Soft Surface Modification of Layered Titanate for Biorecognition

Aug 28, 2012 - Suguru Tsukahara,. †. Nobuaki Soh,. ‡ and Kai Kamada*. ,†. †. Department of Materials Science and Engineering, Faculty of Engin...
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Soft Surface Modification of Layered Titanate for Biorecognition Suguru Tsukahara,† Nobuaki Soh,‡ and Kai Kamada*,† †

Department of Materials Science and Engineering, Faculty of Engineering, Nagasaki University, Nagasaki 852-8521, Japan Faculty of Agriculture, Saga University, Saga 840-8502, Japan

‡

S Supporting Information *

ABSTRACT: We propose a novel and soft surface modification technique of titanate layers with functional biomolecules that can be utilized for bioaffinity reaction. The modification process is mainly divided into two steps, that is, an adsorption of avidin protein on the titanate surface via electrostatic interaction and a binding to biotinylated functional biomolecules based on a tight noncovalent bond of avidin for biotin. The whole procedure is performed in an aqueous solution with a neutral pH, and hence the technique described here accomplishes the conjugation process without any denaturation of biomolecules. The titanate layers supporting the biotinylated antirabbit IgG antibody exhibit a practical recognition performance for the rabbit IgG antigen. Moreover, fluorescent layers are formed by Eudoping into the host titanate, and then fluorescence detection of the recognition succeeded. The present paper clarifies the mechanism of soft surface modification of titanate layers in detail and proves an effectiveness of the functionalized titanates for biological measurements.

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INTRODUCTION Recently, inorganic materials have widely concerned various biological applications, including bioseparation,1−3 magnetic resonance imaging,4−9 targeted drug delivery systems,10−12 radical annihilation,13,14 and thermal tumor therapy.15 These applications efficiently utilize peculiar properties of inorganic materials, such as magnetism, stable fluorescence, and electrical conductivity, that can be attained by selecting an appropriate chemical composition. To exploit inorganic materials for these bioapplications, chemical modification of their surfaces is required in order to add a reliable bioaffinity along with harmlessness, except for the cases that the surfaces directly influence a living system. That is, the addition of “hands”, which can selectively bind target molecules or tissues, is significant to applying the modified materials to the biological uses.16,17 Among inorganic materials with diverse morphologies, layered titanates are two-dimensional (2D) crystals with a thin thickness (∼1 nm) against a relatively large lateral size and play an important role as a state-of-art nanotechnology as well as nanodots (0D) and nanowires (1D). One of important advantages of them is that various guest molecules and ions can be interstratified between the host layers by means of a simple solution process, resulting in formation of numerous layered compounds. In the past decade, several groups reported an intercalation of biomolecules (protein, enzyme, DNA, organic drug molecule) into host inorganic layers (silicate, zirconium phosphate, titanate, etc.), and the physical and/or chemical durability of guest molecules were improved as compared with the naked guests.18−21 Recently, our group also synthesized the enzyme-intercalated layered iron-titanate and reported that the magnetic separation and improved ultraviolet light tolerance of guest enzymes were possible due to ferromagnetism and semiconducting nature of the host layer.22,23 Moreover, the photochemical activity control (photoswitching) of redox © 2012 American Chemical Society

enzymes interstratified with semiconducting layers was accomplished by photoexcitation of the host and subsequent charge transfer to the enzyme.24 The above-mentioned intercalation compounds are spontaneously formed via an electrostatic interaction between charged host layers and guest biomolecules having an opposite surface charge at a certain pH. In general, the guest is strongly bound to the host. For example, the equilibrium binding constant of hemoglobin for layered α-zirconium phosphate exceeded 107 M−1, and the binding capacity of layers with a huge specific surface area was fairly larger than those of other nanomaterials.25 In addition, the immobilized functional biomolecules existing in the interlayer preserve their functions even after the intercalation, because the target (substrate) molecules can access through a semiopen structure along the 2D layers. Hence, the biomolecules bound to the inorganic layers are expected to be an innovative tool that is different from conventional inorganic−bio nanohybrids. To encourage the bioapplication of inorganic layers further, the present study proposes a new surface modification technique to graft biofunctional groups onto the surface of layers. Conventionally, the introduction of functional groups to the inorganic surface has been performed through silane coupling,16,17,26 ligand exchange with bifunctional thiols,27 etc. However, their reaction conditions are typically harsh (e.g., in organic solvent, at nonphysiological pH, or high temperature, etc.)28,29 and thus would be inappropriate as a surface modification of inorganic layers combining fragile biomolecules. To overcome the problem, we have developed a new surface modification method, as shown in Figure 1, which consists of Received: April 28, 2012 Revised: August 19, 2012 Published: August 28, 2012 19285

dx.doi.org/10.1021/jp304107b | J. Phys. Chem. C 2012, 116, 19285−19289

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TBA+ to Ti = 1.0). The colorless and transparent colloidal suspension of single titanate layers (h-TiOx) was formed by stirring the mixture for 2 h at 333 K and subsequent hydrothermal treatment at 373 K for 2 h. The hydrolysis of TTIP containing 1 mol % EuCl3 for total metal content was also performed to synthesize fluorogenic layered titanates (hTiOx(Eu)) using the identical manner. The excess salts in the produced colloidal solution were removed by dialysis for pure water with a membrane filter, and the solution pH was reduced to 7−8 as a result of the dialysis. The particle size of s- and hTiOx was estimated by atomic force microscopy (AFM) and dynamic light scattering (DLS), respectively. The feasibility of surface treatment depicted in Figure 1 was examined by means of a quartz crystal microbalance (QCM) technique. Since the adsorption of avidin molecules on the titanates at the initial step (stage I) was predicted to proceed via an electrostatic interaction, the pH influence on surface charge (zeta potential) of s-TiOx and avidin was evaluated by an electrophoretic laser light scattering method in advance. A 9 MHz AT-cut quartz (8 mm in diameter) sensor covered with the s-TiOx was prepared by dropping the s-TiOx suspension (0.1 mg/mL, 20 μL) and subsequent drying at an ambient atmosphere. The sensor was exposed, in turn, to 0.02 M citrate buffer solutions (pH 6.4) dissolving 5 mg/L of avidin, biotinylated antirabbit IgG antibody, and rabbit IgG antigen with a flow type cell (50 μL/min) at 303 K. Prior to changing the solution, the buffer solution was introduced for a certain duration to remove the solutes from the reaction chamber. A change in resonance frequency, which is inversely proportional to the weight change on the sensor surface, was continuously monitored during the reaction. No significant detachment of sTiOx from the sensor was observed during the QCM measurement. On the contrary, the bioaffinity reaction in Figure 1 was also carried out in the mixed-solution system. The h-TiOx (15 mg/mL, ∼0.1 M, 0.5 mL) was mixed, in turn, with the FITC-avidin (0.05 mg/mL, 1 mL) and biotinylated antibody (1 mg/mL, 0.05 mL) for 2 h each at 303 K; then the rabbit IgG-immobilized agarose gel beads (10 mg/mL, 0.1 mL) were added and shaken overnight at 303 K. The process was conducted in 0.02 M Tris-HCl buffer solution (pH 7.4). After collecting and washing the reacted beads, the fluorescence image of the beads was acquired by confocal laser scanning microscopy. In only the case of h-TiOx(Eu), the bare avidin was used at the first modification step in Figure 1, and the fluorescence intensity of the h-TiOx(Eu) on the bead surface was measured by a microplate reader with band path filters (Ex: 320/20 nm; Em: 590/35 nm).

Figure 1. Schematic illustration of surface modification process of titanate layers with avidin protein and biotinylated antibody for recognition of the corresponding antigen.

physical adsorption of avidin protein on inorganic layers (layered titanate) under an electrostatic interaction in an aqueous solution (stage I), followed by coupling biotin-labeled functional biomolecules via a tight noncovalent bond between avidin and biotin (stage II). The fact that biomolecules can be easily biotinylated means that a variety of biofunctionalizations of inorganic surfaces are realized. In the present study, the antigen recognition performance of physicochemically stable titanate layers treated with biotinylated antibody is evaluated as a test reaction (stage III). Furthermore, the fluorescence detection and visualization of the bioaffinity reaction were attempted by using the rare-earth-doped titanate layer. Since the entire procedure is conducted in a neutral solution under an ambient condition, the present method accomplishes the surface modification under a mild condition as compared with the conventional techniques. It is believed that these results give us an important and fundamental knowledge to apply the inorganic layers for various biological applications.

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EXPERIMENTAL SECTION The precursor of the host titanate layer was synthesized via a conventional solid-state reaction or a hydrolysis reaction of titanium alkoxide, as previously reported. In the former case,30,31 a stoichiometric mixture of Cs2CO3 and TiO2 was calcined at 1073 K for 20 h in air twice with intermediate grinding. The Cs+ in the interlayer of the titanate was substituted for protons during stirring in 1 M HCl for 24 h several times. The protonated titanates were exfoliated to single layers by dispersing into tetrabutyl ammonium hydroxide (TBAOH) solution including a 2-fold molar excess of TBA+ toward the proton-exchange capacity. The obtained basic colloidal solution of single titanate layers (Ti1.825O40.7−, denoted below as s-TiOx) was neutralized and diluted with acetic acid solution (pH 7.0), followed by centrifugation to remove undelaminated titanates. The contents of titanate in the resultant solution were determined by measuring the maximum absorbance at 267 nm. On the other hand, the hydrolysis reaction of titanium(IV) tetraisopropoxide (TTIP)32 was also carried out to fabricate a more stable colloidal solution of titanate layers than the s-TiOx. The TTIP liquid (99.999%) was hydrolyzed with the TBAOH aqueous solution (molar ratio of

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RESULTS AND DISCUSSION As previously reported, the colloidal solution of exfoliated layered s-TiO x (lateral size: 200−500 nm; Figure 1S (Supporting Information)) was easily obtained by vigorously stirring the pristine protonated titanates in the tetrabutylammonium (TBA+) hydroxide solution. The initial step of the surface modification process of titanate layers (stage I in Figure 1) is the physical adsorption of avidin protein as an anchor, which attracts various functional biomolecules. At stage I, the avidin would be immobilized on the titanate surface via electrostatic interaction. Because the charging state of solids strongly depends on the acidity of solution, the pH dependence of the zeta potential of s-TiOx and avidin was evaluated, as shown in Figure 2. While the titanate layers charged negatively over a wide range of pH and the isoelectric point (pI) is located 19286

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linear regression (solid line) is ca. 15 μM−1, comparable to those of other proteins for various inorganic layered materials, such as zirconium phosphates or silicates.25,36 The result demonstrates the high affinity of titanate layers to the avidin; in other words, the titanates are useful as host layers that support the avidin. The QCM method was exploited to investigate whether the antigen recognition illustrated in Figure 1 actually proceeds. The quartz crystal sensor (8 mm in diameter) was covered with the s-TiOx thin layer (2 μg); then the resonance frequency, which is inversely proportional to weight change, was monitored during exposure to avidin (stage I), the biotinylated antirabbit IgG antibody (stage II), and the rabbit IgG antigen (stage III) in 0.02 M Tris-HCl (pH 7.4) at 303 K (Figure 4). As

Figure 2. Effect of pH on zeta potential of s-TiOx (90 mg/L) and avidin (10 mg/L). The pH was adjusted by addition of 0.1 M HCl or 0.1 M NaOH.

at pH ∼ 2,33 the pI of avidin was estimated to be about 8.34 Taking into account these results, it is concluded that the solution pH should be set to pH < 8 for the physical adsorption of avidin. To study the affinity of avidin for the titanate layer, binding parameters were determined by means of a simple centrifugation method described elsewhere. Briefly, the reaction mixtures with various FITC-avidin/s-TiOx weight ratios at pH 7.4 were centrifuged after stirring for 1 h to settle the FITCavidin grafted s-TiOx. The adsorption amount of FITC-avidin was indirectly estimated by measuring the fluorescence of free FITC-avidin remaining in the supernatant (Ex: 485/20 nm; Em: 528/20 nm). As shown in Figure 2S (Supporting Information), the free FITC-avidin concentrations (Cf) were constantly lower than the total concentration (Ct), suggesting that the titanate surface electrostatically adsorbs the avidin at pH < 8. The linear relationship between Ct and Cf means facile control of the adsorption (binding) amount. The binding stoichiometry of avidin (Cb corresponds to Ct at Cf = 0) determined from the vertical intercept in Figure 2S (Supporting Information) was about 0.68 μM (0.046 mg/mL) for [s-TiOx] = 0.33 mM (0.05 mg/mL). The equilibrium binding constant (Kb) of avidin was calculated by the Scatchard equation (eq 1).35 It is known that the equation is frequently used for approximation of the binding constant of a ligand to a specific substrate during complexation or coordination. 1/Cf = nKb(1/r ) − Kb

Figure 4. (a) Variation in resonance frequency of quartz sensor covered with s-TiOx during the surface modification process (stages I− III, represented as I, II, and III in the figure) depicted in Figure 1. (b) Stage II was skipped in the process.

shown in Figure 4a, the introduction of avidin caused a sharp frequency drop, implying the physical adsorption of avidin to sTiOx (stage I), as already demonstrated. Afterward, the biotinylated antirabbit IgG antibody was immobilized via a nearly irreversible noncovalent bond between avidin and biotin (stage II).16,17 As the weight gain was observed during the exposure to the rabbit IgG (stage III), the antigen recognition was accomplished on the surface of s-TiOx. In contrast, the rabbit IgG was introduced just after stage I (skipping stage II) to inspect for a possibility of nonspecific binding. In this case, little weight gain was discernible (Figure 4b), suggesting that the rabbit IgG cannot be reacted with the titanate or the avidin. Consequently, the titanate layer−biomolecules hybrids, which had the molecular recognition property, could be fabricated through the soft surface treatment of titanate layers with avidin and the biotinylated functional biomolecule. Besides the surface reaction using the QCM equipment, the bioaffinity reaction of modified titanates was also undertaken in a practical mixed-solution system. In this experiment, rabbitIgG immobilized agarose gel beads were reacted with the antibody-grafted titanate, where the FITC-avidin was again used to follow the reaction process by a fluorescence microscopy observation. In addition, the h-TiOx, which was

(1)

In eq 1, r and n are the binding density (equivalent to (Ct − Cf)/[s-TiOx]) and saturated adsorption amount of specific binding, respectively. Figure 3 shows the result of Scatchard analysis of avidin binding to the s-TiOx. The Kb calculated from

Figure 3. Scatchard analysis of avidin binding to s-TiOx (0.33 mM). Binding constant (Kb) can be calculated from the y intercept. 19287

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content). As a result, the stable colloidal solution (mean hydrodynamic diameter: 5 nm; Figure 3S (Supporting Information)) was produced as similar to the case without the Eu3+ doping. The dopant Eu3+ should be dissolved into the host layer, because the adsorption of free Eu3+ to the titanate surface brings about an aggregation and poor dispersion of hTiOx(Eu) owing to compensation of the negative charge of the host layers. In fact, the addition of Eu3+ into the colloidal solution of undoped titanate (h-TiOx) rapidly led to the precipitation of h-TiOx. According to the fluorescence spectra of the h-TiOx(Eu) colloidal solution in Figure 5S (Supporting Information), the excitation peak corresponding to band-gap absorption of titanate appeared at 326 nm. On the other hand, the emission peaks assigned to 5D0 → 7F1 and 5D0 → 7F2 transitions of Eu3+ were discernible at 593 and 615 nm, respectively.39,40 Needless to say, no fluorescence was detected for the undoped h-TiOx. These results indicate that the fluorescent layered titanate can be easily produced by the facile hydrolysis reaction. The identification of the rabbit IgG immobilized on the agarose gel beads was carried out using the antirabbit IgG antibody conjugated h-TiOx(Eu). Figure 6a summarizes the

prepared by the one-step hydrolysis of TTIP and had a tiny particle size (mean hydrodynamic diameter: 6 nm; Figure 3S (Supporting Information)), was utilized to improve the dispersibility in the solution. According to the zeta potential measurement, the surface of h-TiOx charged negatively at pH 7.4. However, the higher avidin content than the binding stoichiometry (Cb) brought about an undesired aggregation of h-TiOx due to complete loss of surface charge, and thus the avidin concentration at stage I was set to be lower than the Cb. Figure 5a,b displays the fluorescence images of agarose gel

Figure 5. Optical and fluorescence images of rabbit IgG-immobilized agarose gel beads before (a) and after (b) reaction with antirabbit IgG/FITC-avidin/h-TiOx. The photographs of beads exposed to FITC-avidin/h-TiOx are also displayed in (c).

beads before and after the reaction with the antibodyconjugated h-TiOx, where Figure 5c corresponds to the images of beads exposed to the h-TiOx not labeled with the antibody, as similar to Figure 4b. Figure 5b clearly shows that the bright green fluorescence of FITC was observed mainly at the circumference of beads treated with the antibody-conjugated hTiOx. The X-ray fluorescence (XRF) analysis also indicated the existence of Ti on the beads' surface (Figure 4S, Supporting Information). As a matter of course, the avidin/h-TiOx was not bound to the beads, as indicated in Figure 5c and Figure 4S (Supporting Information). Consequently, it was proved that the antigen recognition process by the modified titanate layers was achieved even in the mixed-solution system. Generally, inorganic materials have a great potential to acquire a novel function depending on their elementary compositions. Inorganic fluorescent materials, such as chalcogenides (quantum dots)37 or doped oxide nanoparticles,38 have a superior stability for photobleaching and pH change as compared with organic molecules and thus have been applied for various biological measurements. With respect to inorganic layered materials, the fluorescence character appears by means of an intercalation of rare earth ions into the interlayer or doping them into the crystal structure of the host layer. Therefore, the h-TiOx host layer was doped with Eu3+ ions (hTiOx(Eu)); then the fluorescence detection of the bioaffinity reaction in Figure 1 was tried without labeling the avidin with organic fluorescence molecules (FITC). The h-TiOx(Eu) was simply synthesized via the hydrolysis reaction of TTIP mixed with an ethanol solution of EuCl3 (1 mol % Eu for total metal

Figure 6. Fluorescence detection of antigen recognition using antibody-conjugated h-TiOx(Eu). (a) Relative fluorescence units of rabbit IgG-immbolized agarose gel beads (control) and those after reaction with avidin/h-TiOx(Eu) or antirabbit IgG/avidin/h-TiOx(Eu) (n = 4). (b) Changes in fluorescence intensity of FITC-avidin/hTiOx(Eu) and avidin/h-TiOx(Eu) under irradiating with an excitation light.

relative fluorescence units (RFUs) of the beads after the reaction. Comparing with the h-TiOx(Eu) combining the avidin only, the RFU of beads exposed to that with the antibody was significantly increased, suggesting that the bioaffinity reaction can be followed by utilizing the fluorescence of the layered titanate. Figure 6b shows the bleaching behavior (RFU variation) of FITC-avidin/h-TiOx and avidin/h-TiOx(Eu) under continuous excitation at 488 and 326 nm, respectively. Except for the initial tiny decline of RFU (