Langmuir 2008, 24, 1625-1628
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Protein Recording Material: Photorecord/Erasable Protein Array Using a UV-Eliminative Linker Koji Nakayama, Takashi Tachikawa, and Tetsuro Majima* The Institute of Scientific and Industrial Research (ISIR), Osaka UniVersity Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan ReceiVed October 27, 2007. In Final Form: December 14, 2007 Protein patterning on solid surfaces is a topic of significant importance in the fields of biosensors, diagnostic assays, cell adhesion technologies, and biochip microarrays. In this letter, we have established a novel, rapid method for the fabrication of a “protein recording material”, which enables us to spatiotemporally regulate the recording, reading, and erasing of a fluorescent protein array as information by a photochemical technique. A photolinker that we synthesized here was used to control the protein array spatiotemporally. The recording process was almost completed after 1 min of photoirradiation to read a clear pattern consisting of a specific protein-ligand complex with high spatiotemporal resolution. The erasing of the protein array was then achieved by photoirradiation onto the entire patterned surface.
Introduction Protein patterning on solid surfaces is a topic of significant importance in the fields of biosensors,1-3 diagnostic assays,4,5 cell adhesion technologies,6-9 and biochip microarrays.10,11 Micro- and nanosized arrays prepared by the superior techniques of lithography and patterning are useful for the screening of trace amounts of analytes.12,13 To produce the array for biosensing, a protein array on a solid surface has the requirement of selective protein interaction14-16 and high spatial resolution.17-19 Recently, a variety of patterning techniques have been established for the practical use of biomaterials, and in particular, photochemical methods have strongly contributed to the fabrication of patterning * Corresponding author. E-mail:
[email protected]. (1) Bieri, C.; Ernst, O. P.; Heyse, S.; Hofmann, K. P.; Vogel, H. Nat. Biotechnol. 1999, 17, 1105-1108. (2) Veiseh, M.; Zareie, M. H.; Zhang, M. Langmuir 2002, 18, 6671-6678. (3) Park, T. J.; Lee, S. Y.; Lee, S. J.; Park, J. P.; Yang, K. S.; Lee, K.-B.; Ko, S.; Park, J. B.; Kim, T.; Kim, S. K.; Shin, Y. B.; Chung, B. H.; Ku, S.-J.; Kim, D. H.; Choi, I. S. Anal. Chem. 2006, 78, 7197-7205. (4) Whitesides, G. M.; Ostuni, E.; Takayama, S.; Jiang, X.; Ingber, D. E. Annu. ReV. Biomed. Eng. 2001, 3, 335-373. (5) Khademhosseini, A.; Suh, K. Y.; Jon, S.; Eng, G.; Yeh, J.; Chen, G.-J.; Langer, R. Anal. Chem. 2004, 76, 3675-3687. (6) Jiang, X.; Ferrigno, R.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 2003, 125, 2366-2367. (7) Ryan, D.; Parviz, B. A.; Linder, V.; Semetey, V.; Sia, S. K.; Su, J.; Mrksich, M.; Whitesides, G. M. Langmuir 2004, 20, 9080-9088. (8) Carrico, I. S.; Maskarinec, S. A.; Heilshorn, S. C.; Mock, M. L.; Liu, J. C.; Nowatzki, P. J.; Franck, C.; Ravichandran, G.; Tirrell, D. A. J. Am. Chem. Soc. 2007, 129, 4874-4875. (9) Veiseh, M.; Veiseh, O.; Martin, M. C.; Asphahani, F.; Zhang, M. Langmuir 2007, 23, 4472-4479. (10) Su, J.; Bringer, M. R.; Ismagilov, R. F.; Mrksich, M. J. Am. Chem. Soc. 2005, 127, 7280-7281. (11) Kim, M. J.; Yang, M.-S.; Kwon, H. T.; Song, J. M. Biomed. MicrodeVices 2007, 9, 565-572. (12) Gauchet, C.; Labadie, G. R.; Poulter, C. D. J. Am. Chem. Soc. 2006, 128, 9274-9275. (13) Phillips, K. S.; Wilkop, T.; Wu, J.-J.; Al-Kaysi, R. O.; Cheng, Q. J. Am. Chem. Soc. 2006, 128, 9590-9591. (14) Khan, F.; He, M.; Taussig, M. J. Anal. Chem. 2006, 78, 3072-3079. (15) Valiokas, R.; Klenkar, G.; Tinazli, A.; Tampe´, R.; Liedberg, B.; Piehler, J. ChemBioChem 2006, 7, 1325-1329. (16) Kang, E.; Park, J.; McClellan, S. J.; Kim, J.; Holland, D. P.; Lee, G. U.; Franses, E. I.; Park, K.; Thompson, D. H. Langmuir 2007, 23, 6281-6288. (17) Krsko, P.; Sukhishvili, S.; Mansfield, M.; Clancy, R.; Libera, M. Langmuir 2003, 19, 5618-5625. (18) Umulis, D. M.; Serpe, M.; O’Connor, M. B.; Othmer, H. G. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 11613-11618. (19) Rundqvist, J.; Mendoza, B.; Werbin, J. L.; Heinz, W. F.; Lemmon, C.; Romer, L. H.; Haviland, D. B.; Hoh, J. H. J. Am. Chem. Soc. 2007, 129, 59-67.
and lithography because fine devices can be fabricated with good spatial resolution and quick processing.20-23 The protein recording material is defined here as a biological device that enables us to spatiotemporally photoregulate the recording, reading, and erasing of information on a solid surface using a protein. In other words, a specific protein can be assembled on a patterned ligand by a protein-ligand interaction and can then be collected by photoelimination of the protein-ligand interaction. In terms of a diagnostic assay, this concept becomes an effective technique for the detection and collection of a specific protein. This is expected to be a useful device not only for biosensing and diagnostic assays but also for record-erasable soft material. Nevertheless, no report has been published on these kinds of materials using the selective interaction and the release of a specific protein controlled by a photochemical technique. In the present study, we demonstrate a novel, rapid method of preparing irreversible protein materials for the recording, reading, and erasing of information. As a first trial for the fabrication of the protein recording material, we used streptavidin, which has a strong association with biotin (Ka ) 4 × 1014 M-1),24 and investigated the physicochemical property of this material in detail. The strategy to obtain the protein recording material is shown in Scheme 1. We synthesized a photoeliminative molecule, 4-(4(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butanoic acid, using an established method.25,26 To obtain the photolinker shown in Scheme 1, biotin and succinimide were introduced into the hydroxyl and carboxyl groups of the photoeliminative molecule with diisopropylcarbodiimide as the coupling reagent, respectively.25,26 The photolinker is stable at room temperature in the dark, although it causes the photoelimination of the biotin moiety with irradiation by >340 nm light. The excitation with >340 nm light should avoid any undesirable damage to the biomolecule; consequently, the present photolinker is suitable for preparing (20) Christman, K. L.; Maynard, H. D. Langmuir 2005, 21, 8389-8393. (21) Howland, M. C.; Sapuri-Butti, A. R.; Dixit, S. S.; Dattelbaum, A. M.; Shreve, A. P.; Parikh, A. N. J. Am. Chem. Soc. 2005, 127, 6752-6765. (22) Sakamoto, M.; Tachikawa, T.; Fujitsuka, M.; Majima, T. AdV. Funct. Mater. 2007, 17, 857-862. (23) Sakamoto, M.; Tachikawa, T.; Kim, S. S.; Fujitsuka, M.; Majima, T. ChemPhysChem 2007, 8, 1701-1706. (24) Green, N. M. AdV. Protein Chem. 1975, 29, 85-113. (25) Holmes, C. P. J. Org. Chem. 1997, 62, 2370-2380. (26) Whitehouse, D. L.; Savinov, S. N.; Austin, D. J. Tetrahedron Lett. 1997, 38, 7851-7852.
10.1021/la703354c CCC: $40.75 © 2008 American Chemical Society Published on Web 01/23/2008
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Scheme 1. Strategy of Synthesizing a Photorecord/Erasable Protein Recording Material Using a Photolinker Immobilized on a Glass Surfacea
Figure 1. Fluorescence imaging of the negative patterns of the Alexa Fluor 532-streptavidin conjugate immobilized on glass surfaces that were prepared by irradiation with >340 nm light for 10 s (A), 30 s (B), 60 s (C), and 300 s (D). The scale bar represents 500 µm.
a (A: recording process) Preparation of a 2D biotin array by UV irradiation through a mask; (B: reading process) reading the fluorescent probe-labeled streptavidin that site-specifically interacted with the biotin array; and (C: erasing process) photoelimination of the streptavidin-biotin complex, caused by exposing both sides of the glass surface to UV light in order to get an erased material (D).
the protein recording material. (A) The photolinker is immobilized on the amino-activated glass surface by a reaction between the succinimide and NH2 groups. The recording of information is carried out by irradiation with >340 nm light through a photomask. (B) The patterned surface is optically visualized by immersion in a buffer solution containing Alexa Fluor 532streptavidin or quantum dot 605-streptavidin conjugates. At this time, the streptavidin having the fluorescent probes should interact only with the biotin moiety of the photolinker. (C) The erasing of the protein array is conducted by irradiation with >340 nm light from both sides of the entire surface. (D) Finally, the eliminated fragments are washed out to obtain the erased surface. Experimental Section Materials. The Alexa Fluor 532-streptavidin conjugate and the quantum dot 605 (CdSe/ZnS core/shell)-streptavidin conjugate were purchased from Invitrogen. 3-Aminopropyltriethoxysilane (APTES) was purchased from Tokyo Kasei. Modification of the Photolinker onto the Coverslip. Glass coverslips (18 × 18 mm2) were cleaned by sonication in heated alkali detergent for 1 h. The cleaned coverslips were amino-silanized by immersion in degassed toluene solution containing 2% APTES
at room temperature for 2 h. The amino-silanized coverslips were reacted with 0.1 mM photolinker dissolved in anhydrous DMF containing 1% N,N-diisopropylethylamine at room temperature for 4 h. Unreacted photolinker was washed out with acetone. Photoirradiation onto the Material. The photoirradiation onto the glass was carried out with a 500 W high-pressure Hg lamp (Ushio USH-500D) through a 340 nm long-pass filter (Toshiba UV34) and/or a homemade photomask. The photoeliminated product was washed out with acetone. In the reading process, the patterned coverslips were immersed in a solution of Alexa Fluor 532streptavidin conjugate (0.1 mM) and quantum dot 605-streptavidin conjugate (10 µM) dissolved in a PBS buffer (10 mM, pH 7.6) containing 0.1% triton X-100 and incubated at room temperature for 1 h. Nonattached fluorescent probe-labeled streptavidin was removed by gently washing with the PBS buffer (pH 7.6, 10 mM) containing 0.1% triton X-100. In the erasing process, photoirradiation on both sides of the coverslip was performed under the above condition, and then the products were washed out by the above buffer. The patterned and erased fluorescence was observed through a filter WIG using a fluorescence microscopy (Olympus BX51). Photobleaching of Fluorescent Probes. The Alexa Fluor 532streptavidin conjugate and the quantum dot 605-streptavidin conjugate were spin-coated onto the coverslips. Photobleaching of the fluorescent probes was performed by photoirradiation using the above method. Quantification of Fluorescence Intensity on the Surface and the Determination of Rate Constants. A histogram of fluorescence intensity showing surface brightness in dark regions (UV-irradiated surface) and bright regions (non-UV-irradiated surface) was estimated. The surface brightness of the masked region was optimized as 100%, which means that the photoelimination of the fluorescent streptavidin does not occur in this region. The normalized fluorescence intensity of the photoirradiated surface was defined as the ratio of the fluorescence intensity of irradiated surface to that of unirradiated surface. To calculate a ratio of unreacted photolinker after photoirradiation (Figure 3 (b)), the photolinker immobilized on the surface was photoirradiated for 10, 30, 60, 180, and 300 s, and the unreacted photolinkers were then visualized by the addition of the quantum dot 605-streptavidin conjugate. To calculate a ratio of unreacted quantum dot 605-streptavidin-photolinker after the photoirradiation (Figure 3 (0)), the fluorescent streptavidin-photolinker immobilized on the surface was photoirradiated for 30, 60, 180, and 300 s. The elimination rate constants of the fluorescent streptavidins were determined from the decays of fluorescence intensity shown in Figure
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Figure 2. Fluorescence imaging of the negative pattern of the Alexa Fluor 532-streptavidin conjugate (A) and the positive pattern of the quantum dot 605-streptavidin conjugate (B). These patterns were erased on both sides by photoirradiation for 5 min. The scale bar represents 500 µm.
Figure 3. Ratio of the remaining fluorescent probes on the surface calculated by the photoelimination of the photolinker without any fluorescent probes (b) and by the photoelimination of the photolinker modified by the quantum dot 605-streptavidin conjugate during the erasing process (0). These data show representative values (there were three repeats). 3. The rate constants of photobleaching were also calculated using the above method.
Results and Discussion In the reading process, the fluorescence intensity of the Alexa Fluor 532-streptavidin conjugates modified on the photoirradiated surface was significantly dependent on the photoirradiation time, which is shown in Figure 1. Interestingly, streptavidin was obviously patterned by photoirradiation for only 10 s. We found that photoirradiation for 10 s induced the 33% elimination of the photolinkers, which is enough to read the recorded information, “ISIR”. The result suggests that the photolinker showed not only highly photoeliminative activity but also a spatiotemporal selectivity for fabricating negative-type protein patterning on the glass surface. Photoirradiation for 30 s made the protein patterning clearer and induced the 55% photoelimination. Eventually, the yield of the elimination was increased to 92% with photoirradiation for 5 min, indicating that the recording process is almost completed within 1 min. In addition, we found that the surface coated with streptavidin showed a high-density and almost homogeneous modification on the nonirradiated area and that the irradiated surface provided a practically homogeneous fluorescence intensity. This phenomenon means that each photolinker on the surface absorbed an almost equal number of photons and the photochemical reaction occurred homogeneously.
Figure 4. Decay of normalized fluorescence intensity of the Alexa Fluor 532-streptavidin conjugate (b) and quantum dot 605streptavidin conjugate (0) by photobleaching. These conjugates, which were spin-coated onto a coverslip, were irradiated with >340 nm light. These data show representative values (there were three repeats).
Figure 2A represents the erasing of the array of the Alexa Fluor 532-streptavidin conjugate, which was prepared by photoirradiation for 30 s through a photomask and the subsequent surface modification, by photoirradiation onto both sides of the recorded surface for 5 min. A bright fluorescent image turned dark overall and reached an unreadable level, although a negligible amount of the fluorescent streptavidin (∼8%) remained on the surface. Such fluorescent streptavidin would be due to nonspecific interaction with the photoreaction products (e.g., 2-nitorosoacetophenone derivatives) on the surface. We also prepared the positive pattern of the quantum dot 605streptavidin conjugate under the same conditions as shown in Figure 2B. It was found that photoirradiation for 10 s and subsequent addition of quantum dot-streptavidin resulted in a sufficiently clear protein array for reading the information, and the yield of the photoelimination was calculated to be 22%. The recorded information was erased by photoirradiation under the above conditions. Thus, we verified all of the processes consisting of recording, reading, and erasing information needed to fabricate the protein recording material using the protein-ligand interaction visualized by two different fluorescent probes. Figure 3 represents the photoirradiation time dependence of the normalized fluorescence intensities (defined as the ratio of the fluoresence intensity of irradiated surface to that of unirradiated surface) on the photoirradiated area before (i.e., recording process) and after (i.e., erasing process) modification of the quantum dot 605-streptavidin conjugates with the photolinkers. Both of the plots before and after the modification were fitted with curves of a single-exponential function, indicating that the photoelimination occurred through a first-order reaction. We determined the rate constant of the photoeliminative reaction without the fluorescent streptavidin to be (3.1 ( 0.6) × 10-2 s-1. Previous reports indicates that its derivative, o-nitrodimethoxyphenylglycine, causes photolysis via a five-step mechanism involving intramolecular hydrogen atom transfer from the benzyl position, cyclization, and subsequent decomposition to produce the 2-nitorosobenzaldehyde derivative.27-29 This photolysis proceeds through a first-order reaction. The elimination of the photolinker that we employed here would proceed through the same mechanism to produce a 2-nitorosoacetophenone derivative. (27) Woodrell, C. D.; Kehayova, P. D.; Jain, A. Org. Lett. 1999, 1, 619-622. (28) Kehayova, P. D.; Woodrell, C. D.; Dostal, P. J.; Chandra, P. P.; Jain, A. Photochem. Photobiol. Sci. 2002, 1, 774-779. (29) Endo, M.; Nakayama, K.; Majima, T. J. Org. Chem. 2004, 69, 42924298.
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To determine the rate constant of the photoelimination of the recorded information during the erasing process, we examined the photobleaching process of Alexa Fluor 532 and quantum dot 605 during photoirradiation. As shown in Figure 4, in the case of Alexa Fluor 532, photobleaching was induced by photoirradiation, and the rate constant of photobleaching was determined to be (2.9 ( 1.2) × 10-3 s-1. However, this value was approximately 10-fold lower than the rate constant of the photoeliminative reaction determined to be (3.1 ( 0.6) × 10-2 s-1. Practically, when the exposure of >340 nm light was carried out for 60 s, the erasing of information due to photobleaching underwent only a 10% decrease in fluorescence intensity whereas selective photoelimination induced an 85% decrease. Consequently, the photoinduced elimination of fluorescent streptavidinbiotin preferentially occurred when compared to its photobleaching in the erasing process although the photoeliminative reaction competed against the photobleaching. However, the photobleaching of the quantum dot 605 was not observed regardless of the photoirradiation for 600 s. This result indicates that the quantum dot showed a resistance against photobleaching. We revealed that the disappearance of fluorescence was initiated by the selective photoreaction of the photolinker but not by the photobleaching of the fluorescent probe. Next, we determined the rate constant of the photoelimination of the quantum dot-streptavidin-biotin complex during the erasing process. The observed time dependence of the normalized fluorescence intensity can fit with a simple single-exponential function as indicated by the broken line in Figure 3. The estimated rate constant was (2.5 ( 0.4) × 10-2 s-1. It should be noted that the rate constant was almost equal to that of the photoelimination prior to the modification with fluorescent streptavidins. Previous reports mentioned that reactive oxygen species, such as singlet molecular oxygen and the OH radical, are generated by the excitation of CdSe quantum dots in water.30,31 As is well known, these species have a high reactivity for oxidizing organic compounds. However, the elimination rate remained unchanged by the excitation of the quantum dot. Therefore, the undesirable dissociation of the quantum dot from the surface due to the oxidation of the streptavidin, biotin, and linker caused by the photochemical reactions during the photoirradiation of the quantum dot is ignored in the present investigation. This interpretation supports the fact that the disappearance of the recorded information should be caused (30) Samia, A. C. S.; Chen, X.; Burda, C. J. Am. Chem. Soc. 2003, 125, 15736-15737. (31) Ipe, B. I.; Lehnig, M.; Niemeyer, C. M. Small 2005, 1, 706-709.
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only by the photoelimination of the specific position in the photolinker; furthermore, the interaction between streptavidin and the photolinker would have a negligible effect on the efficiency of the photoelimination.
Conclusions We established a novel, rapid method for fabricating a protein recording material, which shows recording, reading, and erasing information, by a photochemical technique. The recording process was almost complete after 1 min of photoirradiation to obtain a pattern with high spatiotemporal resolution. The reading process provided a clear protein array induced by a specific proteinligand interaction. The erasing process was achieved by photoirradiation onto an entire patterned surface even though the fluorescent streptavidin conjugates interacted with the biotin of the photolinker. We revealed that selective photoelimination to fabricate the protein recording material preferentially occurred when compared to the photobleaching of the fluorescent probes and the oxidization of materials caused by reactive oxygen species. We expect another protein-ligand interaction instead of the streptavidin-biotin interaction in order to develop biosensors and biological memories. The native chemical ligation,32 the Staudinger ligation,33 and the other ligations34-37 can contribute to the modification of the biomolecules into the photolinker through a covalent bond in order to fabricate a novel biomemory device. The strategy that we demonstrated here will be applicable in biomolecule recording materials using not only native proteins, DNA, and RNA but also photoregulated assembly with inorganic nanomaterials, such as quantum dots. Acknowledgment. This work has been partially supported by a Grant-in-Aid for Scientific Research (Project 17105005 and others) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of the Japanese Government. Supporting Information Available: The surface characterization of the quantum dot-streptavidin immobilized on a glass was carried out using AFM before and after the UV-elimination of the photolinker. This material is available free of charge via the Internet at http://pubs.acs.org. LA703354C (32) Yeo, D. S. Y.; Srinivasan, R.; Chen, G. Y. J.; Yao, S. Q. Chem.sEur. J. 2004, 10, 4664-4672. (33) Ko¨hn, M.; Breinbauer, R. Angew. Chem., Int. Ed. 2004, 43, 3106-3116. (34) Lesaicherre, M.-L.; Lue, R. Y. P.; Chen, G. Y. J.; Zhu, Q.; Yao, S. Q. J. Am. Chem. Soc. 2002, 124, 8768-8769. (35) Camarero, J. A.; Kwon, Y.; Coleman, M. A. J. Am. Chem. Soc. 2004, 126, 14730-14731. (36) Harris, J. L.; Winssinger, N. Chem.sEur. J. 2005, 11, 6792-6801. (37) Lovrinovic, M.; Fruk, L.; Schro¨der, H.; Niemeyer, C. M. Chem. Commun. 2007, 353-355.
