Fabrication of Zinc Oxide Semiconductor Nanoparticles in the

Article pubs.acs.org/crystal

Fabrication of Zinc Oxide Semiconductor Nanoparticles in the Apoferritin Cavity Yoko Suzumoto,† Mitsuhiro Okuda,‡ and Ichiro Yamashita*,‡,§,∥ †

Panasonic Excel Staff Co., Ltd., 612 Suiginya, Shimogyo, Kyoto 600-8411, Japan Advanced Technology Research Laboratories, Panasonic Corporation, 3-4 Hikaridai, Seika, Kyoto 619-0237, Japan § Graduate School of Material Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan ∥ CREST, JST, CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan ‡

ABSTRACT: We developed a liquid phase ZnO nanoparticles (NPs) fabrication method which employed the cage-shaped protein apoferritin as a biotemplate. The suppression of the chemical reaction outside apoferritin by stabilizing Zn2+ with ammonia allowed the selective NP synthesis inside the cavity at room temperature. The composition of synthesized NPs was confirmed to be that of ZnO, and their size was determined to be 5.1 ± 0.8 nm in diameter through energy-dispersive Xray spectroscopy and high-resolution transmission electron microscopy. In-depth studies about the effect of anion species on the NP formation proved that the anion introduction through the channels was competitive and also that the decrease in hydroxide ion (OH−) concentration ratio in the reaction solution led to less ZnO NPs formation.

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INTRODUCTION Zinc oxide (ZnO) is a versatile material studied by many groups because of its remarkable characteristics, such as semiconductivity with a wide band gap of 3.37 eV, a large excitation binding energy (60 meV), and the absorption of ultraviolet light.1 ZnO in the bulk form and thin solid film has been used in various applications, including varistors, phosphors, transparent conducting electrodes, surface acoustic wave devices, piezoelectric transducers, UV protection films, facial powders, and chemical sensors.2−9 ZnO is also attracting researchers’ attention from the environmental point of view and is expected to substitute GaAs and InP for electrode and transistor.10−12 Nowadays, there is a strong demand to produce monodisperse ZnO NPs for applications in nanoelectronics devices which include field-effect transistor (FET) and field emitter devices.13,14 Since the electron energy levels of a ZnO NP are quantized depending on its size, the NPs’ homogeneity is quite important. For this reason, methods for the fabrication of ZnO NPs have been intensively investigated. However, there still remains an important challenge for producing the monodisperse ZnO NPs. We are addressing this issue by means of a biotemplate, the cage-shaped protein, apoferritin. Apoferritin is a spherical protein consisting of 24 polypeptide subunits with a diameter of 12 nm and a 7 nm internal cavity.15 Apoferritin is ubiquitously distributed in living organisms and serves as an iron-storage protein maintaining the homeostasis of iron concentration in cells. Apoferritin in vivo stores iron atoms as a ferrihydrite core. There are eight channels at the 3fold symmetry axis of the protein shell, which consist of negatively charged amino acids (glutamic and aspartic acids). Divalent iron ions, Fe2+, are attracted by the negative charges © 2012 American Chemical Society

on the outer surface surrounding the channels and go through them. In the cavity, Fe2+ ions are condensed and oxidized at negatively charged amino acid areas on the inner surface to form an iron oxide NP.16 There are several reports of the in vitro fabrication of various kinds of NPs in apoferritin, where the cavities worked as chemical reaction vessels with uniform size.17−34 These NP syntheses involved chemically designed reactions. The syntheses of Fe3O4-γ-Fe2O3, MnOOH, CoOOH, CeO2, or Co3O4 typically required the addition of oxidants such as O2 or H2O2.14−18 In the cases of Ca, Ba, In, Ni, and Cr oxoanion compounds, the addition of the oxoanion such as carbonate or phosphate ion was necessary.19−22 The mechanisms of introduction of positively charged ions through the channels were assumed to be the same as that of Fe2+. However, it remains unclear how anions enter the apoferritin cavity. In this context, we precisely designed a slow chemical reaction system (SCRY) which suppressed chemical reactions outside apoferritin and successfully synthesized II−VI compound semiconductor NPs (CdSe, ZnSe, and CdS) in the protein cavity.26−28 The SCRY experiments showed that the introduction of positive metal ions (Zn or Cd) prior to the negative ions (S, Se) is essential for NP syntheses and that suppression of chemical reactions outside apoferritin is indispensable. The results of SCRY experiments suggested that cations should first be condensed at nucleation sites to encourage nucleation which occurs subsequent to the anion introduction. It was also reported that the chemical reaction Received: May 10, 2012 Revised: June 13, 2012 Published: June 18, 2012 4130

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(10000g × 10 min) to remove the bulk precipitates, and then a clear and colorless supernatant solution was recovered. 5. Characterization of ZnO NP inside Apoferritin by Energy Dispersive Spectroscopy (EDS) and High-Resolution TEM (HRTEM). EDS analysis (Hitachi HF-2200) of ZnO NPs synthesized in the standard solution was performed using holey carbon-coated Ti grids. The calibration of EDS measurement was performed by Al Kα (1.49 keV) and Cu Kα (8.05 keV). HR-TEM (Topcon, EM-002B) images of the NPs were taken using the same grid. The acceleration voltage on the TEM is 300 keV.

outside had to be slower than the anion introduction. However, the details have not been clearly understood yet. In addition, it was predicted that it would be difficult to synthesize ZnO due to the anion concentration required for ZnO formation. In fact, an order of 10−3 mol/L anion concentration is expected to be necessary for the synthesis of ZnO with reference to that of CdSe, ZnSe, and CdS. However, the OH− concentration depends on the pH, and if the OH− concentration is fixed as high as 10−3 mol/L (pH 11), most of the protein will be denatured. Therefore, ZnO fabrication in apoferritin is challenging. In this study, we designed a process for ZnO NPs fabrication in the apoferritin cavity based on the SCRY. This is the first report which demonstrates that the monodisperse ZnO NPs were successfully fabricated in the apoferritin cavity by means of SCRY. In addition, introductions of both cation and anion through the ion channels were experimentally investigated.

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RESULTS AND DISCUSSION 1. Concept of the Newly Designed SCRY for the Synthesis of ZnO NPs. The Zn2+ is known to react quickly with OH− under alkaline conditions and form zinc hydroxide (Zn(OH)2), which subsequently dehydrates into ZnO, written as the following equations:

EXPERIMENTAL SECTION

1. Apoferritin Purification. The apoferritin solution (SIGMA, horse spleen apoferritin) was purified by gel filtration chromatography using Sephacryl S-300 columns (GE Healthcare life science, Sephacryl S-300 HR, 2.6 × 60 cm) equilibrated with 100 mmol/L NaCl. The elution flow rate was 1.4 mL/min, and the absorbance was monitored at 280 nm. The fractions containing monomeric apoferritin were collected and condensed using an ultrafilter membrane (Millipore, Centriprep 30). Finally, the obtained solution was stored in 100 mmol/L NaCl. 2. Transmission Electron Microscopy (TEM) and Evaluation of Core Formation Ratio (CFR). The synthesized ZnO NPs were observed by TEM (JEOL, JEM-2200FS), stained with 1.5% (w/v) aurothioglucose, which cannot stain the cavity.25 Carbon-coated Cu grids were used for the TEM observation, and the CFR was calculated by dividing the number of NP-containing apoferritin by the total number of protein. The average NP diameter was determined by averaging the size of 258 ZnO NPs from TEM images. 3. Protein Assay and Evaluation of Yield of Protein Ratio (YPR). The protein concentration in the supernatant solution was measured by the Bradford-assay, and the YPR was calculated by dividing the protein concentration in the supernatant solution by the initial protein concentration. 4. ZnO Mineralization into Apoferritin Cavity. ZnO mineralization experiments were carried out in solutions containing final concentrations of 0.2 mg/mL apoferritin and 50 mmol/L CAPSO− NaOH pH 9.8, varying concentrations of ammonia and zinc nitrate hexahydrate (Zn(NO3)2·6H2O). The final concentrations of Zn(NO3)2 were 3.0, 6.5, and 10.0 mmol/L, and ammonia−water was up to 300 mmol/L. The final pH was fixed at 9.8. We define the solution condition including 6.5 mmol/L Zn(NO3)2 and 150 mmol/L ammonia−water as the “standard solution” or “standard condition” to investigate the effect of each parameter. The time dependence measurements of CFR were performed using the standard solution. To compare solutions of pH 9.8 and pH 8.0, the CAPSO−NaOH in standard solution was replaced with 200 mmol/L HEPES−NaOH. In the investigation of the dependence on anion concentration and the number of valence electrons on the ZnO NPs synthesis, sodium nitrate (NaNO 3) was added as the final concentrations of 50, 100, 150, and 200 mmol/L on each synthesis solution. In the same way, sodium sulfate (Na2SO4) was added to the final concentration of 25, 50, 75, and 100 mmol/L to make negative charges derived from NaNO3 and Na2SO4 equal. As the control experiment, the solution added NaNO3 or Na2SO4 instead of ammonia was prepared (ammonia free experiment). In every experiment, the addition of Zn(NO3)2 was the final step so as to avoid the Zn(OH)2 precipitations being produced in the middle of the fabrication process. The total volume was 1 mL, and the reaction mixture solutions were kept at a constant temperature of 23 °C for 18 h. After 18 h of incubation, the solution was centrifuged

Zn 2 + + 2(OH)− → Zn(OH)2

(1)

Zn(OH)2 → ZnO + H 2O

(2)

These reactions were used for the synthesis of ZnO NPs in apoferritin. Since these two reactions occur both inside and outside apoferritin, the reaction outside should be suppressed to selectively produce a ZnO NP only inside the apoferritin. This system is called SCRY. Figure 1 shows the concept of how the newly designed SCRY scheme works, applied to the synthesis of ZnO NPs.

Figure 1. Schematic for the modified SCRY for the synthesis of ZnO NP inside apoferritin (represented in green): (a) ammnonia stabilized Zn2+ ions are prevented from reacting with OH− so that Zn(OH)2 is not formed outside the cavity; (b) some Zn2+ ions, uncoupled from [Zn(NH3)4]2+ in the vicinity of the 3-fold channels, flow into the apoferritin cavity; (c) the OH− ions subsequently enter the cavity; (d) ZnO NP is synthesized in the cavity through the hydroxylation and dehydration reactions.

First, Zn2+ ions are stabilized by adding an excess of ammonia−water to form [Zn(NH3)4]2+. These ions are stable enough to prevent Zn2+ from reacting with OH−, and then the [Zn(NH3)4]2+ are attracted toward the 3-fold channel area where negatively charged amino acids abound. At this stage, there is a chance for Zn2+, based on the chemical equilibrium, to be uncoupled from the complex and go through the 3-fold channel.35 When Zn2+ ions are introduced in the cavity, they are attracted to the negatively charged amino acid areas on the 4131

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Figure 2. TEM images of ZnO NPs in apoferritin (a) with aurothioglucose staining and (b) without staining. Scale bar is 50 nm. (c) Histogram of the size distribution of NPs. Effects of ammonia and Zn2+ concentrations on (d) the CFR and (e) the YPR.

The CFR peaks could be explained as follows. The addition of Zn2+ into the solution containing ammonia−water sets off two different chemical reactions: eqs 1 and 3

protein inner wall, which are considered to be the nucleation sites. The OH− ions then diffuse into the cavity and react with the highly concentrated Zn2+ much faster at the nucleation sites than outside. Finally, Zn(OH)2 precipitates are produced by hydroxylation according to eq 1 and then an embryo ZnO NP, a ZnO nucleus, is produced by the dehydration reaction (eq 2). 2. Optimization of Ammonia and Zinc Ion Concentration. First, the concentrations of the ammonia and zinc solutions were optimized. Since higher pH provides more OH−, a higher reaction solution pH is better for ZnO synthesis, as long as apoferritin remains stable. On the basis of the preliminary experiments about apoferritin denaturation (losing original shape), we fixed the pH of the solution at 9.8, which would be suitable for ZnO NP synthesis. In the process of ZnO synthesis, Zn(NO3)2 was added last to prevent the reaction between Zn2+ and OH− outside the cavity. After 18 h of incubation, the solutions were observed by TEM and the CFR and the YPR were calculated. Figure 2a shows a TEM image of aurothioglucose-stained ZnO NPs synthesized in apoferritin with standard solution. It shows homogeneous nanodots surrounded by thin white rings with an approximate external diameter of 12 nm. Since it has already been proven that aurothioglucose cannot go through the apoferritin channels and cannot stain the cavity,25 the homogeneous nanodots should be the ZnO NPs formed in the cavity and the white rings surrounding each NP should be the protein shells. Figure 2b is a non-stained TEM image of ZnO NPs which shows clear independent NPs. 285 NPs in the nonstained image were used to determine the average diameter, which was 5.1 ± 0.8 nm (Figure 2c). It is noted that any NPs with more than 8 nm diameter were not confirmed. Parts d and e of Figure 2 show the effects of the ammonia concentrations on the CFR and the YPR, respectively. It is clear that CFR data have maxima without precipitation. In lower ammonia concentrations, considerable amounts of ZnO or Zn(OH)2 bulk precipitates were observed whereas at higher concentrations there were no precipitates.

Zn 2 + + 4NH3 → [Zn(NH3)4 ]2 +

(3)

The ratio of these two chemical reactions depends on Zn2+, OH− and ammonia concentrations. On the basis of the chemical equilibrium constant,36 it was calculated that [Zn(NH3)4]2+ formation accounted for over 90% around all three peak conditions. This explains why no precipitates were observed and that the SCRY scheme worked well. In the cases of lower ammonia concentration, the ammonia molecules were not enough to protect the Zn2+ effectively, which resulted in a considerable amount of Zn(OH)2 bulk precipitation. Zn2+ ions were consumed as Zn(OH)2 bulk precipitates outside the cavity, and the CFR was lowered. Especially in the case of less than or equal to 75 mmol/L of ammonia solution, the Zn(OH)2 bulk precipitation was stronger and the CFR became null. By contrast, higher concentrations of ammonia produced little precipitations and led to the YPR increase; however, they resulted in a lower CFR because Zn2+ ions were overprotected and available Zn2+ ions to form ZnO were too few. The tendency for the CFR maxima to shift toward higher ammonia concentrations when Zn2+ concentration increased is explained the same way. Therefore, there is a suitable ammonia concentration depending on the Zn2+ concentration, where Zn2+ ions are protected appropriately. We also carried out synthesis experiments replacing 50 mmol/L CAPSO−NaOH at pH 9.8 by 200 mmol/L HEPES− NaOH at pH 8.0 to investigate the effect of OH − concentration. The concentrations of all the other components were kept as in the standard solution. After 18 h of incubation, TEM analysis shows no NP formation in the apoferritin cavity. The results indicated that OH− concentration was so low that the Zn(OH)2 could not be synthesized and that alkaline 4132

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conditions, namely a high concentration of OH−, are significant for the synthesis of ZnO NP. 3. Characterization of ZnO NPs inside Apoferritin. ZnO NPs synthesized at standard conditions were characterized by EDS and HR-TEM. The EDS spectrum (Figure 3a)

the 3-fold channel is around 0.4 nm, which is too small for bulky [Zn(NH3)4]2+ to go through.37 Since the channel wall is negatively charged and positively charged ions can easily pass through, therefore, we presumed that positively charged [Zn(NH3)4]2+ ions are attracted around the channel and then Zn2+ ions are produced in the vicinity of the channel by ammonia uncoupling from [Zn(NH3)4]2+ as the chemical equilibrium indicated. After going through the 3-fold channel, Zn2+ and OH− (also diffusing into the cavity), form ZnO. The reaction is much faster than outside, because Zn2+ are highly concentrated at the nucleation sites in addition to the suppression of the reaction outside the cavity. The consumption of source ions, Zn2+ and OH−, in the cavity continues further until the full-development of a ZnO NP. Therefore, it was indicated that the OH− introduction governed the ZnO NP formation. 5. Anion Introduction. Since it was shown that the OH− introduction affects the ZnO NP synthesis, it is of high interest to study whether there are differences in the introduction between monovalent and divalent charged anions. At standard conditions, OH− composes 0.5% of the total anion (0.063 mmol/L OH− calculated from pH 9.8, 13 mmol/ L NO3−, and 0.34 mmol/L Cl− from the purified apoferritin stock solution). To change the ratio of OH−, we added either NaNO3 or Na2SO4 to the standard solution and carried out ZnO NPs synthesis experiments for 18 h at 23 °C. NaNO3 was added with final concentrations of 50, 100, 150, and 200 mmol/ L and Na2SO4 with final concentrations of 25, 50, 75, and 100 mmol/L. Figure 5 shows the CFR obtained from TEM

Figure 3. (a) EDS spectrum of a ZnO NP. (b) HR-TEM image of a ZnO NP and (c) its corresponding FFT image.

showed characteristic Zn (Kα, 8.63 keV; Lα, 1.01 keV) and O (Kα, 0.52 keV) peaks. The other peaks are attributed to Si (1.74 keV) from the measurement device, Ti (4.51 keV) from the TEM grid, C (0.28 keV) from the protein shell, carbon grid, and/or CAPSO, and Cl (2.61 keV) from NaCl. The EDS analysis indicated that synthesized NPs were composed of Zn and O. We analyzed five NPs by HR-TEM, and Figure 3b is a representative image. It shows a clear lattice fringe (2.8 Å), and its fast Fourier transform (FFT) shows a set of clear spots (Figure 3c). This lattice fringe and FFT result correspond to the (000) lattice distance of ZnO (2.81 nm, JCPDS#36-1451), disagreeing with any other zinc oxide or hydroxide such as ZnO2, Zn(OH)2·0.5H2O, or Zn(OH)2 and also Zn metal. The results from EDS analysis and HR-TEM observation confirmed that ZnO was formed in the apoferritin cavities. 4. Time Dependence Experiment of ZnO NP Formation in Apoferritin Cavity. To investigate the mechanism for the formation of ZnO NPs, we measured the time dependence of the CFR at standard conditions. Figure 4

Figure 5. Effects of the anion concentrations on the CFR. The CFR decreases as the concentration of other anions increases. The values on the X-axis show final concentrations of NaNO3 or Na2SO4.

observation. If there is preferable introduction of OH− by the channels, the CFR should remain the same as that of the standard condition. However, the CFR decreased gradually as the concentration of NaNO3 or Na2SO4 increased. This result indicated that the apoferritin channels do not preferably introduce OH− in the cavity. It could be concluded that the ion introduction was competitive for these three kinds of anions. It was also noticeable that even divalent anions could go through the channel, which is composed of negatively charged amino acids having a diameter around 0.4 nm. Since those three anion introductions were competitive, the CFR resulted in a decrease when the ratio of OH− decreased, which was caused by the increase of the total anion number in the reaction solution. On the basis of these results, we could summarize the process of ZnO NP synthesis in the apoferritin cavity as follows. Before

Figure 4. Time dependence of the ZnO NP formation (CFR).

shows the CFR became 30% within 30 s after Zn2+ addition and reached 80% in 5 min. In contrast, the standard solution without apoferritin showed no bulk precipitation for up to 60 min, which means that [Zn(NH3)4]2+ remained stable throughout the experiment. These results showed that Zn2+ were selectively present inside the cavity and formed ZnO NPs. Although the channel wall thermally fluctuates, the diameter of 4133

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the addition of Zn2+, Na+ and anions, including OH−, coexist inside the cavity. This compensates the electrochemical potential difference generated by the negative amino acids on the inner wall of the apoferritin. Once Zn(NO3)2 is added, Zn2+ ions enter the cavity and replace some of Na+ because Zn2+ is more strongly attracted by negatively charged nucleation sites than Na+ due to the difference in valence. Zn2+ will be concentrated at the nucleation sites and then anions diffuse into the cavity, where OH− ions react with the condensed Zn2+ to form ZnO. As the consumption of Zn2+ and OH− generates their ion imbalance between outside and inside the cavity, both ions will continue to flow into the cavity until the ZnO NP is fully developed. The solubilities of zinc nitrate and zinc chloride are 6.75 mol/L and 30.26 mol/L, respectively (depending on solubility product), while Zn(OH)2 is known to be practically insoluble in water (solubility product: 3.5 × 10−17).38 We therefore conclude that Zn(OH)2 is dominantly made and only ZnO is synthesized.

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CONCLUSION We have succeeded in the fabrication of monodisperse ZnO NPs in apoferritin cavities based on the SCRY, where the biotemplate apoferritin worked as a spatially restricted reaction vessel. The method described here is simple, economical, and environmentally friendly compared to the use of expensive mechanical systems. In the standard solution at pH 9.8, Zn(OH)2 was synthesized selectively in the cavity by hydroxylation and Zn(OH)2 subsequently became ZnO by the dehydration reaction. It was suggested that [Zn(NH3)4]2+ decoupled four ammonia molecules in the vicinity of the apoferritin 3-fold channels to allow Zn2+ to flow into the cavity. The anions also diffuse into the cavity even if the inner wall surface of the channel is negatively charged. Meanwhile, the suppression of chemical reactions outside apoferritin remained essentially effective. It was also shown that the anion concentration and anion species affect the NP formation. It is expected that this understanding of the behavior of cations and anions throughout the synthesis will now make it possible to fabricate other engineered NPs inside apoferritin. Furthermore, this understanding will allow us to make various kinds of NPs, not only in apoferritin, but also in smaller or bigger cage shaped-proteins and viruses.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: +81774-98-2516. Fax: +81-774-98-2515 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by the Leading Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan, and Human Frontier Science Program (RGP61/ 2007). Dr. Jean-Charles Eloi is also acknowledged for English proofreading and modifications.

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REFERENCES

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