Continuous Mesoporous Aluminum Oxide Film with Perpendicularly Oriented Mesopore Channels.

Mesoporous aluminum oxide (MAO) films with perpendicularly oriented cylindrical mesopores (pore diameter: ca. 10 nm) were successfully deposited on a glass substrate by a surfactant-templating approach using aluminum nitrate as an aluminum source. The perpendicular orientation of mesopores was confirmed by grazing-incidence small-angle X-ray scattering and neutron reflection experiments. The thickness of the MAO film was around 100 nm, with a surface roughness of less than 6 nm. Since the inner surface of MAO pores was positively charged, negatively charged glucose oxidase molecules could be densely loaded into the cylindrical mesopores without significant loss of enzymatic activity. The present MAO film is potentially useful as an inorganic host material for an enzyme toward the development of a biocatalytic system.

Introduction

Mesoporous silica, which has a uniform pore structure and high adsorption capacity, is a useful host material for biomacromolecules.[1−5] Since the pore wall of mesoporous silica is negatively charged under physiological conditions, biomacromolecules with a low isoelectric point (pI) can be loaded into its pores. In contrast, the loading of negatively charged biomacromolecules requires the modification of the pore wall with amino silanes.[6−9] Titanium oxide,[10] titanium phosphate,[11−13] and aluminum oxide[14−18] have a high pI, and their rigid mesostructured framework will provide a host space for negatively charged biomacromolecules such as glucose oxidase (GOD).

Surfactant-templated mesoporous materials with a high pI are commonly synthesized by the hydrolysis of metal alkoxides.[10−18] These materials have a uniform and ordered pore structure but their pore sizes are usually smaller than the GOD molecule.[10−16] GOD is an important oxidase for glucose sensors and has a molecule that measures 5.4 nm × 6.7 nm × 7.4 nm (PDB ID, 1GAL). A mesopore size of more than 8 nm is required for the pore loading of GOD molecules.

The surfactant-templated sol–gel method has also been applied to deposit a mesoporous thin film on a solid substrate. The resulting mesoporous film usually has a three-dimensional mesopore network or cylindrical mesopore channels oriented parallel to the substrate surface.[19] A perpendicularly oriented cylindrical mesopore channel is favored to create an effective biocatalytic system with an enzyme, but there has been no effective method to synthesize a continuous mesoporous film with perpendicular mesochannels. Yamauchi and co-workers succeeded in forming a continuous mesoporous aluminum oxide (MAO) film composed of vertically oriented nanopillars by using aluminum tri-n-butoxide as the aluminum source.[17,18] However, it is expected that a biomacromolecule tightly confined inside a cylindrical mesopore channel exhibits more enhanced stability than that embedded between nanopillars.[20,21] Recently, Mou and co-workers reported a simple method to deposit a mesoporous silica film with perpendicular nanochannels. The perpendicular silica-nanochannel can be used for the confinement of positively charged protein molecules, but the electrostatic repulsion between GOD and the silica surface would reject the GOD confinement.[22]

Herein, we report the synthesis of a mesoporous aluminum oxide (MAO) film by the surfactant-templated method using aluminum nitrate as the metal precursor. The MAO film possesses a specific pore arrangement that allows perpendicularly oriented cylindrical pore channels to be aligned on a glass substrate. The pore size of the mesopore channel is ca. 10 nm, which is sufficiently larger than the molecular dimensions of GOD. GOD molecules can be highly loaded within the MAO film without the loss of enzymatic activity.

Experimental Section

Materials and Chemicals

A rectangular cover glass slip (24 mm × 45 mm × 0.3 mm, Matsunami Glass Ind., Ltd., Osaka, Japan) was used for the fabrication of the MAO film. Pluronic F127 (PEO106–PPO70–PEO106) and glucose oxidase (GOD) were purchased from Sigma-Aldrich, Japan (Tokyo, Japan). Aluminum nitrate and other chemicals were purchased from Fujifilm Wako Pure Chemical Corp. (Osaka, Japan). Milli-Q water was used for all experiments.

Synthesis and Characterization of the MAO Film

We prepared the MAO film on a glass substrate by using Pluronic F127 as a template surfactant and aluminum nitrate as an aluminum seed. The cover glass slip was sonicated in acetone and water for 5 min each. After drying, the cover glass slip was heated in an oven at 500 °C to remove organic adsorbates. Pluronic F127 (0.1 g) was dissolved in 2 mL of distilled water containing aluminum nitrate (0.1–1.0 M), and the mixture was stirred at room temperature for 4 h. This precursor solution was spin-coated on the clean glass substrate at 600 rpm for 20 s, at an acceleration of 4000 rpm for 60 s. The spin coating was performed under atmospheric conditions (25 °C). The glass substrate was dried overnight at room temperature and then calcined at 500 °C for 6 h in an ambient atmosphere. Hereinafter, we designate the glass substrate with the MAO film as the MAO/glass substrate.

The structural properties of MAO were characterized by field-emission scanning electron microscopy (FE-SEM), neutron reflectometry, and grazing incidence small-angle X-ray scattering (GI-SAXS). The SEM image was measured on a Hitachi S4800. Neutron reflectivity experiments were performed using the BL17 SHARAKU neutron reflectometer installed in the Materials and Life Science Experimental Facility (MLF) at the Japan Proton Accelerator Research Complex (J-PARC).[23] The incident beam power of the proton accelerator was 500 kW, and the neutron data were measured using the time-of-flight technique. The wavelength (λ) range of the incident neutron beam was tuned to approximately λ = 2.2–8.8 Å, and a Qz range of 0.008–0.09 Å–1 was covered, where Qz = (4π/λ)sin θ (where θ represents the incident angle and is equal to 0.3 and 0.9°). All measurements were performed at room temperature. The data were reduced, normalized, and subtracted using a program installed on the BL17 SHARAKU. The Motofit program[24] was used to fit the neutron reflection profiles using a least-squares approach to minimize the deviation of the fit. The thickness, scattering length density, and Gaussian roughness were also evaluated using the Motofit program. GI-SAXS was measured on a Rigaku SmartLab HyPix-3000 system using Cu Kα radiation.

The surface charge on the inner pore surface of the MAO film was examined by observing the adsorption of fluorescein and methylene blue. The MAO/glass substrate was immersed in an aqueous solution containing 10 μM fluorescein and methylene blue overnight. After rinsing with water, the transmission absorption spectrum of the MAO/glass substrate was recorded on a UV/Vis spectrophotometer (JASCO, V570).

Measurement of Activity of GOD within the MAO Film

The MAO/glass substrate was immersed in the GOD solution (1.0 mg/mL) at 5 °C for 20 h. After rinsing with water, the amount of GOD adsorbed at the MAO film was estimated by measuring the transmission absorption spectrum. The MAO/glass substrate with GOD was then immersed in 0.1 M N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) buffer (pH 7.0) containing 150 mM 4-aminoantipyrine, 150 mM phenol, 0.03 g/mL peroxidase, and 0–10 mM glucose. The production of the red dye, a product of the enzymatic reaction, was monitored by observing the time-course absorbance at 500 nm.

Results and Discussion

Figure shows typical SEM images of the MAO film prepared with a precursor solution containing 0.5 M ammonium nitrate. Well-ordered pores with a pore diameter of ca. 10 nm can be observed in the SEM top view (Figure A). As shown in the cross section of the MAO film after cleavage (Figure B), the thickness of the MAO film is uniform (ca. 100 nm), and the pore channels appear to be oriented perpendicular to the substrate surface. The cleavage by hand cannot produce a sharp cleavage plane, and the pore structure was slightly broken. Although the pore structure shown in the cross-sectional SEM view is not clear, the appearance of a vertical structure suggests the perpendicularly oriented cylindrical pore channels. A lower aluminum nitrate concentration in the precursor solution resulted in a disordered pore structure (Figure S1). The MAO film prepared with the 0.5 M aluminum nitrate solution was therefore used for further structural characterization.

Figure 1

Typical (A, B) top and (C) cross-sectional SEM views of the MAO film prepared with a precursor solution containing 0.5 M aluminum nitrate.

Figure A shows the neutron reflection spectrum obtained by irradiating a neutron beam from the upper side of the MAO film. The experimental spectrum could be closely fitted to a single-layer model. The thickness of the MAO film obtained from our neutron reflectivity analysis is 106 nm, with roughnesses of 5.9 and 1.5 nm for the air/MAO and MAO/glass surfaces, respectively. On the assumption that the scattering length density of the alumina matrix was equal to that of the amorphous alumina (5.67 × 10–6 Å–2), the volume fraction of the pores in the MAO film was estimated to be 0.22. When neutron reflection patterns were measured by irradiating a neutron beam from both the film and the glass sides, the MAO film exhibited no Bragg diffraction peaks (Figure B). As shown in Figure , only a vertical mesoscale periodicity was recognized in the GI-SAXS pattern of the MAO film. The d-spacing of the in-plane periodicity is 12.5 nm (Figure B), which is greater than the pore size observed in the SEM top view (ca. 10 nm). We therefore conclude that the perpendicularly oriented cylindrical pore channels are aligned within the MAO film (Figure ).

Figure 2

(A) Neutron reflectivity profiles of the MAO film on a glass substrate and the fitting result. The solid line represents the best fit of the data. (B) Neutron reflection intensity as a function of the detection angle (incident angle: 0.3°, λ: 3.3–3.4 Å). The neutron beam was irradiated from the film side (open circles) and from the glass side (shaded circles). The MAO film was prepared with a precursor solution containing 0.5 M aluminum nitrate.

Figure 3

(A) GI-SAXS pattern and (B) in-plane intensity profile of the MAO film on a glass substrate. The MAO film was prepared with a precursor solution containing 0.5 M aluminum nitrate.

Figure 4

Schematic illustration of the MPA film on a glass substrate.

Jiang et al. reported the formation of a mesoporous amorphous aluminum oxide film with a three-dimensional mesostructure.[17,18] This mesostructure was converted to an array of vertically oriented nanopillars during the crystallization process at high temperatures (above 800 °C). On the other hand, the results of neutron reflection and GI-SAXS experiments indicate that the present MAO film has perpendicularly oriented mesopore channels without conversion of the mesostructure.

The surface charge of the inner pore surface of the MAO film was examined by observing the adsorption of charged dyes. When the MAO/glass substrate was immersed in the dye solution, the adsorption of negatively charged fluorescein was recognized. In contrast, the adsorption of positively charged methylene blue hardly took place (Figure S2A). The conventional mesoporous silica film[25] with a negatively charged surface adsorbed only the methylene blue (Figure S2B). These results indicate a positive charge on the inner pore surface of MAO. This desired surface charge allows efficient GOD adsorption by the MAO film.

We immersed the MAO/glass substrate in GOD solution (1.0 mg/mL) at 5 °C for 20 h. After rinsing, the MAO film with GOD showed an absorption band indicative of cofactor flavin adenine dinucleotide in GOD (Figure S3). The amount of GOD at the MAO film was estimated, from the absorbance at 452 nm, to be 1.9 × 10–6 mol cm–3. By assuming the volume of a single GOD molecule to be 253 nm3, the volume fraction of GOD within the MAO film was calculated to be 0.24, which was close to the volume fraction of pores within the MAO film (0.22). It therefore can be concluded that GOD molecules are efficiently adsorbed into ca. 10 nm-diameter pore channels.

The activity of GOD within MAO was examined using the GOD/peroxidase method. The MAO/glass substrate with GOD was immersed in 0.1 M HEPES buffer (pH 7.0) containing 150 mM 4-aminoantipyrine, 150 mM phenol, 0.03 g/mL peroxidase, and 0–10 mM glucose. The production of the red dye, a product of the enzymatic reaction, was monitored by observing the time-course absorbance at 500 nm. As shown in the inset of Figure , the absorbance of the red dye increased quasi-linearly with time. The apparent Michaelis–Menten constant, Km, derived from the time-course measurements, was 3.76 mM (Figure ). This value was similar to that for GOD in nanocomposite systems: 8.26 mM for carbon nanotubes dispersed in a polymer[26] and 1.4 mM for microporous titanium oxide.[27] GOD within the MAO film thus can maintain a high enzymatic activity.

Figure 5

Dependence of glucose concentration on the reaction rate obtained by the time-course absorbance at 500 nm (inset). The solid line represents the best fit of data to the Michaelis–Menten equation.

Conclusions

In the present study, a continuous mesoporous aluminum oxide film with perpendicularly oriented pore channels was synthesized by using aluminum nitrate as the metal precursor. A conventional mesoporous silica film requires surface modification with amine functional groups for the loading of GOD into the silica mesopores because both silica and GOD surfaces are negatively charged. In contrast, the positively charged inner pore surface of the aluminum oxide film allowed efficient pore loading of GOD without the surface modification. The perpendicularly oriented cylindrical mesopore channel is favored to create an effective biocatalytic system with an enzyme. The present mesoporous aluminum oxide film is thus potentially useful as a rigid host material for an enzyme toward the development of a biocatalytic system.