pubs.acs.org/Langmuir © 2010 American Chemical Society
Selective and Direct Immobilization of Cysteinyl Biomolecules by Electrochemical Cleavage of Azo Linkage Hyun Joo Jung,†,‡ Inseong Hwang,†,‡ Beom Jin Kim,‡ Hyegeun Min,§ Hyunung Yu,§ Tae Geol Lee,*,§ and Taek Dong Chung*,‡ §
‡ Department of Chemistry, Seoul National University, Seoul 151-747, Korea, and Center for Nano-Bio Technology, Division of Convergence Technology, Korea Research Institute of Standards and Science (KRISS). †H. Jung and I. Hwang contributed equally to this work.
Received June 18, 2010. Revised Manuscript Received August 19, 2010 Controlled orientation and reserved activity of biomolecules, when site-selectively immobilized in a highly integrated manner on a minimal time scale, are crucial in designing biosensors for the multiplex detection. Here, we describe a novel method for the orientation-controlled immobilization of biomolecules based on site-selective electrochemical activation of p-hydroxyazobenzene self-assembled monolayer (SAM) followed by one-step coupling of cysteinyl biomolecules. The p-aminophenol, a product of reductive cleavage of p-hydroxyazobenzene, was subsequently oxidized to yield p-quinoneimine which then conjugated with cysteinyl biomolecules through 1,4-Michael addition, thus obviating additional linker agents and the related time consumption. Using this method, we selectively activated the electrode surface and immobilized laminin peptide IKVAV, a neurite promoting motif. When we cultured hippocampal neurons on the electrode, the extended neurites were found only within the electrochemically activated area. Hence, the proposed method represents a new promising platform for the patterning of functional peptides, active proteins, and live cells.
Introduction As the demand for cost-effective, miniaturized, and highdensity biochips has increased, much interest has been focused on the site-selective activation and functionalization of surfaces for the patterning and immobilization of biomolecules.1,2 As such, protein micro- and nanoarrays are now widely applied to various research fields including, but not limited to, drug discovery,3 tissue engineering,4,5 and multiplexed proteomics coupled with immunoassays.6-9 Biomolecules, such as proteins and antibodies, have been immobilized on the activated self-assembled monolayers (SAMs) using reactive quinones,10-12 hetero- and homogeneous bifunctional linkers including hydrazide, carbodiimide, and N-hydroxy*T. G. Lee: Fax þ82-42-868-5032; Phone þ82-42-868-5129; e-mail
[email protected]. T. D. Chung: Fax þ82-2-887-4354; Phone þ82-2-8874362; e-mail
[email protected]. (1) Jonkheijm, P.; Weinrich, D.; Schroder, H.; Niemeyer, C. M.; Waldmann, H. Angew. Chem., Int. Ed. 2008, 47, 9618–9647. (2) Wong, L. S.; Khan, F.; Micklefield, J. Chem. Rev. 2009, 109, 4025–4053. (3) Wingren, C.; Borrebaeck, C. A. K. Drug. Discovery Today 2007, 12, 813–819. (4) Stevens, M. M.; George, J. H. Science 2005, 310, 1135–1138. (5) Robertus, J.; Browne, W. R.; Feringa, B. L. Chem. Soc. Rev. 2010, 39, 354– 378. (6) Zhu, H.; Bilgin, M.; Bangham, R.; Hall, D.; Casamayor, A.; Bertone, P.; Lan, N.; Jansen, R.; Bidlingmaier, S.; Houfek, T.; Mitchell, T.; Miller, P.; Dean, R. A.; Gerstein, M.; Snyder, M. Science 2001, 293, 2101–2105. (7) Kingsmore, S. F. Nat. Rev. Drug Discovery 2006, 5, 310–320. (8) Borrebaeck, C. A. K.; Wingren, C. J. Proteomics 2009, 72, 928–935. (9) Seo, Y. H.; Carroll, K. S. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 16163– 16168. (10) Yousaf, M. N.; Mrksich, M. J. Am. Chem. Soc. 1999, 121, 4286–4287. (11) Chan, E. W. L.; Park, S.; Yousaf, M. N. Angew. Chem., Int. Ed. 2008, 47, 6267–6271. (12) Kim, K.; Jang, M.; Yang, H. S.; Kim, E.; Kim, Y. T.; Kwak, J. Langmuir 2004, 20, 3821–3823. (13) Yeo, W. S.; Mrksich, M. Adv. Mater. 2004, 16, 1352–1356. (14) Kim, K.; Yang, H.; Kim, E.; Han, Y. B.; Kim, Y. T.; Kang, S. H.; Kwak, J. Langmuir 2002, 18, 1460–1462. (15) Buxboim, A.; Bar-Dagan, M.; Frydman, V.; Zbaida, D.; Morpurgo, M.; Bar-Ziv, R. Small 2007, 3, 500–510. (16) Rohde, R. D.; Agnew, H. D.; Yeo, W. S.; Bailey, R. C.; Heath, J. R. J. Am. Chem. Soc. 2006, 128, 9518–9525.
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succinimide (NHS),13-15 azide-alkyne click chemistry,16,17 and biotin-streptavidin.12,15,18 However, target molecules must be chemically modified and activated before employing functional linkers, Diels-Alder conjugation, and click chemistry.10,16,17 Biotin should be present in both SAMs and target biomolecules to use streptavidin as a linker, which doubles the effort in labeling and may lower the sensitivity owing to the elongated SAMs in the case of electrochemical detection. The direct attachment of proteins to a substrate through noncovalent electrostatic interaction can yield partially or seldom active proteins because of the random orientations and aggregations of proteins.19,20 Thus, the direct conjugation of intact biomolecules with activated surfaces, while keeping controlled orientations through chemo- and regioselective immobilization, is of great importance for the fabrication of highly integrated and multiple arrays of biomolecules.2,21-23 Previously, we reported a novel method for the site-selective activation through electrochemically controlled cleavage of an azobenzene monolayer to expose anilines at a low reduction potential for the immobilization of biomolecules and cell patterning.24 Here, we describe an advanced method where onestep conjugation of cysteinyl biomolecules with quinoneimine is demonstrated, eliminating additional steps for the adaptation of linkers; The 1,4-Michael addition of nucleophiles, especially (17) Lin, P. C.; Ueng, S. H.; Tseng, M. C.; Ko, J. L.; Huang, K. T.; Yu, S. C.; Adak, A. K.; Chen, Y. J.; Lin, C. C. Angew. Chem., Int. Ed. 2006, 45, 4286–4290. (18) Kim, K.; Yang, H.; Jon, S.; Kim, E.; Kwak, J. J. Am. Chem. Soc. 2004, 126, 15368–15369. (19) Lee, K. B.; Lim, J. H.; Mirkin, C. A. J. Am. Chem. Soc. 2003, 125, 5588– 5589. (20) Wilson, D. L.; Martin, R.; Hong, S.; Cronin-Golomb, M.; Mirkin, C. A.; Kaplan, D. L. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 13660–13664. (21) Ataka, K.; Giess, F.; Knoll, W.; Naumann, R.; Haber-Pohlmeier, S.; Richter, B.; Heberle, J. J. Am. Chem. Soc. 2004, 126, 16199–16206. (22) Vallina-Garcia, R.; Garcia-Suarez, M. D.; Fernandez-Abedul, M. T.; Mendez, F. J.; Costa-Garcia, A. Biosens. Bioelectron. 2007, 23, 210–217. (23) Balland, V.; Lecomte, S.; Limoges, B. Langmuir 2009, 25, 6532–6542. (24) Jung, H. J.; Min, H.; Yu, H.; Lee, T. G.; Chung, T. D. Chem. Commun. (Camb.) 2010, 46, 3863–3865.
Published on Web 09/01/2010
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Jung et al. Scheme 1. Proposed Experimental Schemes for Immobilization of Cysteinyl Biomolecules
cysteine, to quinoid intermediates is a well-known reaction by which thiols and cysteines can be detected and quantified in physiological conditions.25-27 The low occurrence of cysteine in proteins (0.4-2.3%)28 is beneficial in that it can minimize the possibility of a fixation of multiple sites that causes random orientation, reduced conformational flexibility, and heterogeneous activity of proteins. On the other hand, cysteine is frequently introduced at the N-terminus of a protein expressed and purified through intein-fusion system.29,30 For proteins without intrinsic cysteine, a cysteine-tag can be introduced at either the N- or C-terminus of the proteins.31 In this experiment, surface-bound p-hydroxyazobenzene was initially reduced to p-aminophenol through reductive cleavage involving 4Hþ, 4e-. Subsequent oxidation by depriving p-aminophenol of 2Hþ, 2e- resulted in p-quinoneimine to which cysteinyl nucleophiles can easily be conjugated through 1,4-addition (Scheme 1). We also demonstrate that this approach is applicable to direct cell attachment, growth, and patterning by providing biocompatible electrode surface.
Results and Discussion First, we investigated the redox behavior of p-hydroxyazobenzene SAM on the gold substrate. We observed a single reduction peak appeared at -0.14 V (vs Ag/AgCl) in Britton-Robinson buffer (B-R, pH 2.0) (Figure 1a, solid line). This result is contrasted by the two reduction peaks, at -0.22 V and -0.48 V, for azobenzene without hydroxyl group, showing that the reduction was completed in two sequential steps.24 After the reduction of p-hydroxyazobenzene followed by a subsequent oxidative potential scanning, a new peak appeared near 0.4 V, originating from the oxidation of p-aminophenol groups generated at the first reductive sweep (Figure 1a, solid line). The second potential cycle showed dominant redox behavior of p-aminophenol, confirming (25) White, P. C.; Lawrence, N. S.; Davis, J.; Compton, R. G. Anal. Chim. Acta 2001, 447, 1–10. (26) Roussel, C.; Dayon, L.; Lion, N.; Rohner, T. C.; Josserand, J.; Rossier, J. S.; Jensen, H.; Girault, H. H. J. Am. Soc. Mass Spectrom. 2004, 15, 1767–1779. (27) Roussel, C.; Dayon, L.; Jensen, H.; Girault, H. H. J. Electroanal. Chem. 2004, 570, 187–199. (28) Miseta, A.; Csutora, P. Mol. Biol. Evol. 2000, 17, 1232–1239. (29) Chattopadhaya, S.; Abu Bakar, F. B.; Yao, S. Q. Method Enzymol. 2009, 462, 195–223. (30) Camarero, J. A.; Kwon, Y.; Coleman, M. A. J. Am. Chem. Soc. 2004, 126, 14730–14731. (31) Ichihara, T.; Akada, J. K.; Kamei, S.; Ohshiro, S.; Sato, D.; Fujimoto, M.; Kuramitsu, Y.; Nakamura, K. J. Proteome Res. 2006, 5, 2144–2151.
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Figure 1. Cyclic voltammograms of the reductive cleavage of p-hydroxyazobenzene in (a) B-R (pH 2.0) and (b) PBS (pH 7.4). The scan rate was fixed to 50 mV/s in all experiments.
the complete reduction of p-hydroxyazobenzene in the first reductive sweep (Figure 1a, dashed line around 0.4 V). The complete reductive cleavage was further verified by mass spectrometry (vide infra). On the other hand, the two reduction peaks appeared again, at -0.59 V and -0.82 V, when we reduced p-hydroxyazobenzene at higher pH (phosphate-buffered saline, PBS, pH 7.4) (Figure 1b, solid line). The reduction was delayed to Langmuir 2010, 26(19), 15087–15091
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Figure 2. Representative ToF-SIMS spectra of (a) p-hydroxyazobenzene (Mazo) and (b) p-aminophenol (Mamine) before electrochemical activation (top), after electrochemical activation for 5 s (middle), and 30 s (bottom) in B-R (pH 2.0). The characteristic peaks of gold-clustered p-hydroxyazobenzene (MazoAu-) and deprotonated p-aminophenol (Mamine-H) were aligned with the dashed lines. (c) The normalized intensities of MazoAu- (circle) and (Mamine-H)Au- (square) obtained from three independent spots of the surfaces and plotted against the duration of electrochemical stimulation.
a greater negative potential because of the insufficient protons at high pH compared with Britton-Robinson buffer (pH 2.0). In the second potential scanning after the cleavage, a pair of peaks of reduction and oxidation corresponding to p-aminophenol appeared around 0.05 V. On the basis of these observations, we concluded that the p-hydroxyl group facilitates the two-step reduction even to the level of inseparable reduction peaks, and the lower the pH, the better the process. We then examined chemical species of p-hydroxyazobenzenemodified gold substrates before and after the electrochemical activation in B-R (pH 2.0) using time-of-flight secondary ion mass spectrometry (ToF-SIMS). Figure 2a,b shows mass peaks of gold-clustered p-hydroxyazobenzene (Mazo = C17H19N2OS) before the activation and p-aminophenol (Mamine = C11H16NOS), a cleavage product of p-hydroxyazobenzene, after the activation at -0.3 V for 5 and 30 s. The Langmuir 2010, 26(19), 15087–15091
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Figure 3. One-step immobilization of cysteine on the electrochemically activated surface. (a) Cyclic voltammograms of surfacebound p-aminophenol without cysteine (black), in the presence of 1.0 mM (red) and 2.0 mM (blue) cysteine where oxidative potential of 0.3 V was applied for 60 s after the addition of cysteine. (b,c) Normalized shift of oxidation and reduction peak potential, respectively, after addition of 1.0 mM and 2.0 mM of each amino acid.
MazoAu- peak at m/z 496.0 completely disappeared, while the (Mamine-H)Au- peak at m/z 406.0 appeared in just 5 s, indicating that p-hydroxyazobenzene was fully reduced within 5 s. To monitor the reduction process quantitatively,32 we then obtained mass spectra from three independent spots of the surfaces and compared the normalized intensities during the activation process. The extended duration of electrochemical stimulation gave a slightly reduced intensity of the (Mamine-H)Au- peak when compared with the initial activation for 5 s (see Figure 2c), possibly because of the hydrolysis of p-aminophenol.33 Next, we immobilized cysteine on the activated surface by in situ oxidation of p-aminophenol to p-quinoneimine in PBS (pH 7.4) to facilitate the 1,4-Michael addition of nucleophiles. (32) Kim, Y. P.; Hong, M. Y.; Shon, H. K.; Moon, D. W.; Kim, H. S.; Lee, T. G. Appl. Surf. Sci. 2006, 252, 6801–6804. (33) Hawley, D.; Adamsa, R. N. J. Electroanal. Chem. 1965, 10, 376–386.
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Figure 4. Representative mass peaks for (a) p-hydroxyazobenzene (Mazo), (b) p-aminophenol (Mamine), and (c) cysteine fragment CH2CHNH3COO- (Mcys) obtained in negative mode: top, before electrochemical activation; middle, after electrochemical activation with -0.3 V for 30 s; bottom, subsequent oxidation with 0.3 V for 60 s in PBS (pH 7.4) in the presence of 0.66 mM cysteine. (d) FT-IR spectra of the surfaces before the activation (black), after the electrochemical activation at 0.7 V for 30 s (red), and after the immobilization of cysteine (green). Asterisks indicate characteristic peaks of NH3þ (1715 cm-1) and COO- (1395, 1370 cm-1) groups of amino acid.
After applying an oxidative potential 0.3 V for 60 s, we investigated the surface by cyclic voltammetry in combination with ToFSIMS to confirm the conjugation of cysteine (Figure 3). The more the cysteines were conjugated, the more the redox peak potential shifted toward negative with the dramatic decrease of the peak current (Figure 3a). When we tried the conjugation with the other amino acids and control buffer, such as Gly, Ser, Thr, Lys, His, and PBS, the redox peak potential remained unchanged, while the peak current decreased even less significantly than in the case of Cys (Figure 3b,c, see Figure S1 in Supporting Information for original CV data). The distinguished shift in peak potential indicates generation of a new molecule as a result of specific cysteine conjugation. When we compared the ToF-SIMS spectra of p-hydroxyazobenzene-covered, p-aminophenol-exposed, and cysteine-conjugated surfaces, we could observe that the peak intensity of p-hydroxyazobenzene (Mazo) decreased as the surface was activated (Figure 4a), whereas the p-aminophenol (Mamine), a product of cleaved azo linkage, markedly increased (Figure 4b). After the conjugation of cysteine using in situ oxidation, the peak intensity of p-aminophenol ((Mamine-H)Au-) markedly decreased, yielding Mcys (CH2CHNH3COO-) peak, a fragment of cysteine, in return (Figure 4c, see Figure S2 in Supporting Information for normalized peak intensities). To identify molecular functionalities precisely, we also acquired FT-IR spectra from each surface used for the ToF-SIMS characterization. Figure 4d presents FT-IR spectra of the p-hydroxyazobenzene-modified gold surface that change with the electrochemical reduction in the 3500-2800 and 1800-1100 cm-1 regions. Before cleavage, the peaks associated with j-OH (broad at 3000-3400 cm-1), j-H stretching (1596, 1481 cm-1), and j-N stretching (1245, 1156 cm-1) were observed on a p-hydroxyazobenzene surface (black). After cleavage, new peaks at 3310 cm-1 (NH2 stretching) and 1512 cm-1 (in-plane bending of NH2) appeared, whereas those at 1596 cm-1 (j-H stretching) and 15090 DOI: 10.1021/la102489k
1156 cm-1 (j-N stretching) decreased (red). After the conjugation of cysteine on the surface, an increase in COO- (1395, 1370 cm-1) and NH3þ (1715 cm-1), annotated by asterisks, shows that the terminal groups of cysteine exist on the surface (green). To address practical aspects, we immobilized Cys-Gly-Glylinked laminin Ile-Lys-Val-Ala-Val peptide, CGG-IKVAV, to the electrochemically activated surface without linkers; A peptide containing IKVAV is well-known for promoting neurite outgrowth.24,34 As shown in Figure 5c, healthy hippocampal neurons with extended neurites were found only on the peptide-functionalized surface after two days of seeding. Furthermore, when we partially immersed a gold-deposited and SAM-modified wafer into the solution and allowed electrochemical activation therein, followed by the peptide conjugation by exposing the whole surface to the peptide-containing solution, only the region contacting the solution at the activation step generated neurites (Figure 5d, upper region). This experiment clearly demonstrates the possibility of patterning of neurons in the microscale and the study of cellular functions thereby, which is gaining particular interest as the fundamental steps for understanding of brain function, manufacturing of artificial intelligence, and enhancing the bionics based on the neural interface between human brains and robotics.35,36 In conclusion, we herein suggest a one-step and site-selective immobilization strategy where cysteinyl biomolecules can be immobilized onto a substrate in a chemo- and regioselective manner, preserving their biological activity. It should be noted that our method principally removed additional steps involving various agents typically employed to generate covalent bond, (34) Tashiro, K.; Sephel, G. C.; Weeks, B.; Sasaki, M.; Martin, G. R.; Kleinman, H. K.; Yamada, Y. J. Biol. Chem. 1989, 264, 16174–16182. (35) Kim, D. H.; Viventi, J.; Amsden, J. J.; Xiao, J. L.; Vigeland, L.; Kim, Y. S.; Blanco, J. A.; Panilaitis, B.; Frechette, E. S.; Contreras, D.; Kaplan, D. L.; Omenetto, F. G.; Huang, Y. G.; Hwang, K. C.; Zakin, M. R.; Litt, B.; Rogers, J. A. Nat. Mater. 2010, 9, 511–517. (36) Patolsky, F.; Timko, B. P.; Yu, G. H.; Fang, Y.; Greytak, A. B.; Zheng, G. F.; Lieber, C. M. Science 2006, 313, 1100–1104.
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Figure 5. Growth pattern of hippocampal neurons cultured for two days on the p-hydroxyazobenzene surface (a), on the electrochemically activated p-aminophenol surface (b), and on the surface in situ functionalized with CGG-IKVAV peptide (c). (d) Upper panel, divided by the dashed line, where electrochemical activation was applied by immersing into the solution, contains neurons with extended neurites as in (c). The lower panel, where no activation was administered, shows cell bodies only. Scale bars are 100 μm.
thereby reducing time for immobilization while increasing conjugation yield. Compared with the previously demonstrated azobenzene, p-hydroxyazobenzene could be reduced more easily owing to the electron donating property of p-hydroxyl groups, yielding faster cleavage and reduced side reactions. Therefore, cysteine-tagged or cysteine-containing biomolecules could be selectively and covalently immobilized on substrates within a minute. We believe that this novel method will catalyze the development of high-density, interdigitized array of biochips where the electrochemical activation should be applied to minimize time and space simultaneously. Acknowledgment. This research was supported in part by the Ministry of Knowledge Economy (MKE), Korea, under the Information Technology Research Center (ITRC) support pro-
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gram supervised by the National IT Industry Promotion Agency (NIPA) (NIPA-2010-(C1090-1021-0003)), the Nano/Bio Science & Technology Program (M10536090001-05N3609-00110), and the Bio-Signal Analysis Technology Innovation Program (20100020640) of the Ministry of Education, Science and Technology (MEST), South Korea, and the Converging Research Center Program through the National Research Foundation of Korea (NRF) (2009-0093621) funded by the Ministry of Education, Science and Technology (MEST), South Korea. Supporting Information Available: Details regarding the materials and methods, Figures S1 to S2 and Table S1 with references for the tabulated band assignment of FT-IR in Figure 4d. This material is available free of charge via the Internet at http://pubs.acs.org.
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