity g99.5%, Merck) were used without further purification. Deionized water and commercial baker’s yeast in the form of compressed yeast were used throughout the experiments. Coating of Yeast Cells. According to the method used by Royston et al.42 for coating tobacco mosaic virus (TMV) with silica, TEOS was used as silica source. A mixture of alcohol and water was used as a solvent for coating yeast cells with SiO2. Ammonia was used as a catalyst. Alcohol is necessary as cosolvent in this reaction system since TEOS is insoluble in water. Anyway, a certain amount of water is necessary to suspend the yeast cells. Previous to the coating experiments, the stability of yeast cells in alcohol water mixtures was verified by light scattering experiments and scanning electron microcscopy (SEM). In a typical experiment, approximately 30 g of compressed yeast (equal to approximately 10 g oven-dry) were suspended in 60 mL of solvent (alcohol-water mixture). After 5 mL of 25% ammonia as catalyst and 50 mL of TEOS (225.6 mmol) were added, the suspension was agitated at room temperature for several hours. The product was recovered by centrifugation (5 min at 4000 U/min) and washed twice with alcohol following a washing step with demineralized water. Afterward the precipitate was dried at 105 °C to constant weight. The dried samples were characterized by means of SEM, thermogravimetric analysis (TGA), and density measurements. In addition to that, the light scattering coefficient of a coated plastic sheet was determined. Analytical Methods. Scanning Electron Microscopy (SEM). SEM was performed using a microscope (Jeol, JSM 5600) operated at 15 kV and 20 kV, respectively. The samples were coated with Au prior to SEM examination. Light Scattering Experiments (LS). LS measurements were done using a Mastersizer spectrometer (Malvern Instruments, Model Microplus) equipped with a 633 nm helium-neon laser. Thermogravimetric Analysis (TGA). Thermal stability of the coated yeast cells and removal of the biological core was performed using a thermogravimeter (LECO Instrumente GmbH, TGA701). Samples were dried to constant weight at 105 °C (oven-dry) and heated to 900 °C with 1 K/min in an airstream. Density Measurement. The density of coated yeast cells was determined using a pyknometer and demineralized water as a solvent. Prior to weighing all samples were agitated with ultrasound in order to remove gas bubbles. Measurement of Light Scattering Coefficients. The light scattering coefficient of a coating layer was determined according to the theory
10.1021/cg801143e CCC: $40.75 2009 American Chemical Society Published on Web 03/12/2009
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Figure 1. Particle size distribution of yeast cells in a 50% by weight ethanol-water mixture and SEM image obtained after 10 min.
Figure 2. Yeast cells coated with silica at different concentrations of TEOS. The top left panel corresponds to TEOS concentration of 0.56 mol/L, the top right panel to 1.00 mol/L, the bottom left panel to 1.35 mol/L, and the bottom right panel to 1.64 mol/L. of Kubelka and Munk.43 The fabricated pigment samples were suspended in water to a solids content of approximately 25%. A sheet of transparent plastic film was cut into pieces sized 11 cm × 21 cm each, each piece was weighed, and the reflectance was measured against a black background by means of a spectrophotometer (“ELREPHO”, Lorentzen & Wettre) to get the background reflectance Rb. The preweighed pieces of plastic film were then coated with different amounts of the pigment suspension to produce coat weights from 1 to 40 g/m2 using a laboratory hand coater. Each coated piece of plastic film was dried at 60 °C to constant weight and the area of dry coating was standardized to 10 cm × 10 cm by placing a square metal template on the coating and carefully removing the excessive coating from the plastic films. Each piece of coated film was then weighed again, and
the coat weight w was calculated in kg/m2 from the mass difference and the coating area. Each coated piece of film was then tested for luminous reflectance when placed against a black background (R) and on a pile of coated plastic film pieces (R∞). From these measurements the dimensionless light scattering ability Sw was calculated using the formula:
Sw )
(
(1 - RR∞)(R∞ - Rb) 1 ln (1 - RbR∞)(R∞ - R) (1/R ) R ( ∞ ∞)
)
(1)
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Table 1. Reaction Conditions with Different Alcohol Types and Concentrations alcohol methanol ethanol n-propanol
water/ alcohol
yeast (g/mL)
TEOS (mol/L)
NH3 (mol/L)
water/TEOS (mol/mol)
1:2 1:1 2:1 1:2 1:1 2:1 1:2 1:1 2:1
90.4 90.7 90.3 90.0 89.0 90.3 90.5 90.5 90.7
1.96 1.96 1.96 1.96 1.96 1.96 1.96 1.96 1.96
0.58 0.58 0.58 0.58 0.58 0.58 0.58 0.58 0.58
15.0 12.5 10.1 15.0 12.4 10.1 15.0 12.5 10.2
The light scattering ability Sw was then plotted as a function of the coat weight w, and the Kubelka-Munk light scattering coefficient S in m2/kg was calculated as slope of the graph.
Results and Discussion Stability of Yeast Cells in Ethanol-water Mixtures. Previous to the coating experiments the stability of yeast cells in alcohol water mixtures was verified by LS and SEM. A
sample of 10% by weight yeast suspended in an ethanol-water mixture (50% by weight ethanol) was prepared, and the timedependent particle size distribution of yeast cells was measured using LS. Additionally samples were taken at different times for SEM. The particle size distribution of yeast cells in ethanol-water mixtures (see left of Figure 1) changes only slightly during the first 4 min. After that, the particle size distribution does not change at all indicating that yeast cells are stable and not destroyed in ethanol-water mixtures. SEM images obtained after 10 min (see image on the right in Figure 1) support this assumption as well. Influence of TEOS Concentration. The influence of the concentration of TEOS on the morphology of coated yeast cells was investigated. At a constant ethanol-water ratio of 50% by weight ethanol, the concentration of TEOS in the reaction mixture was varied between 0.56 mol/L and 1.64 mol/L, respectively. In the experiments, 30.0 g of compressed baker’s yeast (10.0 g oven-dry) was suspended in 60 mL of solvent (ethanol-water mixture, 50% by weight ethanol). Ten milliliters of ammonia
Figure 3. Coated yeast cells at different methanol/water mixing ratios of 2:1, 1:1, and 1:2 by weight.
Figure 4. Coated yeast cells at different ethanol: water mixing ratios of 2:1, 1:1, and 1:2 by weight.
Figure 5. Coated yeast cells at different n-propanol/water mixing ratios of 2:1, 1:1, and 1:2 by weight.
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Figure 6. Thermogram and SEM image of coated yeast cells after heating to 900 °C.
Figure 7. Light scattering coefficient as a function of TEOS concentration under similar conditions, using ethanol as solvent.
(25% by weight) and a certain amount of TEOS (10-200 mL) were added to the suspension stirring constantly. After 20 h of agitation the yellowish to slightly brownish precipitates were collected, washed, and dried. Figure 2 shows typical SEM images of the as prepared products. As one can see, polymerization of SiO2 preferrably occurs on the cell surface. Electrostatic interaction of charged and polarized groups of polysaccharides, proteins and lipids (amino groups and basic lysin or arginin side chains) of the cell surface and the silic acid molecules in solution may be one
explanation for the selective growth of SiO2 on the cell surface. The electrostatic interaction leads to an oversaturation and heterogeneous nucleation of SiO2 at the cell surface. At low TEOS concentrations, crinkly structures, reminiscent of collapsed under-pressured balls are formed (top left). These structures have been formed during drying, when liquid evaporates from the interior and the surrounding thin SiO2 layer contracts and shrinks. Hollow spherical microspheres form at higher TEOS concentrations (top right). Further increasing TEOS concentration leads to hollow spherical particles with
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Figure 8. Scattering coefficient of pigments produced at constant TEOS concentration and different alcohol: water ratios, using methanol, ethanol, and n-propanol as cosolvent. The red line indicates the light scattering coefficient of amorphous silica.
thicker walls and higher mechanical stability being formed, which is indicated by a smaller fraction of destroyed particles (bottom left). The thickness of the SiO2 coating around the yeast cells is controlled by the TEOS concentration At even higher TEOS concentrations, particles begin to aggregate and no separate particles are formed. Influence of Alcohol Type and Alcohol-Water Ratio. The influence of water content and alcohol type on the precipitation reaction was investigated as well. Solvent mixtures were prepared consisting of water and alcohol (methanol, ethanol, and n-propanol) at the ratios 1:2, 1:1, and 2:1. The experiments performed are summarized in Table 1. Figures 3-5 present SEM images of samples prepared with different solvent mixtures. An increasing surface texture of the particles is found with increasing water contents for methanol (see Figure 3). Smooth particle surfaces are visible at methanol/water ratios of 2:1 and 1:1. At the highest water content particle surfaces are covered by small SiO2 particles. A similar situation is shown for ethanol/water mixtures in Figure 4. Smooth, closed shells have been formed around the yeast cells at ethanol/water mixing ratios of 2:1 and 1:1. Only isolated small particles are visible on the surfaces. A considerably rougher particle surface is visible for an ethanol/water mixture of 1:2 by weight. In the case of n-propanol, the particle surface becomes rougher with decreasing alcohol contents as well. However, even at low water content the particle surface already comprises small SiO2 particles. In summary, the smoothest surface is obtained by using methanol as cosolvent and a low water content. Increasing alcohol chain lengths or water contents lead to rougher surfaces. The size of particles formed in the Stoeber process tends to increase with increasing molecular weights (chain lengths) of the alcohol solvent.44,45 The final particle size of silica particles synthesized with TEOS in different alcoholic solvents but under otherwise comparable conditions is minimum with methanol and maximum with butanol.44 Kinetic studies of the Stoeber process showed that the growth of colloidal particles is controlled by the initial hydrolysis of TEOS and that the ratelimiting step was largely dependent on water concentration.46,47
More reactive silicic acid molecules are present in the reaction mixture due to the increased hydrolysis rate of silicium alkoxide molecules with increaseing water contents. Consequently, larger particles are formed on yeast cell surfaces because of the fast growth of the few nuclei on the yeast cell surface leading to a rougher coating. Themogravimetric Analysis. In order to probe the stability of the SiO2 shell and to remove the yeast cell template inside of the particles, thermogravimetric analysis was performed. Thermal decomposition of yeast biomass within the particles starts at approximately 200 °C (see thermogram, Figure 6). Compared to the thermogram of pure baker’s yeast, decomposition of yeast biomass begins at slightly higher temperatures, which can be explained by the blocking effect of the SiO2 coating. Decomposition of yeast biomass is completed at approximately 500 °C. SEM images obtained after thermal treatment of the coated particles reveale that the particles are not destroyed by heating. Even crinkly shaped particles with thin SiO2 coatings (shown in Figure 2) remain intact. Burning out the yeast cell templates gave a white powder in all experiments. Density. The density of all hollow silica particles tested was between 40% and 50% lower than the density of solid amorphous silica. Light Scattering Coefficient. Figure 7 shows the light scattering coefficient S of pigments produced under similar conditions using ethanol as cosolvent as a function of TEOS concentration. As one can see, S increases with increasing TEOS concentrations up to a maximum, and then falls again. When comparing the S values with the SEM images one can see that S values are low for the crinkly spheres obtained at low TEOS concentrations and aggregated clusters obtained at high TEOS concentration, whereas S values are higher for smooth or textured particles. Figure 8 shows the light scattering coefficient of the as prepared pigments at constant TEOS concentration and different alcohol/water ratios. If one compares the results of the light scattering factor S with the SEM images shown in Figures 3-5, it is obvious that S increases with increasing surface roughness.
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This indicates that surface structure has a great influence on the light scattering of the particles. Hollow particles with rough surfaces show higher light scattering than smooth particles. A surface texture with dimensions in the range of the wavelength of light increases light scattering significantly. All hollow particles, however, have greater light scattering ability than “bulk” amorphous silica, as hollow particles hold additional air-silica interfaces where light scattering and refraction occur, raising the light scattering coefficient of the pigment. Conclusion In summary, hollow silica particles with spherical morphology were successfully synthesized using the Stoeber process and yeast cells as a template. Morphology and surface roughness of the hollow particles can be controlled by the reaction conditions, that is, alcohol, water, and TEOS concentrations, respectively. The smoothest surface is obtained by using methanol as cosolvent at low water content. Increasing alcohol chain lengths or water contents lead to rougher surfaces. All hollow particles exhibit greater light scattering ability than “bulk” amorphous silica due to their hollow structures and surface textures. The hollow silica particles produced by this new innovative biomimetic process using inexpensive yeast cells as a template could be used as fillers or coating pigments in the paper industry, for example. Their high light scattering ability and low density permits the production of lighter paper with greater opacity. Acknowledgment. This study was part of the IGF Zutech 198ZN research project funded within the program of promoting “pre-competitive joint research (IGF)” by the German Federal Ministry of Economics and Technology BMWi and carried out under the umbrella of the German Federation of Industrial Cooperative Research Associations (AiF) in Cologne. We would like to express our warm gratitude for this support.
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