The elution properties of silver ions from the samples were examined in various solutions to determine the behavior of silver in MRSA environment or in the body. Ag-TNT-TF with dimensions of 20 mm × 20 mm × 2 mm was immersed in 15 mL physiological saline, phosphate buffered saline (+) (PBS(+)), phosphate buffered saline (−) (PBS(−)), and fetal bovine serum solution, maintained at 37 °C for 24 h. Then, the eluates were collected, centrifuged, and filtrated through a 0.22-µm filter. After filtration, Ag concentration in the eluates was determined by inductively coupled plasma mass spectrometry (ICP-MS). In physiological saline, PBS(+), and PBS(−), almost the same average concentration of eluted silver was measured—300, 320, and 440 ppb, respectively. The eluted silver ions may originate from metallic silver and silver titanate. Since the solubility of metallic silver is known to be very small, the large portion of eluted silver was eluted by exchanging silver ions in titanates with Na+, K+, and H+ in the solutions. On the other hand, in fetal bovine serum, the average eluted silver concentration was measured in large amounts—82000 ppb for Ag-TNT-TF. This was because a large quantity of a compound composed of silver and a protein was formed together with AgCl, since the protein that exists in fetal bovine serum has very high affinity with Ag+ through the –SH group or –NH group in the protein, and the amount of exchangeable cations in fetal bovine serum was larger than that in physiological saline and PBS. Moreover, when the silver elution test in fetal bovine serum was performed for a silver metal plate under similar conditions as that for Ag-TNT-TF, silver of 7900 ppb was eluted. This amount was also significantly smaller than that for of Ag-TNT-TF. In the

Synthesis of Titanate and Titanium Dioxide

nanocomposite thin film.

**nanocomposite thin film**

Nanotube Thin Films and Their Applications to Biomaterials 57

Fig. 12. Repeated silver ion elution test of silver nanoparticle/silver titanate nanotube

The modified Japanese Industrial Standard test (JIS Z 2801) was performed as an antibacterial test as follows. To approximate an infection environment within an actual organism, an inactivated bovine serum (0.4 mL) was used as a solvent of bacterial suspension to create a eutrophic condition, and the antibacterial test was conducted using MRSA with a biofilm-forming gene. A bacterial suspension (0.4 mL) was dropped on a 50 mm × 50 mm × 2 mm test piece, covered with a 40 mm × 40 mm polyethylene film (Elmex Corp.), and cultured at 37 °C for 24 h. The test piece was washed, and the viable MRSA count was determined. The antibacterial test was performed thrice for each of the samples of Na-TNT-TF and Ag-TNT-TF, to obtain averaged values of viable MRSA counts. The

*R* = {log(*B*/*A*) − log(*C*/*A*)} = log(*B*/*C*) Here, *A*, *B*, and *C* are the average viable MRSA counts just after inoculation, after 24 h for a blank and after 24 h for a sample, respectively. The viable MRSA count just after the inoculation was 2.1 × 105 CFU/sample, and for a blank, the average viable MRSA count after 24 h increased to 5.9 × 108 CFU/sample. In Na-TNT-TF, the average viable MRSA count after 24 h was slightly less than that of the blank: 1.1 × 107 CFU/sample. On the other hand, in Ag-TNT-TF, the average viable MRSA count after 24 h was markedly small: 3.3 × 102 CFU/sample. *R* increased from 1.7 for Na-TNT-TF to 6.3 for Ag-TNT-TF through the silver ion-exchange treatment. These results indicate that the silver ion-exchanged titanate thin films display high antibacterial activity against MRSA. It was also revealed that although the crystal structure of titanate itself does not have a large antibacterial effect, higher antibacterial activity arises in the silver in the titanate. The conversion of sodium titanates into antibacterial materials through the silver ion-exchange treatment can apply to other nanostructured sodium titanates. For example, by the same silver ion-exchange treatment, porous sodium titanate film calcined at 600 °C reported by Kim et al. (Kim et al., 1997) can also be converted into silver nanoparticle / silver titanate nanocomposite thin film

**3.2 Antibacterial property of silver nanoparticle / silver titanate nanotube**

antibacterial activity value (*R*) for the sample was calculated as follows.

with high antibacterial activity for MRSA of *R*=6.7.

silver elution test of Ag-TNT-TF, although silver is eluted from silver nanoparticles deposited on the surface of titanate, its elution amount is thus considered small. Consequently, in the XRD and TF-XRD patterns of the sample obtained by the silver elution test of Ag-TNT-TF, diffraction peaks appeared near 2θ = 10°, 24° and 28°, which were not observed in Ag-TNT-TF. These diffraction peaks of the sample obtained by the silver elution test of Ag-TNT-TF appeared at the same locations as the diffraction peaks observed in Na-TNT-TF. This indicates that the crystal structure of the sample obtained by the silver elution test of Ag-TNT-TF is similar to that of Na-TNT-TF. The likely reasons are as follows: (1) the layered structure of titanate of Na-TNT-TF transformed into a three-dimensional structure because Ag+ ions were inserted into the interlayer of titanate by the silver ion-exchange treatment to form silver titanate, (2) Ag+ ions were eluted from the silver titanate and Na+ ions were reinserted into the titanate during the silver elution test; and (3) the threedimensional structure of titanate returned to the original condition as in Na-TNT-TF. Thus, it is clearly demonstrated that insertion (intercalation) and elimination (deintercalation) of Ag+ ions occurs during the silver ion-exchange treatment and the silver elution test, respectively. Therefore, this experiment indicated that Ag+ ions in silver titanate greatly contributed to the elution of Ag+. Diffraction peaks attributable to AgCl were also observed in the sample obtained by the silver elution test of Ag-TNT-TF, because AgCl particles were formed by the reaction between eluted Ag+ and Cl in fetal bovine serum. Since fetal bovine serum solution is considered as the system most similar to MRSA environment, silver elution tests in fetal bovine serum solution were repeated (Fig. 12). In Ag-TNT-TF, the elution concentration slowly decreased from 94000 ppb for the first test to 11000 ppb for the tenth, indicating that a large amount of eluted silver was measured in the tenth test for Ag-TNT-TF. The 2-step elution curve was obtained from Ag-TNT-TF. A rapid elution of a large amount of silver at the initial stage of the repeated elution test (between the first and third time) was considered to be mainly due to Ag+ ion elution from the silver titanate based on the ion-exchange reaction. Since the elution reaction (ion-exchange reaction) of Ag+ from silver titanate is rapid, the elution of Ag+ is considered to be almost completed at the initial stage of the repeated elution test. These discussions are also supported by the above described TF-XRD data, indicating that crystal structure of the sample obtained by the silver elution test of Ag-TNT-TF is similar to that of Na-TNT-TF. Therefore, a slow elution of a small amount of silver after the forth repetition of the elution test was considered to be mainly because of silver elution from the silver nanoparticles. This two-step elution curve is difficult to explain if it is considered that only silver nanoparticles are formed in the thin film, but it is reasonably explained if two types of Ag (silver nanoparticles and silver titanate) exhibiting different elution behaviors are present in Ag-TNT-TF. The silver titanates loading silver nanoparticles would be promising as a novel antibacterial material, because they have two silver sources. The silver-ion elution property of silver titanate would be different from that of the silver nanoparticles, i.e., the elution speed of silver ions from silver titanate would be greater than that from silver nanoparticles. Therefore, silver titanate would be effective for short-term bacterial killing, and silver nanoparticles would be effective for long-term antibacterial action. Since we have already found that a thicker (i.e., 20 µm thick) titanate nanotube film can be formed after a longer reaction time or 20 h in NaOH solution, it would be possible to prolong the elution period of silver ions and to increase the amount of eluted silver ions or the duration.

silver elution test of Ag-TNT-TF, although silver is eluted from silver nanoparticles deposited on the surface of titanate, its elution amount is thus considered small. Consequently, in the XRD and TF-XRD patterns of the sample obtained by the silver elution test of Ag-TNT-TF, diffraction peaks appeared near 2θ = 10°, 24° and 28°, which were not observed in Ag-TNT-TF. These diffraction peaks of the sample obtained by the silver elution test of Ag-TNT-TF appeared at the same locations as the diffraction peaks observed in Na-TNT-TF. This indicates that the crystal structure of the sample obtained by the silver elution test of Ag-TNT-TF is similar to that of Na-TNT-TF. The likely reasons are as follows: (1) the layered structure of titanate of Na-TNT-TF transformed into a three-dimensional structure because Ag+ ions were inserted into the interlayer of titanate by the silver ion-exchange treatment to form silver titanate, (2) Ag+ ions were eluted from the silver titanate and Na+ ions were reinserted into the titanate during the silver elution test; and (3) the threedimensional structure of titanate returned to the original condition as in Na-TNT-TF. Thus, it is clearly demonstrated that insertion (intercalation) and elimination (deintercalation) of Ag+ ions occurs during the silver ion-exchange treatment and the silver elution test, respectively. Therefore, this experiment indicated that Ag+ ions in silver titanate greatly contributed to the elution of Ag+. Diffraction peaks attributable to AgCl were also observed in the sample obtained by the silver elution test of Ag-TNT-TF, because AgCl particles were

serum solution is considered as the system most similar to MRSA environment, silver elution tests in fetal bovine serum solution were repeated (Fig. 12). In Ag-TNT-TF, the elution concentration slowly decreased from 94000 ppb for the first test to 11000 ppb for the tenth, indicating that a large amount of eluted silver was measured in the tenth test for Ag-TNT-TF. The 2-step elution curve was obtained from Ag-TNT-TF. A rapid elution of a large amount of silver at the initial stage of the repeated elution test (between the first and third time) was considered to be mainly due to Ag+ ion elution from the silver titanate based on the ion-exchange reaction. Since the elution reaction (ion-exchange reaction) of Ag+ from silver titanate is rapid, the elution of Ag+ is considered to be almost completed at the initial stage of the repeated elution test. These discussions are also supported by the above described TF-XRD data, indicating that crystal structure of the sample obtained by the silver elution test of Ag-TNT-TF is similar to that of Na-TNT-TF. Therefore, a slow elution of a small amount of silver after the forth repetition of the elution test was considered to be mainly because of silver elution from the silver nanoparticles. This two-step elution curve is difficult to explain if it is considered that only silver nanoparticles are formed in the thin film, but it is reasonably explained if two types of Ag (silver nanoparticles and silver titanate) exhibiting different elution behaviors are present in Ag-TNT-TF. The silver titanates loading silver nanoparticles would be promising as a novel antibacterial material, because they have two silver sources. The silver-ion elution property of silver titanate would be different from that of the silver nanoparticles, i.e., the elution speed of silver ions from silver titanate would be greater than that from silver nanoparticles. Therefore, silver titanate would be effective for short-term bacterial killing, and silver nanoparticles would be effective for long-term antibacterial action. Since we have already found that a thicker (i.e., 20 µm thick) titanate nanotube film can be formed after a longer reaction time or 20 h in NaOH solution, it would be possible to prolong the elution period of silver ions and to

in fetal bovine serum. Since fetal bovine

formed by the reaction between eluted Ag+ and Cl-

increase the amount of eluted silver ions or the duration.

Fig. 12. Repeated silver ion elution test of silver nanoparticle/silver titanate nanotube nanocomposite thin film.

#### **3.2 Antibacterial property of silver nanoparticle / silver titanate nanotube nanocomposite thin film**

The modified Japanese Industrial Standard test (JIS Z 2801) was performed as an antibacterial test as follows. To approximate an infection environment within an actual organism, an inactivated bovine serum (0.4 mL) was used as a solvent of bacterial suspension to create a eutrophic condition, and the antibacterial test was conducted using MRSA with a biofilm-forming gene. A bacterial suspension (0.4 mL) was dropped on a 50 mm × 50 mm × 2 mm test piece, covered with a 40 mm × 40 mm polyethylene film (Elmex Corp.), and cultured at 37 °C for 24 h. The test piece was washed, and the viable MRSA count was determined. The antibacterial test was performed thrice for each of the samples of Na-TNT-TF and Ag-TNT-TF, to obtain averaged values of viable MRSA counts. The antibacterial activity value (*R*) for the sample was calculated as follows.

$$R = \{\log(B/A) - \log(C/A)\} = \log(B/C)$$

Here, *A*, *B*, and *C* are the average viable MRSA counts just after inoculation, after 24 h for a blank and after 24 h for a sample, respectively. The viable MRSA count just after the inoculation was 2.1 × 105 CFU/sample, and for a blank, the average viable MRSA count after 24 h increased to 5.9 × 108 CFU/sample. In Na-TNT-TF, the average viable MRSA count after 24 h was slightly less than that of the blank: 1.1 × 107 CFU/sample. On the other hand, in Ag-TNT-TF, the average viable MRSA count after 24 h was markedly small: 3.3 × 102 CFU/sample. *R* increased from 1.7 for Na-TNT-TF to 6.3 for Ag-TNT-TF through the silver ion-exchange treatment. These results indicate that the silver ion-exchanged titanate thin films display high antibacterial activity against MRSA. It was also revealed that although the crystal structure of titanate itself does not have a large antibacterial effect, higher antibacterial activity arises in the silver in the titanate. The conversion of sodium titanates into antibacterial materials through the silver ion-exchange treatment can apply to other nanostructured sodium titanates. For example, by the same silver ion-exchange treatment, porous sodium titanate film calcined at 600 °C reported by Kim et al. (Kim et al., 1997) can also be converted into silver nanoparticle / silver titanate nanocomposite thin film with high antibacterial activity for MRSA of *R*=6.7.

Synthesis of Titanate and Titanium Dioxide

TiO2-NT-FT.

Nanotube Thin Films and Their Applications to Biomaterials 59

films for 4 days, the SEM images showed a stretch of the dome-shaped form consisting of foliaceous particles peculiar to apatite (Figs. 13c, d). XRD patterns of the two films showed diffraction peaks attributable to apatite, respectively. These results indicate that the surfaces of these two thin films are completely covered with apatite and that the apatite-forming ability of the two films is greater than that of Na-TNT-TF having layered structure. In contrast, for TiO2-NT-FT after immersing the film in SBF for 2 days, the surface is thinly covered with apatite. However immersing Ag-TNT-TF for 2 days, the surface is almost covered with a dome-shaped form consisting of foliaceous particles peculiar to the apatite (Figs. 13e, f). The apatite-forming ability of Ag-TNT-TF is, thus, slightly higher than that of

We then investigated the newly observed high apatite-forming ability of silver nanoparticle/silver titanate nanocomposite thin film (Ag-TNT-TF). After immersing the film in SBF for 4 days, we observed bulky particles of a few micrometers in diameter together with apatite. XRD pattern shows diffraction peaks attributable to silver chloride. EDX element mapping analysis shows the bulky particles to be composed of Ag and Cl (Fig. 14) and, therefore, to be silver chloride particles. Therefore, Ag+ ions are eluted from silver titanate mainly by ion-exchange reaction with cations, resulting in deposition of silver chloride particles. After immersing the film in SBF for 1 day, SEM images reveal only bulky AgCl particles on the film surface, however immersing the film for 2 days, the surface is almost covered with a dome-shaped form consisting of foliaceous particles peculiar to the apatite (Figs. 13e, f), as mentioned above. We clarified whether silver nanoparticle or silver titanate contributes more to the apatite formation by investigating the apatite-forming ability of a silver metal plate. After immersing the plate in SBF for 4 days, no apatite formation was evident; thus, the silver titanate nanotubes are responsible for the high apatite-forming ability. Researchers have reported that the effects of crystal structure (Uchida et al., 2003) and surface hydroxyl groups such as Ti-OH (Kasuga et al., 2002) influence the apatite formation on the titanium compounds immersed in SBF. Kokubo et al. reported that the apatite-forming ability is improved by the crystal structure transformation of sodium titanate into titanium dioxide (Fujibayashi et al., 2001; Uchida et al., 2002; Takemoto et al., 2006). Although the detailed crystal structure of silver titanate is not yet known, we speculate that the surface atomic arrangement and surface functional groups of silver titanate might be suitable for rapid apatite formation. We further investigated the high apatite forming ability by considering –OH groups that influence apatite formation using the FT-IR measurements. As shown in Fig. 15, Na-TNT-TF and TiO2-NT-FT exhibited similar absorption spectra in a wide range of 3000−3700 cm−1. These absorption spectra are considered to be mainly due to water molecules adsorbed on the inner and outer surfaces of nanotubes and partially due to –OH groups on the surface. Unlike Na-TNT-TF and TiO2- NT-FT, strong absorption was observed at 3000−3400 cm−1 in addition to 3400−3700 cm−1 in Ag-TNT-TF. This absorption at 3000−3400 cm−1 is considered to indicate the existence of surface –OH groups due to silver titanate. A surface atomic arrangement peculiar to silver titanate would arise and a large number of –OH groups would be generated on the

nanotube surfaces, which would stimulate apatite formation.

Oh et al. (Oh et al., 2005) and Tsuchiya et al. (Tsuchiya et al., 2006) reported anatase-type titanium dioxide nanotube thin films synthesized by anodization and heat treatment of the titanium metal. We compared the apatite-forming ability of these nanotube thin films with

## **4. Apatite-forming ability of titanate and titanium dioxide nanotube thin films**

The three thin films (Na-TNT-TF, TiO2-NT-FT (anatase type titanium dioxide nanotube thin film formed by the H+ ion-exchange treatment and calcination at 450 °C of Na-TNT-TF), Ag-TNT-TF) formed on a titanium metal were immersed in simulated body fluid (SBF) and monitored the development of apatite formation on their surfaces. In accordance with ISO 23317, "Implants for surgery-*In vitro* evaluation for apatite-forming ability of implant materials," we evaluated the apatite-forming ability on the surface of the coating in SBF. A plate was placed in 96.0 mL SBF at 36.5°C. After 2, 4, and 14 days, the plate was removed and gently rinsed with water. The surface of the plate was dried in air.

For Na-TNT-TF, after immersing the film for 4 days, the SEM images showed only nanotubes and no substances with foliaceous morphology peculiar to apatite; the XRD patterns remained unchanged. However, when the SBF immersion was extended to 14 days, the SEM images showed the surface of the film to be completely covered with a domeshaped form consisting of foliaceous particles peculiar to apatite (Figs. 13a, b); the XRD pattern showed diffraction peaks attributable to apatite. Thus, apatite is confirmed to be formed on the sodium titanate thin films. In addition, after immersing the hydrogen titanate nanotube thin film formed by exchanging Na+ ions between the layers of the layered sodium titanate for H+ ions in SBF for 4 days, no apatite was evident. This lack of apatite indicates that ions (Na+ and H+) between the titanate layers do not particularly contribute to the acceleration of the apatite formation. In contrast, for TiO2-NT-FT formed by the 450°C calcination of the hydrogen titanate nanotube thin film and Ag-TNT-TF, after immersing the

Fig. 13. Low magnification (a, c, e) and high magnification (b, d, f) SEM images of Na-TNT-TF (a, b), TiO2-NT-FT (c, d), and Ag-TNT-TF (e, f) after immersions in SBF.

**4. Apatite-forming ability of titanate and titanium dioxide nanotube thin films**  The three thin films (Na-TNT-TF, TiO2-NT-FT (anatase type titanium dioxide nanotube thin film formed by the H+ ion-exchange treatment and calcination at 450 °C of Na-TNT-TF), Ag-TNT-TF) formed on a titanium metal were immersed in simulated body fluid (SBF) and monitored the development of apatite formation on their surfaces. In accordance with ISO 23317, "Implants for surgery-*In vitro* evaluation for apatite-forming ability of implant materials," we evaluated the apatite-forming ability on the surface of the coating in SBF. A plate was placed in 96.0 mL SBF at 36.5°C. After 2, 4, and 14 days, the plate was removed

For Na-TNT-TF, after immersing the film for 4 days, the SEM images showed only nanotubes and no substances with foliaceous morphology peculiar to apatite; the XRD patterns remained unchanged. However, when the SBF immersion was extended to 14 days, the SEM images showed the surface of the film to be completely covered with a domeshaped form consisting of foliaceous particles peculiar to apatite (Figs. 13a, b); the XRD pattern showed diffraction peaks attributable to apatite. Thus, apatite is confirmed to be formed on the sodium titanate thin films. In addition, after immersing the hydrogen titanate nanotube thin film formed by exchanging Na+ ions between the layers of the layered sodium titanate for H+ ions in SBF for 4 days, no apatite was evident. This lack of apatite indicates that ions (Na+ and H+) between the titanate layers do not particularly contribute to the acceleration of the apatite formation. In contrast, for TiO2-NT-FT formed by the 450°C calcination of the hydrogen titanate nanotube thin film and Ag-TNT-TF, after immersing the

and gently rinsed with water. The surface of the plate was dried in air.

a c e

b d f

TF (a, b), TiO2-NT-FT (c, d), and Ag-TNT-TF (e, f) after immersions in SBF.

Fig. 13. Low magnification (a, c, e) and high magnification (b, d, f) SEM images of Na-TNT-

films for 4 days, the SEM images showed a stretch of the dome-shaped form consisting of foliaceous particles peculiar to apatite (Figs. 13c, d). XRD patterns of the two films showed diffraction peaks attributable to apatite, respectively. These results indicate that the surfaces of these two thin films are completely covered with apatite and that the apatite-forming ability of the two films is greater than that of Na-TNT-TF having layered structure. In contrast, for TiO2-NT-FT after immersing the film in SBF for 2 days, the surface is thinly covered with apatite. However immersing Ag-TNT-TF for 2 days, the surface is almost covered with a dome-shaped form consisting of foliaceous particles peculiar to the apatite (Figs. 13e, f). The apatite-forming ability of Ag-TNT-TF is, thus, slightly higher than that of TiO2-NT-FT.

We then investigated the newly observed high apatite-forming ability of silver nanoparticle/silver titanate nanocomposite thin film (Ag-TNT-TF). After immersing the film in SBF for 4 days, we observed bulky particles of a few micrometers in diameter together with apatite. XRD pattern shows diffraction peaks attributable to silver chloride. EDX element mapping analysis shows the bulky particles to be composed of Ag and Cl (Fig. 14) and, therefore, to be silver chloride particles. Therefore, Ag+ ions are eluted from silver titanate mainly by ion-exchange reaction with cations, resulting in deposition of silver chloride particles. After immersing the film in SBF for 1 day, SEM images reveal only bulky AgCl particles on the film surface, however immersing the film for 2 days, the surface is almost covered with a dome-shaped form consisting of foliaceous particles peculiar to the apatite (Figs. 13e, f), as mentioned above. We clarified whether silver nanoparticle or silver titanate contributes more to the apatite formation by investigating the apatite-forming ability of a silver metal plate. After immersing the plate in SBF for 4 days, no apatite formation was evident; thus, the silver titanate nanotubes are responsible for the high apatite-forming ability. Researchers have reported that the effects of crystal structure (Uchida et al., 2003) and surface hydroxyl groups such as Ti-OH (Kasuga et al., 2002) influence the apatite formation on the titanium compounds immersed in SBF. Kokubo et al. reported that the apatite-forming ability is improved by the crystal structure transformation of sodium titanate into titanium dioxide (Fujibayashi et al., 2001; Uchida et al., 2002; Takemoto et al., 2006). Although the detailed crystal structure of silver titanate is not yet known, we speculate that the surface atomic arrangement and surface functional groups of silver titanate might be suitable for rapid apatite formation. We further investigated the high apatite forming ability by considering –OH groups that influence apatite formation using the FT-IR measurements. As shown in Fig. 15, Na-TNT-TF and TiO2-NT-FT exhibited similar absorption spectra in a wide range of 3000−3700 cm−1. These absorption spectra are considered to be mainly due to water molecules adsorbed on the inner and outer surfaces of nanotubes and partially due to –OH groups on the surface. Unlike Na-TNT-TF and TiO2- NT-FT, strong absorption was observed at 3000−3400 cm−1 in addition to 3400−3700 cm−1 in Ag-TNT-TF. This absorption at 3000−3400 cm−1 is considered to indicate the existence of surface –OH groups due to silver titanate. A surface atomic arrangement peculiar to silver titanate would arise and a large number of –OH groups would be generated on the nanotube surfaces, which would stimulate apatite formation.

Oh et al. (Oh et al., 2005) and Tsuchiya et al. (Tsuchiya et al., 2006) reported anatase-type titanium dioxide nanotube thin films synthesized by anodization and heat treatment of the titanium metal. We compared the apatite-forming ability of these nanotube thin films with

Synthesis of Titanate and Titanium Dioxide

**5. Conclusion**

Nanotube Thin Films and Their Applications to Biomaterials 61

to that of the thin film with 2-μm long nanotube synthesized by Tsuchiya et al. (Tsuchiya et al., 2006) and clearly superior to that of the nanotube thin film synthesized by Oh et al. (Oh et al., 2005) and the thin film with 500-nm long nanotube synthesized by Tsuchiya et al. (Tsuchiya et al., 2006). While comparing the ratio of the void parts to the anatase-type titanium dioxide part on the surface using SEM images, we found the proportion consisting anatase-type titanium dioxide to be larger in the surface of our synthesized thin film as compared to that in the surfaces of the thin films reported by Oh et al. and Tsuchiya et al.

In this study, novel procedure of synthesis and fixation of Na-TNT onto titanium metals with various morphologies such as plate, wire, mesh, tube, and sphere was reported. Especially, since the Na-TNT/Ti composite wires have softness and flexibility peculiar to metal titanium because of the existence of titanium metal in its core part, this wire can be fabricated into various shapes of cloth, fiber, etc. with centimeter or meter size by using conventional spinning techniques. The Na-TNT thin films can be transformed into anatasetype titanium dioxide nanotube thin films. Another advantage of the proposed procedure is that the thickness of the thin films produced is greater than that of the thin films reported by other researchers. Therefore, Na-TNT's applications mentioned in the introduction would be remarkably expanded. In addition, the novel growth of the Na-TNT film on substrates such as Co–Cr alloy and SUS316L and simple patterning of the Na-TNT phase by the hydrothermal transcription method were also reported. As these substrates including titanium metal, Co-Cr alloy, and SUS316L have superior mechanical properties and corrosion resistance, they are frequently used as implants such as artificial joints. Generally, the coating of films to implants with complex shapes requires thin films with uniform and controlled thickness, and a high fixing strength to the implants. Because of the direct growth of the nanotubes from the substrate, our proposed method is very simple and the fixing strength to the substrate is expected to be higher. Therefore, the method proposed in the

Next, through a silver ion-exchange treatment, Na+ ions in sodium titanate nanotube were exchanged with Ag+ ions in silver acetate solution, along with the loading of silver nanoparticles on the titanate surfaces, and the layered structure of titanate transformed into a new three-dimensional crystal structure. Results of silver ion elution tests of the obtained thin films in fetal bovine serum solution indicate that the release period and the number of silver ions released from the silver titanate thin films can be controlled. The silver ionexchanged titanate thin films showed high antibacterial activity against MRSA. It was also revealed that although the crystal structure of titanate itself has no large antibacterial effect, higher antibacterial activity mainly arises from the silver ions held in the titanate. The samples coated with apatite containing silver and silver plate have already been reported as possessing antibacterial properties through metallic silver with low solubility. In contrast, in this study, the antibacterial properties were mainly caused by the elution of silver ions from a titanate with an ion-exchange property. Since the thin film obtained by this study has a higher silver-ion elution speed, greater and more rapid antibacterial effects than in metallic silver can be expected. Since we have also revealed that the morphology, thickness, and crystal structure of the titanate phase can be controlled, we think that this can also promise

Hence, the apatite-forming ability of our film is also correspondingly higher.

present study has excellent potential for these biomedical applications.

Fig. 14. Elemental mapping performed on Ag-TNT-TF after immersion in SBF for 4 days using EDX analysis.

Fig. 15. FT-IR spectra of Na-TNT-TF (a), TiO2-NT-FT (b), and Ag-TNT-TF (c).

that of TiO2-NT-FT obtained in this study by immersing the film in SBF for 2 days. SEM images show not only a small amount of the dome-shaped form consisting of foliaceous particles peculiar to apatite, but also several slightly swelled and whitish areas. EDX element mapping on this thin film revealed that titanium dioxide nanotubes exist in the blackish areas and apatite exists in the whitish areas. At this point, after 2 days of immersion, the apatite phase has grown slightly but not yet achieved its dome-shaped form. Therefore, the apatite-forming ability of TiO2-NT-FT is slightly superior though still similar to that of the thin film with 2-μm long nanotube synthesized by Tsuchiya et al. (Tsuchiya et al., 2006) and clearly superior to that of the nanotube thin film synthesized by Oh et al. (Oh et al., 2005) and the thin film with 500-nm long nanotube synthesized by Tsuchiya et al. (Tsuchiya et al., 2006). While comparing the ratio of the void parts to the anatase-type titanium dioxide part on the surface using SEM images, we found the proportion consisting anatase-type titanium dioxide to be larger in the surface of our synthesized thin film as compared to that in the surfaces of the thin films reported by Oh et al. and Tsuchiya et al. Hence, the apatite-forming ability of our film is also correspondingly higher.

## **5. Conclusion**

60 Smart Nanoparticles Technology

Ca

O

Fig. 14. Elemental mapping performed on Ag-TNT-TF after immersion in SBF for 4 days

Ag Cl

Fig. 15. FT-IR spectra of Na-TNT-TF (a), TiO2-NT-FT (b), and Ag-TNT-TF (c).

that of TiO2-NT-FT obtained in this study by immersing the film in SBF for 2 days. SEM images show not only a small amount of the dome-shaped form consisting of foliaceous particles peculiar to apatite, but also several slightly swelled and whitish areas. EDX element mapping on this thin film revealed that titanium dioxide nanotubes exist in the blackish areas and apatite exists in the whitish areas. At this point, after 2 days of immersion, the apatite phase has grown slightly but not yet achieved its dome-shaped form. Therefore, the apatite-forming ability of TiO2-NT-FT is slightly superior though still similar

P

using EDX analysis.

SEM image

0.5 m μ

In this study, novel procedure of synthesis and fixation of Na-TNT onto titanium metals with various morphologies such as plate, wire, mesh, tube, and sphere was reported. Especially, since the Na-TNT/Ti composite wires have softness and flexibility peculiar to metal titanium because of the existence of titanium metal in its core part, this wire can be fabricated into various shapes of cloth, fiber, etc. with centimeter or meter size by using conventional spinning techniques. The Na-TNT thin films can be transformed into anatasetype titanium dioxide nanotube thin films. Another advantage of the proposed procedure is that the thickness of the thin films produced is greater than that of the thin films reported by other researchers. Therefore, Na-TNT's applications mentioned in the introduction would be remarkably expanded. In addition, the novel growth of the Na-TNT film on substrates such as Co–Cr alloy and SUS316L and simple patterning of the Na-TNT phase by the hydrothermal transcription method were also reported. As these substrates including titanium metal, Co-Cr alloy, and SUS316L have superior mechanical properties and corrosion resistance, they are frequently used as implants such as artificial joints. Generally, the coating of films to implants with complex shapes requires thin films with uniform and controlled thickness, and a high fixing strength to the implants. Because of the direct growth of the nanotubes from the substrate, our proposed method is very simple and the fixing strength to the substrate is expected to be higher. Therefore, the method proposed in the present study has excellent potential for these biomedical applications.

Next, through a silver ion-exchange treatment, Na+ ions in sodium titanate nanotube were exchanged with Ag+ ions in silver acetate solution, along with the loading of silver nanoparticles on the titanate surfaces, and the layered structure of titanate transformed into a new three-dimensional crystal structure. Results of silver ion elution tests of the obtained thin films in fetal bovine serum solution indicate that the release period and the number of silver ions released from the silver titanate thin films can be controlled. The silver ionexchanged titanate thin films showed high antibacterial activity against MRSA. It was also revealed that although the crystal structure of titanate itself has no large antibacterial effect, higher antibacterial activity mainly arises from the silver ions held in the titanate. The samples coated with apatite containing silver and silver plate have already been reported as possessing antibacterial properties through metallic silver with low solubility. In contrast, in this study, the antibacterial properties were mainly caused by the elution of silver ions from a titanate with an ion-exchange property. Since the thin film obtained by this study has a higher silver-ion elution speed, greater and more rapid antibacterial effects than in metallic silver can be expected. Since we have also revealed that the morphology, thickness, and crystal structure of the titanate phase can be controlled, we think that this can also promise

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control of the antibacterial properties, i.e., the duration and amount of antibacterial activity, for the future. The obtained results should aid the development of more convenient and inexpensive antibacterial implants.

Furthermore, the present study compares the apatite-forming ability of a sodium titanate nanotube thin film, an anatase-type titanium dioxide nanotube thin film, and a silver nanoparticle/silver titanate nanotube nanocomposite thin film. Of these, the apatite-forming ability of the silver titanate nanotube was higher than that of the titanium dioxide nanotubes or the sodium titanate nanotubes, in that order. This superior apatite-forming ability of the silver nanoparticle/silver titanate nanotube nanocomposite thin film is a novel phenomenon and is presumably due to the surface atomic arrangement of silver titanate, the large amount of Ti-OH formed on the nanotube surface, or both. In conclusion, the silver nanoparticle/silver titanate nanotube nanocomposite thin film, which have the antibacterial property and the ability to form bone-like apatite, i.e., the osteoconductive property, may have bright prospects for future use in implant materials such as artificial joints.

## **6. Acknowledgment**

This research was partially supported by KAKENHI (16685021, 19750172) and Saga University Dean's Grant 2010 For Promising Young Researchers. Figures are reproduced with permission from American Chemical Society, Elsevier, and John Wiley & Sons.

#### **7. References**

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Furthermore, the present study compares the apatite-forming ability of a sodium titanate nanotube thin film, an anatase-type titanium dioxide nanotube thin film, and a silver nanoparticle/silver titanate nanotube nanocomposite thin film. Of these, the apatite-forming ability of the silver titanate nanotube was higher than that of the titanium dioxide nanotubes or the sodium titanate nanotubes, in that order. This superior apatite-forming ability of the silver nanoparticle/silver titanate nanotube nanocomposite thin film is a novel phenomenon and is presumably due to the surface atomic arrangement of silver titanate, the large amount of Ti-OH formed on the nanotube surface, or both. In conclusion, the silver nanoparticle/silver titanate nanotube nanocomposite thin film, which have the antibacterial property and the ability to form bone-like apatite, i.e., the osteoconductive property, may

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**4**

*Japan*

**Self-Organization of Silver-Core Bimetallic**

Metal nanoparticles have received much attentions as a building block of advanced materials for nanoscience and nanotechnology (Bönnemann & Richards, 2001). Their optical, (Fukumi et al., 1994; Lu et al., 1999; Link et al., 1999; Shipway et al., 2000), magnetic (Sun et al., 1999; Teranishi & Miyake, 1999), and catalytic (Kiely et al., 1998; Pileni, 1998; Bradley, 1994; Harriman, 1990; Lee et al., 1995; Toshima et al., 1995; Bonilla et al., 2000; Siepen et al., 2000) properties have been reported with great interests. The character of metal nanoparticle can be altered by the addition of other metals. Bimetallic nanoparticles, composed of two different metallic elements, have been reported to show outstanding characters different from the corresponding monometallic nanoparticles (Harriman, 1990; Yonezawa & Toshima, 1993; Toshima & Hirakawa, 1997, 1999; Toshima & Wang, 1994; Lee et al., 1995). For example, catalytic activities of gold (Au)-core structured bimetallic nanoparticles, gold/platinum (Au/Pt) (Harriman, 1990; Yonezawa & Toshima, 1993; Toshima & Hirakawa, 1999), gold/palladium (Au/Pd) (Toshima & Hirakawa, 1999; Lee et al., 1995), and gold/rhodium (Au/Rh) (Toshima & Hirakawa, 1999), for hydrogenation and/or water reduction are higher than platinum (Pt), palladium (Pd), and rhodium (Rh) monometallic nanoparticles, respectively. Surprisingly, in some cases, a physical mixture of monometallic nanoparticles such as Pt and ruthenium (Ru) nanoparticles in solution shows higher catalytic activity than the corresponding monometallic nanoparticles under a certain condition (Toshima et al., 1995; Toshima & Hirakawa, 1997). This suggests that an interaction between two kinds of monometallic nanoparticles can produce novel nanoparticles. Further, it has been reported that physical mixture of silver (Ag) and other metal nanoparticles, such as Pt, Rh, and Pd, spontaneously forms the bimetallic nanoparticles with Ag-core structure in aqueous solution. This reaction can be used to construct the core-shell structured novel bimetallic nanoparticles. The formed nanoparticles

In this chapter, the simple method of the preparation of core-shell structured bimetallic nanoparticles by the physical mixing and the application of the formed novel metal nanoparticles for catalytic reaction are described. The topics of the catalytic reaction presented in this chapter are the visible light induced hydrogen generation (Toshima &

demonstrate superior character for certain catalytic reactions.

**1. Introduction**

**Nanoparticles and Their Application**

**for Catalytic Reaction**

*Faculty of Engineering, Shizuoka University*

Kazutaka Hirakawa

## **Self-Organization of Silver-Core Bimetallic Nanoparticles and Their Application for Catalytic Reaction**

Kazutaka Hirakawa *Faculty of Engineering, Shizuoka University Japan*

## **1. Introduction**

66 Smart Nanoparticles Technology

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Plate, Wire, Mesh, Microsphere, and Microtube Composed of Sodium Titanate

Formation of Sodium Titanate Nanotube Films by Hydrothermal Transcription.

Apatite-forming ability of titanium compound nanotube thin films formed on a titanium metal plate in a simulated body fluid. *Colloids and Surfaces B: Biointerfaces*,

Nanohole Arrays by a Liquid Phase Deposition Method. *Journal of Alloys and*

composition, structure and formation process of nanotube Na2Ti2O4(OH)2. *Journal*

Phase Transition between Nanostructures of Titanate and Titanium Dioxides via Simple Wet-Chemical Reactions. *Journal of the American Ceramic Society*, Vol. 127, Metal nanoparticles have received much attentions as a building block of advanced materials for nanoscience and nanotechnology (Bönnemann & Richards, 2001). Their optical, (Fukumi et al., 1994; Lu et al., 1999; Link et al., 1999; Shipway et al., 2000), magnetic (Sun et al., 1999; Teranishi & Miyake, 1999), and catalytic (Kiely et al., 1998; Pileni, 1998; Bradley, 1994; Harriman, 1990; Lee et al., 1995; Toshima et al., 1995; Bonilla et al., 2000; Siepen et al., 2000) properties have been reported with great interests. The character of metal nanoparticle can be altered by the addition of other metals. Bimetallic nanoparticles, composed of two different metallic elements, have been reported to show outstanding characters different from the corresponding monometallic nanoparticles (Harriman, 1990; Yonezawa & Toshima, 1993; Toshima & Hirakawa, 1997, 1999; Toshima & Wang, 1994; Lee et al., 1995). For example, catalytic activities of gold (Au)-core structured bimetallic nanoparticles, gold/platinum (Au/Pt) (Harriman, 1990; Yonezawa & Toshima, 1993; Toshima & Hirakawa, 1999), gold/palladium (Au/Pd) (Toshima & Hirakawa, 1999; Lee et al., 1995), and gold/rhodium (Au/Rh) (Toshima & Hirakawa, 1999), for hydrogenation and/or water reduction are higher than platinum (Pt), palladium (Pd), and rhodium (Rh) monometallic nanoparticles, respectively. Surprisingly, in some cases, a physical mixture of monometallic nanoparticles such as Pt and ruthenium (Ru) nanoparticles in solution shows higher catalytic activity than the corresponding monometallic nanoparticles under a certain condition (Toshima et al., 1995; Toshima & Hirakawa, 1997). This suggests that an interaction between two kinds of monometallic nanoparticles can produce novel nanoparticles. Further, it has been reported that physical mixture of silver (Ag) and other metal nanoparticles, such as Pt, Rh, and Pd, spontaneously forms the bimetallic nanoparticles with Ag-core structure in aqueous solution. This reaction can be used to construct the core-shell structured novel bimetallic nanoparticles. The formed nanoparticles demonstrate superior character for certain catalytic reactions.

In this chapter, the simple method of the preparation of core-shell structured bimetallic nanoparticles by the physical mixing and the application of the formed novel metal nanoparticles for catalytic reaction are described. The topics of the catalytic reaction presented in this chapter are the visible light induced hydrogen generation (Toshima &

Self-Organization of Silver-Core Bimetallic

0

L-1, 50 mL) nanoparticles were mixed.

after indicated periods.

0.5

1

Absorbance

1.5

2

absorption band completely.

Nanoparticles and Their Application for Catalytic Reaction 69

extinguished within 10 min, suggesting that influences of Rh nanopartilces on Ag nanoparticles depend on the size of the Ag nanoparticles. When relatively smaller molar quantity of Rh to Ag was added, the plasmon absorption was not completely extinguished. More than 40 atom-mol% of Rh against to Ag was required to extinguish the plasmon

> 0 min 5 min 10 min 20 min 30 min 24 h

300 400 500 600

Wavelength / nm

nanoparticles. The aqueous solutions of Ag (1 atom-mmol L-1, 50 mL) and Rh (1 atom-mmol

Figure 3 shows transmission electron microscopy (TEM) photographs of the physical mixtures of Ag and Rh monometallic nanoparticles. The samples for TEM measurement were prepared by drying the aqueous dispersions of the physical mixtures of Ag and Rh nanoparticles under vacuum in 0, 10, and 30 min, and 24 h, respectively, after mixing. Relatively large particles are attributed to Ag nanoparticles, and rather small ones are Rh nanoparticles. The TEM photographs showed that Rh particles gathered around Ag particle to surround within several minutes, comparable period of the extinction of plasmon absorption. Interestingly, these aggregated particles changed into homogeneous small particles (average diameter =2.7 nm) after 24 h. Preliminary study has shown that the

0 min 10 min 24 h Fig. 3. TEM photographs of the physical mixtures of Ag and Rh nanoparticles. The aqueous solutions of Ag and Rh nanoparticles (1/1, atom-mol/atom-mol) were mixed, and dried

Fig. 2. UV-Vis spectral change of the physical mixtures of dispersions of Ag and Rh

**2.1.2 Transmission electron microgram of the siver-core bimetallic nanoparticles**

Hirakawa, 2003), the removal of reactive oxygen species (Hirakawa & Sano, 2009), and its application to the chemoprevention of ultraviolet induced biomolecules damage (Hirakwa et al., 2008, 2009).

## **2. Spontaneous formation of silver-core bimetallic nanoparticles**

Much attention has been paid to bimetallic nanoparticles, especially those having a core/shell structure (Toshima et al., 2007). From the view point of Au catalysts, bimetallic nanoparticles have received much attention recently. On the other hand, a physical mixture of monometallic nanoparticles such as Pt and Ru nanoparticles in solution shows higher catalytic activity than the corresponding monometallic nanoparticles under a certain condition (Toshima et al., 1995; Toshima & Hirakawa, 1997). Further, it has been reported that physical mixture of Ag and other metal nanoparticles, such as Pt, Rh, and Pd, spontaneously forms the bimetallic nanoparticles with Ag-core structure in aqueous solution (Figure 1). In this section, the spontaneous formation of the Ag-core bimetallic nanoparticles is reviewed.

## **2.1 Siver-core/rhodium-shell bimetallic nanoparticles**

The interaction between Ag and Rh monometallic nanoparticles in solution by physical mixing was reported. The main reason for using Ag and Rh nanoparticles is the reported prominent characteristics of Rh nanoparticles as a catalyst (Toshima & Hirakawa, 1999), and the expected electronic effect of Ag similar to Au upon enhancement of the catalytic activity of Rh. Furthermore, Ag is inexpensive metal compared with Au. The colloidal dispersions of Ag and Rh monometallic nanoparticles protected by poly(*N*-vinyl-2-pyrrolidone) (PVP), a water soluble polymer, were prepared by an alcohol reduction method (Hirai et al., 1979). Average diameters of Ag and Rh monometallic nanoparticles were 7.5 nm and 2.2 nm, respectively.

### **2.1.1 Surface plasmon absorption of siver-core bimetallic nanoparticles**

Colloidal sol of Ag nanoparticles shows characteristic plasmon absorption aeound 400 nm (Henglein, 1979). The plasmon absorption band of Ag nanoparticles decreased by addition of Rh nanoparticles and was almost completely extinguished within 30 min after mixing (Figure 2). The parts of plasmon absorption in larger wavelength region were preferentially

Hirakawa, 2003), the removal of reactive oxygen species (Hirakawa & Sano, 2009), and its application to the chemoprevention of ultraviolet induced biomolecules damage (Hirakwa

Much attention has been paid to bimetallic nanoparticles, especially those having a core/shell structure (Toshima et al., 2007). From the view point of Au catalysts, bimetallic nanoparticles have received much attention recently. On the other hand, a physical mixture of monometallic nanoparticles such as Pt and Ru nanoparticles in solution shows higher catalytic activity than the corresponding monometallic nanoparticles under a certain condition (Toshima et al., 1995; Toshima & Hirakawa, 1997). Further, it has been reported that physical mixture of Ag and other metal nanoparticles, such as Pt, Rh, and Pd, spontaneously forms the bimetallic nanoparticles with Ag-core structure in aqueous solution (Figure 1). In this section, the spontaneous formation of the Ag-core bimetallic

Fig. 1. Schematic diagram of the spontaneous formation of Ag-core bimetallic nanoparticles

Self-aggregation <sup>X</sup>

Ag-core/X-shell nanoparticles

The interaction between Ag and Rh monometallic nanoparticles in solution by physical mixing was reported. The main reason for using Ag and Rh nanoparticles is the reported prominent characteristics of Rh nanoparticles as a catalyst (Toshima & Hirakawa, 1999), and the expected electronic effect of Ag similar to Au upon enhancement of the catalytic activity of Rh. Furthermore, Ag is inexpensive metal compared with Au. The colloidal dispersions of Ag and Rh monometallic nanoparticles protected by poly(*N*-vinyl-2-pyrrolidone) (PVP), a water soluble polymer, were prepared by an alcohol reduction method (Hirai et al., 1979). Average diameters of Ag and Rh monometallic nanoparticles were 7.5 nm and 2.2 nm,

Colloidal sol of Ag nanoparticles shows characteristic plasmon absorption aeound 400 nm (Henglein, 1979). The plasmon absorption band of Ag nanoparticles decreased by addition of Rh nanoparticles and was almost completely extinguished within 30 min after mixing (Figure 2). The parts of plasmon absorption in larger wavelength region were preferentially

**2.1.1 Surface plasmon absorption of siver-core bimetallic nanoparticles**

**2.1 Siver-core/rhodium-shell bimetallic nanoparticles**

**Ag**

X: for example Rh, Pt, Pd

**X**

**2. Spontaneous formation of silver-core bimetallic nanoparticles**

et al., 2008, 2009).

nanoparticles is reviewed.

respectively.

extinguished within 10 min, suggesting that influences of Rh nanopartilces on Ag nanoparticles depend on the size of the Ag nanoparticles. When relatively smaller molar quantity of Rh to Ag was added, the plasmon absorption was not completely extinguished. More than 40 atom-mol% of Rh against to Ag was required to extinguish the plasmon absorption band completely.

Fig. 2. UV-Vis spectral change of the physical mixtures of dispersions of Ag and Rh nanoparticles. The aqueous solutions of Ag (1 atom-mmol L-1, 50 mL) and Rh (1 atom-mmol L-1, 50 mL) nanoparticles were mixed.

## **2.1.2 Transmission electron microgram of the siver-core bimetallic nanoparticles**

Figure 3 shows transmission electron microscopy (TEM) photographs of the physical mixtures of Ag and Rh monometallic nanoparticles. The samples for TEM measurement were prepared by drying the aqueous dispersions of the physical mixtures of Ag and Rh nanoparticles under vacuum in 0, 10, and 30 min, and 24 h, respectively, after mixing. Relatively large particles are attributed to Ag nanoparticles, and rather small ones are Rh nanoparticles. The TEM photographs showed that Rh particles gathered around Ag particle to surround within several minutes, comparable period of the extinction of plasmon absorption. Interestingly, these aggregated particles changed into homogeneous small particles (average diameter =2.7 nm) after 24 h. Preliminary study has shown that the

Fig. 3. TEM photographs of the physical mixtures of Ag and Rh nanoparticles. The aqueous solutions of Ag and Rh nanoparticles (1/1, atom-mol/atom-mol) were mixed, and dried after indicated periods.

Self-Organization of Silver-Core Bimetallic

**2.2 Silver-core/noble metal-shell bimatallic nanoparticles**

**2.3 Application to the preparation of trimetallic nanoparticles**

the order of Rh/Ag > Pd/Ag > Pt/Ag.

nanoparticles.

photocatalyst.

Nanoparticles and Their Application for Catalytic Reaction 71

required to completely extinguish the plasmon absorption, which is reasonably supporting the above assumption. These observations suggest that the physical mixture of Ag and Rh nanoparticles spontaneously generates Ag/Rh bimetallic nanoparticles with an Agcore/Rh-shell structure. The disappearance of the XRD peak of Ag nanoparticles suggests that the core of this bimetallic nanoparticles is not complete Ag, but possibly has a partial Ag/Rh alloy structure. The driving force of the formation of this Ag/Rh bimetallic nanoparticles may be due to the larger binding energy between Ag and Rh atoms than between Rh atoms (Peiner & Kopitzki, 1998). Reduction of diameter of the nanoparticle increases not only its surface energy but also number of the binding sites between Ag and Rh atoms, which stabilizes the total energy. Therefore, the shrinking of Ag/Rh bimetallic nanoparticles might be explained by the balance between the binding energy and the surface energy. The size and the rate of formation of the bimetallic nanoparticles can be controlled by the kind and concentration of protective agents. The self-assembling formation of bimetallic nanoparticle using Ag nanoparticle is applicable to construction of novel

The above mentioned procedure can be used to prepare the Ag-core/noble metal shell nanoparticles, other than Ag-core/Rh-shell nanoparticles. The physical mixing of Ag and other metal nanoparticles, such as Au, Pt, Rh, and Pd particles, produces Ag-core bimetallic particles. The interaction rate between Ag and other metal nanoparticles was determined by the extinction of the surface plasmon absorption of Ag nanoparticle. The initial step of this reaction was investigated by isothermal titration calorimetry (Toshima et al., 2005). This study revealed that the strength of the interaction between Ag and other metals increases in

The formed Ag-core/Pt-shell nanoparticle catalyzed the decomposition of hydrogen peroxide (described later). On the other hand, Au and Au/Ag nanoparticles showed an activity of photocatalytic decomposition of methylene blue (Hirakawa, 2007), although their activities were significantly smaller than that of well-known titanium dioxide photocatalyst (Fujishima et al., 2000, 2008). The physical mixing method is simple and useful to prepare novel bimetallic nanoparticles. These nanoparticles may be used as catalyst and

This method can be applied to the preparation of trimetallic nanoparticles (Toshima et al., 2007, 2011). It has been reported that the synthesis of trimetallic nanoparticles having a Aucore structure by a combination of the preparation of bimetallic nanoparticles by coreduction with the formation of core/shell-structured bimetallic nanoparticles by selforganization in physical mixture (Figure 5). The formation of trimetallic nanoparticles has been suggested by UV–Vis spectral change, TEM image change, FT-IR spectra of adsorbed carbon monoxide, XPS spectra and calorimetric studies. The catalytic activity of trimetallic nanoparticles in the molar ratio of Au/Pd/Rh = 1/4/20 was higher than the corresponding monometallic and bimetallic nanoparticles for hydrogenation of methyl acrylate. This high catalytic activity can be understood by sequential electronic charge transfer from surface Rh

atoms to interlayered Pt atoms and then to core Au atoms (Toshima et al., 2011).

increase of Rh/Ag molar ratio reduces the average diameter and the size distribution of the nanoparticles. The elemental analysis using characteristic X-ray in high-resolution TEM measurement has shown that the particles produced from their physical mixtures in 24 h are composed of Ag and Rh.

#### **2.1.3 X-ray diffraction of the of siver-core bimetallic nanoparticles**

Figure 4 shows X-ray diffraction (XRD) patterns of poly(*N*-vinyl-2-pyrrolidone)-protected Ag and Rh monometallic nanoparticles, and their physical mixture. The sample of the physical mixture of Ag and Rh nanoparticles was prepared by drying the mixtures of their aqueous solutions under vacuum for 24 h after mixing. The XRD pattern of the mixtures of Ag and Rh nanoparticles was similar to that of Rh nanoparticle, suggesting that the surface of the particle produced by mixing Ag and Rh nanoparticles is composed of Rh. Similarly, Au-core/Pt-shell and Au-core/Pd-shell structured nanoparticles have shown the XRD pattern quite similar to that of their surface metals (Yonezawa & Toshima, 1995). These findings suggest that the aggregation of Rh particles around the Ag particle is involved in the extinction of the plasmon absorption.

#### **2.1.4 Mechanism of the formation of the siver-core/rhodium shell bimetallic nanoparticles**

Henglein *et al*. reported that lead (Pb) atoms transfer from Pb colloidal particle onto the surface of Ag colloidal particle in physical mixing of Ag and Pb colloidal sols (Henglein et al., 1992). If the extinction of the plasmon absorption is due to coating of the surface of Ag particle by Rh atoms transferred from Rh nanoparticle, at least 28 mol% of Rh to Ag is required assuming that a Ag particle (average diameter = 7.5 nm) is uniformly coated by Rh atoms in a one-atom layer. In the present experiments about 40 atom-mol% of Rh to Ag was

Fig. 4. XRD patterns of Ag and Rh monometallic nanoparticles, and their physical mixture (1/1, atom-mol/atom-mol)

increase of Rh/Ag molar ratio reduces the average diameter and the size distribution of the nanoparticles. The elemental analysis using characteristic X-ray in high-resolution TEM measurement has shown that the particles produced from their physical mixtures in 24 h are

Figure 4 shows X-ray diffraction (XRD) patterns of poly(*N*-vinyl-2-pyrrolidone)-protected Ag and Rh monometallic nanoparticles, and their physical mixture. The sample of the physical mixture of Ag and Rh nanoparticles was prepared by drying the mixtures of their aqueous solutions under vacuum for 24 h after mixing. The XRD pattern of the mixtures of Ag and Rh nanoparticles was similar to that of Rh nanoparticle, suggesting that the surface of the particle produced by mixing Ag and Rh nanoparticles is composed of Rh. Similarly, Au-core/Pt-shell and Au-core/Pd-shell structured nanoparticles have shown the XRD pattern quite similar to that of their surface metals (Yonezawa & Toshima, 1995). These findings suggest that the aggregation of Rh particles around the Ag particle is involved in

**2.1.3 X-ray diffraction of the of siver-core bimetallic nanoparticles**

**2.1.4 Mechanism of the formation of the siver-core/rhodium shell bimetallic**

Henglein *et al*. reported that lead (Pb) atoms transfer from Pb colloidal particle onto the surface of Ag colloidal particle in physical mixing of Ag and Pb colloidal sols (Henglein et al., 1992). If the extinction of the plasmon absorption is due to coating of the surface of Ag particle by Rh atoms transferred from Rh nanoparticle, at least 28 mol% of Rh to Ag is required assuming that a Ag particle (average diameter = 7.5 nm) is uniformly coated by Rh atoms in a one-atom layer. In the present experiments about 40 atom-mol% of Rh to Ag was

37.9˚

(1 1 1) surface of hcp structure 40.7˚

Ag

Rh

Ag + Rh

(1/1, atom-mol/atom-mol)

(1 1 1) surface of hcp structure

47.6˚

(2 1 1) surface of hcp structure

30.00 40.00 50.00 60.00 2 / ˚

Fig. 4. XRD patterns of Ag and Rh monometallic nanoparticles, and their physical mixture

composed of Ag and Rh.

**nanoparticles**

the extinction of the plasmon absorption.

required to completely extinguish the plasmon absorption, which is reasonably supporting the above assumption. These observations suggest that the physical mixture of Ag and Rh nanoparticles spontaneously generates Ag/Rh bimetallic nanoparticles with an Agcore/Rh-shell structure. The disappearance of the XRD peak of Ag nanoparticles suggests that the core of this bimetallic nanoparticles is not complete Ag, but possibly has a partial Ag/Rh alloy structure. The driving force of the formation of this Ag/Rh bimetallic nanoparticles may be due to the larger binding energy between Ag and Rh atoms than between Rh atoms (Peiner & Kopitzki, 1998). Reduction of diameter of the nanoparticle increases not only its surface energy but also number of the binding sites between Ag and Rh atoms, which stabilizes the total energy. Therefore, the shrinking of Ag/Rh bimetallic nanoparticles might be explained by the balance between the binding energy and the surface energy. The size and the rate of formation of the bimetallic nanoparticles can be controlled by the kind and concentration of protective agents. The self-assembling formation of bimetallic nanoparticle using Ag nanoparticle is applicable to construction of novel nanoparticles.

## **2.2 Silver-core/noble metal-shell bimatallic nanoparticles**

The above mentioned procedure can be used to prepare the Ag-core/noble metal shell nanoparticles, other than Ag-core/Rh-shell nanoparticles. The physical mixing of Ag and other metal nanoparticles, such as Au, Pt, Rh, and Pd particles, produces Ag-core bimetallic particles. The interaction rate between Ag and other metal nanoparticles was determined by the extinction of the surface plasmon absorption of Ag nanoparticle. The initial step of this reaction was investigated by isothermal titration calorimetry (Toshima et al., 2005). This study revealed that the strength of the interaction between Ag and other metals increases in the order of Rh/Ag > Pd/Ag > Pt/Ag.

The formed Ag-core/Pt-shell nanoparticle catalyzed the decomposition of hydrogen peroxide (described later). On the other hand, Au and Au/Ag nanoparticles showed an activity of photocatalytic decomposition of methylene blue (Hirakawa, 2007), although their activities were significantly smaller than that of well-known titanium dioxide photocatalyst (Fujishima et al., 2000, 2008). The physical mixing method is simple and useful to prepare novel bimetallic nanoparticles. These nanoparticles may be used as catalyst and photocatalyst.

## **2.3 Application to the preparation of trimetallic nanoparticles**

This method can be applied to the preparation of trimetallic nanoparticles (Toshima et al., 2007, 2011). It has been reported that the synthesis of trimetallic nanoparticles having a Aucore structure by a combination of the preparation of bimetallic nanoparticles by coreduction with the formation of core/shell-structured bimetallic nanoparticles by selforganization in physical mixture (Figure 5). The formation of trimetallic nanoparticles has been suggested by UV–Vis spectral change, TEM image change, FT-IR spectra of adsorbed carbon monoxide, XPS spectra and calorimetric studies. The catalytic activity of trimetallic nanoparticles in the molar ratio of Au/Pd/Rh = 1/4/20 was higher than the corresponding monometallic and bimetallic nanoparticles for hydrogenation of methyl acrylate. This high catalytic activity can be understood by sequential electronic charge transfer from surface Rh atoms to interlayered Pt atoms and then to core Au atoms (Toshima et al., 2011).

Self-Organization of Silver-Core Bimetallic

ratio of Au/Pt = 2/3 is the most active catalyst.

Similar results reported on the other catalytic reactions.

**3.2 Application of siver-core/rhodium-shell bimetallic nanoparticles**

0

0.2

*k*H2 / s-1

mixture of Ag and Rh monometallic nanoparticles.

**metal nanoparticle**

(Toshima et al., 1995) (Figure 8).

0.4

0.6

Nanoparticles and Their Application for Catalytic Reaction 73

the presence of water-soluble polymers and non-ionic surfactant-micelles, respectively. The UV-Vis spectra and the transmission electron micrographs suggest that the polymerprotected Au/Pt bimetallic systems are composed of bimetallic alloy clusters, but the micelle-protected ones are mostly composed of the mixtures of the monometallic Au and Pt particles. The *in-situ* UV-Vis spectra during the reductions can elucidate the formation processes of the bimetallic dispersions which are different from each other depending on the protective reagent. The Au/Pt bimetallic systems can be used as the catalyst for visible lightinduced hydrogen generation. The bimetallic system stabilized by the polymer at a molar

It has been reported that the catalytic activity of the Ag/Rh bimetallic nanoparticles for visible-light-induced hydrogen generation (Toshima & Hirakawa, 1999) in an aqueous solution composed of ethylenediaminetetraacetic acid, tris(bipyridine)ruthenium(II), methyl viologen, and metal nanoparticle catalyst. The activity is clearly higher than the corresponding monometallic nanoparticles and alloy-structured Ag/Rh nanoparticles, suggesting that the Ag-core shows an electronic effect on the surface Rh as in the case of the Au-core (Yonezawa & Toshima, 1993) and enhances the catalytic activity of the surface Rh. The highest catalytic activity was observed at 1:9 ratio of Ag and Rh atoms (Figure 7).

> 0 20 40 60 80 100 Molar ratio of Ag (%)

Fig. 7. Hydrogen generation rate coefficient (*k*H2) depending on the molar ratio of Ag of Ag/Rh bimetallic nanoparticles. The *k*H2 indicates the number of generated H2 molecules on a surface metal atom per one second. The average is the calculated activity of the simple

**3.3 Carbon dioxide reduction by visible-light-induced electron transfer system using**

A photochemical reduction of CO2 can be applied to a novel energy storage process for the utilization of solar energy in the future. The above mentioned catalytic system can be applied to CO2 reduction. The strategy is the catalytic reduction of CO2 using electrons gathered by an electron transfer system (Willner et al., 1987, Toshima et al., 1995). It has been reported that nanoparticles catalyzes the reduction of CO2 and the generation of methane

Average

Random Alloy structure

Core-Shell structure

Fig. 5. Schematic diagram of the formation of the trimetallic Au/Pt/Rh nanoparticles

## **3. Visible-light-induced hydrogen generation by metal nanoparticle catalytic system**

Metal nanoparticles are very important materials for nanoscience and nanotechnology (Fukumi et al., 1994; Lu et al., 1999; Link et al., 1999; Sun et al., 1999; Teranishi & Miyake, 1999; Akinaga, 2002). A particularly large number of reports have been published on their applications to catalysts (Kiely et al., 1998; Pileni, 1998; Bradley, 1994; Widegren & Finke 2003; Willner et al., 1987; Toshima et al., 1995; Yonezawa & Toshima, 1993). As the catalyst in the homogeneous system, the colloidal dispersions of metal nanoparticles have the advantage that they are soluble or homogeneous in an aqueous solution and transparent to visible light (Kiely et al., 1998; Pileni, 1998; Bradley, 1994; Widegren & Finke 2003; Willner et al., 1987). Thus, colloidal metal nanoparticles are useful for photocatalytic reaction systems. For example, colloidal metal nanoparticles catalyze the water reduction in the visible-lightinduced electron transfer system composed of ethylenediaminetetraacetic acid disodium salt (EDTA), tris(bipyridine)ruthenium(II) dichloride ([Ru(bpy)3]2+), and 1,1'-dimethyl-4,4' bipyridium dichloride (methyl viologen, MV2+) (Yonezawa & Toshima, 1993) (Figure 6).

Fig. 6. Schematic diagram of the visible-light induced hydrogen generation using the electron transfer system and metal nanoparticle catalyst

#### **3.1 Catalytic activity of gold-core/platinum-shell bimetallic nanoparticles**

The bimetallization of metal nanoparticle can improve the catalytic activity of surface metal. Especially, core-shell structured nanoparticles are important. Several study demonstrated the Au-core/Pt shell metal nanoparticles show higher catalytic activity for the visible-lightinduced hydrogen generation than Pt monometallic nanoparticles. The following study is an example of the hydrogen generation using Au/Pt nanoparticle catalyst (Yonezawa & Toshima, 1993). In this study, the Au/Pt bimetallic systems stabilized by polymer and micelle were obtained by alcohol- and photo-reduction of the corresponding metal ions in

HAuCl4 + H2PtCl6 gold ion platinum ion

rhodium ion

**system**

Electron Donor

Oxidized Product

*h* **Rh**

RhCl3 *d* = 2.9 nm

**3. Visible-light-induced hydrogen generation by metal nanoparticle catalytic**

Metal nanoparticles are very important materials for nanoscience and nanotechnology (Fukumi et al., 1994; Lu et al., 1999; Link et al., 1999; Sun et al., 1999; Teranishi & Miyake, 1999; Akinaga, 2002). A particularly large number of reports have been published on their applications to catalysts (Kiely et al., 1998; Pileni, 1998; Bradley, 1994; Widegren & Finke 2003; Willner et al., 1987; Toshima et al., 1995; Yonezawa & Toshima, 1993). As the catalyst in the homogeneous system, the colloidal dispersions of metal nanoparticles have the advantage that they are soluble or homogeneous in an aqueous solution and transparent to visible light (Kiely et al., 1998; Pileni, 1998; Bradley, 1994; Widegren & Finke 2003; Willner et al., 1987). Thus, colloidal metal nanoparticles are useful for photocatalytic reaction systems. For example, colloidal metal nanoparticles catalyze the water reduction in the visible-lightinduced electron transfer system composed of ethylenediaminetetraacetic acid disodium salt (EDTA), tris(bipyridine)ruthenium(II) dichloride ([Ru(bpy)3]2+), and 1,1'-dimethyl-4,4' bipyridium dichloride (methyl viologen, MV2+) (Yonezawa & Toshima, 1993) (Figure 6).

MV2+

MV•+

**Metal**

2H+

H2

Chemical reduction

Fig. 5. Schematic diagram of the formation of the trimetallic Au/Pt/Rh nanoparticles

[Ru(bpy)3]2+

[Ru(bpy)3]3+

electron transfer system and metal nanoparticle catalyst

Fig. 6. Schematic diagram of the visible-light induced hydrogen generation using the

The bimetallization of metal nanoparticle can improve the catalytic activity of surface metal. Especially, core-shell structured nanoparticles are important. Several study demonstrated the Au-core/Pt shell metal nanoparticles show higher catalytic activity for the visible-lightinduced hydrogen generation than Pt monometallic nanoparticles. The following study is an example of the hydrogen generation using Au/Pt nanoparticle catalyst (Yonezawa & Toshima, 1993). In this study, the Au/Pt bimetallic systems stabilized by polymer and micelle were obtained by alcohol- and photo-reduction of the corresponding metal ions in

**3.1 Catalytic activity of gold-core/platinum-shell bimetallic nanoparticles**

*d* = 2.3 nm

**Au Pt**

**Rh** *<sup>d</sup>* = 3.6 nm

Physical mixing

**Au**

**Pt**

the presence of water-soluble polymers and non-ionic surfactant-micelles, respectively. The UV-Vis spectra and the transmission electron micrographs suggest that the polymerprotected Au/Pt bimetallic systems are composed of bimetallic alloy clusters, but the micelle-protected ones are mostly composed of the mixtures of the monometallic Au and Pt particles. The *in-situ* UV-Vis spectra during the reductions can elucidate the formation processes of the bimetallic dispersions which are different from each other depending on the protective reagent. The Au/Pt bimetallic systems can be used as the catalyst for visible lightinduced hydrogen generation. The bimetallic system stabilized by the polymer at a molar ratio of Au/Pt = 2/3 is the most active catalyst.

## **3.2 Application of siver-core/rhodium-shell bimetallic nanoparticles**

It has been reported that the catalytic activity of the Ag/Rh bimetallic nanoparticles for visible-light-induced hydrogen generation (Toshima & Hirakawa, 1999) in an aqueous solution composed of ethylenediaminetetraacetic acid, tris(bipyridine)ruthenium(II), methyl viologen, and metal nanoparticle catalyst. The activity is clearly higher than the corresponding monometallic nanoparticles and alloy-structured Ag/Rh nanoparticles, suggesting that the Ag-core shows an electronic effect on the surface Rh as in the case of the Au-core (Yonezawa & Toshima, 1993) and enhances the catalytic activity of the surface Rh. The highest catalytic activity was observed at 1:9 ratio of Ag and Rh atoms (Figure 7). Similar results reported on the other catalytic reactions.

Fig. 7. Hydrogen generation rate coefficient (*k*H2) depending on the molar ratio of Ag of Ag/Rh bimetallic nanoparticles. The *k*H2 indicates the number of generated H2 molecules on a surface metal atom per one second. The average is the calculated activity of the simple mixture of Ag and Rh monometallic nanoparticles.

#### **3.3 Carbon dioxide reduction by visible-light-induced electron transfer system using metal nanoparticle**

A photochemical reduction of CO2 can be applied to a novel energy storage process for the utilization of solar energy in the future. The above mentioned catalytic system can be applied to CO2 reduction. The strategy is the catalytic reduction of CO2 using electrons gathered by an electron transfer system (Willner et al., 1987, Toshima et al., 1995). It has been reported that nanoparticles catalyzes the reduction of CO2 and the generation of methane (Toshima et al., 1995) (Figure 8).

Self-Organization of Silver-Core Bimetallic

reduction of 12CO2 originated from EDTA.

is not miscible with liposome in water.

reduction.

**3.3.3 Liposome-protected metal nanoparticle catalyst**

**3.3.2 Methane generation from carbon dioxide reduction**

Nanoparticles and Their Application for Catalytic Reaction 75

The formation of methane was then clearly detected by gas chromatography (about 19 nmol in the case of the liposome protected Pt nanoparticles system). In order to confirm the methane generation from CO2, isotope experiments were carried out using NaH13CO3 as a CO2 source and analyzed by a gas chromatograph mass-spectrometer. Since NaHCO3 is equilibrated with CO2 in solution and easily treated, it was a good source of CO2 in the present experiments. In this experiment, 13CH4 was clearly detected, though the produced methane was not pure 13CH4 and it did contain 12CH4. In the same experiment, the mole ratio of 13CO2 to 12CO2 in the gas phase was about 57:43, which is nearly the same as the isotopic ratio of the generated methane. EDTA works as an electron donor in the system and is known to decompose into CO2. Therefore, 12CH4 generation possibly occurs through the reduction of 12CO2 generated from EDTA. The effect of EDTA on methane generation was examined in the Pt-liposome system. Methane was detected on visible-light irradiation of the system involving EDTA without CO2 or NaHCO3 but could not be detected in the absence of EDTA. These results suggest that the detected 12CH4 is generated by the

Liposome was better than other protective-colloid of Pt nanoparticles for methane generation. This is probably explained by assuming that liposome can form a larger and stronger hydrophobic region to concentrate CO2 around a Pt nanoparticle than C12EO micelle and poly(*N*-vinyl-2-pyrrolidone). In addition, Ru-C12EO showed higher catalytic activity than Pt-C12EO. Thus, Ru-liposome was considered to be an active catalyst for methane generation in the system tested here. The synthesis of Ru-liposome was tried in a way similar to that of Pt-liposome, but the suspension of the Ru-liposome was not active as a catalyst. The resulting Ru-liposome was not as homogeneous, probably because the Ru ion

**3.3.4 Summary of the carbon dioxide photo-reduction by metal nanoparticle catalyst**  The Pt and Ru nanoparticle catalysts, which were prepared by a photoreduction method of metal salt in water without ethanol, successfully generated methane from CO2. The methane generation suggests that the eight-electron reduction of CO2 easily proceeds on metal nanoparticles possibly due to a thermodynamic advantage. This is different from an electrochemical CO2 reduction using Pt electrodes, on which CO2 is reduced to CO with adsorbed hydrogen atoms. In the present system using metal nanoparticles, the competition reaction, i.e., the kinetically favorably hydrogen generation, inhibits the methane generation. An increase of CO2 concentration, the electron supply rate, or both may enhance CO2

**4. Catalytic decomposition of hydrogen peroxide by metal nanoparticle**

The modification of biomacromolecules upon exposure to reactive oxygen species, including hydrogen peroxide (H2O2), dioxide(1-) (superoxide O2•-), hydroxyl radical (HO•), and singlet oxygen (1O2), is the likely initial event involved in the induction of the mutagenic and lethal effects of various oxidative stress agents (Kawanishi et al. 2001; Cadet et al., 2003;

Fig. 8. Schematic diagram of the visible-light induced CO2 reduction using the electron transfer system and metal nanoparticle catalyst

The possible reaction scheme of the CO2 reduction is as follows:

$$\rm{CO\_2 + 8H^+ + 8e^- \to CH\_4 + 2H\_2O} \tag{1}.$$

This eight-electron reduction of CO2 is advantageous process compared with other possible CO2 reduction process from the thermodynamic point of view. Although it is not a study using the silver–core bimetallic nanoprticles, this topic is closely related to the applications of bimetallic nanoparticles to catalytic reaction. Thus, the topic of the CO2 reduction using metal nanoparticle catalyst is presented here.

Typical reactions were performed by the similar manner to the hydrogen generation. A 20 cm3 Pyrex Schlenk tube was charged with a 10 cm3 aqueous solution, containing EDTA (a sacrificial electron donor), [Ru(bpy)3]2+ (a photosensitizer), MV2+ (an electron mediator), NaHCO3 (a pH adjuster and a CO2 source), and colloidal dispersion of metal nanoparticles. The mixtures were degassed by freeze-thaw cycles and the tubes were then filled with 1 atm of CO2. The photo-irradiation was carried out for 3 or 4 h with a 500 W super-high-pressure mercury lamp through a UV cut filter (> 390 nm) in a water bath maintained at 30 C. About 100 μmol of methane was detected in this system (Toshima et al., 1995). However, it has not been confirmed that methane was actually the reduction product of CO2.

#### **3.3.1 Strategy for the demonstration of the methane generation from carbon dioxide**

In a heterogeneous system, photoreduction of CO2 was confirmed by experiments using an isotope (Ishitani et al. 1993). To our knowledge, however, the isotopic method has not been applied to the confirmation of the photoreduction of CO2 in a homogeneous system using the colloidal dispersion of metal nanoparticles. To confirm the above mentioned methane generation, the following study was carried out. In this study, photoreduction of CO2 was carried out in a similar system to one reported previously (Toshima et al., 1995), and the generation of methane from CO2 was confirmed by isotopic experiments. As the catalysts, novel metal nanoparticles, *i.e*., liposome-protected Pt nanoparticles, were prepared and used in the present system. Colloidal dispersions of Pt and Ru nanoparticles were prepared by photoreduction without using ethanol (Yamaji et al. 1995). Preparation of nanoparticles without ethanol is required, because the coexisting ethanol is decomposed during the photochemical reaction, leading to the formation methane. This methane formation cannot be distinguished from the actual methane generation from CO2. Protective agents used for the metal nanoparticles were poly(*N*-vinyl-2-pyrrolidone), C12EO, and liposome. The products in the gas phase were analyzed with a gas chromatograph. The characterization of gaseous products was carried out with a gas chromatograph mass-spectrometer.

MV2+

MV•+

**Metal**

CO2 + H+

CH4 + H2

[Ru(bpy)3]

*h*

Electron Donor

Oxidized Product

[Ru(bpy)3]

The possible reaction scheme of the CO2 reduction is as follows:

transfer system and metal nanoparticle catalyst

metal nanoparticle catalyst is presented here.

2+

3+

been confirmed that methane was actually the reduction product of CO2.

Fig. 8. Schematic diagram of the visible-light induced CO2 reduction using the electron

 CO2 + 8H+ + 8e- → CH4 + 2H2O (1). This eight-electron reduction of CO2 is advantageous process compared with other possible CO2 reduction process from the thermodynamic point of view. Although it is not a study using the silver–core bimetallic nanoprticles, this topic is closely related to the applications of bimetallic nanoparticles to catalytic reaction. Thus, the topic of the CO2 reduction using

Typical reactions were performed by the similar manner to the hydrogen generation. A 20 cm3 Pyrex Schlenk tube was charged with a 10 cm3 aqueous solution, containing EDTA (a sacrificial electron donor), [Ru(bpy)3]2+ (a photosensitizer), MV2+ (an electron mediator), NaHCO3 (a pH adjuster and a CO2 source), and colloidal dispersion of metal nanoparticles. The mixtures were degassed by freeze-thaw cycles and the tubes were then filled with 1 atm of CO2. The photo-irradiation was carried out for 3 or 4 h with a 500 W super-high-pressure mercury lamp through a UV cut filter (> 390 nm) in a water bath maintained at 30 C. About 100 μmol of methane was detected in this system (Toshima et al., 1995). However, it has not

**3.3.1 Strategy for the demonstration of the methane generation from carbon dioxide**  In a heterogeneous system, photoreduction of CO2 was confirmed by experiments using an isotope (Ishitani et al. 1993). To our knowledge, however, the isotopic method has not been applied to the confirmation of the photoreduction of CO2 in a homogeneous system using the colloidal dispersion of metal nanoparticles. To confirm the above mentioned methane generation, the following study was carried out. In this study, photoreduction of CO2 was carried out in a similar system to one reported previously (Toshima et al., 1995), and the generation of methane from CO2 was confirmed by isotopic experiments. As the catalysts, novel metal nanoparticles, *i.e*., liposome-protected Pt nanoparticles, were prepared and used in the present system. Colloidal dispersions of Pt and Ru nanoparticles were prepared by photoreduction without using ethanol (Yamaji et al. 1995). Preparation of nanoparticles without ethanol is required, because the coexisting ethanol is decomposed during the photochemical reaction, leading to the formation methane. This methane formation cannot be distinguished from the actual methane generation from CO2. Protective agents used for the metal nanoparticles were poly(*N*-vinyl-2-pyrrolidone), C12EO, and liposome. The products in the gas phase were analyzed with a gas chromatograph. The characterization of

gaseous products was carried out with a gas chromatograph mass-spectrometer.

## **3.3.2 Methane generation from carbon dioxide reduction**

The formation of methane was then clearly detected by gas chromatography (about 19 nmol in the case of the liposome protected Pt nanoparticles system). In order to confirm the methane generation from CO2, isotope experiments were carried out using NaH13CO3 as a CO2 source and analyzed by a gas chromatograph mass-spectrometer. Since NaHCO3 is equilibrated with CO2 in solution and easily treated, it was a good source of CO2 in the present experiments. In this experiment, 13CH4 was clearly detected, though the produced methane was not pure 13CH4 and it did contain 12CH4. In the same experiment, the mole ratio of 13CO2 to 12CO2 in the gas phase was about 57:43, which is nearly the same as the isotopic ratio of the generated methane. EDTA works as an electron donor in the system and is known to decompose into CO2. Therefore, 12CH4 generation possibly occurs through the reduction of 12CO2 generated from EDTA. The effect of EDTA on methane generation was examined in the Pt-liposome system. Methane was detected on visible-light irradiation of the system involving EDTA without CO2 or NaHCO3 but could not be detected in the absence of EDTA. These results suggest that the detected 12CH4 is generated by the reduction of 12CO2 originated from EDTA.

## **3.3.3 Liposome-protected metal nanoparticle catalyst**

Liposome was better than other protective-colloid of Pt nanoparticles for methane generation. This is probably explained by assuming that liposome can form a larger and stronger hydrophobic region to concentrate CO2 around a Pt nanoparticle than C12EO micelle and poly(*N*-vinyl-2-pyrrolidone). In addition, Ru-C12EO showed higher catalytic activity than Pt-C12EO. Thus, Ru-liposome was considered to be an active catalyst for methane generation in the system tested here. The synthesis of Ru-liposome was tried in a way similar to that of Pt-liposome, but the suspension of the Ru-liposome was not active as a catalyst. The resulting Ru-liposome was not as homogeneous, probably because the Ru ion is not miscible with liposome in water.

## **3.3.4 Summary of the carbon dioxide photo-reduction by metal nanoparticle catalyst**

The Pt and Ru nanoparticle catalysts, which were prepared by a photoreduction method of metal salt in water without ethanol, successfully generated methane from CO2. The methane generation suggests that the eight-electron reduction of CO2 easily proceeds on metal nanoparticles possibly due to a thermodynamic advantage. This is different from an electrochemical CO2 reduction using Pt electrodes, on which CO2 is reduced to CO with adsorbed hydrogen atoms. In the present system using metal nanoparticles, the competition reaction, i.e., the kinetically favorably hydrogen generation, inhibits the methane generation. An increase of CO2 concentration, the electron supply rate, or both may enhance CO2 reduction.

## **4. Catalytic decomposition of hydrogen peroxide by metal nanoparticle**

The modification of biomacromolecules upon exposure to reactive oxygen species, including hydrogen peroxide (H2O2), dioxide(1-) (superoxide O2•-), hydroxyl radical (HO•), and singlet oxygen (1O2), is the likely initial event involved in the induction of the mutagenic and lethal effects of various oxidative stress agents (Kawanishi et al. 2001; Cadet et al., 2003;

Self-Organization of Silver-Core Bimetallic

*σ*= 2.0 nm).

oxygen as follows:

colloidal dispersion was purified with an ultra-filter.

Reactive Oxygen Species

**4.2.2 Method of the detection of hydrogen peroxide**

Folic acid H2O2

Fig. 9. Fluorometry of reactive oxygen species (hydrogen peroxide) using folic acid

measured using a fluorescence spectrophotometer with 350-nm excitation.

**4.2.3 Platinum nanoparticles effectively scavenge hydrogen peroxide**

The generated H2O2 was measured by a previously reported method using folic acid (Hirakawa, 2006). This assay is based on the fluorescence enhancement of less-fluorescent folic acid via oxidative decomposition by H2O2 and copper(II) ion into strong-fluorescent 2 amino-4-oxo-3*H*-pterine-6-carboxylic acid (Figure 9). The concentration of H2O2 ([H2O2]) can be determined using a calibration curve. A reaction mixture containing folic acid, copper(II) chloride, and the H2O2 sample (or H2O2 generator 4) with or without the metal nanoparticle in a sodium phosphate buffer (pH 7.6) was incubated in a microtube for 30 min. After incubation at 37 C, the fluorescence intensity of the reaction mixture at 450 nm was

Platinum nanoparticles effectively scavenged H2O2 in a dose-dependent manner and showed the highest activity among the metal nanoparticles used in this study (Figure 10). A sample solution of 5 μM/atom Pt nanoparticles, among which 1 μg Pt metal is included, exhibits comparable activity for H2O2 decomposition to that of 10 units of catalase. One unit of catalase can remove 1.0 μmol H2O2 per min in water (pH 7.0, 25 C). Poly(*N*-vinyl-2 pyrrolidone) itself did not scavenge H2O2. This experiment confirmed that poly(*N*-vinyl-2 pyrrolidone)-protected Pt nanoparticles can remove H2O2. The mechanism of H2O2 removal by Pt nanoparticles can be explained by catalytic decomposition into water and molecular

Nanoparticles and Their Application for Catalytic Reaction 77

nanoparticles (Shiraishi & Toshima, 1999) was prepared from reduction of 1 mM AgNO3 with NaBH4 in the presence of 40 mM poly(*N*-vinyl-2-pyrrolidone). The obtained Ag

These poly(*N*-vinyl-2-pyrrolidone)-protected metal nanoparticles formed water-soluble sols. The average diameters (*d*) and standard deviations (*σ*) of monometallic nanoparticles determined by TEM measurement were as follows: Pt (*d* = 2.2 nm, *σ*= 1.0 nm), Pd (*d* = 2.0 nm, *σ*= 0.9 nm), Rh (*d* = 2.2 nm, *σ*= 1.0 nm), Ag (*d* = 10.0 nm, *σ*=1.9 nm), and Au (*d* = 10.2 nm,

**OFF**

Wavelength / nm

350 450 550

**ON**

Intensity

0

100

200

300

400

Fluorescence

Drechsel & Patel, 2008). Therefore, the activity of reactive oxygen species generation by various chemical compounds is closely related to their toxicity, carcinogenicity, or both. For example, hydroquinone, a metabolite of carcinogenic benzene, causes DNA damage via H2O2 generation (Hirakawa et al., 2002). Many studies have addressed the role of antioxidants, such as vitamins (Slaga, 1995; Sohmiya et al., 2004) and catechins (Weyant et al., 2001), in protection against cancers and cardiovascular diseases. These antioxidants can scavenge reactive oxygen species and protect against cancer occurrence. On the other hand, every antioxidant is in fact, a redox agent, protecting against reactive oxygen species in some circumstances and promoting free radical or secondary reactive oxygen species generation in others. Indeed, an excess of these antioxidants elevates the incidence of cancer (Nitta et al. 1991; Omenn et al., 1996). Solovieva et al. reported that antioxidants, ascorbic acid (Solovieva et al., 2007) and dithiothreitol (Solovieva et al., 2008), exhibit cytotoxicity via H2O2 generation. Relevantly, it has been reported that vitamins A (Murata & Kawanishi, 2000) and E (Yamashita et al., 1998) and catechins (Oikawa et al., 2003) induce DNA oxidation through H2O2 generation during their oxidation. H2O2 is a long-lived reactive oxygen species which plays an important role in biomacromolecular damage induced by various chemical compounds (Kawanishi et al., 2001; Hirakawa et al., 2002).

## **4.1 Metal catalyzes decomposition of hydrogen peroxide**

Various studies have demonstrated the catalytic decomposition of H2O2 by noble metals such as Pt (Keating et al., 1965; McKee, 1969; Bianchi et al., 1962), Pd (Keating et al., 1965; McKee, 1969; Bianchi et al., 1962; Eley & Macmahon, 1972) Ag (Baumgartner et al., 1963; Goszner et al., 1972; Goszner & Bischof, 1974), and Au (Eley & Macmahon, 1972; Goszner & Bischof, 1974). These metals themselves are hardly oxidized by reactive oxygen species, however, it is difficult to use metal powder or foils as anti-oxidative drugs. Recently, Kajita et al. reported that Pt nanoparticles catalyze the decomposition of reactive oxygen species (Kajita et al., 2007). These nanoparticles can be dispersed in water and used as homogenous solutions. Because this removal mechanism is catalytic decomposition, no oxidized product is formed through this reaction. Platinum metal is used as a food additive and is not considered to be a toxic material. This result led us to the idea that inorganic materials, in particular noble metals, rather than organic antioxidants, can be used as novel chemopreventive agents against reactive oxygen species-mediated biomolecules damage. In this section, the examination of the removal of H2O2 generated from a chemical compound, hydroquione, using water-soluble polymer-protected Pt and Ag/Pt nanoparticles are reviewed.

## **4.2 Catalytic activity of monometallic nanoparticles**

## **4.2.1 Preparation of metal nanoparticles for reactive oxygen scavenger**

Colloidal dispersions of poly(*N*-vinyl-2-pyrrolidone)-protected Pt, Pd, Rh, and Au nanoparticles were prepared using an alcohol reduction method (Hirai et al., 1979). 50 mL of water/ethanol (1/1, v/v) solution containing 1 mM metal salts and 40 mM poly(*N*-vinyl-2 pyrrolidone) (monomer unit) was refluxed for 2 h, resulting in the formation of typical colored sols of metal nanoparticles. The solvent was removed by vacuum evaporation, and the nanoparticles were dispersed into water to prepare 1 mM/atom (atomic concentration) metal colloidal sols. An aqueous solution of poly(*N*-vinyl-2-pyrrolidone)-protected Ag

Drechsel & Patel, 2008). Therefore, the activity of reactive oxygen species generation by various chemical compounds is closely related to their toxicity, carcinogenicity, or both. For example, hydroquinone, a metabolite of carcinogenic benzene, causes DNA damage via H2O2 generation (Hirakawa et al., 2002). Many studies have addressed the role of antioxidants, such as vitamins (Slaga, 1995; Sohmiya et al., 2004) and catechins (Weyant et al., 2001), in protection against cancers and cardiovascular diseases. These antioxidants can scavenge reactive oxygen species and protect against cancer occurrence. On the other hand, every antioxidant is in fact, a redox agent, protecting against reactive oxygen species in some circumstances and promoting free radical or secondary reactive oxygen species generation in others. Indeed, an excess of these antioxidants elevates the incidence of cancer (Nitta et al. 1991; Omenn et al., 1996). Solovieva et al. reported that antioxidants, ascorbic acid (Solovieva et al., 2007) and dithiothreitol (Solovieva et al., 2008), exhibit cytotoxicity via H2O2 generation. Relevantly, it has been reported that vitamins A (Murata & Kawanishi, 2000) and E (Yamashita et al., 1998) and catechins (Oikawa et al., 2003) induce DNA oxidation through H2O2 generation during their oxidation. H2O2 is a long-lived reactive oxygen species which plays an important role in biomacromolecular damage induced by

various chemical compounds (Kawanishi et al., 2001; Hirakawa et al., 2002).

Various studies have demonstrated the catalytic decomposition of H2O2 by noble metals such as Pt (Keating et al., 1965; McKee, 1969; Bianchi et al., 1962), Pd (Keating et al., 1965; McKee, 1969; Bianchi et al., 1962; Eley & Macmahon, 1972) Ag (Baumgartner et al., 1963; Goszner et al., 1972; Goszner & Bischof, 1974), and Au (Eley & Macmahon, 1972; Goszner & Bischof, 1974). These metals themselves are hardly oxidized by reactive oxygen species, however, it is difficult to use metal powder or foils as anti-oxidative drugs. Recently, Kajita et al. reported that Pt nanoparticles catalyze the decomposition of reactive oxygen species (Kajita et al., 2007). These nanoparticles can be dispersed in water and used as homogenous solutions. Because this removal mechanism is catalytic decomposition, no oxidized product is formed through this reaction. Platinum metal is used as a food additive and is not considered to be a toxic material. This result led us to the idea that inorganic materials, in particular noble metals, rather than organic antioxidants, can be used as novel chemopreventive agents against reactive oxygen species-mediated biomolecules damage. In this section, the examination of the removal of H2O2 generated from a chemical compound, hydroquione, using water-soluble polymer-protected Pt and Ag/Pt nanoparticles are

**4.1 Metal catalyzes decomposition of hydrogen peroxide**

**4.2 Catalytic activity of monometallic nanoparticles**

**4.2.1 Preparation of metal nanoparticles for reactive oxygen scavenger**

Colloidal dispersions of poly(*N*-vinyl-2-pyrrolidone)-protected Pt, Pd, Rh, and Au nanoparticles were prepared using an alcohol reduction method (Hirai et al., 1979). 50 mL of water/ethanol (1/1, v/v) solution containing 1 mM metal salts and 40 mM poly(*N*-vinyl-2 pyrrolidone) (monomer unit) was refluxed for 2 h, resulting in the formation of typical colored sols of metal nanoparticles. The solvent was removed by vacuum evaporation, and the nanoparticles were dispersed into water to prepare 1 mM/atom (atomic concentration) metal colloidal sols. An aqueous solution of poly(*N*-vinyl-2-pyrrolidone)-protected Ag

reviewed.

nanoparticles (Shiraishi & Toshima, 1999) was prepared from reduction of 1 mM AgNO3 with NaBH4 in the presence of 40 mM poly(*N*-vinyl-2-pyrrolidone). The obtained Ag colloidal dispersion was purified with an ultra-filter.

These poly(*N*-vinyl-2-pyrrolidone)-protected metal nanoparticles formed water-soluble sols. The average diameters (*d*) and standard deviations (*σ*) of monometallic nanoparticles determined by TEM measurement were as follows: Pt (*d* = 2.2 nm, *σ*= 1.0 nm), Pd (*d* = 2.0 nm, *σ*= 0.9 nm), Rh (*d* = 2.2 nm, *σ*= 1.0 nm), Ag (*d* = 10.0 nm, *σ*=1.9 nm), and Au (*d* = 10.2 nm, *σ*= 2.0 nm).

Fig. 9. Fluorometry of reactive oxygen species (hydrogen peroxide) using folic acid

## **4.2.2 Method of the detection of hydrogen peroxide**

The generated H2O2 was measured by a previously reported method using folic acid (Hirakawa, 2006). This assay is based on the fluorescence enhancement of less-fluorescent folic acid via oxidative decomposition by H2O2 and copper(II) ion into strong-fluorescent 2 amino-4-oxo-3*H*-pterine-6-carboxylic acid (Figure 9). The concentration of H2O2 ([H2O2]) can be determined using a calibration curve. A reaction mixture containing folic acid, copper(II) chloride, and the H2O2 sample (or H2O2 generator 4) with or without the metal nanoparticle in a sodium phosphate buffer (pH 7.6) was incubated in a microtube for 30 min. After incubation at 37 C, the fluorescence intensity of the reaction mixture at 450 nm was measured using a fluorescence spectrophotometer with 350-nm excitation.

## **4.2.3 Platinum nanoparticles effectively scavenge hydrogen peroxide**

Platinum nanoparticles effectively scavenged H2O2 in a dose-dependent manner and showed the highest activity among the metal nanoparticles used in this study (Figure 10). A sample solution of 5 μM/atom Pt nanoparticles, among which 1 μg Pt metal is included, exhibits comparable activity for H2O2 decomposition to that of 10 units of catalase. One unit of catalase can remove 1.0 μmol H2O2 per min in water (pH 7.0, 25 C). Poly(*N*-vinyl-2 pyrrolidone) itself did not scavenge H2O2. This experiment confirmed that poly(*N*-vinyl-2 pyrrolidone)-protected Pt nanoparticles can remove H2O2. The mechanism of H2O2 removal by Pt nanoparticles can be explained by catalytic decomposition into water and molecular oxygen as follows:

Self-Organization of Silver-Core Bimetallic

0

1

2

Absorbance

Average diameter: *d* = 2.2 nm Standard Deviation:

nanoparticles 24 hour after mixing.

catalytic activity per unit atom.

**nanoparticles**

= 1.0 nm

3

4

2007).

Nanoparticles and Their Application for Catalytic Reaction 79

nanopaticles (Toshima & Hirakawa, 2003). These findings suggest the formation of selforganized Ag/Pt bimetallic nanoparticles. These metal nanoparticles are stable in water for several months. The Ag/Pt (Ag-atom/Pt-atom, 1/1) bimetallic nanoparticles were prepared using a self-organization method to mix Pt and Ag monometallic nanoparticles according to previous reports (Toshima & Hirakawa, 2003; Toshima et al., 2002, 2005; Matsushita et al.,

> 300 400 500 600 Wavelength / nm

50nm 50nm 50nm

*d* = 10.0 nm = 1.9 nm

*d* = 2.6 nm = 1.6 nm

Fig. 11. Absorption spectral change of the physical mixture of dispersions of Ag and Pt nanoparticles. The aqueous solutions of Ag (1 mM/atom, 10 mL) and Pt (1 mM/atom, 10 mL) nanoparticles were mixed and measured at 0, 10, 20, and 30 min, and 24 h after mixing.

Fig. 12. TEM photographs of metal nanoparticles. The sample of Ag/Pt nanoparticles was prepared by drying the mixtures of the aqueous solutions of Pt and Ag monometallic

**4.3.2 Hydrogen peroxide formation from hydroquinone and its removal by metal**

Hydroquinone, which is a metabolite of carcinogenic benzene, was used as H2O2 source. This compound can generate H2O2 through autooxidation (Figure 13) (Hirakwa et al., 2002). Under these experimental conditions, hydroquinone generated H2O2 in a dose-dependent manner (Figure 14). Twenty units/mL catalase effectively removed H2O2 generated from this system, and 10 μM/atom (2 μg/mL) Pt nanoparticles exhibited a comparable activity to that of this catalase. Silver nanoparticles showed apparently weaker activity for H2O2 removal than Pt nanoparticles. The bimetallization of Pt with Ag apparently suppressed the

(A) Pt (B) Ag (C) Ag/Pt

Time 0 min 10 min 20 min 30 min 24 h

$$\text{H}\_2\text{O}\_2 \to \text{H}\_2\text{O} + 1/2\,\text{O}\_2\tag{2}$$

The generation of O2 gas through the H2O2 decomposition was confirmed with a gas-burette as following procedure. The 10 mL of aqueous solution containing 0.1 M H2O2 was treated by 10 μg Pt nanoparticles and generated O2 gas was measured with a gas-burette. The volume of detected gas coincided with that of the theoretically calculated value of O2 generation from the decomposition of H2O2 in the sample solution.

Fig. 10. Removal of H2O2 by metal nanoparticles and catalase. The 1 mL of sample solution containing 100 μM H2O2, 10 μM folic acid, 20 μM copper(II) chloride, and indicated concentration of metal nanoparticles or catalase was incubated for 30 min. The concentration of H2O2 was estimated from the fluorescence measurement.

#### **4.3 Application of siver-core/platinum-shell bimetallic nanoparticles to catalytic decomposition of hydrogen peroxide generated by chemical compound**

#### **4.3.1 Preparation of silver-core bimetallic nanoparticles for hydrogen peroxide scavenger**

The catalytic activity of Pt and its modified particles with Ag (Ag/Pt) on the decomposition of H2O2 generated from chemical compounds was evaluated, since Pt showed the highest activity. The Ag/Pt nanoparticles were prepared from the following procedure. The absorption spectrum of the sol of Pt nanoparticles is a flat curve (Figure 11), indicating the formation of homogenous particles. Ag nanoparticles exhibited a typical yellow color due to surface plasmon absorption around 400 nm. It has been reported that a physical mixture of Ag and Pt nanoparticles spontaneously forms bimetallic nanoparticles, possibly Ag-core/Ptshell structured particles (Toshima et al., 2005). The time-course of the absorption spectra of this physical mixture showed the extinction of Ag surface plasmon absorption, and the absorption was completely extinguished within 24 h (Figure 11), suggesting that the surface of the formed bimetallic nanoparticles is composed of Pt atoms. Typical TEM images showed the formation of relatively small particles of Pt and large particles of Ag (Figure 12). TEM photographs showed that the large Ag particles disappeared through interaction with Pt particles, resulting in the formation of bimetallic particles smaller than the parent Ag particles (Figure 12). A similar result has been observed in the case of Ag/Rh bimetallic

 H2O2 → H2O + 1/2 O2 (2). The generation of O2 gas through the H2O2 decomposition was confirmed with a gas-burette as following procedure. The 10 mL of aqueous solution containing 0.1 M H2O2 was treated by 10 μg Pt nanoparticles and generated O2 gas was measured with a gas-burette. The volume of detected gas coincided with that of the theoretically calculated value of O2

0 20 40 60 80 100

[Catalase] / units mL-1

0 2 4 6 8 10

[Nanoparticle] / M/atom

Fig. 10. Removal of H2O2 by metal nanoparticles and catalase. The 1 mL of sample solution containing 100 μM H2O2, 10 μM folic acid, 20 μM copper(II) chloride, and indicated

concentration of metal nanoparticles or catalase was incubated for 30 min. The concentration

The catalytic activity of Pt and its modified particles with Ag (Ag/Pt) on the decomposition of H2O2 generated from chemical compounds was evaluated, since Pt showed the highest activity. The Ag/Pt nanoparticles were prepared from the following procedure. The absorption spectrum of the sol of Pt nanoparticles is a flat curve (Figure 11), indicating the formation of homogenous particles. Ag nanoparticles exhibited a typical yellow color due to surface plasmon absorption around 400 nm. It has been reported that a physical mixture of Ag and Pt nanoparticles spontaneously forms bimetallic nanoparticles, possibly Ag-core/Ptshell structured particles (Toshima et al., 2005). The time-course of the absorption spectra of this physical mixture showed the extinction of Ag surface plasmon absorption, and the absorption was completely extinguished within 24 h (Figure 11), suggesting that the surface of the formed bimetallic nanoparticles is composed of Pt atoms. Typical TEM images showed the formation of relatively small particles of Pt and large particles of Ag (Figure 12). TEM photographs showed that the large Ag particles disappeared through interaction with Pt particles, resulting in the formation of bimetallic particles smaller than the parent Ag particles (Figure 12). A similar result has been observed in the case of Ag/Rh bimetallic

**4.3 Application of siver-core/platinum-shell bimetallic nanoparticles to catalytic**

**4.3.1 Preparation of silver-core bimetallic nanoparticles for hydrogen peroxide**

**decomposition of hydrogen peroxide generated by chemical compound**

0

20

40

60

80

Ag Rh Au Pd Pt Catalase 100

generation from the decomposition of H2O2 in the sample solution.

0

of H2O2 was estimated from the fluorescence measurement.

20

40

[H

**scavenger**

O2 2] / M

60

80

100

nanopaticles (Toshima & Hirakawa, 2003). These findings suggest the formation of selforganized Ag/Pt bimetallic nanoparticles. These metal nanoparticles are stable in water for several months. The Ag/Pt (Ag-atom/Pt-atom, 1/1) bimetallic nanoparticles were prepared using a self-organization method to mix Pt and Ag monometallic nanoparticles according to previous reports (Toshima & Hirakawa, 2003; Toshima et al., 2002, 2005; Matsushita et al., 2007).

Fig. 11. Absorption spectral change of the physical mixture of dispersions of Ag and Pt nanoparticles. The aqueous solutions of Ag (1 mM/atom, 10 mL) and Pt (1 mM/atom, 10 mL) nanoparticles were mixed and measured at 0, 10, 20, and 30 min, and 24 h after mixing.

Fig. 12. TEM photographs of metal nanoparticles. The sample of Ag/Pt nanoparticles was prepared by drying the mixtures of the aqueous solutions of Pt and Ag monometallic nanoparticles 24 hour after mixing.

## **4.3.2 Hydrogen peroxide formation from hydroquinone and its removal by metal nanoparticles**

Hydroquinone, which is a metabolite of carcinogenic benzene, was used as H2O2 source. This compound can generate H2O2 through autooxidation (Figure 13) (Hirakwa et al., 2002). Under these experimental conditions, hydroquinone generated H2O2 in a dose-dependent manner (Figure 14). Twenty units/mL catalase effectively removed H2O2 generated from this system, and 10 μM/atom (2 μg/mL) Pt nanoparticles exhibited a comparable activity to that of this catalase. Silver nanoparticles showed apparently weaker activity for H2O2 removal than Pt nanoparticles. The bimetallization of Pt with Ag apparently suppressed the catalytic activity per unit atom.

Self-Organization of Silver-Core Bimetallic

0

20

40 60

[H

estimated from the fluorescence measurement.

O2 2] / % 80

100

hydroquinone.

**nanoparticles**

modification with Ag.

Nanoparticles and Their Application for Catalytic Reaction 81

absorbance reader. The oxidized form of hydroquinone can be reduced into the parent hydroquinone by NADH (Hirakwa et al., 2002). The concentration of NADH was gradually decreased through the redox of hydroquinone and Pt nanoparticles hardly inhibited NADH consumption (data not shown). This result indicated that Pt nanoparticles do not inhibit the H2O2 generation itself, because H2O2 is produced through the autooxidation of

0 20 40 60 80 100

[Nanoparticle] / M/atom

Fig. 15. Removal of H2O2 generated through the autooxidation of hydroquinone by metal nanoparticles and catalase. The 1 mL of sample solution containing 10 μM folic acid, 20 μM

**4.4 Summary and possible mechanism of hydrogen peroxide decomposition by metal**

Poly(*N*-vinyl-2-pyrrolidone)-protected metal nanoparticles, in particular Pt nanoparticles, exhibited a removal effect on H2O2 generated through autooxidation of hydroquinone (Figure 16). The removal of H2O2 by these metal nanoparticles can be explained by a catalytic reaction similar to that by catalase, which decomposes H2O2 into H2O and O2. The formation of H2O2 during autooxidation of hydroquinone is through O2•-, which is generated from a reduction of O2 by hydroquinone (Hirakawa et al., 2002). Because the lifetime of O2•-, which dismutates into H2O2 through reaction with H+, is short (~ 0.1 ms), the scavenging of O2•- by a metal nanoparticle can be negligible. The H2O2 removal activity per metal atom of these metal nanoparticles occurred in the following order: Pt > Ag ≈ Ag/Pt. The activities of H2O2 decomposition per metal atom consisting of these metal nanoparticles (μM-H2O2/μM-nanometal) have been estimated, and the resulting values are 4.2, 12.2, and 3.8 for Ag, Pt, and Ag/Pt, respectively. Further, the activity on the surface area of the Ag/Pt nanoparticles (17 μM-H2O2/cm2-nanometal) was also smaller than that of Pt (49 μM-H2O2/cm2-nanometal). These findings showed that the Pt nanoparticles have the highest catalytic activity for H2O2 decomposition in the metal nanoparticles used in this experiment and the activity of Pt nanoparticles is suppressed by

copper(II) chloride, 50 μM hydroquinone, and indicated concentration of metal nanoparticles or catalase was incubated for 30 min. The concentration of H2O2 was

0 20 40 60 80 100

[Catalase] / units mL-1

Ag/Pt Ag Catalase Pt

Fig. 13. Schematic diagram of hydrogen peroxide formation by the autooxidation of hydroquinone

Fig. 14. H2O2 generation through autooxidation of hydroquinone in the absence or presence of metal nanoparticles and catalase. The 1 mL of sample solution containing 10 μM folic acid, 20 μM copper(II) chloride, and indicated concentration of hydroquinone with or without 10 μM/atom metal nanoparticles or 20 units/mL catalase was incubated for 30 min. The concentration of generated H2O2 was estimated from the fluorescence measurement.

#### **4.3.3 Activity of silver-core/platinum-shell nanoparticles on hydrogen peroxide decomposition**

Figure 15 shows the removal activity of H2O2 generated from a high concentration of hydroquinone (50 μM) by metal nanoparticles. These metal nanoparticles and catalase scavenged H2O2 in a dose-dependent manner. The activity of the 10 μM/atom (2 μg/mL) Pt nanoparticles was comparable to that of 20 units/mL catalase, and Pt completely scavenged H2O2 over 20 μM/atom (4 μg/mL). The activity per atom of the Ag/Pt bimetallic nanoparticles was almost the same as that of the Ag monometallic nanoparticles.

To investigate the effect of Pt nanoparticles on H2O2 generation through the autooxidation of hydroquinone, NADH consumption during this autooxidation was measured. The consumption of NADH during the autooxidation of hydroquinone was measured by a previously reported method (Oikawa et al., 2003). A sample solution containing 100 µM NADH, 50 µM hydroquinone, and 20 µM copper(II) chloride was incubated at 37 C in the absence or presence of 20 µM/atom Pt nanoparticles. The concentration of NADH was determined by the measurement of absorbance of NADH at 340 nm using a microplate

O

O

O

+ Ag

+ Ag/Pt

+ Pt + catalase

OH

0 20 40 60 80 100 [Hydroquinone] / M