pubs.acs.org/Langmuir © 2010 American Chemical Society
Virus-Templated Photoimprint on the Surface of an Azobenzene-Containing Polymer Taiji Ikawa,*,† Yuji Kato,‡ Takeshi Yamada,‡ Masahito Shiozawa,† Mamiko Narita,† Makoto Mouri,† Fumihiko Hoshino,† Osamu Watanabe,† Masahiro Tawata,‡ and Hiroshi Shimoyama‡ † ‡
Toyota Central Research & Development Laboratories, Inc., Nagakute, Aichi 480-1192, Japan, and Department of Electrical and Electronic Engineering, Meijo University, Shiogamaguchi, Tenpaku-ku, Nagoya 468-8502, Japan Received March 29, 2010. Revised Manuscript Received June 8, 2010
A photoimprint-based immobilization process is presented for cylindrical viruses on the surface of an azobenzenebearing acrylate polymer by using atomic force microscopy (AFM). Tobacco mosaic virus (TMV), 18 nm in diameter and ca. 300 nm in length, was employed as a model virus. First, a droplet of an aqueous solution containing TMV was placed on the acrylate polymer surface. After drying the droplet, the polymer surface was irradiated with light at a wavelength of 470 nm from blue-light-emitting diodes. Finally, the surface was washed by aqueous solution with detergents. The polymer surface was observed at each step by AFM. TMV was shown to embed itself gradually on the polymer surface during photoirradiation in a time scale of tens of minutes because of the formation of the surface groove complementary to the shape of TMV. Analysis of immobilization efficiency of TMV on the polymer surface by the immunological enzyme luminescence indicated that efficiency increased proportional to the photoirradiation time. In these experimental conditions, the absorption band of the azobenzene moiety remained constant before and after the photoirradiation. These results show that TMV is physically held on the complementary groove formed on the polymer surface by the photoirradiation.
Introduction Viruses causing diseases ranging from smallpox to the common cold come in a wide diversity of sizes, shapes, and surface chemicals. They have either DNA or RNA genes wrapped in a protective shell of proteins called a capsid. Their sizes range from 10 nm to several hundred nm, and their shapes are classified into four main morphological types: helical, icosahedral, enveloped, and complex.1 For a wide variety of viruses, immobilization and stabilization technologies on a solid phase substrate have become important for the use in analysis,2 sensors, separations,3 and vaccinations that focus on medicine, therapy, healthcare, and food safety. Regarding the separation and/or isolation of viruses, the most studied systems for virus immobilization have been based on the use of antibodies, and antibody-based virus sorbents have been constructed.3,4 Likewise, molecularly imprinted polymers (MIPs) against viruses could be attractive and inexpensive alternatives for the virus sorbents.5,6 Special consideration must be taken to ensure the formation of complementary cavities when large particles such as viruses are imprinted on MIPs. This is because their binding capability for viruses is not very efficient due to the formation of polymer-virus aggregation
*To whom all correspondence should be addressed: e-mail e1056@mosk. tytlabs.co.jp; tel þ81-561-71-7761.
(1) Collier, L.; Balows, A.; Sussman, M. In Topley and Wilson’s Microbiology and Microbial Infections, 9th ed.; Hodder Arnold: Oxford, 1998; Vol. 1. (2) Kuznetsov, Y. G.; Malkin, A. J.; Lucas, R. W.; Plomp, M.; McPherson, A. J. Gen. Virol. 2001, 82, 2025. (3) Bennett, A. R.; Davids, F. G. C.; Vlahodimou, S.; Banks, J. G.; Betts, R. P. J. Appl. Microbiol. 1997, 83, 259. (4) Ozanne, G.; d’Halewyn, M. A. J. Clin. Microbiol. 1992, 30, 564. (5) Bolisay, D. L.; Culver, J. N.; Kofinas, P. Biomacromolecules 2007, 8, 3893– 3899. (6) Tai, D. F.; Lin, C. Y.; Wu, T. Z.; Chen, L. K. Anal. Chem. 2005, 77, 5140.
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and difficulties of template removal after cross-linking of the polymer matrix. In a series of our previous works, we have presented a novel photoimprint-based immobilization method for several different substances, for example, polystyrene particles,7,8 DNA,7 immunoglobulin G (IgG),7,9,10 and assembled actin filaments11 on the surface of an azobenzene-bearing acrylate polymer (azopolymer). In the case of polystyrene particles, scanning electron microscopy (SEM) and atomic force microscopy (AFM) study reveal that the azopolymer surface deforms along with the shape of the particles with diameters ranging from 20 to 2000 nm during photoirradiation.7,12-14 Likewise, in the case of IgG, the formation of the complementary cavity is on the azopolymer surface after the photoirradiation, while the cavities are very small (2-3 nm) to obtain precise images even by using tapping-mode AFM.7 From these experimental results, we propose photoimprint-based immobilization mechanism such that the azopolymer surface deforms along with the shape of the substances on the polymer surface during photoirradiation. Consequently, the polymer surface (7) Ikawa, T.; Hoshino, F.; Matsuyama, T.; Takahashi, H.; Watanabe, O. Langmuir 2006, 22, 2747. (8) Watanabe, O.; Ikawa, T.; Kato, T.; Tawata, T.; Shimoyama, H. Appl. Phys. Lett. 2007, 88, 204107. (9) Narita, M.; Hoshino, F.; Mouri, M.; Tsuchimori, M.; Ikawa, T.; Watanabe, O. Macromolecules 2007, 40, 623. (10) Narita, M.; Ikawa, T.; Mouri, M.; Tsuchimori, M.; Hoshino, F.; Watanabe, O. Jpn. J. Appl. Phys. 2008, 47, 1332. (11) Ikawa, T.; Hoshino, F.; Watanabe, O.; Li, Y.; Pincus, P.; Safinya, C. R. Phys. Rev. Lett. 2007, 98, 018101. (12) Ikawa, T.; Mitsuoka, T.; Hasegawa, M.; Tsuchimori, M.; Watanabe, O.; Kawata, Y.; Egami, C.; Sugihara, O.; Okamoto, N. J. Phys. Chem. B 2000, 104, 9055. (13) Hasegawa, M.; Ikawa, T.; Tsuchimori, M.; Watanabe, O.; Kawata, Y. Macromolecules 2001, 34, 7471. (14) Ikawa, T.; Mitsuoka, T.; Hasegawa, M.; Tsuchimori, M.; Watanabe, O.; Kawata, Y. Phys. Rev. B 2001, 64, 195408.
Published on Web 07/07/2010
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Figure 1. Chemical structure of the azopolymer used in this study.
physically holds and immobilizes them. The immobilization method has the distinct potential for biological applications including protein chip application,9,10 biological imaging,11 and MIPs because it provides a nonchemical and nonheated process. It also provides a new concept for fabrication of two-dimensional arrays of spherical particles for photonic devices.8 In this article, we demonstrate the distinct evidence that the photoimprint mechanism can immobilize the cylindrical virus on the azopolymer surface. Tobacco mosaic virus (TMV) was employed as a model virus. TMV has a typical helical structure 300 nm in length and 18 nm in diameter15 and a protein cylinder arranged around a helical RNA core.16 Its isoelectric point is 3.5, and the virus particle is negatively charged at neutral pH conditions.17 In the experiment, TMV is photoimprinted on the azopolymer surfaces by photoirradiation, using AFM to analyze the process. Next, the immobilization efficiency of TMV on the azopolymer surface is analyzed by the immunological enzyme luminescence method. From these results, it can be seen that TMV gradually embeds itself on the azopolymer surface during photoirradiation, in direct proportion to the immobilization efficiency of TMV. Promising applications of this phenomenon for AFM imaging and virus MIPs are discussed.
Experimental Section Materials. Azopolymer. Poly{40 -[[[2-(methacyloyloxy)ethyl]ethyl]amino]-4-cyanoazobenzene-co-methyl methacrylate} (15 mol % azobenzene moieties) was obtained by free radical polymerization according to a previous report.7,9 Chemical structure of the azopolymer is shown in Figure 1. The number- and weight-average molecular weights (Mn and Mw) of the azopolymer were 18 500 and 35 000, measured by gel permeation chromatography (eluent CHCl3, Shodex. GPC-101 with K-805 L, Showa Denko) with calibration using standard polystyrene. The glass transition temperature (Tg) was 121 °C, determined by differential scanning calorimetry (TA Instruments Q1000). The wavelength of the absorption band of the azobenzene moiety in the azopolymer was ranging from 400 to 600 nm, and the wavelength of maximum absorption (λmax) was 450 nm. Films of the azopolymer on the glass substrate were prepared from pyridine solution (12.5 g/L) by the spin-coating technique (rotation speed was 4000 rpm and spinning time was 30 s). The films were then placed in a vacuum oven at 140 °C for 24 h to obtain solvent-free samples. The typical thickness of the film was 40 nm. TMV was purchased from BIOREBA AG. Purification of TMV was performed by a Beckman XL-90 ultracentrifuge system.18 20 mg of TMV powder was dissolved in 0.5 mL of phosphate-buffered saline (PBS) solution. The TMV solution was centrifuged at 3500 rpm for 30 min at 4 °C. The TMV solution supernatant was then pelleted by ultracentrifuge at 28 000 rpm by a Beckman 50.4Ti rotor for 0.5 h at 4 °C. The pellet was resuspended in PBS solution. This procedure was repeated thrice. Final virus concentration was (15) (16) (17) (18)
Caspar, D. L. D. Adv. Protein Chem. 1963, 18, 37. Namba, K.; Stubbs, G. Science 1986, 232, 738. Scheele, R. B.; Lauffer, M. A. Biochemistry 1967, 6, 3076. Boedtker, H.; Simmonds, N. S. J. Am. Chem. Soc. 1958, 80, 2550.
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determined by measuring absorbance at a wavelength of 260 nm using a NanoDrop ND 1000 spectrophotometer and the extinction coefficient at 260 nm of the absorption band of TMV.18 Sample Preparation and AFM Imaging. Stock solution of TMV was desalted and diluted by buffer exchange using Milipore Microcon YM-10 filter units. The final concentration of the TMV solution was 5.5 μg/mL of TMV and 10 μM of PBS. 2 μL of working solution was spotted on the azopolymer film, and then the solution was dried naturally. The film was then irradiated with light at a wavelength of 470 nm from an array of blue-lightemitting diodes (LEDs). The absorbance at a wavelength of 470 nm for the film was 10% lower than the absorbance at the wavelength of 450 nm (λmax). Optical power density at the polymer surface was measured by an Ophir optical power meter and was around 25 mW/cm2. The film surface was observed by a contact-mode AFM (Nanoscope E, Digital Instruments) with a silicon nitrite cantilever (ORC8, the typical tip radius of curvature was 15 nm; the spring constant was 0.05 N/m, Veeco). After AFM imaging, the film was rinsed overnight in an aqueous solution of 1 wt % sodium dodecyl sulfate to remove TMV from the film surface and was observed by AFM again. UV-vis absorption spectra of the film before and after photoirradiation were obtained by a Shimadzu UV-2000 spectrophotometer. Immobilization Efficiency Analysis. The immobilization efficiency of TMV on azopolymer was analyzed by the immunological chemiluminescence method (see the Supporting Information). First, 2 μL of TMV solution with concentration ranging from 5 to 100 μg/mL was spotted on azopolymer films. Some films were dried in a vacuum chamber to dry off the spotting solution (dry process); others were sealed in small chambers (Hybridization cassettes, Arrayit Co) with saturated water vapor to avoid drying off the spotting solution (wet process). These films were irradiated with light at a wavelength of 470 nm and optical power density of 25 mW/cm2 from blue LEDs for 0, 10, 20, and 30 min. After the irradiation process, the films were rinsed in a PBS solution containing 0.5% Tween 20 for 30 min, and then the solution was blown off from the film surface by a N2 blower. The film surface was covered with a spaced cover glass that had printed protuberances of 20 μm thick at the four corners (Takara spaced cover glass). 1 μg/mL alkaline phosphatase-linked anti-TMV immunoglobulin G (BIOREBA AG) was penetrated into the gap between the film and the spaced cover glass, and the films were kept at room temperature for 1 h. The film with the spaced cover glass was immersed in PBS solution, and the cover glass was removed carefully. The film was kept in the PBS solution for 5 min. After blowing the PBS solution from the film, it was covered with the spaced cover glass again, and the chemiluminescent substrate solution (ready-to-use CDP-star in Emerald II, Applied Biosystemes) was penetrated into the gap. The chemiluminescence signal from the film surface was quantified by a cooled chargecoupled-device camera (LumiVision PRO, Aisin/Taitec Inc.). An occupation area of TMV in a spot formed by the dry process (ATMV) was calculated using the following equation: ATMV = CVNAaTMV/Mw, in which C is the weight concentration of the spotting solution, V is the volume of the spotting solution (2 μL), NA is Avogadro’s constant (6.02 1023), aTMV is the molecular area of one TMV molecule (300 nm 18 nm), and Mw is the molecular weight of TMV (40 106). From this equation, the area of a circular spot with 1 mm radius (3.14 10-6 m2), formed by 2 μL of the TMV solution, will be occupied by TMV solution with a concentration of around 20 μg/mL (ATMV = 3.25 10-6 m2).
Results AFM Analysis. Figure 2 shows contact-mode AFM images of the azopolymer surface treated with TMV. Shown in Figure 2a-c are the images of the sample in the presence of TMV for different photoirradiation times of 0, 10, and 20 min, respectively. All images show rod-shaped particles with a typical length of around 300 nm; however, the rods are not of uniform length. Some rods Langmuir 2010, 26(15), 12673–12679
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Figure 2. AFM images of the azopolymer surface: (a-c) azopolymer surface in the presence of TMV; (d-f) azopolymer surface after washing the surface by detergent. (a, d) Without photoirradiation, (b, e) 10 min photoirradiation, and (c, f) 20 min photoirradiation.
are shorter than 300 nm, and some are connected at each end. Several aggregations of granular particles are also observed in Figure 2c. The granular particles on Figure 2c were also observed on the samples of which preparation conditions were the same with Figure 2a,b. Frequency of the appearance of the granular particles depended on the sample preservation time; the frequency increased with increasing the preservation time after purification of the TMV stock solution. The granular particles were, therefore, not formed by the photoirradiation but formed during the preservation. These structures coincide with the structure seen by electron microscopy; the rods and the granular particles are attributed to TMV and decomposed materials from TMV.15,19,20 Comparing Figure 2a with Figure 2b,c, strong image noises appear, and most of the rod-shaped particles are aligned parallel to the horizontal scanning direction in Figure 2a. This strongly suggests that the rod-shaped particles on the surface are moved and aligned by the probe tip during scanning due to the unstable adsorption of TMV on the azopolymer surface. On the other hand, clear images of the rod-shaped particle are obtained in Figure 2b,c. The heights of the rod-shaped particles gradually decrease with the photoirradiation time. (The details are shown in Figures 3 and the following paragraph.) This suggests that the rod-shaped particles are embedded into the azopolymer surface during the photoirradiation; therefore, the rods are immobilized firmly against the scanning of the probe tip. It must be noted that most of the rod-shaped particles are located in diagonal directions in Figure 2b. This is not due to the scanning of the probe tip, but rather to the flow of the buffer solution during the sample preparation, because the direction of the particles is different at each position even on the same sample. The TMV particles might be aligned along the convection flow during evaporating the solvent; for example, the convection flow was along the diagonal direction during the sample preparation in Figure 2b. (19) Shenton, W.; Douglas, T.; Young, M.; Stubbs, G.; Mann, S. Adv. Mater. 1999, 225, 11. (20) Schramm, G.; Wiedeman, M. Z. Naturforsch. B 1951, 6, 379.
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Shown in Figure 2d-f are contact-mode AFM images of the azopolymer surface after removal of TMV by washing with detergents; the samples were fabricated for different photoirradiation times of 0, 10, and 20 min, respectively. The flat surface with some small debris is observed in Figure 2d. No deformation is caused on the azopolymer surface without photoirradiation. On the other hand, the observed structure of the grooves in Figure 2e,f complements that of the rodlike particles shown in Figure 2b,c, indicating that TMV shape is imprinted on the azopolymer surface by the photoirradiation. Figure 3 represents a detailed AFM analysis of the rod-shaped particles and the grooves on the azopolymer. The lateral and longitudinal cross sections of the typical rod-shaped particles on the azopolymer surface are shown in Figure 3a,b. From these cross sections, the rod-shaped particles are found to lay flat on the azopolymer surface, and the particles are gradually embedded while keeping their sides parallel to the azopolymer surface during the photoirradiation. Figure 3c shows a change in the average height of the rod-shaped particles as a function of the photoirradiation time. The height shown in the inset in Figure 3c is averaged from 100 particles in several AFM images of the same sample. The average height decreases from 12 to 5 nm as the photoirradiation time increases from 0 to 30 min. In the lateral cross sections in Figure 3a, the full width at halfmaxima (fwhm) of the peaks are around 30 nm, which is twice as large as the 18 nm diameter of TMV. This is due to the common imaging artifacts from a convolution of the probe geometry and the samples.2 Likewise, the average height at 0 min photoirradiation is 12 nm in Figure 3c, which is smaller than the TMV diameter of 18 nm. This must be due to the strong interaction between the probe tip and TMV by the contact mode AFM operation, together with the unstable adsorption of TMV during the scanning in the AFM operation as shown in Figure 2a. Although the cross sections contain these artifacts, it is evident that the height of the particles gradually decreases on a time scale of minutes during the photoirradiation. DOI: 10.1021/la101229p
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Figure 3. AFM analysis of TMV on the azopolymer surface. (a, b) Cross sections of TMV on the azopolymer surface along with (a) lateral and (b) longitudinal directions. (c) Change in the height of the surface object on the azopolymer as a function of the photoirradiation time. The error bars indicate the standard deviations. (d, e) Cross sections of the grooves formed on the azopolymer surface after removal of TMV along with (d) lateral and (e) longitudinal directions. (f) Change in the depth of the groove formed on the azopolymer surface as a function of the photoirradiation time. The error bars indicate the standard deviations.
The lateral and longitudinal cross sections of the typical grooves on the azopolymer surface after removal of the rodshaped particles are shown in Figure 3d,e. The lateral cross sections in Figure 3d show that the depth of the groove increases with the photoirradiation time accompanied by an uplift of the rim of the groove. The distance between the peaks of the rim is around 50 nm, which is 3 times larger than the diameter of TMV. The observed distance between the rims is accurate because the geometry of the sample surface, rather than of the probe, establishes the line profile of the peaks of the groove. On the other hand, the depth of the groove possibly appears small due to the probe tip not reaching the bottom of the groove. A change in the average depth of the grooves formed on the azopolymer surface as a function of the photoirradiation time is shown in Figure 3f. The depth, defined in the inset in Figure 3f, is averaged from 100 grooves in several AFM images of the same sample. By increasing the photoirradiation time to 30 min, the average depth of the grooves was increased to 5 nm. Sums of the average height in Figure 3c and the average depth in Figure 3f at the same photoirradiation time are kept constant at around 10 nm, which is smaller than the 18 nm diameter of TMV. The groove depth might be slightly underestimated due to the probe tip not reaching the bottom of the groove. Based on the AFM analysis, Figure 4 summarizes the behavior of the azopolymer surface with TMV during the photoirradiation. As the photoirradiation time increases, TMV gradually embeds into the azopolymer surface because of the formation of the complement groove just beneath TMV. The groove depth increases with the uplift of the groove rim during the photoirradiation, but the distance between the groove rims stays constant at 12676 DOI: 10.1021/la101229p
Figure 4. Surface deformation of the azopolymer by the photoirradiation with TMV. Reconstituted from AFM analysis in Figure 3. The azopolymer surface shapes are normalized at beneath TMV.
around 50 nm, 3 times larger than the diameter of TMV. To be precise, the geometry of the grooves on the azopolymer surface is slightly different from that of TMV. Finally, over half of the TMV body embeds into the groove after 30 min of photoirradiation. Overall, these results indicate that both the azopolymer surface deform along the shape of TMV during the photoirradiation. As a result, the contact-mode scanning of the probe tip in the AFM operations supports TMV in the groove formed on the azopolymer surface. Immobilization Efficiency Analysis. The immobilization efficiency of TMV on the azopolymer was analyzed by the immunological chemiluminescence method. Figure 5a shows the efficiency of the immobilization of TMV in dry and wet processes (see Experimental Section). In the dry process, as the TMV concentration of the spotting solution increases, the chemiluminescence intensity increases and saturates over 0.025 mg/mL of TMV. Based on the basic calculation of the molecular area (see Experimental Section), the whole area of the spot will be covered with TMV at a concentration of 20 μg/mL. The calculation Langmuir 2010, 26(15), 12673–12679
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Figure 5. Immobilization efficiency of TMV on the azopolymer surface. (a) Intensity of the chemiluminescence from the TMV spots formed on the azopolymer surface as a function of the concentration. Open and closed squares are wet and dry process, respectively (see Experimental Section). (b) Intensity of the chemiluminescence from the TMV spots formed on the azopolymer surface in the dry process as a function of the photoirradiation time.
matches the experimental data of the saturate concentration. Comparing with the dry processes, the chemiluminescence intensity of the spots in the wet process is 2 orders of magnitude weaker, although the intensity increases with increasing concentration of TMV. In addition, on the sample surface in the wet process, AFM shows no groove formation. From this experiment, the adsorption of TMV on the azopolymer surface in the aqueous solution is found to be very weak. The inherent attractive force between the azopolymer and TMV is not strong. Figure 5b shows a change in the intensity of the chemiluminescence from the TMV spots on the azopolymer surface as a function of the photoirradiation time in the dry process. The intensity of the chemiluminescence increases with increasing photoirradiation time. This means that the number of TMV remaining on the azopolymer surface increases with the photoirradiation, which is almost proportional to the groove formation behavior on the azopolymer surface, as shown in Figures 3 and 4. To clarify the possibility of the photochemical reaction in this process, changes in the absorption spectra of the azopolymer before and after photoirradiation are examined. The absorption spectra are unchanged in these experimental conditions, meaning that there is no photochemical reaction of the azobenzene moiety.
Discussion Deformation Mechanism. As shown in Figures 2;4, the azopolymer surface deforms along with the shape of TMV during the photoirradiation, although the exact geometry of the azopolymer Langmuir 2010, 26(15), 12673–12679
surface is slightly different from that of TMV. In this section the deformation mechanism of the azopolymer surface is discussed. Polymers that contain azobenzene show various photoresponses.21 Especially, the polymer containing a pseudostilbenetype azobenzene (the 4- and 40 -positions of the two azobenzene rings are substituted by electron-donating and electron-withdrawing groups) is well-known for the formation of artificial surface relief structures by exposure to an interference pattern of the two beams of light or a single laser beam.21-23 The formation of the surface relief structure is considered to originate from the trans-cistrans photoisomerization cycle of the pseudostilbene-type azobenzene and subsequent mass transport of polymer matrix.21,24 In this process, there is no volume loss during the deformation, and the possibility of photochemical reactions is negligible because the absorption band of the azobenzene moiety is unchanged before and after the photoirradiation. Thus, it is reasonable that the deformation is a mass transport effect. Recently, Karageorgiev et al. reported that the polymer containing a pseudostilbene-type azobenzene behaves like a viscoelastic fluid during photoirradiation.25 The result strongly suggests that the photoisomerization motion plasticizes the polymer matrix during the photoirradiation, which should be the first step of the mass transport of the polymer matrix. The photoisomerization cycle of azobenzene and subsequent polymer migration explain the groove formation on the azopolymer surface with TMV because the total volume of the azopolymer surface and the absorption band of the azobenzene were not changed before and after the photoirradiation. The next question to consider is the driving force of the mass transport. Various driving forces behind the surface deformation by two-beam interference have been proposed, such as internal pressure, light intensity gradient, and intermolecular interaction.21-24 However, the nature of the driving force has not been directly confirmed. We have already reported the mechanism on the azopolymer surface deformation with submicrometer-sized polystyrene particles.14 In that case, the azopolymer surface beneath the particles depressed and its sides heaved up during the photoirradiation, while the geometries of the azopolymer surface and the particles were different. This means that the interfacial force itself is not the only factor for the azopolymer surface deformation with the particles. On the basis of the calculation of the electromagnetic field distribution around the polystyrene particles, we found that the electromagnetic field localized on the surface of the particles (the optical near-field)26 could cause the polymer migration. Comparing TMV with the polystyrene particles, the deformation behavior of the azopolymer surface was found to be almost the same as shown in Figures 3 and 4, and the electromagnetic field of a cylindrical particle like TMV is essentially similar to the spherical particles.27 Hence, the electromagnetic field localized on the surface of TMV is considered to be one of the origins of the groove formation. From these considerations, we propose the following mechanism on the azopolymer surface deformation by TMV. First, light (21) Natansohn, A.; Rochon, P. Chem. Rev. 2002, 102, 4139. (22) Rochon, P.; Batalla, E.; Natansohn, A. Appl. Phys. Lett. 1995, 66, 136. (23) Kim, D. Y.; Li, L.; Kumar, J.; Tripathy, S. K. Appl. Phys. Lett. 1995, 66, 1166. (24) Pedersen, T. G.; Johansen, P. M.; Holm, N. C. R.; Ramanujam, P. S.; Hvilsted, S. Phys. Rev. Lett. 1997, 80, 89. (25) Karageogiev, P.; Neher, D.; Schulz, B.; Stiller, B.; Pietsch, U.; Giersig, M.; Brehmer, L. Nature Mater. 2005, 4, 699. (26) Ohtsu, M.; Kobayashi, K. In Optical Near Fields: Introduction to Classical and Quantum Theories of Electromagnetic Phenomena at the Nanoscale; Advanced Texts in Physics; Springer-Verlag: Berlin, 2004. (27) Moreno, F.; Saiz, J. M.; Gonzalez, F. In Light Scattering and Nanoscale Surface Roughness; Maradudin, A. A., Ed.; Nanostructure Science and Technology; Springer: New York, 2007; Vol. 12, pp 305-339.
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absorption induces the photoisomerization cycle of the azobenzene moiety, and then the photoisomerization motion causes the softening of the polymer matrix. Second, the polymer chain migrates, following the optical near-field of TMV. Finally, when the photoirradiation stops, the cessation of the photoisomerization motion causes the fixing of the deformed structure. Immobilization Mechanism. Because of the deformation of the azopolymer surface, TMV is immobilized on the azopolymer surface. From the immobilization efficiency analysis in the wet process (Figure 5a), the attractive force between TMV and the azopolymer itself are found to be very weak in the aqueous solution. This must be due to a difference in the surface characteristics between TMV and the azopolymer. The azopolymer has no charged and hydrophobic surface (the main component of the azopolymer is methyl methacrylate, and the contact angle of water to the azopolymer is 75°). In contrast, TMV has a highly negatively charged (isoelectric point is 3.5) and hydrophilic surface.17 For this reason, the attractive forces between TMV and the azopolymer are only intermolecular forces (weak bonds) such as van der Waals forces and hydrogen-bonding interaction,28 which are lower than the fluctuating force caused by the pushing molecules of the surrounding fluid (Brownian motion of thermal energy kT). Comparing with the Brownian motion of TMV in the fluid on a time scale of microseconds, the deformation of the azopolymer surface is a very slow process on a time scale of tens of minute. Thus, the azopolymer surface will probably not deform along with TMV in the wet process. If TMV decomposes, the hydrophobic interior of TMV will adsorb on the azopolymer surface because of the hydrophobic interaction. However, we did not observe the image of the decomposed material like Figure 2C in the wet process. It is reasonable to suppose that TMV is stable enough not to decompose in the wet process. On the other hand, in the dry process, the thermal motion of TMV on the azopolymer surface is negligible. TMV particles can remain stationary on the azopolymer surface in spite of the unstable adsorption, and therefore the azopolymer surface can deform along with the TMV, as shown in Figures 2;4. It could be argued that TMV was fixed on the azopolymer surface by photochemical reactions; however, the possibility of a photochemical reaction is very low because the absorption spectra of the azopolymer were confirmed to be unchanged in these experimental conditions. In addition, TMV is completely removed from the azopolymer surface only by washing with surfactants, even after 30 min photoirradiation (Figure 2f ). The immobilization process is believed to be a noncovalent physical process, rather than a covalent binding process. From these experimental data, the increase in immobilization efficiency by photoirradiation is attributed to the increase in the contact area between the azopolymer and TMV surfaces due to the formation of the complement groove, although the groove structure does not perfectly follow the shape of the TMV. The increase of the contact area can increase a number of weak bonds (intermolecular bonds). If a weak bond has an energy of approximately a kT and desorption requires all bonds to be broken simultaneously, the probability of desorption is roughly given by ∼e-N, where N is the number of weak bonds.7,29 The desorption rate of TMV decreases exponentially with increasing a number of weak bonds. Based on these considerations, the increase in the contact area and resulting increase in a number (28) Israelachivili, J. N. Intermolecular and Surface Forces; Academic Press: London, 1985. (29) Ramsden, J. J. Q. Rev. Biophys. 1993, 27, 41.
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of weak bonds between TMV and the azopolymer surfaces should be the principal factor responsible for the photoimmobilization of TMV on the azopolymer. Applications for AFM and MIPs. The procedure described above is well-suited for AFM imaging of biologically derived macromolecules and supramolecular assembly because it can reduce unfavorable probe-sample interactions during AFM operations, which is an important factor in determining distinct structures of virus particles.2 As shown in Figure 2, clear TMV images appeared on the azopolymer surface after the photoirradiation, even by using contact-mode AFM where interaction between the probe tip and the sample is relatively strong. The procedure can also provide a nonreactive, nonionic, and flat surface eliminating the possibility of chemical denaturation of the immobilization objects; therefore, the structure and morphology of the objects can easily be observed by this technique without interference from the substrate. There is an important disadvantage when compared with chemically modified mica30,31 as a substrate for AFM imaging: the information about the height of the object is spoiled by its embedding into the azopolymer surface. However, comparing the observed height of the object with the groove formed on the azopolymer surface, the true height can be estimated. Another disadvantage is that the azopolymer used in this experiment is inadequate for the immobilization of highly charged objects like TMV in the aqueous condition as shown in Figure 5b. (Note that general hydrophilic proteins, for example IgG, green fluorescent protein, and F-actin, could be immobilized on the same azopolymer surface in aqueous media.7,11) To immobilize highly charged objects like TMV in the aqueous condition, introduction of a counter charged functional group to the azopolymer is essential. To use the azopolymer for MIPs, the introduction of an appropriate functional group which allows ionic and hydrogen bonding in the target macromolecule is also crucial. Recently, we reported that the introduction of COOH and NMe2 into the azopolymers, which can introduce surface charges, strongly affected the immobilization properties of IgG, e.g., the efficiency of immobilization and the activity of the immobilized IgG.32 The introduction of COOH promoted a more active orientation of the immobilized IgG. This functional group in the azopolymer is expected to create a specific recognition cavity for the macromolecules. The cavity is only created at the polymer surface, which enables the smooth removal of the template macromolecules even if the macromolecules are aggregated. Furthermore, the isomerization motion of the azobenzene moiety might be an important factor for the formation of the recognition cavity. We have reported that the photoimmobilization efficiencies of IgG onto two types of azopolymers containing 4-amino-40 -cyanoazobenzene (CNazopolymer) and aminoazobenzene (H-azopolymer) were different.9 Although their original hydrophobicities and adsorption efficiencies were almost the same, the photoimmobilization efficiency on the H-azopolymer was higher than that on the CNazopolymer. The cis state of azobenzene in the H-azopolymer is stable over 160 h; on the other hand, that in the CN-azopolymer is relatively unstable up to 30 min. This suggests that changes in the dipole moment or in the basicity due to the isomerization of the azobenzene moiety are taken into account for the increase in (30) Bezanilla, M.; Manne, S.; Laney, D. E.; Lyubchenko, Y. L.; Hansma, H. G. Langmuir 1995, 11, 655. (31) Lamture, J. B.; Beattie, K. L.; Burke, B. E.; Eggers, M. D.; Ehrlich, D. J.; Fowler, R.; Hollies, M. A.; Kosicki, B. B.; Reich, R. K.; Smith, S. R.; Varma, R. S.; Hogan, M. E. Nucleic Acids Res. 1994, 22, 2121. (32) Mouri, M.; Ikawa, T.; Narita, M.; Hoshino, F.; Watanabe, O. Macromol. Biosci. 2010, 10, 612.
Langmuir 2010, 26(15), 12673–12679
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Article
immobilization efficiency on the azopolymer surface by the photoirradiation. The efficient use of isomerization motion of azobenzene would be effective for creating the specific cavity for the use of the azopolymer as a MIP. As shown in Figure 4, the geometry of the formed cavity is slightly different from that of TMV. This might be disadvantage for the generation of selectivity in MIP applications. The introduction of the appropriate functional group as stated above will improve the creation of the cavity with more precise geometry.
Conclusion A detailed analysis of the photoimmobilization phenomenon for the cylindrical virus TMV on photoresponsive polymers containing an azobenzene moiety is presented. The formation of the cavity complementary to TMV on the polymer surface and the subsequent increase of the physical interfacial forces between TMV and polymer surfaces explain the immobilization phenomenon. The phenomenon provides a new technique for the immobilization of large-scale macromolecules especially in AFM operation because of the nonreactive, nonionic, and flat surface together
Langmuir 2010, 26(15), 12673–12679
with reducing unfavorable probe-sample interactions. The phenomenon also offers a new concept of the molecular imprinting method for large-scale macromolecules. The photophysically induced change in the surface shape and the isomerization of the azo-dyes make it possible to imprint both the topographical feature and the surface characteristics of the macromolecules. Introduction of appropriate functional groups such as ionic and hydrogen-bonding groups to the azopolymer is expected to create the specific recognition site for the target macromolecules, which would be very appropriate for the separation and the detection systems for pathogens. Acknowledgment. This work is supported in part by MEXT KAKENHI (18550163). We thank Y. Murakami for useful discussions and their support. Supporting Information Available: Method of immobilization efficiency analysis. This material is available free of charge via the Internet at http://pubs.acs.org.
DOI: 10.1021/la101229p
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