Hydrothermal synthesis of ZnO hollow spheres using spherobacterium as biotemplates

Hydrothermal synthesis of ZnO hollow spheres using spherobacterium as biotemplates

Microporous and Mesoporous Materials 100 (2007) 322–327 www.elsevier.com/locate/micromeso Hydrothermal synthesis of ZnO hollow spheres using spheroba...

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Microporous and Mesoporous Materials 100 (2007) 322–327 www.elsevier.com/locate/micromeso

Hydrothermal synthesis of ZnO hollow spheres using spherobacterium as biotemplates Han Zhou, Tongxiang Fan, Di Zhang

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State Key Lab of Metal Matrix Composites, Shanghai Jiaotong University, Shanghai 200030, PR China Received 20 June 2006; received in revised form 26 October 2006; accepted 14 November 2006 Available online 2 January 2007

Abstract By using spherobacterium Streptococcus thermophilus as a natural biotemplate, ZnO hollow spheres have been synthesized in a simple hydrothermal method based on the surface biofunctionality of the microorganism, followed by calcination. The as-obtained products are characterized by techniques of XRD, FESEM, TEM and N2 adsorption. Furthermore, a possible formation mechanism involving a twostep encapsulation process is proposed which has an effect on the bimodal pore structure of the products with pores in the mesoporous range. The research introduces a new concept to synthesize porous hollow spheres by using spherobacterium as a biological template and opens up a new pathway to synthesize hollow nanospheres, nanotubes and other kinds of 3D nanostructures with bacterium as the template via simple chemical routes. Ó 2006 Elsevier Inc. All rights reserved. Keywords: ZnO; Bacteria; Template; Hollow spheres; Porous

1. Introduction In recent years, inorganic hollow spheres with nanometer-to-micrometer dimensions have attracted great interests because of their various applications in catalysis, drug delivery, battery materials or photonic crystals [1–4]. The template-directed method is the most commonly used method for the preparation of hollow spheres. To date, a series of inorganic hollow spheres have been prepared by employing silica, PS and carbonaceous polysaccharide microspheres as the templates [5]. In spite of these successes, there still exist some problems to be overcome. One is that the processes usually require a core surface modification to ensure successful coating of shell substances (as are typically required for polystyrene microspheres) which is fussy and complicated. For instance, many oxide hollow spheres prepared by templating of PS latices have to deposit several layers of polyelectrolyte film

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Corresponding author. Tel.: +86 21 62932122; fax: +86 21 62822012. E-mail address: [email protected] (D. Zhang).

1387-1811/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.micromeso.2006.11.020

on it to modify the surface [6]. Moreover, traditional template-directed method always requires several steps to synthesize templates first, which is time-consuming, high cost and probably ‘‘environment-unfriendly’’ [7]. Thus, establishing a general method using inexpensive, efficient and ‘‘environment-friendly’’ template to synthesize various hollow spheres is highly appealing. Both living and dead cells of microorganisms are capable of directing the synthesis and assembly of crystalline inorganic materials because of their active biomolecules on the cell walls [8–12]. Living cells retain all of their biofunctionality whereas dead cells may still retain active biomolecules with functional groups. Microorganisms including bacterial superstructures [8], virus [9–11] and yeast [12] have been used as templates to direct the deposition, assembly, and patterning of inorganic nanoparticles and microstructures. Consequently, spherobacterium is a good candidate as the biotemplate to prepare hollow spheres with some distinct advantages over other methods: firstly, since the abundant functional groups on the cell walls are inherited from the bacteria which are able to bind metal cations or polar molecules through coordination or

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electrostatic interactions, no surface modification or activation steps are required, reducing the number of processing steps. Additionally, bacterium is abundant in nature and can be easily obtained in large amounts in a short time with no environment pollution. Comparing with other templates, bacterium is economical, ‘‘environment-friendly’’, safe and timesaving. Furthermore, this bacteria-templating method could be extended to the preparation of various hollow structures since bacterium possesses a variety of well-defined morphologies controlled at the micro- or even nanoscopic level such as cocci, bacillus and spirillum. Etc. all of which serving as templates can lead to the formation of corresponding hollow structures (e.g. hollow nanorods, nanotubes, nanohelixs, nanocables, diplo-spheres, chain spheres and other kinds of 3D nanostructures) while other biotemplates usually lack the diversity of morphologies. This would open up possibilities for extensive study of the physical and chemical properties of these hollow structures and extend their application potentials in industrial catalysis, separation technology, environmental protection, electrochemistry, membranes, sensors, optical devices, etc. Here, we report a simple hydrothermal method to prepare ZnO hollow spheres using spherobacterium Streptococcus thermophilus as a biological template based on the interaction between the inherent functional groups on the cell walls and the reactants. A possible formation mechanism involving a two-step encapsulation process is proposed which has an effect on the porosity of the products. This bacteria-templating method provides an economical, green, and convenient strategy comparing with traditional template-directed method, which may open up a new pathway to synthesize porous hollow inorganic spheres in a facile bio-assisted method.

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5.0 kV (Acc. V), and the spot size (SE) was 3.0; the work distance (WD) was 13.5 mm. Transmission electron microscopy (TEM) measurements were performed on a JEM-2010 transmission electron microscope operated at an accelerating voltage of 200 kV. Fourier-transform infrared (FTIR) spectroscopy measurements were recorded on a Nicolet NEXUS-670 instrument. Nitrogen adsorption– desorption isotherms were measured with a Micromeritics ASAP 2010 adsorption analyzer (Micromeritics Instrument Corp., Norcross, GA). 3. Results

2. Experimental

Fig. 1 displays the X-ray diffraction patterns of the as-prepared samples before and after calcination. The XRD pattern of bacteria/ZnO core-shell spheres without calcination in Fig. 1a is similar to that of calcined at 600 °C in Fig. 1b except for lower intensity of the diffraction peaks, confirming that the shells of the two samples are composed of wurtzite ZnO (JCPDS card No. 36˚ , c = 5.206 A ˚ ) nanocrystals. 1451, a = 3.249 A The morphology and structure of the samples are further investigated by field emission scanning electron microscopy (FESEM). Fig. 2a indicates that the original morphology of Str. theromophilus is approximately spherical with the diameter varying between 0.5 lm and 0.9 lm. As shown in Fig. 2b, the as-obtained bacteria/ZnO is spherical with diameters ranging from 1.2 lm to 1.5 lm. The thickness of ZnO shell is estimated to be about 200– 400 nm. The spheres with approximately the same size assemble into close-packed arrays in short-range as seen in Fig. 2c. Careful observation shows that the surfaces of these spheres are constructed by nanoparticles with diameters ranging from 20 nm to 40 nm as shown in a magnified image in Fig. 2d. Fig. 2e is the FESEM image of the ZnO hollow spheres after the removal of bacteria templates by

Lactobacillus powder of Streptococcus thermophilus (Str. theromophilus) were provided by Beijing Zhuanger Company. Zinc acetate, triethanoiamine, ammonia, ethanol were provided by Shanghai Chemical Company. In a typical procedure, 1 g Lactobacillus powder of about 109 Str. theromophilus were dissolved in 20 ml distilled water, then 0.2 g Zinc acetate and 4 ml triethanolamine were added to the solution simultaneously. The pH of the system was adjusted to 9 by ammonia. The system was then continuously magnetic stirred at 90 °C for 3 h, and aged. The resulting mixture was centrifuged and washed with distilled water and ethanol for several times, then dried in vacuum at 60 °C. Finally, the powders were calcined at 600 °C and cooled to room temperature naturally in air. X-ray diffraction (XRD) measurements were carried out on a Bruker-AXS D8 Advance instrument operating at a voltage of 40 kV and a current of 40 mA with Cu Ka radi˚ ). Field-emission scanning electron ation (k = 1.5406 A microscopy (FESEM, FEI Sirion 200) was operated under

Fig. 1. XRD patterns of (a) bacteria/ZnO core-shell spheres before calcination. (b) ZnO hollow spheres after calcination at 600 °C.

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Fig. 2. FESEM images of (a) original Str. theromophilus template, with the inset of higher magnification. (b–d) Bacteria/ZnO core-shell spheres observed under different magnifications. (e) ZnO hollow spheres after removal of bacteria templates by calcination at 600 °C, with the inset of an individual broken hollow sphere.

calcination at 600 °C, showing the conservation of the spherical shape. The cracked spheres with apparent cavity demonstrate the hollow nature of the products. The hollow structure of the as-prepared ZnO spheres is further confirmed by transmission electron microscopy (TEM) equipped with electron diffraction (ED). Fig. 3a

reveals that the bacteria/ZnO spheres are spherical with the diameter between 1.2 lm and 1.5 lm; some cells are at the stage of cell division as shown in the inset of Fig. 3a. Fig. 3b indicates ZnO hollow spheres after the removal of bacteria template, the pale center together with the dark edge is the evidence of the hollow structure of the

Fig. 3. TEM images of (a) bacteria/ZnO core-shell spheres. Inset shows some cells are at the stage of cell division. (b) ZnO hollow spheres after calcination. Inset shows the corresponding ED pattern. (c) An individual ZnO hollow sphere. (d and e) Broken ZnO hollow spheres. Inset of (d) is the higher magnification image of the pane part showing the crystalline nature of the ZnO nanoparticles. The scale bar of inset in (a) is 1 lm.

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microsphere. After calcination the hollow structures are still retained, and there is no collapse in the spherical symmetry. The electron diffraction pattern of the hollow ZnO spheres shows diffuse rings, indicating the ZnO spheres are polycrystalline. Fig. 3c shows an individual ZnO hollow sphere with the shell thickness of about 50 nm. Some hollow nanospheres are broken, and the fragments of the cracked one are present in Fig. 3d and e, further confirming the hollow structure. The higher magnification image shows that the size of nanoparticles is 30–40 nm. 4. Discussion The mechanism for the formation of bacteria/ZnO coreshell spheres via a two-step encapsulation process is proposed in Scheme 1. The cell wall of Str. theromophilus and many gram-positive bacteria is primarily made up of peptidoglycan (PG), which is a polymer of N-acelglucosamine and N-acetylmuramic acid. The two other important constituents are teichoic acid and teichuronic acid [13]. The determined reactive functional groups are essentially carboxyls, amines, hydroxyls and phosphoryls which are able to bind metal anions through coordination or electrostatic interactions. Carboxylic and phosphatic groups act as metal binding sites below pH of 5 [14]. At pH > 8, the basic „NH+ is the only and dominant site absorbing Zn [15], which is consistent with the IR spectrum studies in Fig. 4. The spectrum of the biomass treated with acetone to dissolve the intracellular matter displays a shoulder peak at 1656 cm 1 corresponding to the [email protected] stretching vibration of the amide I band of proteins. On contacting Zn2+, the biomass exhibits spectrum with a clear shift from 1656 cm 1 to 1637 cm 1 due to the interaction of Zn2+ with the nitrogen of the acidamide group. Another change can be observed at the peak attributed to the N–H stretching vibration shifting from 3406 cm 1 to 3429 cm 1due to the interaction of Zn2+ with the nitrogen of the amidogroup. In our experiment, the basic „NH+ site is the only

Fig. 4. FTIR spectrum of (a) original template treated with acetone to dissolve the intracellular matter. (b) Original template treated with Zn2+ at the pH of 9 without triethanolamine: mmax/cm 1 3406 (NH), 1656 (CO), 1531 (CN), and 1390 (COOH).

and dominant Zn sorbent, though the absorbed Zn is little. These functional groups are also active sites since triethanolamine molecules can be absorbed onto the cell walls and further modify the bacterial surface. In our experiment, there is neglectable Zn absorbed on the cell walls as shown in the IR spectrum. Furthermore, once we replaced triethanolamine with NaOH, there was no ZnO nanoparticle observed on the cell walls. So, the functional groups are mainly combined with triethanolamine molecules by the covalence of –COOET, –OET, and „POET as shown in Scheme 1, though the exact bonding mechanism is quite difficult to know. The whole encapsulation process can be defined as a two-step encapsulation process. In the first-step, triethanolamine molecules are absorbed onto the cell walls combining with functional groups of carboxyls, hydroxyls and

Scheme 1. Schematic illustration of bacteria/ZnO core-shell spheres formation via a two-step encapsulation process. (TEOH represents triethanolamine, N(CH2CH2OH)3).

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Fig. 5. (a) FESEM image of bacteria/ZnO spheres indicating the two-step encapsulation process, respectively. (b) TEM image of an individual ZnO hollow sphere indicating three parts of the sphere.

phosphoryl of the cell walls, and further modify the bacterial surface. Zn(AC)2 in the solution will react with triethanolamine covered on the cell walls to form the first rough ZnO encapsulation layer. Simultaneously, TEOH functions as a base which will react with Zn(AC)2 to produce ZnO molecular nanoclusters. In the second-step, ZnO clusters formed are inclined to grow by rapidly colliding with other ZnO clusters according to the ripening and aggregation theory [16]. Additionally, the first-encapsulation products have a larger surface area than a single ZnO cluster, which means ZnO cluster formed on the cell walls has a larger efficiency and probability to collide with other clusters in an appropriate concentration. As a result, ZnO covers the cell walls of bacteria. As shown in Fig. 5a, two obvious encapsulation steps could be clearly identified. The A sphere is at the stage of the first-step encapsulation whose layer is rather rough while the B sphere is in the process of the second-step which coincides with the mechanism above exactly. The encapsulated spheres were calcined at 600 °C to remove the bacteria template, resulting in ZnO hollow spheres. As shown in Fig. 5b, a pale circle can be obviously seen which indicates the cell wall trace of the original template. The generation of the pale circle is derived from the first-step encapsulation of TEOH on the cell walls. After calcination, the thin layer of TEOH disappeared and the pale circle retaining the profile of the cell wall formed, which further strengthen the mechanism above. As stated above, we can draw the conclusion that the bacteria have played three important roles in the process. Firstly, it serves as a hard template to direct the morphology of the products and also as a support for the deposition of nanoparticles. Secondly, the abundant functional groups inherited from the cells are active sites to interact with the reactants. Thirdly, the biomolecules on the cell walls exert an influence on restraining the growth of ZnO nanoparticles. The two-step encapsulation process has an effect on the porosity of the products. Fig. 6 gives the nitrogen adsorption–desorption isotherm of the 600 °C calcined sample. It shows type IV-like isotherm, indicating the presence of mesoporosity in the hollow spheres. The Barrett–Joyner–

Fig. 6. Nitrogen adsorption–desorption isotherms and the corresponding pore size distribution (inset) for the ZnO hollow spheres.

Halenda (BJH) method used to calculate the pore size distribution (inset) indicates that the as-synthesized samples have a bimodal pore structure due to the two-step encapsulation process: smaller pores with a diameter of 2.5 nm and bigger pores of approximately 11 nm in diameter. The 2.5 nm pores most probably are the voids that which are left between the clustered particles via the second-step encapsulation and the bigger pores are assigned to the pore structure of the inner parts assigned to the first-step one. 5. Conclusions In conclusion, ZnO hollow spheres have been synthesized in a simple hydrothermal method by using spherobacterium Str. thermophilus as a green, economical and easily obtained biotemplate based on the interaction between the inherent functional groups on the cell walls and the reactants. The possible formation mechanism involving a two-step encapsulation process which has an effect on the bimodal pore structure of the products with pores in the mesoporous range has been investigated. These hollow

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spheres are likely to find applications in adsorption, gas sensors, optical devices and photonic crystals. This biotemplating strategy is generally extendable to the synthesis of other inorganic hollow spheres. Moreover, bacterium of various shapes such as cocci, bacillus and spirillum acting as templates can lead to the formation of various porous hollow structures (e.g. hollow nanotubes, nanohelixs, diplo-spheres, chain spheres and other kinds of 3D nanostructures) in a relative simple way to general chemistry methods and this would open up possibilities for extensive study of the physical and chemical properties of these hollow structures and extend their application potentials. Acknowledgments This work is supported by National Natural Science Foundation of China (No. 50371055), Major Project on Basic research of Shanghai (No. 04DZ14002), Fok Ying Tung Education Fund (No. 94010), Program for New Century Excellent Talents in University (NCET-04-0387), Nano-research Program of Shanghai (No. 0452nm045 and No. 05nm05020) and National Basic Research Program of China (No. 2006CB601200). References [1] Z.Q. Li, Y. Ding, Y.J. Xiong, Q. Yang, Y. Xie, Chem. Commun. (2005) 918.

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