Article pubs.acs.org/Langmuir
Photocatalytic Properties of TiO2 Composites Immobilized with Gold Nanoparticle Assemblies Using the Streptavidin−Biotin Interaction Hirofumi Harada,† Akira Onoda,*,† Taro Uematsu,†,‡ Susumu Kuwabata,† and Takashi Hayashi*,† †
Department of Applied Chemistry and ‡Frontier Research Base for Global Young Researchers, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan S Supporting Information *
ABSTRACT: A method using biomolecules to precisely fabricate the morphology of metal nanoparticles immobilized on the surface of a semiconductor using biomolecules is described. A biotin moiety (Biot) is introduced onto the surface of a gold nanoparticle (AuNP) by covalent coupling with α-lipoic acid to assemble AuNPs in the presence of streptavidin (STV). The assembly of Biot-AuNP/STV is immobilized on the surface of TiO2 chemically modified with 1-(3-aminopropyl)silatrane (APS) to provide a positively charged surface. The Au content immobilized on the surface of TiO2 is clearly increased to 9.5 wt % (Au) as a result of the STV−biotin interaction and the electrostatic interaction between negatively charged Biot-AuNPs and the positively charged surface of APS/TiO2. Transmission electron microscopy (TEM) analysis reveals that the composite has an ordered surface geometry in which Biot-AuNPs are spread over the composite surface in two dimensions. The photocatalytic activity toward decomposition of methyl orange dye promoted by this composite is 55%, which is higher than that of the other composites. The Biot-AuNP/STV@APS/TiO2 composite efficiently reduces O2 molecules at Eonset = −0.23 V vs Ag|AgCl, which is more positive than that of other composites (Eonset = −0.40 to −0.32 V). The result suggests that an increased number of AuNPs immobilized in close contact with the TiO2 surface facilitates photoinduced charge transfer. This strategy, which takes advantage of the specific interactions provided by biomolecules and the chemical modification on the surface, has remarkable potential for efficient fabrication of metal nanoparticles on the surface of the semiconductor, which accelerates the reduction of oxygen molecules.
M
impregnation, and vacuum deposition.20−22 However, it remains challenging to immobilize monodispersed MNPs in controlled distances on the semiconductor surface using conventional methods. We therefore focused on developing a general method to immobilize MNP assemblies on the surface of SCs. The approaches to assemble MNPs using biomolecules have been used to develop sensors and plasmonic devices because the colorimetric changes of plasmonic MNPs, which depend on the interparticle distance, are advantageous for such applications.23−29 The interactions resulting from specific and tight recognition provided by biomolecules have been harnessed for fabrication of MNP assemblies, and applied to tailor the interparticle distances while maintaining the original sizes and shapes of the MNPs.30−32 Streptavidin (STV) is a tetrameric protein, which has four binding pockets for a biotin molecule (Kd = 10−15 M). The two neighboring binding pockets space each other apart and direct toward the opposite direction (Figure S1), thereby STV could be a suitable protein for a linear connection with a biotinylated compound.33,34 The interaction between STV and biotin (Biot) has been
etal nanoparticle−semiconductor (MNP−SC) composites have attracted significant interest as an emerging class of photocatalysts for a wide range of chemical reactions and photoelectrochemical conversions.1−9 In particular, an interfacial structure between two materials in such hybrid composites has been identified as an important feature that is expected to improve the reactivities and photochemical properties of MNP−SCs. When SCs absorb incident light corresponding to the band gap energy, an excited electron and an oxidative hole are generated in the SC materials. The electrons excited to the conduction band of SC can be transferred to MNPs of noble metals such as gold, silver, and copper immobilized on the surface. The enhancement of charge separation has been studied in gold nanoparticle−titanium dioxide composites (AuNP−TiO2), where AuNPs are directly immobilized on the surface of TiO2.10,11 The MNPs immobilized in a well-controlled fashion on the surface have been proposed to be the factor that determines the physicochemical properties of MNP−SC materials.12−14 The properties of immobilized MNPs including surface plasmon resonance (SPR) absorption, refractive index, and light scattering, which are affected by MNP composition, size, and shape, are important parameters to explore in the development of new features in MNP−SC materials.12,14−19 In general, AuNP−TiO2 composites are prepared by sol−gel synthesis, © XXXX American Chemical Society
Received: March 18, 2016 Revised: May 24, 2016
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were modified with α-lipoic acid (LA), which was further coupled to a biotin derivative, 1 (Figure 1). This molecule retains sufficient binding affinity with STV (Figure S2). First, the formation of the assembly including Biot-AuNPs and STV proteins (Biot-AuNP/STV) was analyzed by SPR absorption (Figure 2a). The SPR peaks of AuNP and Biot-AuNP are observed at 523 and 525 nm, respectively. The surface modification using the biotin moieties has a small effect on SPR absorption of AuNP. The red color of Biot-AuNP changes to purple in the presence of STV, and two absorption peaks arise at 524 and 612 nm. The red-shifted absorption indicates that AuNPs are assembled and that the average interparticle distance between Biot-AuNPs decreases upon addition of STV. The assembly of Biot-AuNP/STV was confirmed by gel electrophoresis (Figure 2b). LA-modified AuNPs do not undergo a significant shift with addition of excess STV to the AuNPs, indicating the absence of the strong interaction between AuNP and STV. The decreased band intensity suggests the formation of the aggregate undetectable in the gel electrophoresis as a result of the nonspecific interaction between STV and the negatively charged AuNPs (lanes 1−4). By contrast, the bands for Biot-AuNP are shifted with addition of increasing amounts of STV (lane 5−10). This proves that Biot-AuNPs are assembled via the strong and specific interaction between STV and the biotin moiety. The formation of the assembly reaches a maximum in the presence of 50 equiv of STV (lane 10). This shift band disappears, and a new shift band assignable to the assembly with the smaller molecular weight appears with continued addition of over 50 equiv of STV (Figure S3, lane 7−14). This indicates a transition from the large assembly to isolated AuNP in which several STV proteins are bound on the surface. The shift band drops to a plateau in the presence of 200 equiv of STV. We thus estimate that approximately 200 molecules of Biot are modified on the surface of a single equivalent of Biot-AuNP. This is consistent with the evidence that the Biot-AuNP/STV assembly forms to the maximum extent in the presence of 50 equiv of STV, considering that STV contains four biotin binding sites. Therefore, roughly 200 units of biotin is covalently modified on each equivalent of AuNP, indicating that ca. 5% of lipoic acid (LA) molecules on the surface of AuNP are chemically modified.40 The Biot-AuNP/STV assembly was also visualized by transmission electron microscopy (TEM) (Figure 2c). The size (ca. 15 nm) and shape of the AuNPs are retained in BiotAuNP and Biot-AuNP/STV. These results indicate that the Biot-AuNPs are assembled via a supramolecular interaction between STV and the biotin moieties. Preparation and Characterization of AuNP−TiO2. A series of AuNP−TiO2 composites was prepared by soaking TiO2 into an aqueous solution of AuNPs. The AuNP modified with lipoic acid has a negatively charged surface, which was experimentally confirmed by the measurement of ζ potential. The values of ζ potential measured for AuNP, Biot-AuNP, and Biot-AuNP/STV are −68 mV, − 49 mV, and −13 mV, respectively (Table 1). The surface of TiO2 (zeta potential ζ = −14 mV) was chemically modified with 1-(3-aminopropyl)silatrane (APS) containing an amino group to enhance the electrostatic interaction between the positively charged surface (ζ = +13 mV) of APS-modified TiO2 (APS/TiO2) and the negatively charged AuNPs modified with lipoic acid.41 Immobilization efficiency was evaluated using the Au content of the TiO2 composites determined by ICP-AES (Figure 3).
demonstrated to be useful in the assembly of AuNPs and gold nanorods.35−39 Here, we report a new method for preparing AuNP−TiO2 composites immobilized with AuNP assemblies connected using the specific tight interaction between STV and biotin in the lateral direction against the surface plane of TiO2 (Figure 1).
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RESULTS AND DISCUSSION AuNP Assembly Conjugated Using the Streptavidin− Biotin Interaction. Biotinylated AuNPs (Biot-AuNPs) were prepared via chemical modification of citrate-stabilized AuNPs with an average diameter of ca. 15 nm. Citrate-stabilized AuNPs
Figure 1. (a) Preparation of a TiO2 composite immobilized with AuNP assemblies via the streptavidin−biotin interaction. (b) Chemical structure of APS-modified TiO2 surface. B
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Figure 2. Characterization of biotinylated gold nanoparticles (Biot-AuNPs) and the assembly using STV (Biot-AuNP/STV). (a) UV−vis spectra of LA-modified AuNPs (blue), Biot-AuNPs (green), and Biot-AuNP/STV (red). Photographs of Biot-AuNPs and Biot-AuNP/STV in an aqueous solution are shown. (b) Agarose gel electrophoresis of LA-modified AuNPs (lane 1−4) and Biot-AuNPs (lane 5−10). The concentration of STV increases from lane 1 to 4 (0, 0.5, 1, 10 equiv vs AuNP) and from lane 5 to 10 (0, 0.25, 0.5, 2, 10, 50 equiv vs AuNP). The yield of the assembly reaches a maximum when the ratio of biot:biotin-binding pocket of STV is 1:1 in the presence of 50 equiv of STV. (c) TEM images of Biot-AuNP and Biot-AuNP/STV. The scale bar is 100 nm.
Table 1. Average Size and ζ Potential of AuNPs and TiO2 with Different Chemical Modifications AuNP Biot-AuNP Biot-AuNP/STV STV TiO2 APS/TiO2 PAA/TiO2b
size (r. nm)a
ζ potential (mV)
9.5 14 1.8 × 102 5.1 4.7 × 102 7.3 × 102 2.6 × 102
−68 −49 −13 2.5 −14 13 −42
a Size was determined by DLS. bNegatively charged AuNPs are not efficiently immobilized on negatively charged TiO2 (ζ = −42 mV) coated with poly(acrylic acid) (PAA/TiO2).
The Au content of AuNP@TiO2 (0.65 wt % (Au)) was found to be increased to 3.1 wt % in AuNP@APS/TiO2. This suggests that the positively charged APS-modified surface is advantageous for immobilization of negatively charged AuNPs by the electrostatic interaction. The Au content of Biot-AuNP/STV@ TiO2 is 8.8 wt % (Au), which is 10 times greater than that of AuNP@TiO2 (0.65 wt %(Au)), although the ζ potential of Biot-AuNP/STV is positively shifted to −13 mV, being alleviated by the positively charged STV (ζ = +2.5 mV). This suggests that the formation of the AuNP assembly by the streptavidin−biotin interaction significantly increases the amount of immobilized AuNPs on the composite against the weaker electrostatic interaction between Biot-AuNP/STV and the surface of TiO2. In addition, the Au content of Biot-AuNP/ STV@APS/TiO2 (9.5 wt % (Au)) is slightly increased relative to that of Biot-AuNP/STV@TiO2 (8.8 wt % (Au)), indicating that the positively charged surface of TiO2 is effective for immobilizing the AuNP assembly including STV. The immobilization of AuNPs onto the TiO2 surface was thus promoted by both the streptavidin−biotin interaction and the APS modification of TiO2.
Figure 3. Au content (wt % (Au)) determined by ICP-AES and photographs of AuNP−TiO2 composites: From left to right; TiO2, APS/TiO2, AuNP@TiO2, AuNP@APS/TiO2, Biot-AuNP/STV@ TiO2, and Biot-AuNP/STV@APS/TiO2.
Diffuse transmission spectra of TiO 2 and APS/TiO 2 composites were measured to evaluate the morphology of AuNPs immobilized on the TiO2 surface, which was affected by the streptavidin−biotin interaction. The results obtained for the composites immobilized with AuNP and Biot-AuNP/STV on the two substrates, TiO2 and APS/TiO2, are shown in Figure 4. The SPR signals of the isolated and closely positioned AuNPs were expected to be observed in the vicinity of 520 and 620 nm, C
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Figure 4. Diffuse transmission spectra of AuNP@TiO2 (blue dashed line), AuNP@APS/TiO2 (blue solid line), Biot-AuNP/STV@TiO2 (red dashed line), and Biot-AuNP/STV@APS/TiO2 (red solid line).
respectively, which are similar to those observed in solution (Figure S4). In fact, AuNP@TiO2 and AuNP@APS/TiO2 both exhibit a main absorption at approximately 520 nm. A more intense absorption at 620 nm for AuNP@APS/TiO2 was observed, suggesting that the number of immobilized AuNPs with negative charges is increased on the positively charged APS surface. This trend leads to an increase in the number of AuNPs within closer distances of each other. The absorption at 620 nm for Biot-AuNP/STV@TiO2 is also intense, suggesting that some portions of AuNP are assembled on the surface by the supramolecular connection with STV in the case of BiotAuNP/STV@TiO2. In addition, the absorption at approximately 620 nm is more prominent in Biot-AuNP/STV@APS/ TiO2. This result indicates that the positively charged surface of APS/TiO2 is a suitable platform for immobilizing the assembly of negatively charged AuNPs connected with the streptavidin− biotin interaction. The morphology of AuNPs on the surface of the composites is visualized by TEM analysis (Figure 5). A few individual AuNPs with diameters of ca. 15 nm are observed on the surface of AuNP@TiO2 (Figure 5a). In the case of APS/TiO2, the amount of AuNPs on the surface is clearly increased relative to that of AuNP@TiO2 (Figure 5b), suggesting that AuNPs are sparsely immobilized on the surface of AuNP@APS/TiO2. By contrast, AuNPs are intensely aggregated and immobilized in a sufficient amount in a three-dimensional fashion on the surface of Biot-AuNP/STV@TiO2 (Figure 5c). Therefore, STV proteins are expected to link the units of Biot-AuNP to form the assembly. Interestingly, larger amounts of AuNP assemblies for Biot-AuNP/STV were observed on the surface of the positively charged APS/TiO2 surface and, more importantly, are spread in a two-dimensional fashion in the case of BiotAuNP/STV@APS/TiO2 (Figure 5d). The TEM analysis clarified that APS modification of TiO2 induces efficient immobilization of AuNPs and that the streptavidin−biotin interaction promotes the assembly of Biot-AuNPs on the composites. The biotin molecules are linked with a Au−S bond, which has been known to slide along the surfaces of AuNPs. The movable nature of the Au−S bond enables reorganization of the three-dimensional assembly to the two-dimensional assembly on the positively charged surface of APS/TiO2. The
Figure 5. Representative TEM images of (a) AuNP@TiO2, (b) AuNP@APS/TiO2, (c) Biot-AuNP/STV@TiO2, and (d) Biot-AuNP/ STV@APS/TiO2. All scale bars indicate 100 nm.
APS-modified surface was found to provide a preferential platform to tightly adsorb the Biot-AuNP/STV assemblies in a favorable morphology. Photocatalytic Activity. The photocatalytic activity of the series of composites was analyzed by photodecomposition of methyl orange (MO) in an effort to characterize uniquely assembled AuNPs on the surfaces of the composites. The composites were dispersed in an oxygen-saturated aqueous MO solution. After UV−vis light irradiation (300−800 nm, 147 mW cm−2) using a 500 W Xe lamp, decomposition of MO was determined by measuring its absorption at 463 nm. The time course profiles of the MO concentration and the conversion of MO after 5 min for the AuNP−TiO2 composites are shown in Figure 6a,b, respectively. The conversion of MO was increased to 18.3% in the presence of AuNP@TiO2 composites relative to the values of 2.4% for the TiO2 and 6.8% for APS/TiO2 catalyst without AuNPs. AuNP@APS/TiO2 exhibits higher activity (44.3%) due to the greater amount of immobilized AuNPs (3.1 wt % (Au)). Therefore, the positively charged TiO2 surface ((ζ = +13 mV), which enables tight binding of the AuNPs on ASP/TiO2, is important for the higher photocatalytic activity. The conversion for Biot-AuNP/STV@TiO2 dropped to 13.2%, although sufficient AuNPs were immobilized on TiO2 (8.8 wt % (Au)). Surprisingly, the conversion promoted by Biot-AuNP/STV@APS/TiO2 (9.5 wt % (Au)) was found to be 55.8%, which represents a 4-fold increase over that of Biot-AuNP/STV@TiO2, despite the similar Au content. The activity of Biot-AuNP/STV@APS/TiO2 is also slightly higher than that of AuNP@APS/TiO2, although the activity in terms of unit % of Au is lower. Electrocatalytic Activity for O2 Reduction Reaction. The electrocatalytic activity toward the oxygen reduction reaction (ORR) was next investigated using TiO2 composites fabricated on FTO (Florine-doped tin oxide) electrodes. Cyclic voltammetry of the TiO2 composites coated on the FTO electrode was performed in O2- or N2-saturated neutral D
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Figure 7. Cyclic voltammograms of TiO2 composite-coated FTO electrodes in (a) O2- and (b) N2-saturated 0.5 M NaClO4, pH 7.0 at 25 °C. APS/TiO2 (gray line), AuNP@APS/TiO2 (blue line), BiotAuNP/STV@TiO2 (red dotted line), Biot-AuNP/STV@APS/TiO2 (red solid line). Scan rate is 0.1 V sec−1. The measurements were carried out in the dark. The reduction peaks at −0.64 V and −0.87 V are assigned to two- and four electron reduction of O2.42,43
Table 2. Electrochemical Data for TiO2 Composites in Oxygen Reduction Reaction Biot-AuNP/STV@APS/TiO2 Biot-AuNP/STV@TiO2 AuNP@APS/TiO2 APS/TiO2 TiO2 FTO
Figure 6. (a) Time course profiles of photocatalytic decomposition of MO in the presence of AuNP−TiO2 composites TiO2, AuNP@TiO2, AuNP@APS/TiO2, Biot-AuNP/STV@TiO2, and Biot-AuNP/STV@ APS/TiO2. (b) Conversion of MO decomposition in 5 min. The photodecomposition of MO does not proceed in the absence of the composite catalyst nor in the presence of AuNPs without TiO2.
Eonset (V vs Ag/AgCl)
Jmax (mA cm−2)
−0.23 −0.32 −0.32 −0.47 −0.45 −0.40
−0.47 −0.51 −0.60 −0.44 −0.65 −0.43
respectively, which are similar to the potential for bare FTO electrodes (−0.40 V). While the Eonset for the electrodes for AuNP@APS/TiO2 and Biot-AuNP/STV@TiO2 shifts more positively to −0.32 V, suggesting that the AuNPs accelerate the O2 reduction. Furthermore, a remarkable positive shift in Eonset (−0.23 V) was observed in the case of Biot-AuNP/STV@APS/
aqueous media (Figure 7). The remarkably larger cathodic current observed under aerated conditions for the TiO2-coated electrodes obviously corresponds to O2 reduction. The onset potential (Eonset) and current density (Jmax) values for the TiO2coated electrodes are listed in Table 2. The Eonset values for the TiO2 and APS/TiO2 electrodes are −0.47 V and −0.45 V, E
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photocatalytic activity. The TEM analysis provides a clear indication of the AuNP assembled in proximity using STV− biotin interaction on the APS-modified TiO2. By contrast, the composite with AuNPs accumulated on the bare TiO2 surface with the STV−biotin interaction was found to be less active, although the Au content (wt %) is within a similar range. The results provide strong evidence that immobilization of AuNPs in close contact with each other on the surface of TiO2 plays a significant role in improvement of photocatalytic activity because of the efficient electron transfer to the AuNPs. The formation of unique two-dimensional assembly of AuNPs on the surface of APS-modified TiO2 can be explained as follows. Thiol ligands are known to move and diffuse on a gold surface,52,53 and exchange their position via dissociation and association on the surface.54−56 Therefore, AuNP assemblies have, in general a dynamic nature owing to mobile bonds between the gold surface and the thiolate ligand. This feature is expected to be similar in our assembly, although the AuNPs are connected through the biotin and STV that are linked by a strong intermolecular interaction (Kd = ∼ 10−15 M). The assembly of Biot-AuNP/STV itself would be more stabilized in a spherical form due to a smaller surface-tovolume ratio as we confirmed the three-dimensional structure by TEM imaging. When the AuNP assembly interacts with the positively charged surface of APS/TiO2, the flexible and threedimensional assembly would preferentially reorganize their shape by maximizing the number of sites that connect the negatively charged surface of AuNPs and the positively charged surface on APS/TiO2 by electrostatic interaction. The remarkable surface morphology of the TiO2 composite with the AuNP assemblies provides a reaction platform that enables efficient electron transfer to improve electrocatalytic reduction of O2 (Figure 9). Hydrogen peroxide and hydroxyl radicals generated from hydrogen peroxide under UV radiation would not be effectively consumed to decompose MO in the photodecomposition, although we observed considerably higher electrocatalytic activity in the ORR. The diffusion of MO to the surface of AuNP, where STV is also immobilized, might be limited in Biot-AuNP/STV@APS/TiO2, or the reactive oxygen species might react with the molecules used for immobilization, thereby decreasing the conversion in photodecomposition of MO.
TiO2. The results suggest that the arrangement of AuNPs assembled densely on the surface of the Biot-AuNP/STV@ APS/TiO2 on the FTO electrode is responsible for efficient reduction of O2. Contribution of the AuNP Assembly to Photocatalytic and Electrocatalytic Activity. Excited electrons generated within the conduction band (CB) of TiO2 under UV irradiation rapidly recombine with the holes generated within the valence band (VB) of TiO2. The excited electrons are less likely to be transferred to molecules that can be reduced. By contrast, when AuNPs are immobilized on the surface of TiO2, the excited electrons within CB of TiO2 are effectively captured by AuNPs overcoming the Schotteky barrier.10,11,22 The charge recombination between photogenerated electrons and holes within TiO2 would be suppressed. This electron transfer would be facilitated by AuNPs in close proximity to each other on the surface of TiO2. Notably, the AuNP assembly that enables dense immobilization within a short distance on the surface of Biot-AuNP/STV@APS/TiO2 would provide a significant increase in the amount of electrons transferred to AuNPs. In fact, the TiO2 composite with the AuNP assembly catalyzes the ORR at a significantly low overpotential in the dark during electrochemical analyses, suggesting that the TiO2 surface with the AuNP assembly considerably accelerates the ORR. The photo- and electrocatalytic ORR of immobilized AuNPs have been thoroughly investigated and found to be sufficiently potent to produce reactive oxygen species, such as H2O2.44−50 We therefore expect that the electrons photogenerated by TiO2 are efficiently transferred to the AuNPs, where efficient generation of H2O2 proceeds.51 Together with the MO decomposition by hydroxyl radicals photogenerated by the valence band holes of TiO2, the mechanism proposed here explains the higher catalytic activity observed in the photodecomposition of MO for Biot-AuNP/STV@APS/TiO2, in which the AuNP assemblies are immobilized and twodimensionally spread, relative to that for Biot-AuNP/STV@ APS/TiO2 where aggregated AuNPs are immobilized on the surface (Figure 8). In this study, we found that the morphology of the AuNPs immobilized on the surface of TiO2 affects the photocatalytic activity of AuNP−TiO 2 composites. The AuNP−TiO 2 composite with AuNP assemblies that are effectively dispersed on the surface by the STV−biotin interaction has maximum
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CONCLUSION We prepared an AuNP−TiO2 composite-immobilized biotin with effectively dispersed AuNP assemblies, which was connected using the supramolecular interaction between streptavidin (STV) protein and biotin, on the TiO2 surface, and investigated the photocatalytic and electrocatalytic activity of the composites. The negatively charged AuNPs modified with a biotin moiety were confirmed to be assembled via the STV-Biot interaction and the assembly was immobilized as a single layer on the positively charged APS-modified TiO2 surface (Biot-AuNP/STV@APS/TiO2). The Biot-AuNP/ STV@APS/TiO2 composite is more effective than the other composites investigated with respect to photodecomposition of MO and electroreduction of O2. The results indicate that the increased number of AuNPs in close contact with TiO2 greatly improves electron transfer to AuNP and the photocatalytic activity exhibited by the AuNP−TiO2 composite. This work demonstrates that fabricated nanoparticle assemblies on semiconductor materials can be precisely designed as photocatalysts. It is expected that further studies of such metal
Figure 8. Proposed mechanism of photocatalysis in Biot-AuNP/ STV@APS/TiO2. F
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H-7650 system. The size distribution of AuNPs among ca. 150 particles was estimated using ImageJ software. ICP-AES was conducted using a Shimadzu ICPS-7510 instrument. Photocatalytic activity of the composites was investigated using irradiation from a 500 W xenon arc lamp (Optical Modulex, SX-U1501XQ, USHIO Inc.). Electrochemical measurements were conducted using a Compactstat potentiostat (Ivium Technology, The Netherlands). The size and ζ potential of nanoparticles and composites were analyzed using Zetasizer Nano ZS (Malvern Instruments, UK). Preparation of Biotinylated AuNP and AuNP Assembly. Biotinylated AuNPs (Biot-AuNPs) were prepared according to the method reported in the literature with minor optimization modifications.38,39 Briefly, HAuCl4 (41.2 mg, 0.10 mmol) in 100 mL of water was refluxed for 10 min, and trisodium citrate dihydrate (114 mg, 0.39 mmol) in 10 mL of water was quickly added to the boiling solution at once. After refluxing for 20 min, the reaction mixture was allowed to cool to room temperature to give citrate-stabilized AuNPs. Lipoic acid (LA) (2.06 mg, 10 mmol) in 2 mL of methanol and 1 M NaOHaq was alternately added dropwise to maintain the pH of the solution at 8.0, and the mixed solution was stirred for 1 h at room temperature. LA-modified AuNPs were separated by centrifugation (14800 rpm, 20 min, 4 °C), washed three times with NaOHaq (pH 10.2), and stored in the same solution. LA-modified AuNPs (ε520 = 2.47 × 108 cm−1 M−1)57 were then dispersed in 50 mM MES buffer (pH 5.5). The LA-modified AuNP solution (3.0 nM (per particle), 10 mL) was mixed with EDC (2.49 mg, 13 mmol) and 1 (54.5 μg, 0.13 μmol) in 1 mL of 50 mM MES buffer (pH 5.5), and the mixture was stirred for 32 h at room temperature. Black precipitates were collected by centrifugation (3000 rpm, 5 min, 4 °C) and stirred in 10 mL of NaOHaq (pH 12.0) for 6 h at room temperature for complete dispersal in the solution. Biot-AuNPs were stored in NaOHaq (pH 10.2). The assembly of STV and Biot-AuNPs (Biot-AuNP/STV) was prepared by mixing the Biot-AuNP solution (50 nM (per particle), 10 mL) and aqueous solution of STV (10 μM, 500 μL). APS-Modification of the TiO2 Surface. TiO2 (300 mg) was suspended in aqueous APS solution (17.7 mM, 30 mL) and the mixed suspension was stirred for 30 min at room temperature. The supernatant was removed, and the same procedure was repeated three times. The resultant muddy precipitate was washed twice with deionized water to give APS-modified TiO2 (APS/TiO2). APS/TiO2 dispersed in solution was stored before use. PAA-Modification of the TiO2 Surface. Poly(acrylic acid) (Mw = 5000, Wako) in deionized water (20 mM) was mixed with TiO2 to provide a final concentration of 100 mg/mL. The suspension was stirred for 30 min at 4 °C. The suspension was centrifuged at 3000 rpm for 10 min at 4 °C and washed twice with deionized water. The procedure for PAA-modification was repeated three times, and the resultant muddy precipitate was carefully washed with deionized water. Preparation of AuNP−TiO2 Composites. All composites were prepared by mixing the suspension of TiO2 and AuNP solutions. For example, TiO2 (100 mg) was suspended in 10 mL of 300 nM (per particle) LA-modified AuNP, and the mixture was incubated for 48 h at 4 °C. The suspension was centrifuged at 4000 rpm, and the precipitate was washed twice with deionized water and lyophilized for 2 days. The composite with AuNP assembly, Biot-AuNP/STV@APS/ TiO2, was prepared by mixing the suspension of APS/TiO2 and BiotAuNP/STV solution with the same procedure. Fifty equivalents of STV were added to the Biot-AuNP solution to form the AuNP assembly to the maximum extent and immediately used for the immobilization. AuNP−TiO2 Composites Immobilized on FTO. FTO-coated glass plates (9.79 Ω/sq., AGC Fabritech Co., Ltd.) was washed with SC1 (30% ammonia: 35% H2O2: H2O = 1:1:5) activated at 60 °C for 30 min and then rinsed with deionized water and ethanol. TiO2 in aqueous solution (100 mg/mL) containing 10% glycerol was homogenized using a ball-mill (AN1−515, Nitto Kagaku) for 24 h. The resultant dispersion of TiO2 was spin coated on the FTO electrode (10 × 25 mm) at 3000 rpm for 15 s. The electrode was heated at 450 °C for 1 h. The fabricated electrode, TiO2@FTO, was soaked in aqueous APS solution (17.7 mM) for 30 min, and then
Figure 9. A schematic representation of the surface morphology of (a) Biot-AuNP/STV@TiO2 and (b) Biot-AuNP/STV@APS/TiO2.
nanoparticle−semiconductor composites will significantly improve our understanding of the photochemical behavior of nanomaterials. Efforts to construct metal nanoparticle−semiconductor composites with various properties using this strategy are now in progress.
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EXPERIMENTAL METHODS
Materials. All reagents were used without purification unless otherwise noted. Hydrogen tetracholoroaurate(III) trihydrate was purchased from Kishida Chemicals. α-lipoic acid (LA) and 1-ethyl-3(3-(dimethylamino)propyl)carbodiimide (EDC) were purchased from Tokyo Chemical Industry. The precursor of biotin unit 1 was purchased from Thermo Scientific. TiO2 (99.9% rutile, ∼ 5 μm) and a standard solution of gold (1000 ppm) for inductively coupled plasma atomic emission spectroscopy (ICP-AES) were purchased from Wako Chemicals. Streptavidin (STV) protein was kindly gifted from Prof. T. R. Ward at the University of Basel. General. All aqueous solutions were prepared using Milli-Q water (>18.2 MΩ cm−1). Agarose gel electrophoresis was run with 0.5% agarose gel in 44.5 mM Tris-borate buffer including 1 mM EDTA for 1 h. UV/vis absorption spectra were obtained on a Shimadzu UV-3150 spectrophotometer equipped with a thermostated cell holder (±0.1 °C) or on a Shimadzu BioSpec-nano spectrophotometer. Diffuse transmission spectroscopy was performed using a JASCO V670 instrument equipped with an integral sphere. The pH values were monitored with a Horiba F-52 pH meter. Transmission electron microscopy (TEM) was performed using a Hitachi High-Technologies G
DOI: 10.1021/acs.langmuir.6b01073 Langmuir XXXX, XXX, XXX−XXX
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Langmuir washed twice with water and the same procedure was repeated twice. These TiO2-immobilized electrodes were soaked in 100 nM (per particle) AuNP or Biot-AuNP/STV solution at 4 °C for 2 days and gently rinsed three times with water. ICP-AES. The composites were weighed individually in glass containers and heated at 100 °C in aqua regia for 1 h until AuNPs and organic compounds were dissolved in the solution. The Au content of each of the composites in the solution was determined by ICP-AES measurements. Photocatalytic Activity. Photocatalytic activity of the composites was investigated by decomposition of methyl orange (MO).58 Each composite (0.10 mg/mL) was suspended in an aqueous MO solution (10 μM) and the reaction mixture was purged with O2 gas (>99.9%) for at least 20 min before the reaction. The reaction temperature was kept at 25 °C with a cryostat system (USP-203IR-B, Unisoku Co. Ltd.) during the irradiation of UV/vis light. The light intensity was 147 W cm−2. Aliquots of reaction mixture were collected, and the catalyst was removed by centrifugation (15000 rpm, 20 min) and filtration using a 0.22 μm filter (Millex-GV, Millipore). Conversion was determined by UV−vis absorbance of MO at 464 nm. Electrochemical Measurement. The FTO electrodes immobilized with TiO2 composite were incorporated into a cell as the working electrode sealing with an o-ring with a diameter of 7 mm. An Ag|AgCl electrode with a saturated KClaq and Pt-mesh electrode were employed as a reference and a counter electrode, respectively. Cyclic voltammetry of AuNP−TiO2 composite were measured in O2- or N2-saturated aqueous solution (0.5 M NaHClO4, pH 7.0 at 25 °C). Onset potential (Eonset) was determined as the potential at which the cathodic current becomes larger than −10 μA cm−2. All measurements were carried out in the dark with the scan rate of 0.1 V/sec.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b01073. Crystal structure of STV, competition binding assay of a Biot molecule, agarose gel electrophoresis of Biot-AuNP, and diffuse transmission spectra in solution (PDF)
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AUTHOR INFORMATION
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by Grants-in-Aid for Scientific Research (Innovative Areas “Element Block”, area 2401, KAKENHI 25102527 and 15H00746) from MEXT, the Japan Society for the Promotion of Science (JSPS), and JST, Strategic International Collaborative Research Program, SICORP. A.O. acknowledges support from the Japan Association of Chemical Innovation (JACI). H.H. acknowledges support from the Program for Leading Graduate Schools for Osaka University: Interdisciplinary Program for Biomedical Sciences (IPBS). We thank Prof. Thomas R. Ward for his generous gift of mature streptavidin, as well as Dr. Takami Akagi and Prof. Mitsuru Akashi for their assistance with measurements of the size and ζ potential of nanoparticles.
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REFERENCES
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DOI: 10.1021/acs.langmuir.6b01073 Langmuir XXXX, XXX, XXX−XXX
Article
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