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Langmuir 2002, 18, 4526-4529
Three-Dimensionally Ordered Macroporous Polymer Materials: An Approach for Biosensor Applications Weiping Qian,†,‡ Zhong-Ze Gu,† Akira Fujishima,§ and Osamu Sato*,† Kanagawa Academy of Science and Technology, KSP Bldg. East 412, 3-3-1 Sakado, Takatsu, Kawasaki-shi, Kanagawa 213-0012, Japan, National Laboratory of Molecular and Biomolecular Electronics, Southeast University, Si Pai Lou 2, Nanjing 210096, People’s Republic of China, and Department of Applied Chemistry, School of Engineering, The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-8565, Japan Received December 17, 2001. In Final Form: March 12, 2002
Biosensor experiments involve immobilizing one reactant on a surface and monitoring its interaction with a second component in solution. In general, biosensors consist of two components: a highly specific recognition element and a transducer that converts the molecular recognition event into a quantifiable signal. Signal transduction has been accomplished with electrochemical,1 field-effect transistor,2 optical absorption, fluorescence, surface plasmon resonance,3 interferometric,4,5 and other devices.6 In these devices, biomolecules such as antibodies or oligonucleotides are immobilized on a solid substrate by numerous steps and used to detect the presence of a target antigen or oligonucleotide. Asher et al. developed a sensing material that reported on analyte concentrations via diffraction of visible light.7 Their material was a mesoscopically periodic crystalline colloidal array (CCA) of polymer spheres polymerized within a hydrogel that swelled and shrank reversibly in the presence of certain analytes. The CCA diffracts light at wavelengths determined by the lattice spacing, which gives rise to an intense color. The chemical molecular-recognition events (i.e., crown ethers for metal ions) cause the gel to swell owing to an increased osmotic pressure, which increases the mean separation between the colloidal spheres and so shifts the Bragg peak of the diffracted light to longer wavelengths. We present here a new method for biosensor fabrication, in which three-dimensionally ordered macroporous (3DOM) polystyrene films were used as an immobilizing and transducing matrix. Proteins were simply immobilized on the pore surfaces of 3DOM polystyrene substrates by physical adsorption. The signal transduction was achieved by monitoring the diffraction peak shifts * To whom correspondence should be addressed. E-mail: sato@ fchem.chem.t.u-tokyo.ac.jp. † Kanagawa Academy of Science and Technology. ‡ Southeast University. § The University of Tokyo. (1) Hickman, J. J.; Ofer, D.; Laibinis, P. E.; Whitesides, G. M.; Wrighton, M. S. Science 1991, 252, 688. (2) McConnell, H. M.; Owicki, J. C.; Parce, J. W.; Miller, D. L.; Baxter, G. T.; Wada, H. G.; Pitchford, S. Science 1992, 257, 1906. (3) Rich, R. L.; Myszka, D. G. Curr. Opin. Biotechnol. 2000, 11, 54. (4) Lin, V. S.; Motesharei, K.; Dancil, K. P.; Sailor, M. J.; Ghadiri, M. R. Science 1997, 278, 840. (5) Yu, F.; Yao, D. F.; Qian, W. P. Clin. Chem. 2000, 46, 1489. (6) Sensors Update; Baltes, H., Gopel, W., Hesse, J., Eds.; WileyVCH: Weinhein, Germany, 1989-1996; Vols. 1-9. (7) (a) Holtz, J. H.; Asher, S. A. Nature 1997, 389, 829. (b) Holtz, J. H.; Holtz, J. S. W.; Munro, C. H.; Asher, S. A. Anal. Chem. 1998, 70, 780.
upon the change in refractive index of the solution near the pore surface that occurs during analyte binding. Interestingly, 3DOM polystyrene films for biosensor applications not only tap their diffraction properties but also employ other characteristics of the material, including high surface area and simplicity of biomolecular immobilization. This approach to the detection of ligandreceptor binding does not require labeling of the analyte, can eliminate disturbance due to nonspecific binding, and is sufficiently simple and general that it may find use in biochemical assays and immunoassays. Recent developments using colloidal crystal templating allow the preparation of 3DOM materials8-17 that display the existence of optical stop bands, in which strong diffraction effects limit the optical transmission of the films. Under normal incidence, the diffraction peak position and the shift of the film can be estimated from Bragg’s law:
λ ) 1.663dna
(1)
where λ is the peak wavelength, d represents the centerto-center distance, and na is the average reflective index of the film. As in the case of enzyme-linked immunosorbent assays, polystyrene became the choice of a solid substrate in our experiments because of the hydrophobic nature of its surface and excellent optical properties. Proteins could be simply immobilized on the pore surfaces of 3DOM polystyrene substrates by physical adsorption. In our biosensor (Figure 1), binding of an analyte to its corresponding recognition partner, immobilized on the 3DOM polystyrene substrate, results in a change in the average refractive index of the layer medium and is detected as a corresponding shift in the diffraction peak position. Assuming that the average refractive index is determined from the volume average of the dielectric constants of different compositions, we obtain
λ ) 1.633d ×
xVpolystyrenenpolystyrene2 + Vproteinnprotein2 + Vwaternwater2
(2)
where npolystyrene, nprotein, and nwater are the reflective indices of polystyrene, protein, and water, respectively, fixed at their typical bulk values npolystyrene ) 1.59, nprotein ) 1.42, and nwater ) 1.33. Vpolystyrene, Vprotein, and Vwater are the volume fractions of polystyrene, protein, and water, respectively. The shift in the diffraction peak position with protein binding can be used to quantitatively estimate the amount of bound protein. We formed colloid crystal templates of silica spheres (∼190 nm in diameter; Nissan Chemical Ind., Ltd., Japan) (8) Velev, O. D.; Jede, T. A.; Lobo, R. F.; Lenhoff, A. M. Nature 1997, 389, 447. (9) Holland, B. T.; Blanford, C. F.; Stein, A. Science 1998, 281, 538. (10) Wijnhoven, J. E. G. J.; Vos, W. L. Science 1998, 281, 802. (11) Velev, O. D.; Tessier, P. M.; Lenhoff, A. M.; Kaler, E. W. Nature 1999, 401, 548. (12) Jiang, P.; Cizeron, J.; Bertone, J. F.; Colvin, V. L. J. Am. Chem. Soc. 1999, 121, 7957. (13) Park, S. H.; Xia, Y. N. Adv. Mater. 1998, 10, 1045. (14) Park, S. H.; Xia, Y. N. Chem. Mater. 1998, 10, 1745. (15) Johnson, S. A.; Ollivier, P. J.; Mallouk, T. E. Science 1999, 283, 963. (16) Jiang, P.; Hwang, K. S.; Mittleman, D. M.; Bertone, J. F.; Colvin, V. L. J. Am. Chem. Soc. 1999, 121, 11630. (17) Jiang, P.; Bertone, J. F.; Colvin, V. L. Science 2001, 291, 453.
10.1021/la0118199 CCC: $22.00 © 2002 American Chemical Society Published on Web 04/26/2002
Notes
Figure 1. (A) Schematic diagrams of the solvent evaporation methods used to fabricate large-area 3DOM polystyrene films in a free-standing form. (B) and (C) show SEM images, at different magnifications, of 3DOM polystyrene films, prepared using a ∼190 nm SiO2 colloidal crystal as a template and the scheme in Figure 1A. Note the excellent ordering in macroporous polystyrene films.
on microslides using literature procedures18,19 with some modifications and improvements. We simultaneously prepared multiple colloidal crystal templates with same quality. A glass trough together with a stand was used as the experimental cell. An important feature of our method is that all of the microslides were mounted vertically in a stand and kept parallel to each other. Careful control over the growth conditions makes it possible for us to obtain high-quality colloidal crystal templates. The detailed description of our method can be found in another manuscript.20 We infiltrated a toluene solution of polystyrene (average molecular weight of 312 000; Wako Pure Chemical Industries, Ltd., Japan) into the vacant space between the spheres. After 2-3 days, the polystyrene film together with the template was spontaneously separated from the microslide, and then the template was removed by treatment with 4% hydrofluoric acid, producing largearea polystyrene films with three-dimensional ordering of pores in a free-standing form. A schematic outline of the procedure for producing 3DOM polystyrene films is shown in Figure 1A. The 3DOM polystyrene films used in this study were carefully washed with ethanol. Figure 1B,C shows typical SEM images of a 3DOM polystyrene film with a ∼190 nm diameter. The images exhibit an ordered void structure from the silica spheres. These large cavities are not isolated but rather are interconnected to each other. The experimental cell was assembled from two microslides, one silicone rubber spacer and one polystyrene film. A “U” type silicone rubber spacer was mounted on the macroporous side of a polystyrene film, and then the film together with the space was sandwiched between two microslides. After the fabrication, the experimental cell was precisely mounted on the holder supplied by the producer. A schematic for a 3DOM polystyrene film based (18) Dushkin, C. D.; Nagayama, K.; Miwa, T.; Kralchevsky, P. A. Langmuir 1993, 9, 3695. (19) Jiang, P.; Bertone, J. F.; Hwang, K. S.; Colvin, V. L. Chem. Mater. 1999, 11, 2132. (20) Qian, W. P.; Gu, Z.-Z.; Fujishima, A.; Sato, O. Langmuir, submitted.
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Figure 2. Schematic representation of a biosensor fabrication. (A) An aqueous solution is filled in the pores of a 3DOM polystyrene film. (B) The ligand is immobilized in the pores. (C) The places where no ligand immobilization has occurred are blocked with BSA. (D) The analyte binds on the pore surfaces.
biosensor is shown in Figure 2. To immobilize proteins on the pore surfaces in 3DOM polystyrene films, we filled the pores with ethanol first because of the hydrophobic nature of the polystyrene surface and then replaced the ethanol solution with an aqueous solution (Figure 2A). Ligand (600 µL) at a concentration of 2.0 mg/mL in 50 mmol/L PBS at pH 7.2 was added to each sample cell. After 1 h incubation at room temperature and overnight incubation at 4 °C, the ligand solutions were removed. After the ligand was immobilized in the pores (Figure 2B), we blocked the places where no protein adsorption had occurred with bovine serum albumin (BSA, 10 mg/ mL), to prevent nonspecific adsorption of proteins during subsequent steps (Figure 2C).21 The pores were subsequently rinsed and filled with the sample containing the putative target analyte (Figure 2D). In situ analyses of the ligand immobilization, BSA blocking, and analyte binding on the pore surfaces were performed using a Shimadzu UV-3101 PC with transmission mode. All transmission measurements were performed at normal incidence and constant regions. The instrument resolution is 0.1 nm, and the spot size of the detecting light was 11 mm × 4 mm. Each peak wavelength maximum was acquired from the recorded transmission spectrum using the software IGOR Pro version 4.0 (WaveMetric, Inc., USA). We used polystyrene films immobilized with Staphylococcal protein A (SpA) and goat anti-human IgG (goat anti-hIgG) to test the validity, sensitivity, and selectivity of the biosensors. Human IgG (hIgG) was used as an analyte, because it can be captured by both immobilized SpA and goat anti-hIgG. SpA is a membrane-bound protein from Staphylococcus aureus that binds to the Fc fragment of IgG.22,23 A whole range of antibodies with different specificities can also be dissociated from the SpA surface simply by lowering the pH of the solutions.24 Goat anti(21) Qian, W. P.; Yao, D. F.; Yu, F.; Xu, B.; Zhou, R.; Bao, X.; Lu, Z. Clin. Chem. 2000, 46, 1456. (22) Lindmark, R.; Thoren-Tolling, K.; Sjoquist, J. J. Immunol. Methods 1983, 62, 1. (23) Forsgren, A.; Sjoquist, J. J. Immunol. 1966, 97, 822.
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Figure 3. (A) In situ transmission spectra showing the immobilization of SpA, the blocking of BSA, and the binding of hIgG with SpA immobilized on the pore surfaces. (B) Correlation between the added hIgG concentrations and the shifts induced by formed hIgG-SpA complexes and hIgG-goat anti-hIgG complexes; insets show the possible orientations of hIgG molecules.
hIgG immobilized in the polystyrene microtiter plates can be used to detect hIgG in an enzyme-linked immunosorbent assay. Addition of a protein solution into the pores results in a red shift of the diffraction peak. This shift is attributed to two factors. First, replacement of some of the aqueous phase with the protein solution will change the mean refractive index of the film and be observed as a shift. The second factor that contributes to the shift is ascribed to the adsorption of protein on the pore surfaces. At this stage, the film is rinsed thoroughly with the same aqueous phase to ensure that refractive index changes arise only from the second factor. In our experiments, addition of a 2.0 mg/mL SpA solution with a subsequent water rinsing resulted in a shift of ∼2.1 nm (Figure 3A). Only ∼0.1 nm shift was observed in the following blocking process with BSA (Figure 3A) because of the dense distribution of SpA molecules previously adsorbed on the polystyrene surface. Once SPA was immobilized, binding of any additional biomolecules could lead to a shift of the diffraction peak that directly scales with analyte mass. Addition of a 5.0 mg/mL IgG solution resulted in a shift of 4.1 nm (Figure 3A). This detection is consistent with the formation of molecular packing of hIgG on the pore surface. IgG is a protein composed of two heavy chains (MW 50 kDa) and two light chain (25 kDa) attached by disulfide bridges that form a Y-like structure; their shape is usually compared to a disk with an average diameter of 12 nm
Notes
and a thickness of 4 nm.25,26 If a 12 nm thickness of the IgG layer is assumed, a shift maximum of 6.6 nm can be estimated from Bragg’s law. However, it is impossible for the thickness of the bound IgG layer to reach this level due to limitations in the number of binding sites and steric hindrance. So a shift of 4.1 nm for 5 mg/mL of IgG concentration is reasonable. Protonation of the binding sites on SpA by decreasing the pH of the solution releases IgG from SpA.27 Thus, replacing the solution in the pores with a solution containing 0.1 M acetic acid (pH 2.78) resulted in an almost instantaneous blue shift of the diffraction peak close to a level corresponding to uncomplexed SpA. A subsequent rinse with water returned the diffraction peak to the original position measured before the introduction of IgG. We probed a series of hIgG solutions at various concentrations (Figure 3B). The shifts induced by hIgG bound to immobilized SpA increased proportionally with increasing concentrations of added hIgG from 0.01 to 2.5 mg/mL and then reached a plateau (Figure 3B). For increasing concentrations from 2.5 to 10 mg/mL, only a slight change was observed in the peak position. This was because most of the hIgG binding sites of immobilized SpA had been saturated by hIgG at the concentration of 2.5 mg/mL. Similar detection had been observed in the measurements of the immobilized goat anti-hIgG (Figure 3B), but the shifts induced by goat anti-hIgG binding with hIgG at the same concentrations were smaller than the shifts induced by SpA-hIgG. This is because goat antihIgG binds to the different regions of hIgG, and also the binding capacities of SpA and goat anti-hIgG are different. The possible orientations of hIgG molecules on the immobilized SpA and goat anti-hIgG surfaces are shown in the insets of Figure 3B. Our biosensor shows concentration-dependent binding from 0.01 to 2.5 mg/mL and a sensitivity of 0.01 mg/mL for hIgG. In recent years, several optical sensor techniques have been developed for the direct monitoring of biomolecular recognition processes at the surface of a sensor. Among them, surface plasmon resonance (SPR) methods have made an important contribution to the quantification of biomolecular interactions. The sensitivity for the measurement of analyte(s) in the optical biosensors is dependent on the molecular weight of the analyte. On the basis of the thickness of the protein corona, we can quantitatively predict the correlation of the shift with the thickness of binding pairs, as shown in the Supporting Information. Considering that our instrument resolution is 0.1 nm, a ∼0.1 nm thickness change of the binding IgG layer can be discriminated in our biosensor. Such a detection limit is comparable to that of conventional SPR.28 A larger concentration range of IgG from 0.01 to 2.5 mg/ mL could be detected using a simple diffractive optical biosensor. Using this approach, we demonstrate proof of principle of a label-free optical biosensor to quantify biomolecular interactions on a surface in a commercially available UV-visible spectrophotometer. This is very important for biological analyses. Our biosensor was also found to completely eliminate disturbance due to nonspecific binding. The response of (24) Palmer, D. A.; French, M. T.; Miller, J. N. Analyst 1994, 119, 2769. (25) Delamarche, E.; Sundarababu, G.; Biebuyck, H.; Michel, B.; Gerber, C.; Sigrist, H.; Wolf, H.; Ringsdorf, H.; Xanthopoulos, N.; Mathieu, H. J. Laugmuir 1996, 12, 1997. (26) Qian, W. P.; Yao, D. F.; Xu, B.; Yu, F.; Lu, Z. H.; Knoll, W. Chem. Mater. 1999, 11, 1399. (27) Dancil, K. S.; Greiner, D. P.; Sailor, M. J. J. Am. Chem. Soc. 1999, 121, 7925. (28) Homola, J.; Yee, S. S.; Gauglitz, G. Sens. Actuators, B 1999, 54, 3.
Notes
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Figure 4. Transmission spectra showing the comparison of the binding of immobilized SpA with whole hIgG molecules, Fc, F(ab′)2, and Fab fragments. (A) IgG (3.2 nm red shift). (B) Fc fragments (1.1 nm red shift). (C) F(ab′)2 (no shift). (D) Fab (no shift).
the SpA-immobilized polystyrene films was tested with whole IgG molecules, Fc, F(ab′)2, and Fab fragments. IgG is susceptible to proteolytic attack by enzymes such as pepsin and papain. Cleavage at the hinge region of IgG produces fragments known as Fc and either F(ab′)2 or Fab depending upon the enzyme used. The Fc fragment contains the binding domain recognized by SpA. The F(ab′)2 or Fab fragments contain the antigen binding regions and do not bind specifically to SpA. Additions of 1.0 mg/mL solutions of the whole IgG molecules and the Fc fragments resulted in red shifts of 3.2 and 1.1 nm, respectively (Figure 4A,B). Upon introduction of solutions of 1.0 mg/mL of F(ab′)2 and Fab fragments, respectively, no wavelength shifts were observed (Figure 4C,D). 3DOM polystyrene substrate based biosensors measure the change in refractive index of the solution near the pore surface that occurs during complex formation.
Transduction can be achieved by monitoring the diffraction peak shift, which scales with the mass of analyte bound on the pore surfaces. Consequently, our method is simple and general and can be used to detect a variety of biomolecular complexes, including oligonucleotides, antibody-antigen interactions, enzyme-substrate interactions, and lectin-glycoprotein interactions. Supporting Information Available: Correlation between the thickness of the protein corona and the shift in the peak position estimated by Bragg’s law (Supporting Figure 1), and figures showing the experimental cell (Supporting Figure 2) and the selection of the immobilization concentration of ligands (Supporting Figure 3). This material is available free of charge via the Internet at http://pubs.acs.org. LA0118199
