Gold Nanoparticle Inclusion into Protein Nanotube as a Layered Wall

Article pubs.acs.org/Langmuir

Gold Nanoparticle Inclusion into Protein Nanotube as a Layered Wall Component Shun Goto,† Yusuke Amano,† Motofusa Akiyama,† Christoph Böttcher,‡ and Teruyuki Komatsu*,† †

Department of Applied Chemistry, Faculty of Science and Engineering, Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo 112-8551, Japan ‡ Research Center of Electron Microscopy, Institute of Chemistry and Biochemistry, Freie Universität Berlin, Fabeckstrasse 36a, 14195 Berlin, Germany S Supporting Information *

ABSTRACT: We describe the synthesis, structure, and catalytic activity of human serum albumin (HSA) nanotubes (NTs) including gold nanoparticles (AuNPs) as a layered wall component. The NTs were fabricated as an alternating layerby-layer assembly of AuNP and HSA admixture (a negatively charged part) and poly-L-arginine (PLA, a positively charged part) into a track-etched polycarbonate membrane (400 nm pore diameter) with subsequent dissolution of the template. SEM images showed the formation of uniform hollow cylinders of (PLA/AuNP-HSA)3 with a 426 ± 12 nm outer diameter and 65 ± 7 nm wall thickness. Transmission electron microscopy and energy dispersive X-ray measurements revealed high loading of AuNPs in the tubular wall. HSAs bind strongly onto the individual AuNP (K = 1.25 × 109 M−1), generating a core−shell AuNP-HSA corona, which is the requirement of the robust NT formation. Calcination of the (PLA/ AuNP-HSA)3 NTs at 500 °C under air yielded red solid NTs composed of thermally fused AuNPs. From the mass decrease by heat treatment, we calculated the weight of the organic components (PLA and HSA) and thereby constructed a six-layer model of the tube. The (PLA/AuNP-HSA)3 NTs serve as a heterogeneous catalyst for reduction of 4-nitrophenol with sodium borohydrate. Furthermore, implantation of the stiff (PLA/AuNP-HSA)3 NTs vertically onto glass plate produced uniformly cylindrical tube arrays.

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INTRODUCTION Nanoscale hollow cylinders, nanotubes (NTs), comprising biomaterials such as amphiphilic lipids,1,2 polyelectrolytes,3−11 DNAs,10,12 peptides,13,14 and proteins15−25 have attracted considerable attention because of their potential applications for drug delivery, molecular separation, and enzymatic reactions. Proteins, which represent the highest level of sophisticated functions in nature, are particularly promising building blocks to create smart NT. Martin et al. were the first to report the synthesis of glucose oxidase NTs using layer-bylayer (LbL) polymerization in an anodic Al2O3 membrane in which each protein layer was cross-linked by glutaraldehyde.15 However, many tubes appeared to collapse upon the membrane dissolving process if the porous and hard template was removed by chemical etching using 5% phosphoric acid. We recently demonstrated the supramolecular synthesis of protein NTs using an alternating LbL assembly of negatively charged proteins and positively charged poly(amino acid)s into a track-etched polycarbonate (PC) membrane with subsequent dissolution of the template in N,N-dimethylformamide (DMF).20 The typical cylinder is prepared using a pair of human serum albumin (HSA) and poly-L-arginine (PLA), (PLA/HSA)3 NTs. Since this achievement, we have synthesized © XXXX American Chemical Society

protein NTs of several kinds and have characterized their nanostructures and chemical reactivities.20−25 For instance, HSA NTs having an antibody layer as an internal wall can trap infectious hepatitis B virus in the (one-dimensional) 1D pore space interior (yield: 100%),24 and solid NTs comprising αFe2O3 nanoparticles prepared from ferritin protein show unique magnetic and catalytic properties.21 These results encourage further research to prepare new NT architectures incorporating various combinations of materials. If inorganic nanomaterials such as metal nanoparticles and semiconductor quantum dots are blended in the biocylindrical wall, then the functionalities of the tubes might be markedly enhanced. The obtained hybrid NT combines the merits of both biomolecules and inorganic nanomaterials with the potential for widely diverse applications. We chose gold NP (AuNP) for this study because it has received an enormous amount of interest as a result of its biocompatibility, catalytic activity and tunable optical and electronic properties. 26−29 As building blocks for the preparation of a range of molecular devices, AuNPs are Received: August 24, 2013 Revised: October 21, 2013

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therefore widely exploited. This report describes for the first time the synthesis and structure of protein NTs including citrate-reduced AuNPs as layered wall components, (PLA/ AuNP-HSA)3 NTs. High-temperature treatment of this NT eliminated the organic component, thereby yielding solid NT made of thermally stacked AuNPs. Furthermore, the (PLA/ AuNP-HSA)3 NTs showed enzyme activity for 4-nitrophenol (4Nip) reduction and can be immobilized vertically on the glass surface.

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Technologies Inc.) with a temperature control unit (89090A; Agilent Technologies Inc.). Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) Observations. For SEM measurements of the NTs, the freeze-dried sample was fixed on double-sided conductive carbon tape and sputter-coated with Pd−Pt using an ion sputter (E-1045; Hitachi Ltd.). Then SEM observations were conducted using a scanning electron microscope (S-4300; Hitachi Ltd.) with accelerating voltage of 10 kV. To ascertain the average size of the outer diameter and wall thickness, at least 40 different NTs were evaluated. Energy dispersive X-ray (EDX) spectra were measured using a detection unit (HIT S3000N; EDAX Inc.) attached to a scanning electron microscope (S-3000N; Hitachi Ltd.). For TEM observations of the NTs, 6 μL of the aqueous dispersion of the sample was dropped onto an elastic carbon-coated copper grid (100 mesh; Okenshoji Co. Ltd., Japan) and air-dried at 25 °C with no staining. The grid’s surface was hydrophilized by glow charge in advance using a hydrophilic treatment device (HDT-400; JEOL Ltd.). These specimens were observed using a transmission electron microscope (JEM-1011; JEOL Ltd.) with accelerating voltage of 100 kV. For TEM, measurements of AuNP-HSA conjugate were conducted as follows. The aqueous AuNP solution (1.0 mg/mL) was mixed with HSA (1.0 mg/mL) ([HSA]/[AuNP] = 183, mol/mol) and incubated for 24 h at 4 °C. The resultant solution was centrifuged (13 600 × g) for 60 min. Subsequently, the obtained red precipitate was redispersed in deionized water. Droplets of the sample solution were applied to amorphous carbon film-covered 200-mesh grids, which had been hydrophilized before use by plasma treatment (8 W, 60 s) in a sputter coater (Bal-Tec Med 020; Leica Microsystems). The excess fluid was then blotted with filter paper and an aqueous solution of 1 m/v % uranyl acetate was applied for another 45 s. The grids were eventually left to air-dry after fluid blotting again. Then the specimens were observed using a transmission electron microscope equipped with a field-emission gun (Tecnai F20 microscope; FEI Co.) with accelerating voltage of 160 kV. Images were recorded using a CCD camera (Eagle 4k-CCD device; FEI Co.). To avoid Fresnel fringe formation at the particle edges the images were recorded in the Gaussian focus. FT-IR Spectroscopy. For FT-IR spectroscopic measurements, small KBr discs were prepared with of NT powder. Spectra at 4 cm−1 resolution were recorded using an FT-IR spectrophotometer (FT/IR4200; Jasco Corp.) at 25 °C. X-ray Photoelectron Spectroscopy (XPS). XPS measurements of the calcinated NTs were conducted using an X-ray photoelectron spectrometer (AXIS-HSi; KRATOS Analytical Ltd.) equipped with a monochromatic Al Kα X-ray source (1486.6 eV; anode operating at 15 kV and 10 mA). The base pressure in the analysis chamber was maintained at about 2 × 10−9 Torr. The pass energy values were 160 eV for survey scan and 40 eV for narrow scan. The powder sample was mounted on stainless plate with carbon tape. The area of analysis was about 600 × 800 μm. The binding energies were calibrated based on C 1s peak at 285.0 eV. Catalytic Reduction of 4-Nitrophenol (4Nip) with NaBH4. The (PLA/AuNP-HSA)3 NTs (500 μg) were suspended in pure water (2.78 mL) by brief sonication using a bath-type sonicator. The obtained dispersion was transferred to a quartz cuvette (10 mm optical path length) after which 4Nip (10 mM, 20 μL) was added. Then, aqueous NaBH4 solution (100 mM, 0.2 mL) was injected into the sample cuvette ([4Nip] = 66.7 μM, [NaBH4] = 6.67 mM). Thereafter, the dispersion was mixed by magnetic stirring under air at 25 °C. Concentration of 4Nip in the solution was assayed by measuring the absorbance of the initially observed peak at 400 nm. Comparison experiments were conducted under the same conditions using AuNTs (300 μg) or without catalyst. Template-Assisted Synthesis of Protein NT Array on Glass Plate. A hydrophilic coated glass slide with high amino-group density (MAS coated glass slide; Matsunami Glass Ind. Ltd., Japan) was immersed into DMSO solution of bifunctional cross-linker, 1,8disuccinimidyl suberate (DSS, 0.8 μM) for 15 min at 25 °C with

EXPERIMENTAL SECTION

Materials and Apparatus. Human serum albumin (HSA) was purchased from Benesis Corp. Poly-L-arginine hydrochloride (PLA, Mw: ca. 70 000) and HAuCl4·4H2O were purchased from SigmaAldrich Corp. Sodium citrate tribasic dehydrate and 4-nitrophenol (4Nip) were purchased from Wako Pure Chemical Industries Ltd. The 1,8-disuccinimidyl suberate (DSS) was purchased from Thermo Fisher Scientific Inc. The water was deionized (18.2 MΩcm) using water purification systems (Elix UV and Milli Q Reference; Millipore Corp.). Synthesis of AuNP. The citrate-reduced AuNPs were prepared using the general procedure.30,31 Briefly, to the refluxed aqueous HAuCl4·4H2O solution (0.4 g/L, 100 mL), sodium citrate tribasic dihydrate in water (24 mg/mL, 5 mL) was added and continuously refluxed for 20 min with vigorous stirring. After cooling slowly to 25 °C, the obtained AuNP solution was concentrated to 1 mg/mL (5 mM Au0) using an ultrafilter (Q0100, 10 kDa Mw cutoff; Advantec Toyo Kaisha Ltd.) in an UHP-76K ultraholder. The resultant dark red AuNP solution was filtrated through a cellulose acetate membrane (DISMIC 0.2 μm; Advantec Toyo Kaisha Ltd.) and stored in a refrigerator at 4 °C. The size distribution of the AuNPs was ascertained using dynamic light scattering measurements with a particle size analyzer (Zetasizer Nano; Malvern Instruments, Ltd.). Template Synthesis of (PLA/AuNP-HSA)3 NTs. First, for mixing AuNP and HSA well, HSA solution (50 mg/mL, 200 μL) was added to phosphate buffer (PB) solution (10 mM, pH 7.1, 9.8 mL) of AuNP (0.92 mg/mL). The resultant mixture was incubated for 18 h at 4 °C. The obtained solution was filtered using a cellulose acetate membrane (DISMIC 0.2 μm) and was then stored in a refrigerator at 4 °C. The NTs were synthesized according to our previously reported procedure.20 The PB solution (pH 7.1, 10 mM, 10 mL) of PLA (1.0 mg/mL) containing 0.1 M NaCl was filtered through a tracketched polycarbonate (PC) membrane (isopore membrane, 25 mm, pore diameter (Dp) 400 nm; Millipore Corp.) (0.25 mL/min) using a syringe pump (PHD-2000; Harvard Apparatus). Excess PLA absorbed on the pore wall was removed by water filtration (10 mL, 1.0 mL/min) with subsequent drying under reduced pressure for 10 min. Then the PB solution (pH 7.1, 10 mM, 10 mL) of AuNP (0.9 mg/mL) containing HSA (1.0 mg/mL) was filtered through the membrane (0.5 mL/min) to deposit the second layer of negatively charged AuNPHSA composite. After washing with water (10 mL, 1.0 mL/min), the membrane was dried again in vacuo for 10 min. These pressure infiltrations were repeated for a total of three cycles. The PC membrane surface was wiped using a wet cotton-swab and dried under air. To isolate the cylindrical cores from the PC template, the membrane was immersed into a DMF solution in the glass test-tube. The PC support was dissolved immediately. Then the supernatant was carefully pipetted out, and the same amount of DMF was added. After washing four times, the precipitates were freeze-dried rapidly in vacuo, yielding uniform (PLA/AuNP-HSA)3 NTs as a red-brown powder. Calcination of (PLA/AuNP-HSA)3 NTs. The lyophilized (PLA/ AuNP-HSA)3 NTs were calcinated in a crucible under air within a muffle furnace (FP32; Yamato Scientific Co. Ltd.). The temperature was increased gradually to 500 °C during 1 h, maintained for another 1 h, and decreased to 25 °C over a period of 2 h. The obtained red powder (AuNT) was stored in an automatic low-humidity chamber (Super Dry; Toyo Living Co. Ltd., Japan, humidity ≤1%). UV−vis Absorption Spectroscopy. UV−vis absorption spectra were measured using a UV−visible spectrophotometer (8453; Agilent B

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subsequent repeated rinsing with DMSO. The PC membrane embedded with (PLA/AuNP-HSA)3 NTs was cut into three pieces, one of which (1.27 cm2) was carefully placed onto the DSS-modified glass plate. Before placement, about 10 μL of PB solution (pH 7.1, 10 mM) was dropped onto the surface. Then the PC membrane-pasted glass was dried in an automatic low-humidity chamber for 15 min at 25 °C to attach the NT edge and the glass plate. To remove the PC support, the glass slide with the membrane was put into a DMF solution (ca. 9 mL) and washed with DMF. Subsequently, the glass plate was dropped into liquid N2 and freeze-dried in vacuo, yielding the lyophilized (PLA/AuNP-HSA)3 NT array as a red-brown thin film on the planar surface. The (PLA/HSA)3 NT array was prepared using the same procedure.

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RESULTS AND DISCUSSION Synthesis and Structure of (PLA/AuNP-HSA)3 NTs. HSA NTs including AuNPs were fabricated by LbL assembly technique using nanoporous PC membrane.20 The diameter of the citrate-reduced AuNPs used in this study was 12.6 ± 3.7 nm, as measured using dynamic light scattering (DLS). The aqueous AuNP solution showed a characteristic band of surface plasmon resonance (SPR) at 520 nm (Supporting Information Figure S1), which is well consistent with data reported for 10− 20 nm AuNP.30−32 Three-cycle injections of the PLA solution (1 mg/mL) followed by AuNP and HSA solution admixture ([HSA] = 1 mg/mL, [AuNP] = 0.9 mg/mL) to the PC membrane [pore diameter (Dp); 400 nm] using a syringe pump formed multilayered thin-films on channel surfaces. The obtained hybrid membrane was then immersed in DMF to dissolve the PC framework. The precipitates were quickly freeze-dried, providing a lyophilized red-brown powder. Subsequently obtained SEM images showed uniform (PLA/ AuNP-HSA)3 NTs (Figure 1A,B). The outer diameter and wall thickness were determined respectively as 426 ± 12 and 65 ± 7 nm. The maximum tube length is ca. 9 μm, which corresponds to the PC membrane pore depth. TEM images of the air-dried sample of an aqueous NT dispersion exhibited high contrast without using staining material (Figure 1C,D), which is attributable to the fact that AuNPs form part of the cylindrical wall. High-magnification pictures showed individual AuNP dots of 13 nm. The (PLA/AuNP-HSA)3 NTs swelled considerably in water, and their wall thickness doubled if compared to the dried state (65 nm →110 nm) (Figure 1D). The swelling was directed toward the inner tube channel, although the outer diameter remained unaltered. As a result, drastic reduction of the channel diameter was observed (296 nm → 206 nm). It is noteworthy that the surface part of the cylinder with ca. 8 nm thickness (bright seam) does not include AuNPs (Figure 1C (inset),D). This observation presumably indicates that the NT exterior consists of the initially deposited PLA layer.33 Furthermore, EDX mapping of the SEM images showed the homogeneous distribution of AuNPs in the multilayered wall (Figure 1E,F). Under the assumption of a six-layered structure of the (PLA/ AuNP-HSA)3 NT in which an AuNP-HSA layer has single AuNP thickness (13 nm), the width of one PLA layer is estimated as 8.7 nm. This value coincides with a PLA layer thickness of (PLA/HSA)3 NT without AuNP and is intermediate between the previously reported results of polyelectrolyte films fabricated in the nanoporous membrane under pressure conditions.4,5,7−10 Attempts to prepare similar NT using negatively charged poly-L-glutamic acid (PLG) instead of HSA failed. Only extremely fragile tubes were harvested with wall thickness of

Figure 1. Electron microscopic images of (PLA/AuNP-HSA)3 NTs prepared using a PC template (Dp: 400 nm). (A,B) SEM images, (C,D) TEM images, and (E,F) EDX mapping [E, original SEM image; F, Au M mapping image (blue parts indicate the presence of Au)].

less than 30 nm (Supporting Information Figure S2). Furthermore, a pair of PLA and AuNP did not engender a tubular structure at all.34 Many AuNPs aggregated on top of the PC template, thereby forming an insoluble paste that blocked the pores. How can we interpret the fact that the combination of PLA and AuNP/HSA can generate robust NTs and why are AuNPs loaded efficiently into the HSA layer of the tube wall? We inferred that AuNPs form a supramolecular core−shell composite with HSA. Core−Shell AuNP-HSA Conjugate. Recently, many reports in the literature have described AuNP-protein (or other NP) corona.32,35−40 Pineda et al. suggested that interactions between AuNP and HSA in the AuNP-HSA corona are mainly electrostatic.32 Chanana and Liz-Marzan et al. demonstrated that the AuNP-BSA corona has high colloidal stability.35 We then investigated interaction between AuNP and HSA. The aqueous HSA solution (1.0 mg/mL) was incubated with AuNP (1.0 mg/mL) ([HSA]/[AuNP] = 183, mol/mol) for 24 h at 4 °C and centrifuged at 13 600 × g. After removal of the supernatant, the red precipitate was redispersed in deionized water. The resultant AuNPs were characterized using TEM. The image of the negatively stained sample showed a bright seam of ca. 3 nm on the individual AuNP (Figure 2A,B). We recorded the images in the Gaussian focus to avoid Fresnel fringe formation at the particle edges. The overall diameter was estimated as 20.1 ± 2.9 nm, which contrasts sharply to the very smooth surface of naked AuNPs (14.0 ± 1.1 nm diameter; Figure 2C,D). Closer inspection of the TEM micrographs revealed that each AuNP core cannot directly contact to the nearest neighbors. It always maintains a distance because of the protein layer. This interparticle spacing avoids C

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Figure 3. Electron microscopy images of calcinated NTs prepared from (PLA/AuNP-HSA)3 NTs (shown in Figure 1): (A) SEM and (b) TEM.

bending vibrations) at 1654 cm−1 and 1545 cm−1 (Supporting Information Figure S3).41 In contrast, calcinated NTs showed no IR absorption in this region. EDX spectra demonstrated that the C and N peaks based on the polypeptides disappeared after combustion (Supporting Information Figure S4). In light of these results, we concluded that the calcination of the (PLA/ AuNP-HSA)3 NTs at 500 °C for 1 h burned HSA and PLA completely, thereby yielding solid NTs comprising thermally merged AuNPs. Longer treatment or higher temperature (500 °C for 4 h or 600 °C for 1 h) deformed the hollow cylinder structure, probably because of the low melting point of AuNP compared to that of bulk Au (1063 °C). The X-ray photoelectron spectroscopy (XPS) pattern of the calcinated NTs showed predominant double peaks of Au0, Au 4f7/2 (83.7 eV), and Au 4f5/2 (87.3 eV) (Figure 4),42 and absolutely no N 1s peak at 399 eV (Supporting Information Figure S5), which also implies that all organic components fade away completely because of the heating.

Figure 2. TEM images of (A,B) core−shell AuNP-HSA conjugate and (C,D) bare AuNPs. The spacer between the red bars represents the thickness of the absorbed HSAs on AuNP surface. The samples were negatively stained with uranyl acetate (1.0 m/v%).

unfavorable flocculation and confers perfect colloidal dispersion of AuNPs in solution. By DLS measurement, the average diameter of the HSA-coated AuNP was determined as 21.1 ± 7.6 nm, which agrees well with the TEM result. The visible absorption spectrum of AuNP in HSA solution (λmax: 525 nm) exhibits a marked bathochromic shift (6 nm) compared to that of the original AuNP (Supporting Information Figure S1), which suggests that the refractive index of the medium or area near the particle was increased by protein adhesion onto the AuNP.32 A binding constant (K) of HSA to AuNP was determined using florescence spectroscopy. The fluorescence emission intensity of HSA in PBS solution (λem: 340 nm) was quenched upon addition of AuNPs. The calculated K value of 1.25 × 109 M−1 was consistent with the result reported by Pineda et al. (K = 6.65 × 108 M−1).32 On the basis of these results, we concluded that strong binding of HSA on the individual AuNP generates core−shell AuNP-HSA corona, which is the requirement for the formation of robust NT in template synthesis. We first prepared LbL assembly of AuNP-protein corona in nanotubular form. Calcination of (PLA/AuNP-HSA)3 NTs. We previously reported that heat treatment of (PLA/ferritin)3 NTs eliminates the protein cage, thereby yielding solid NTs composed of iron oxide (α-Fe2O3) NPs.21 The obtained α-Fe2O3 NTs showed super paramagnetic properties and photocatalytic activity for 4chlorophenol degradation. As expected, calcination of the (PLA/AuNP-HSA)3 NTs under air at 500 °C for 1 h produced a red powder. Furthermore, SEM and TEM images demonstrate that the hollow tubular structure was retained intact, although a certain degree of shrinkage of the morphology was observed: 195 ± 10 nm outer diameter, 41 ± 4 nm wall thickness, and ca. 4 μm tube length (Figure 3). A detailed inspection revealed that the cylindrical wall consists of numerous nanoscale particles with diameter of ca. 13 nm, which might be the remaining AuNPs. The complete disappearance of the HSA and PLA components after calcination was confirmed using FT−IR and energy dispersive X-ray (EDX) spectroscopy. The FT−IR spectrum of (PLA/AuNP-HSA)3 NTs respectively showed the typical protein amide I band (CO stretching vibrations) and the amide II band (C−N stretching coupled with N−H

Figure 4. XPS spectrum of calcinated NTs: AuNTs.

Calcination of the fragile (PLA/AuNP-PLG)3 NTs provided only amorphous AuNP aggregate. The use of AuNP-HSA corona, realizing the homogeneous distribution of AuNPs in the tube wall, is necessary to prepare structure-defined AuNTs by heat treatment. LbL Structure Modeling of (PLA/AuNP-HSA)3 NT. The mass of the red-brown (PLA/AuNP-HSA)3 NTs powder harvested from an individual PC membrane template (diameter 25 mm, possessing 3.03 × 108 effective channels) was regularly 494 ± 18 μg. The weight of its calcinated red powder (AuNTs) was 296 ± 14 μg. Therefore, the Au content in the (PLA/ AuNP-HSA)3 NT is 60 wt %. From the mass decrease by D

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spherical polyelectrolyte brushes.47 It is interesting that Lu et al. demonstrated that AuNP in lysozyme protein crystal is useful as a unique catalyst and that its activity is modulated by tuning the crystal growth.48 Here we investigated the catalytic performance of (PLA/AuNP-HSA)3 NTs toward the reduction of 4Nip to 4-aminophenol (4Amp) using NaBH4. To the aqueous dispersion of (PLA/AuNP-HSA)3 NTs, 4Nip and NaBH4 were added. Then the mixture was stirred continuously at 25 °C. The conversion of 4Nip was assayed by measuring the absorption intensity at 400 nm. Without catalyst, the substrate remained unaltered for 12 h (Figure 6). In contrast, marked

calcination, we can construct a six-layer (PLA/AuNP-HSA)3 NT model. First, we hypothesized a simple hollow column made of solid Au with a dimension of 195 nm outer diameter, 41 nm wall thickness, and 4.0 μm length corresponding to the dimensions of our AuNT. The weight of this Au column is calculated as 1.5 × 10−12 g. On the one hand, the weight of one AuNT prepared from (PLA/AuNP-HSA) 3 NT by heat treatment was determined as 9.8 × 10−13 g (296 μg/3.03 × 108 pieces). This finding suggests that the tube wall to a great extent comprises AuNP assemblies with a packing fraction of 64%, a value which is similar to that of a body-centered cubic lattice structure. The net volume of AuNPs in one AuNT (VAuNP) was therefore determined as 5.08 × 10−20 m3. Second, the mass of the organic components (PLA and HSA) in one (PLA/AuNP-HSA)3 NT was predicted by the following eq 1 ⎡ ⎧⎛ D ⎞ 2 ⎛ D ⎤ ⎞2 ⎫ ⎢π ⎨⎜ ⎟ − ⎜ − 3TPLA − 3TAuNP ‐ HSA ⎟ ⎬L − VAuNP⎥d ... ⎝2 ⎠⎭ ⎢⎣ ⎩⎝ 2 ⎠ ⎥⎦ (1)

Therein, D is the outer diameter of (PLA/AuNP-HSA)3 NT, TPLA and TAuNP‑HSA respectively represent the thicknesses of the PLA layer (8.7 nm) and AuNP-HSA layer (13 nm), L is the tube length, and d stands for the polypeptide density. The calculated mass was 6.13 × 10−13 g, so that the expected weight of (PLA/AuNP-HSA)3 NT obtained from one PC membrane would become 483 μg. This calculation agrees well with the measured value of 494 μg and supports our LbL (six-layer) structure model. Third, the density of AuNP in the HSA layers was determined. The AuNP packing factor in the “HSA layer” is approximately 0.13.43 If the AuNPs are dispersed homogeneously in the three HSA layers, then the quantities of AuNPs in the second, fourth, and sixth layers of (PLA/AuNP-HSA)3 NT were estimated as 1.65 × 104, 1.47 × 104, and 1.29 × 104, respectively. On the basis of these analyses, we portrayed the schematic illustration of (PLA/AuNP-HSA)3 NT, as shown in Figure 5.

Figure 6. Conversion of 4Nip in aqueous NT dispersions with NaBH4 at 25 °C.

breeching of the yellow color of 4Nip and increase of absorption at 300 nm based on 4Amp production were observed in the (PLA/AuNP-HSA)3 NT dispersion (after 20 min). The AuNTs obtained using the heat treatment showed the same catalytic activity, but the efficiency was much less than that of (PLA/AuNP-HSA)3 NT. Most likely, the superior performance of the (PLA/AuNP-HSA)3 NTs is attributed to the more homogeneous distribution of AuNPs in the swollen HSA layers.49 The reduction process apparently involves (i) diffusion of 4Nip in the multilayered walls of the tube, (ii) adsorption of the substrate onto the AuNP surface, (iii) interfacial electron transfer, and (iv) desorption of the product (4Amp) from the tube wall. HSA is the most abundant plasma protein in the circulatory system and acts as a transporter for a range of insoluble small molecules.50 Thus, the role of the HSA in the NT could be not only to disperse the AuNP units homogeneously in the tube wall but also to concentrate the substrate into the cylinder from the bulk aqueous solution. This (PLA/AuNP-HSA)3 NT catalyst can be used repeatedly without particular decline of the activity (Figure 7). The conversion of 4Nip retained 94% even after the third reaction cycle. On the one hand, the AuNT activity became half of the initial value at the fourth time. The activity was only 34% of the efficacy of (PLA/AuNP-HSA)3 NT. In case of the AuNT, the product might adsorb onto the AuNP grains and diminishes the active surface area for a catalytic reaction. NT Arrays Immobilized on Glass Plates. Another advantage of the template synthesis is potential implantation of the core structure onto the solid substrates.51,52 Such a construct confers a uniform high-density array of NT on planar

Figure 5. Schematic illustration of (PLA/AuNP-HSA)3 NT in which 4.4 × 104 pieces of AuNP are included in the HSA layers. This is a speculative model involving several assumptions.

Catalytic Activity. Actually, AuNPs are well-known to show several catalytic activities.44,45 In particular, the reduction of nitrophenols to corresponding amino derivatives with sodium borohydride (NaBH4) has often been used as a model reaction to examine the catalytic property of AuNP.46−49 Recently, previous reports have described that AuNPs dispersed in polymer materials showed excellent catalytic activity for the 4-nitrophenol (4Nip) reduction. Pal et al. fabricated AuNPs immobilized on polystyrene-based anion exchange resins as an effective catalyst.46 Ballauff et al. prepared AuNP embedded E

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liquid N2 followed by freeze-drying in vacuo. After lyophilization, a thin red-brown film remained on the surface. SEM images of the red-brown area showed the formation of a uniform NT array in which the individual cylinders were fixed perpendicularly onto the substrate (Figure 8B,C). The dimension of uprightly oriented NTs was identical to that of free tubes extracted directly from the template. The outer diameter was 425 ± 12 nm with wall thickness of 66 ± 7 nm. The maximum height was again ca. 9 μm. As expected, the NT density (7.9 × 107 tubes/cm2) showed good agreement with the porosity number of the PC membrane (7.9 × 107 pores/ cm2). In contrast, a similarly implanted (PLA/HSA)3 NT array without AuNP on the glass plate was weak. We inferred that the incorporation of AuNPs as a layered wall component can enhance the tube’s structural stability and rigidity. The stiff (PLA/AuNP-HSA)3 NTs can be immobilized on the glass plate by covalent bonding between the amino groups (lysine) at the tube’s terminal and amino groups on the glass.

Figure 7. Conversion of 4Nip in aqueous NT dispersions with NaBH4 as a function of cycles at 25 °C.

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CONCLUSIONS We have shown the template-assisted synthesis of multilayered NTs of PLA and HSA-coated AuNPs. The core−shell AuNPHSA corona in which the protein is physically or chemically bound to the NP surface plays a crucial role in robust NT formation. Heat treatment of the tubes yielded neat and solid AuNTs. The polypeptide components were removed completely, whereas the tube dimension almost halved. The tube walls were composed of thermally agglutinated AuNP grains of 13 nm diameter with a packing fraction estimated as around 64%. The mass difference after the calcination enabled the deduction of a six-layer model of the (PLA/AuNP-HSA)3 NT. Furthermore, the hybrid hollow cylinders exhibit catalytic activity for the reduction of 4Nip with NaBH4. The substrate can diffuse into the swollen walls of the tube and can be reduced to the corresponding aniline (4Amp) on the AuNP surface. Results suggest that blending of other metal NPs or semiconductors with HSA can enable preparation of a new generation of nanotubular architectures. Moreover, we demonstrated the template-assisted synthesis of a protein NT “forest” comprising (PLA/AuNP-HSA)3 hollow cylinders covalently attached onto the planar glass surface.

surfaces, which would become a unique molecular trap device for bioseparation chemistry and a novel biochip for use as a chemical reactor or sensor. A hydrophilic coated glass slide with a high amino-group density (MAS coated glass) was first modified by bifunctional cross-linker 1,8-disuccinimidyl suberate (DSS). Then a PC membrane embedded with (PLA/ AuNP-HSA)3 NTs was pasted onto the surface to attach the tube edge and glass plate (Figure 8A). The PC framework was subsequently dissolved in DMF. The glass was plunged into

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ASSOCIATED CONTENT

S Supporting Information *

UV−vis absorption spectra of AuNP and AuNP-HSA conjugate in water (Figure S1), SEM image of (PLA/AuNP-PLG)3 NTs (Figure S2), IR and EDX spectra of (PLA/AuNP-HSA)3 NTs and calcinated NTs (Figure S3, S4), and XPS spectrum of calcinated NTs (Figure S5). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

* E-mail: [email protected]. Tel/Fax: +81 3-3817-1910. Notes

The authors declare no competing financial interest.

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Figure 8. (A) Schematic illustrations of immobilization process of (PLA/AuNP-HSA)3 NTs on glass surface: (a) pasting and drying, (b) PC template dissolution and freeze-drying. (B,C) SEM images of (PLA/AuNP-HSA)3 NT arrays immobilized covalently on a planar glass plate. *Hydrophilic-coated glass slide with high amino-group density.

ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific Research on Innovative Area (“Coordination Programming” Area 2107, No. 21108013) from MEXT Japan. Skillful F

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experiments on synthesis of NTs and catalytic reactions conducted by Miss Eri Ishikawa are gratefully acknowledged.

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