Deposition of Polyelectrolyte Multilayer Film on a Nanoporous

Oct 25, 2012 - the incident light satisfies the phase-matching conditions, the optical waveguide ... Bedford, MA) was used for all experiments. Poly(a...
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Deposition of Polyelectrolyte Multilayer Film on a Nanoporous Alumina Membrane for Stable Label-Free Optical Biosensing Kazuhiro Hotta,† Akira Yamaguchi,*,‡ and Norio Teramae*,† †

Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980-8578, Japan College of Science, Ibaraki University, 2-1-1 Bunkyo, Mito, Ibaraki 310-8512, Japan

‡

S Supporting Information *

ABSTRACT: The stabilization of inorganic nanostructures is a key challenge in developing label-free optical biosensors using inorganic nanoporous films. In the present study, a polyelectrolyte multilayer film (PMF) composed of poly(acrylic acid) and poly(allylamine hydrochloride) was deposited on porous anodic alumina (PAA) film (pore diameter = 40 nm) by the layer-by-layer technique. The characteristics of the PMF as a protective coating layer against dissolution of the PAA film were examined by means of in situ optical waveguide spectroscopy (OWS), ex situ scanning electron microscope/energy-dispersive spectroscopy (SEM/ EDS), and infrared reflection−adsorption spectroscopy (IR-RAS). The results obtained by OWS and SEM/EDS indicated the formation of PMF at both the alumina pore surface and the PAA film surfaces. Processing the PMF-deposited PAA (PMF−PAA) film at 180 °C to promote thermal cross-linking allowed PMF to act as a strong protective coating layer in aqueous buffer solutions (pH 2.5−8.7) but also in common alumina matrix etchant. Residual amino groups in the cross-linked PMF could also be used to conjugate single-probe DNA at the PMF−PAA film for OWS-based DNA−DNA hybridization assay. From the results obtained in this study, it was concluded that the deposition of PMF followed by thermal cross-linking is potentially useful not only as a protective coating and but also for immobilizing biorecognition elements in the PAA film.

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INTRODUCTION Anodically prepared inorganic nanoporous films have tunable and well-defined one-dimensional cylindrical pores and thus have attracted much attention in the fields of separation,1−3 gas sensing,4,5 photonics,6,7 catalysis,8,9 and nanostructure fabrication.10−13 More recently, their use as a sensing platform for label-free optical biosensors has been studied extensively, since sensitive biosensors can be designed by controlling the anodization and post-treatment conditions and by functionalizing the inorganic nanoporous film.14−25 Optical biosensors based on inorganic nanoporous film are a type of refractometric sensor. Changes in the refractive index of a film accompanied by adsorption of an analyte into the nanopores have been detected by means of Fabry−Perot fringes,14−16 photonic band gaps,17,18 and optical waveguide modes.19−25 Since the sensitivity of refractometric sensors tends to be lower than that of fluorometric sensors, considerable efforts have been directed toward enhancing the sensitivity of optical biosensors using inorganic nanoporous film. For example, a porous silicon film fabricated by electrochemical etching followed by thermal oxidation has been demonstrated to be a sensitive optical interferometric substrate.14 A porous anodic alumina (PAA) membrane with a caplike gold layer was fabricated as a sensitive label-free DNA sensor based on localized surface plasmon resonance coupled with interferometry.16 In optical biosensors based on optical waveguide spectroscopy (OWS), the sensor sensitivity could be enhanced by engineering the nanostructures and tuning the optical properties of the PAA film.24 © 2012 American Chemical Society

Although a number of improvements to sensor sensitivity have been achieved, a major impediment to the use of inorganic nanoporous film for optical biosensors lies in the chemical instability of inorganic nanoporous structures in aqueous solution. For example, porous silicon film is chemically stable under acidic conditions but dissolves in physiological to alkaline solutions.14,26 PAA film is relatively stable in physiological solution, but due to its amphoteric character, it dissolves in acidic and alkaline solutions.21,27 An optical biosensor with an inorganic nanoporous film is sensitive to the film structure, refractive index of the film, and the number of biorecognition elements immobilized on the film.19−25 Dissolution of the inorganic nanoporous film would therefore cause unfavorable drift of the baseline and detachment of capture probes from the film, resulting in a deterioration of their biosensing performance. To overcome this problem with chemical instability, it is possible to choose an intrinsically stable nanoporous film as a sensor substrate. Mun et al.15 employed a chemically stable nanoporous TiO2 film as an interferometric biosensor that demonstrated stable sensor responses over a wide range of pH conditions. Another possible approach would be the use of a protective coating layer on the surface of the nanoporous structures, as shown in Figure 1. If the protective coating layer Received: September 3, 2012 Revised: October 15, 2012 Published: October 25, 2012 23533

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Figure 1. Schematic illustrations of PMF−PAA film (upper panel) and of DNA−DNA hybridization assay using PMF−PAA film (lower panel).

inhibits the accessibility of water molecules and ions to the inorganic surface, dissolution of the inorganic matrix could be suppressed. Layer-by-layer (LbL) deposition is a versatile bottom-up technique for forming a polyelectrolyte multilayer film (PMF) on a charged surface.28 Since the LbL technique enables PMF to be deposited on the inner and outer surfaces of inorganic nanopores,29 it is a good candidate to act as a protective coating of the inorganic nanoporous film. The functional groups in the PMF could also be used to immobilize biorecognition elements onto the nanopore surface.29 In this study, we examined the LbL deposition of polyelectrolytes on the PAA film and demonstrated that thermally cross-linked PMF could act as a strong protective coating layer to prevent dissolution of the PAA film. The PAA film used in this study had a packed array of cylindrical pores (ϕ = ca. 40 nm). Poly(acrylic acid) (paa) and poly(allyl amine hydrochloride) (pah) were chosen as building blocks for the PMF, since paa/pah-based PMF is known to exhibit high stability by the thermal cross-linking of the polymer chains.30,31 In addition, paa/pah-based PMF has reactive carboxyl and amino groups that can be used for covalent immobilization of biorecognition elements.29 We studied the deposition process of the PMF at the PAA film and the chemical stability of the PMF-coated PAA (PMF− PAA) film using in situ optical waveguide spectroscopy (OWS), in which the reflection spectra (reflectivity vs wavelength) of the PAA film on a thin Al layer were measured in the Kretschmann configuration as shown in Figure 2.22−25 When the incident light satisfies the phase-matching conditions, the optical waveguide mode is evanescently excited in the PAA film and a sharp attenuation dip appears in the reflection spectra. The wavelength position of the waveguide coupling dip is sensitive to changes in the refractive index of the PAA film. The refractive index of the PAA film varies with dissolution of the alumina matrix and by adsorption of polyelectrolytes at the PAA film. Accordingly, OWS is suitable for observing the PMF deposition process and the chemical stability of the PMF−PAA film. The PMF was also characterized by ex situ measurements, such as infrared reflection−absorption spectroscopy (IR-RAS)

Figure 2. Schematic illustration of the setup for optical waveguide spectroscopy (OWS).

and scanning electron microscopy (SEM) equipped with energy-dispersive X-ray spectroscopy (EDS). In a previous study, we confirmed that PAA film can be utilized as a platform for a sensitive biosensor based on OWS.24 Hence, the applicability of the PMF−PAA film to a label-free optical biosensor was examined from DNA−DNA hybridization assay. The informative nature of the OWS sensorgram, obtained by measuring change in the OWS response vs time, for immobilization and hybridization of single-strand DNA was demonstrated. From the results obtained in this study, we conclude that protective coating with PMF followed by thermal cross-linking of the polymer chains is useful for constructing a stable label-free optical biosensor using inorganic nanoporous film.

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EXPERIMENTAL SECTION Materials. A square cover glass slip (25 × 25 × 0.3 mm) was purchased from Matsunami Glass Ind., Ltd. (Osaka, Japan). An Al wire (99.99%; Nilaco Co., Tokyo, Japan) was used to thermally deposit the Al film. Milli-Q water (Millipore Corp., Bedford, MA) was used for all experiments. Poly(acrylic acid) (paa; Mw ≈ 100 000) and poly(allyl amine hydrochloride) (pah; Mw ≈ 15 000) were obtained from Aldrich, Inc. (Tokyo, Japan). Sulfosuccinimidyl-6′-(biotinamido)-6-hexanamido hex23534

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was characterized by IR-RAS measurements using an FTIR spectrometer (JASCO; FT/IR-4200 equipped with a RAS attachment, model PRO410-H). Hereinafter, we denote crosslinked PMF as cPMF (Table 1). Optical Waveguide Spectroscopy. We have given the details of the OWS experimental apparatus in previous reports.23−25 In brief, the reflection spectra of a sample substrate with the PAA film or the PMF−PAA film were measured in the Kretschmann configuration (Figure 2). A white light from a Xe lamp (Hamamatsu Photonics; L8254) was collimated using a spatial filter assembly (ϕ = 1 mm), and s-polarized light was chosen using a polarizer (Sigma Koki Co., Ltd.; SPF). The light was then directed at the sample substrate attached to a BK7 equilateral glass prism at a certain angle of incidence, and the reflected light intensity from the PAA film was measured using a photonic multichannel analyzer (Hamamatsu Photonics; PMA-11). A PDMS flow cell with a cell volume of 2 μL (16 mm × 1.3 mm × 100 μm) was set on the sample substrate. The sample solutions were injected into the flow cell using a syringe pump. Stability Tests of the PMF−PAA Film. The chemical stability of the PMF−PAA film was examined both by in situ OWS and by ex situ SEM/EDS experiments. In the SEM/EDS experiments, the structure and composition of the PMF−PAA film were observed before and after immersion of the PMF− PAA film in a 10 wt % phosphoric acid solution, which is a common alumina matrix etchant. In the OWS experiments, the OWS response was monitored under a continuous flow of buffer solutions (citrate buffer for pH 2−4, phosphate buffer for pH 5−9, and glycine−NaOH buffer for pH 9.8). All the buffer solutions contained 10 mM buffer and 0.1 M NaCl. The flow rate of each solution was 15 μL/min. The stability tests were carried out at 25 °C. DNA−DNA Hybridization Assay. The PMF4−PAA film after thermal cross-linking (cPMF4−PAA film, Table 1) was used for the DNA−DNA hybridization assay. The cPMF4− PAA film was functionalized by a single-stranded probe DNA using biotin−streptavidin coupling, as shown schematically in Figure 1. First, the residual amino groups in cPMF4−PAA were modified with an amine-reactive biotin reagent (Sulfo-NHSLC-LC-biotin) by passing the 0.2 M biotin reagent, in a buffer solution (buffer A; 10 mM phosphate buffer containing 0.1 M NaCl, pH 7.1), into the flow cell for 30 min. Then, the solution in the flow cell was replaced by buffer A to remove the unreacted biotin reagent. Subsequently, 0.1 and 1 μM streptavidin in buffer A were loaded into the flow cell to immobilize the streptavidin by means of the biotin−avidin coupling reaction. Then, after rinsing the flow cell with another buffer solution (buffer B: 10 mM phosphate buffer, 0.2 M NaCl and 1 mM EDTA, pH 7.3), the biotinylated probe DNAs were attached to the streptavidin by flowing 10 μM biotinylated probe DNA in buffer B. The flow rate was 15 μL/min for each immobilization step. Finally, the 100 nM target DNA in buffer B was passed into the flow cell to hybridize the probe DNA with the target DNA. The flow rate during hybrization was 10 μL/min. OWS was applied to monitor the functionalization and hybridization processes. The temperature during the DNA− DNA hybridization assay was 25 °C.

anoate (Sulfo-NHS-LC-LC-biotin) was obtained from Pierce, Inc. (Rockford, IL). The other reagents were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan) and were used as received. Single-stranded DNAs were customsynthesized and purified by HPLC (>97%) by Nihon Gene Research Laboratories Inc. (Sendai, Japan). Their sequences were as follows. Probe, 5′-biotin-TTT TTT ATT TGG GTG AGA TTG CTC ACA-3′; complementary target (T1), 5′-TGT GAG CAA TCT CAC CCA AAT-3′; and noncomplementary target (T2), 5′-GCC TAC GCC CTC AGC TCC AAC-3′. Deposition of PMF on PAA Films. A PAA film was fabricated by partial anodization of an Al film deposited on a cover glass substrate as described in our previous report.24 The resultant sample substrate is comprised of PAA film and Al thin film on the glass substrate as shown in Figure 2. The thicknesses of the PAA film and the Al thin film were 500 and 14 nm, respectively. The average pore diameter of the cylindrical alumina pores of the PAA film was ca. 40 nm (see Figure 4, left-hand images). For the in situ OWS experiments to monitor the LbL deposition, a PDMS flow cell was set on the sample substrate attached to a BK7 equilateral glass prism (Figure 2). The OWS responses from the sample substrate were measured under alternate flow of the polyelectrolyte solution and rinsing water. The polyelectrolyte (paa and pah) solutions were prepared as described in the literature.29 The paa solution contained 20 mM paa and 0.5 M NaCl, with its pH adjusted to 4.0 by addition of concentrated NaOH solution. The pah solution contained 20 mM pah and 0.5 M NaCl, and its pH was adjusted to 4.0 by adding concentrated HCl solution. The flow rate of the polyelectrolyte solutions and rinsing water was 15 μL/min. For the stability tests and DNA−DNA hybridization assay, LbL deposition was carried out by immersing the sample substrate in the polyelectrolyte solutions. First, the sample substrate was immersed in the paa solution for 5 min to adsorb negatively charged paa onto the positively charged alumina surface, followed by immersion of the PAA/Al film in pure water for 1 min to remove excess paa. The substrate was then immersed in the pah solution for 5 min, followed by pure water for 1 min. These processes were repeated until the desired number of polyelectrolyte layers had been deposited on the PAA film. Finally, the sample substrate was thoroughly rinsed with water and dried in a nitrogen stream. Hereinafter, we denote the PAA film with n layers of PMF as PMFn−PAA film (e.g., a PAA film coated with 3 layers composed of paa/pah/paa is denoted as PMF3−PAA film) (Table 1). The structure and Table 1. Abbreviations for the Sample Substrates Prepared in This Study abbreviation PAA film PMFn−PAA film cPMFn−PAA film

PAA film prepared by anodization PAA film with as-deposited n-layers of PMF PMFn−PAA film after thermal cross-linking

composition of the PMF−PAA film were characterized by a field-emission SEM (Hitachi S-4300) equipped with an EDS (EDAX Genesis 7000). The LbL deposition was carried out at 25 °C. Thermal cross-linking between the paa and pah multilayers was performed by heating PMF−PAA film in an oven under atmospheric conditions. The degree of cross-linking was controlled by adjusting the heating time and temperature and

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RESULTS AND DISCUSSION Deposition of PMF on the PAA Film. The OWS experiments were carried out to monitor the LbL deposition process of PMF on the PAA film. In the reflection spectrum of 23535

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the PAA film measured in contact with water, a clear waveguide coupling dip corresponding to the excitation of a TE0 waveguide mode can be seen (spectrum 0 in Figure 3a). For

deposition. Then, loading of the paa or pah solution and rinsing with water were carried out repeatedly (Figure 3). The shift of λOWG for the formation of the second/pah, third/paa, fourth/ pah, and fifth/paa layers were 66, 126, 124, and 141 nm, respectively. The red-shift values tended to increase as deposited layers were added, suggesting greater adsorption volume by the polyelectrolyte layer as deposited layers were added. This deposition behavior is well-known in the LbL deposition of polyelectrolytes.32,33 Figure 4 shows typical SEM images of PAA films before and after the deposition of PMF. The effective pore diameter

Figure 3. (a) Changes in reflection spectra of the sample substrate with PAA film laid down by the LbL deposition of polyelectrolytes (paa and pah). (b) Time courses of λOWG during the alternative injection of polyelectrolyte solution (paa or pah solution) and water. Black, red, and blue arrows indicate the injection of rinsing water, paa solution, and pah solution, respectively. Numbers in the reflection spectra correspond to time points for spectra capturing in the time course profiles (b). In these experiments, the incident angles of the probing light were set at 62.8°, 63.8°, 65.0°, and 66.8° for monitoring first/paa and second/pah deposition, third/paa deposition, fourth/pah deposition, and fifth/paa deposition, respectively.

Figure 4. Top and cross-sectional SEM images of the PAA film coated with 0, 4, and 5 layers of the paa/pah multilayer film. Scale bars correspond to 200 nm.

tended to be narrower on increasing the number of deposited layers, indicating the deposition of PMF on the outer surface and inner pore surface of the PAA film, as schematically shown in Figure 1. When Fresnel calculations were carried out, assuming the deposition of PMF on only the outer surface of the PAA film,24 the total shift of λOWG after the fifth-layer deposition corresponded to the formation of a greater than 100 nm thick PMF. However, such a thick PMF was not detected in the SEM images (Figure 4, right). The Fresnel calculation also suggested the deposition of PMF on the outer surface and inner pore surface of the PAA film. As shown in Figure 3b, loading of the polyelectrolyte solution induced large and abrupt red-shifts of λOWG within a few minutes, indicating that deposition of the polyelectrolyte layer approached saturation within a few minutes. In the removal of excess polyelectrolyte, abrupt blue-shifts of λOWG took place within 1 min of loading of the rinsing water, indicating rapid desorption of the excess polyelectrolytes. The adsorption kinetics of the polyelectrolyte suggest that exposing the sample substrate with PAA to the polyelectrolyte solutions for 5 min is sufficient for polyelectrolytes to be deposited on the PAA film. Rinsing for 1 min appears to almost completely remove any excess polyelectrolyte. Accordingly, the PMF−PAA films used for the following experiments were prepared by successive exposure of the sample substrate to polyelectrolyte solutions (5 min) and rinsing water (1 min). Chemical Stability of the PMF−PAA Film. The chemical stability of the PMF5−PAA/Al film was first examined by exposing the sample substrate to a 10 wt % phosphoric acid solution, a common alumina matrix etchant.24 Figure 5 shows typical SEM images obtained for three kinds of PAA films: (a) a bare PAA film without PMF coating, (b) PMF5−PAA film, and (c) cPMF5−PAA/Al prepared by heating at 180 °C for 1 h (Table 1). After the thermal cross-linking, the amidation between carboxyl groups of the paa layer and amino groups of

deposition of the PMF, the polyelectrolyte solution and rinsing water were alternatively loaded into the flow cell, and the wavelength-position of the waveguide coupling dip (λOWG) was monitored over time (Figure 3b). In the OWS experiments, the incident angle of probing light was adjusted between 62.8° and 66.8° to bring the waveguide coupling dip within the measurable wavelength range of our OWS experimental apparatus (360−760 nm). When pure water was passed into the flow cell with the bare PAA film, λOWG was 425 nm at the incident angle of 62.8° (spectrum 0 in Figure 3a). On the first loading of the paa solution, an abrupt red-shift of λOWG from 425 to 533 nm occurred within a few minutes (Figure 3b). Then, on rinsing with pure water to remove any excess paa from the PAA film, λOWG was abruptly blue-shifted (Figure 3b), reaching a steady state at 477 nm (spectrum 1 in Figure 3a). In OWS, the redshift in λOWG is due to the increase in refractive index of the PAA film that results from the adsorption of the analyte by the PAA film. The shift of λOWG (ΔλOWG) can be approximately expressed as follows:23 ΔλOWG = aqads

(1)

where a is a constant and qads is the total amount of adsorbed analyte. Accordingly, the net shift of 52 nm (425−477 nm) in Figure 3b can be ascribed to the deposition of the first/paa layer on the PAA film. Next, loading of a pah solution into the flow cell and rinsing with water were successively carried out, as a result of which λOWG showed a red-shift from 477 nm (spectrum 1 in Figure 3a) to 543 nm (spectrum 2 in Figure 3a). This red-shift can be ascribed to deposition of the second/pah. After measurement of spectrum 2 in Figure 3a, the incident angle of probing light was varied from 62.8° to 63.8°, and spectrum 2′ was measured prior to monitoring the third/paa 23536

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chemically stable, even when exposed to alumina matrix etchant. The stability of the cPMF4−PAA film in buffer solutions was examined by OWS and SEM/EDX experiments. In the OWS experiments, the time courses of λOWG were observed for successive loading of buffer solution (pH 2.5−9.8) and pure water into the flow cell attached to (a) the bare PAA film and (b) the cPMF4−PAA film (Figure 6). In the time-course

Figure 5. SEM images of (a) PAA film, (b) PMF5−PAA film, and (c) cPMF5−PAA film before/after immersion of the films in a 10 wt % phosphoric acid solution. Scale bars correspond to 200 nm.

the pah layer was confirmed by the appearance of amide peaks at 1550 and 1660 cm−1 in the IR-RAS spectrum (see Figure S1 in Supporting Information).30,31 The chemical compositions of each film were characterized by quantitative analysis of EDS spectra. Table 2 summarizes the mass ratio of aluminum to

Figure 6. Time courses of λOWG found for (a) PAA film and (b) cPMF4−PAA film under alternating flows of buffer solutions and pure water. Red and black arrows indicate injection of the buffer solutions and water, respectively. The pH values of buffer solutions are shown under the red arrows. The buffer solutions contained 10 mM buffer [phosphate buffer (pH 2.5−3.8), citrate buffer (pH 4.8−8.7), and glycine−NaOH buffer (pH 9.8)] and 100 mM NaCl. The inset in the upper graph shows the time course of λOWG after flow of the glycine− NaOH buffer (pH 9.8).

Table 2. EDS Quantitative Analysis Data for PAA, PMF5− PAA, and cPMF5−PAA Films before and after Immersion in 10 wt % Phosphoric Acid Solution sample

immersion time/min

C/Si

Al/Si

bare PAA PMF5−PAA

0 0 30 0 180

0.17 0.52 0.22 0.54 0.56

0.26 0.26 0.17 0.18 0.15

cPMF5−PAA

profiles of λOWG, abrupt red-shifts of λOWG were induced by changing the solution in the flow cell from pure water to buffer solutions because the refractive indices of the buffer solutions containing electrolytes (10 mM buffer and 100 mM NaCl) were higher than that of pure water. During the flow of buffer solutions, except those at pH 8.0 and 8.5, a gradual blue-shift in λOWG was seen for the bare PAA film (Figure 6a). This can be ascribed to the unfavorable dissolution of the alumina matrix. For pH 8.0 and 8.5 buffer solutions, no noticeable blue-shift is observed, indicating the resistance of the alumna matrix to solutions at around the isoelectric point of the alumina surface (pH 8−9).34,35 On the other hand, no significant blue-shift in λOWG is seen for the flow of each buffer solution (pH 2.5−8.7) for the cPMF4−PAA film (Figure 6b). As seen in the OWS experiment, the nanoporous structure of the cPMF4−PAA film did not change after flow of the buffer solutions (pH 2.5−8.7). At a buffer solution pH of 9.8, a gradual blue-shift in λOWG is seen for the cPMF4−PAA film. However, biosensing with DNA or protein as a biorecognition element is generally performed at pH values below 9. It can therefore be concluded that the thermally cross-linked PMF works as an effective protective coating of the PAA film, thus opening the path to biosensor applications. The thermal cross-linking temperature was varied between 120 and 180 °C. When thermal cross-linking was carried out at 120 °C, OWS experiments revealed slow dissolution of the alumina matrix and/or detachment of PMF. On the other hand,

silicon (Al/Si) and carbon to silicon (C/Si) found for each film. The silicon peak originates from the glass substrate under the PAA and Al films. For the bare PAA film, the nanoporous alumina structure had completely collapsed after 5 min exposure to the phosphoric acid solution, due to dissolution of the alumina matrix (Figure 5a). For the PMF5−PAA film, its nanoporous structure appeared to be maintained after 30 min exposure to the phosphoric acid solution (Figure 5b). However, the EDS results suggest slight dissolution of the alumina matrix and detachment of the PMF; both the Al/Si and C/Si ratios decreased after 30 min of exposure (Table 2). Further dissolution of the alumina matrix and detachment of PMF were observed after 90 min exposure of the PMF5−PAA film (Figure 5b). However, the PMF after thermal cross-linking acted as a strong protective coating. As shown in Figure 5c, the thermally cross-linked PMF5−PAA (cPMF5−PAA) film maintained its nanoporous structure even after 3 h exposure while preserving the mass ratio of Al/Si and C/Si (Table 1). It can be considered that, after thermal cross-linking, the PMF is tightly bound to the PAA surface.30,31 This tightly bound PMF appears to inhibit the accessibility of water and ions to the alumina surface, making the thermally cross-linked PMF5−PAA film 23537

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no such dissolution or detachment was seen for cPMF4−PAA film cross-linked at above 140 °C. The IR peak intensity ratio of the amide group to the residual carboxyl group increases with higher cross-linking temperatures (Figure S2), suggesting the formation of highly cross-linked PMF that is suitable as an effective protective coating. We also confirmed that thermal cross-linking at above 160 °C was favored for immobilization of streptavidin at the PMF4−PAA (Table S1): the amount of streptavidin bound to the cPMF4−PAA film cross-linked at 180 °C was about 3 times greater than that cross-linked at below 140 °C. The temperature for thermal cross-linking of PMF was therefore set at 180 °C. DNA−DNA Hybridization Assay. The usefulness of the cPMF4−PAA film for a label-free optical biosensor was demonstrated by DNA−DNA hybridization assay based on OWS. The OWS experiments were carried out to monitor the immobilization process of single-stranded probe DNA at the cPMF4−PAA film and the hybridization reactions of the probe DNA with complementary or noncomplementary target DNA (see Figure 1). Each process for immobilization and hybridization is shown as OWS sensorgrams (Figure 7).

streptavidin through strong biotin−avidin coupling. After removing excess streptavidin by washing the cell with a rinsing buffer solution, λOWG changed to 526.6 nm (Figure 7a). Accordingly, the total red-shift of λOWG for the immobilization of streptavidin was 56.6 nm, which was greater than that for sulfo-NHS-LC-LC-biotin due to the higher molecular weight of streptavidin (ca. 60 000). The successive loading of the probe DNA solution and the rinsing buffer resulted in a red-shift of λOWG (ΔλOWG = ca. 10 nm) due to the conjugation of probe DNA molecules with streptavidin (Figure 7b). After the immobilization of the probe DNA molecules at the cPMF4−PAA film, introducing the noncomplementary target (T2) DNA solution induced no red-shift of λOWG. The slight blue-shift before and after introducing the T2 solution would be due to slow desorption of the probe DNA molecules nonspecifically bound to the cPMF4−PAA film. In contrast, a red-shift of λOWG (ca. 8 nm) was clearly seen on loading of the complementary target (T1) DNA solution (Figure 7b). These OWG responses indicate selective adsorption of T1 DNA due to hybridization of the probe DNA and T1 DNA at the cPMF4−PAA film. It can thus be concluded that the cPMF4− PAA film is applicable to DNA−DNA hybridization assay and that the residual amino groups in the cross-linked PMF4 can be used as active sites for immobilization of probe molecules.

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CONCLUSION The PMF composed of paa/pah multilayers was deposited on the PAA film (pore diameter: ca. 40 nm) to improve the chemical stability of the PAA film. Successive immersion of the polyelectrolyte solutions and rinsing with water caused PMF to be formed at the outer surface and inner pore surface of the PAA film. The deposited PMF, after thermal cross-linking at 180 °C for 1 h, served as an effective protective coating of the PAA film by inhibiting dissolution of the alumina matrix and detachment of the PMF. The PAA film with cross-linked PMF was able to survive in phosphoric acid solution, a common alumina matrix etchant, for 3 h. Its chemical stability was also confirmed in aqueous buffer solutions at pH values ranging from 2.5 to 8.7. It was possible to immobilize probe DNA on the PAA film for DNA−DNA hybridization assays based on OWS. The results obtained in this study confirm that PMF coating is potentially useful for constructing optical biosensors employing an inorganic nanoporous film.

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Figure 7. OWS sensorgrams obtained for the cPMF4−PAA film: (a) immobilization of biotin and streptavidin and (b) immobilization of probe DNA and hybridization of probe DNA with noncomplementary (T2) and complementary (T1) target DNAs. The concentrations of the probe and target DNAs are 10 μM (probe) and 100 nM (T1 and T2), respectively. The incident angles of probing light were (a) 69° and (b) 70°.

ASSOCIATED CONTENT

S Supporting Information *

IR-RAS spectra of the PMF5−PAA films; IR-RAS spectra of the PMF4−PAA films cross-linked at different temperatures; OWS response for immobilization of streptavidin. This material is available free of charge via the Internet at http://pubs.acs.org.

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First, the sulfo-NHS-LC-LC-biotin solution was loaded to the flow cell with cPMF4−PAA film, and then the cell was washed in the rinsing buffer, for which λOWG shifted to red from 465.5 to 470.0 nm (Figure 7a). This red-shift of λOWG (ΔλOWG = 3.5 nm) can be attributed to covalent conjugation of the biotin units at the cross-linked PMF4 as schematically shown in Figure 1. The immobilization of streptavidin was performed by considering an affinity constant (7.3 × 107 M−1) for streptavidin and biotin bound to a gold surface.36 The successive loading of 0.1 and 1 μM streptavidin solutions induced a stepwise red-shift of λOWG due to immobilization of

AUTHOR INFORMATION

Corresponding Author

*Phone +81-22-795-6549; Fax +81-22-795-6552; e-mail [email protected] (A.Y.), [email protected] (N.T.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by Grants-in-Aid for Scientific Research (No. 21685009 and No. 22225003) from the Ministry 23538

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(33) Schlenoff, J. B.; Dubas, S. T. Macromolecules 2001, 34, 592−598. (34) Fukuzaki, S.; Urano, H.; Nagata, K. J. Ferment. Bioeng. 1996, 81, 163−167. (35) Tombacz, E.; Szekeres, M. Langmuir 2000, 17, 1411−1419. (36) Tang, Y.; Mernaugh, R.; Zeng, X. Anal. Chem. 2006, 78, 1841− 1848.

of Education, Culture, Sports, Science and Technology of Japan.

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dx.doi.org/10.1021/jp308724m | J. Phys. Chem. C 2012, 116, 23533−23539