12116
Langmuir 2008, 24, 12116-12118
Carbohydrate Microarrays by Microcontact “Click” Chemistry Olaf Michel and Bart Jan Ravoo* Organic Chemistry Institute and Center for Nanotechnology (CeNTech), Westfa¨lische Wilhelms-UniVersita¨t Mu¨nster, Corrensstrasse 40, 48149 Mu¨nster, Germany ReceiVed July 18, 2008 Carbohydrate microarrays can be prepared by microcontact printing of carbohydrate alkyne conjugates on azide self-assembled monolayers (SAMs). The carbohydrates are immobilized by a “click” reaction in the contact area between the stamp and the substrate. The immobilized carbohydrates retain their characteristic selectivity toward lectins.
It is widely recognized that, among the biological polymers, the carbohydrates are both richest in information and most difficult to analyze and synthesize. Carbohydrates mediate many biological recognition and signaling processes, and the preparation of carbohydrate microarrays is of relevance to the high-throughput analysis of carbohydrate-protein interactions in the rapidly growing field of glycomics.1 Carbohydrate microarrays can be made by ink-jet printing of carbohydrate probes on activated transparent substrates.2 This method is similar to the state-ofthe-art preparation of DNA microarrays.3 It is fast and cheap and amenable to microarrays with many thousands of spots that can be read out by using fluorescence microscopy. However, the resolution of ink-jet printing is limited, the probe density is not easily reproducible, and the spots tend to be very heterogeneous (“coffee-stain effect”). Here, we present an alternative approach to carbohydrate microarrays using microcontact printing.4 Microcontact printing is an emerging replication method for biological microarrays, including protein5 and DNA microarrays6 for proteomics and genomics, respectively. Advantages of microcontact printing include high edge resolution (better than 100 nm), reproducible probe density, and homogeneous spots. Interesting approaches that allow the printing of multiple biological inks simultaneously have been recently reported.7 In this Letter, we demonstrate that a carbohydrate microarray composed of micropatterned monolayers of simple carbohydrates can be printed on glass and on Si wafers. The carbohydrates are immobilized by a Cu(I)* To whom correspondence should be addressed. E-mail: b.j.ravoo@ uni-muenster.de. (1) (a) Love, K. R.; Seeberger, P. H. Angew. Chem., Int. Ed. 2002, 41, 3583– 3586. (b) Feizi, T.; Fazio, F.; Chai, W.; Wong, C. H. Curr. Opin. Struct. Biol. 2003, 13, 637–654. (c) Seeberger, P. H.; Werz, D. B. Nature 2007, 446, 1046– 1051. (2) (a) Park, S.; Shin, I. Angew. Chem., Int. Ed. 2002, 41, 3180–3182. (b) Ratner, D. M.; Adams, E. W.; Su, J.; O‘Keefe, B. R.; Mrksich, M.; Seeberger, P. H. ChemBioChem 2004, 5, 379–383. (3) (a) Heise, C.; Bier, F. F. Top. Curr. Chem. 2006, 261, 1–25. (b) Pirrung, M. C. Angew. Chem., Int. Ed. 2002, 41, 1276–1289. (4) (a) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 550–575. (b) Ruiz, S. A.; Chen, C. S. Soft Matter 2007, 3, 168–177. (5) (a) Bernard, A.; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H. R.; Biebuyck, H. Langmuir 1998, 14, 2225–2229. (b) Ludden, M. J. W.; Mulder, A.; Schulze, K.; Subramaniam, V.; Tampe´, R.; Huskens, J. Chem.sEur. J. 2008, 14, 2044–2051. (6) Lange, S. A.; Benes, V.; Kern, D. P.; Ho¨rber, J. K. H.; Bernard, A. Anal. Chem. 2004, 76, 1641–1647. (7) (a) Renault, J. P.; Bernard, A.; Juncker, D.; Michel, B.; Bosshard, H. R.; Delamarche, E. Angew. Chem., Int. Ed. 2002, 41, 2320–2323. (b) Rozkiewicz, D. I.; Brugman, W.; Kerkhoven, R. M.; Ravoo, B. J.; Reinhoudt, D. N. J. Am. Chem. Soc. 2007, 129, 11593–11599.
Chart 1. Molecular Structures of Carbohydrate Inks 1-4
catalyzed “click” reaction8 that occurs exclusively in the contact area between the stamp and substrate (“microcontact chemistry”). The printed carbohydrate microarrays display the expected selectivity in a lectin binding assay. Although it has been shown that carbohydrates can be “clicked” onto self-assembled monolayers (SAMs) on glass and gold substrates,9 to the best of our knowledge, there are no examples of microcontact printing of carbohydrates in the literature. It should be noted that carbohydrates are not compatible with alkoxysilanes so that an indirect immobilization and patterning method is required for glass substrates. Microcontact chemistry on an intermediate SAM as described here fulfills this requirement. Carbohydrate conjugates 1-4 (Chart 1) were synthesized according to literature methods.10 Experimental details and analytical data are provided in the Supporting Information. Carbohydrate conjugates 1-4 combine alkyne end groups for “click” chemistry, a triethyleneglycol spacer and a β-glucoside (8) (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004–2021. (b) Bock, V. D.; Hiemstra, H.; van Maarseveen, J. H. Eur. J. Org. Chem. 2006, 51–68. (9) (a) Sun, X. L.; Stabler, C. L.; Cazalis, C. S.; Chaikof, E. L. Bioconjugate Chem. 2006, 17, 52–57. (b) Zhang, Y.; Luo, S. Z.; Tang, Y. J.; Yu, L.; Hou, K. Y.; Cheng, J. P.; Zeng, X. Q.; Wang, P. G. Anal. Chem. 2006, 78, 2001–2008. (10) (a) McPhee, M.; Kerwin, S. M. J. Org. Chem. 1996, 61, 9385–9393. (b) Bouillon, C.; Meyer, A.; Vidal, S.; Jochum, A.; Chevolot, Y.; Cloarec, J. P.; Praly, J. P.; Vasseur, J. J.; Morvan, F. J. Org. Chem. 2006, 71, 4700–4702. (c) Mori, M.; Ito, Y.; Ogawa, T. Carbohydr. Res. 1986, 155, 57–72.
10.1021/la802304w CCC: $40.75 2008 American Chemical Society Published on Web 10/07/2008
Letters
Figure 1. C 1s peak (285 eV) in the XPS spectrum of (A) an azide SAM on a Si wafer; (B) β-glucoside 1 printed on an azide SAM; and (C) acetyl-protected β-glucoside printed on an azide SAM.
(1), β-galactoside (2), R-mannoside (3), and β-maltoside (4), respectively, for lectin binding. Azide-terminated SAMs on glass and on Si wafers were prepared by surface adsorption of 11bromo-undecyltriethoxysilane followed by substitution of bromide for azide according to literature procedures.11,12 Carbohydrates 1-4 were used as inks in microcontact printing on azide SAMs on glass and on Si wafers. To this end, poly(dimethylsiloxane) (PDMS) stamps were oxidized in ozone atmosphere and inked with a solution of conjugates 1-4 (10 mM in ethanol). The ink solution also contained 0.1 mM CuSO4 and 2 mM ascorbic acid to provide the Cu(I) catalyst for the “click” reaction between the alkyne ink and azide SAM in the contact regions between the stamp and substrate. The stamps were contacted with the azide SAM for 10 min. By printing with flat stamps, the carbohydrate inks were immobilized homogeneously throughout the substrate surface. The presence of the carbohydrate on the surface was verified by a number of analytical methods. First, the water contact angle decreased from 81/72° (adv/rec) for the azide SAM to 31/6° for the carbohydrate SAMs. Importanty, if acetyl-protected β-glucoside was printed, the contact angle decreased to only 69/22°. The contact angles reflect the hydrophilicity of the immobilized carbohydrates and the apolarity of the protected β-glucoside. Second, the azide peak in the IR spectrum of the azide SAM (2093 cm-1) was no longer (11) Balachander, N.; Sukenik, C. Langmuir 1990, 6, 1621–1627. (12) Rozkiewicz, D. I.; Janczewski, D.; Verboom, W.; Ravoo, B. J.; Reinhoudt, D. N. Angew. Chem., Int. Ed. 2006, 45, 5292–5296.
Langmuir, Vol. 24, No. 21, 2008 12117
Figure 2. Carbohydrate arrays by microcontact printing. (A) Fluorescence image (75 µm × 100 µm) of an array of R-mannoside 3 exposed to FITC-labeled Con A. (B) AFM friction image (20 µm × 20 µm) of an array of β-maltoside 4 exposed to Con A.
observed after printing the carbohydrate inks, indicating that the azide had reacted to a triazole. Third, the X-ray photoelectron spectroscopy (XPS) spectrum of the azide SAM (Figure 1A) displayed a sharp C 1s peak around 285 eV as expected for a hydrocarbon chain containing mainly C-C and C-H bonds (except the terminal C-atoms), while the XPS spectrum of the β-glucoside 1 SAM (Figure 1B) displayed two overlapping C peaks for C-C/C-H and C-O. Importantly, if acetyl-protected β-glucoside was printed, the XPS spectrum (Figure 1C) displayed three overlapping peaks for C-C/C-H, C-O, and CdO. We note that although we have previously demonstrated that simple alkynes12 as well as complex DNA-alkyne conjugates13 can be printed on azide SAMs without using the Cu(I) catalyst, the printing of carbohydrates 1-4 was not successful in the absence of catalyst. We speculate that the hydrophilic oligo(ethylene glycol) chain hampers the surface reaction in the absence of catalyst. Carbohydrate conjugates 1-4 were also printed with patterned stamps (5 µm lines spaced 25 µm apart). By filling the remaining area with a rhodamine alkyne conjugate (LRA),12 it was observed by fluorescence microscopy that the line pattern of the relief stamp was faithfully reproduced as a line pattern of carbohydrate (5 µm dark lines) separated by rhodamine (25 µm red lines). Images are provided in the Supporting Information. This is indirect but convincing evidence that the carbohydrate conjugates can be printed in microarrays using “click” chemistry. (13) Rozkiewicz, D. I.; Gierlich, J.; Burley, G. A.; Gutsmiedl, K.; Carell, T.; Ravoo, B. J.; Reinhoudt, D. N. ChemBioChem 2007, 8, 1997–2002.
12118 Langmuir, Vol. 24, No. 21, 2008
Finally, the carbohydrate conjugates 1-4 were printed with patterned stamps (5 µm lines spaced 10 µm apart) and exposed to fluorescein-labeled lectin Concanavalin A (FITC-labeled Con A, 1 mg in 10 mL of PBS solution at pH 7.4). It is known that Con A binds to R-mannose and β-maltose (which is terminated by R-glucose) but not to β-glucose or β-galactose.14 Indeed, it was observed with fluorescence microscopy that FITC-labeled Con A binds selectively to the R-mannoside 3 and β-maltoside 4 arrays (resulting in 5 µm green lines spaced 10 µm apart; see Figure 2A) while no binding can be observed with β-glucoside 1 or β-galactoside 2. The lectin-carbohydrate microarrays were also investigated by using atomic force microscopy (AFM) (Figure 2B), and the carbohydrate-bound Con A can be readily detected on the R-mannoside 3 and β-maltoside 4 arrays but not on the β-glucoside 1 or β-galactoside 2 array. Hence, the carbohydrates retain their characteristic molecular recognition of Con A when immobilized in a microcontact printed microarray. (14) (a) Park, S.; Lee, M.; Pyo, S.; Shin, I. J. Am. Chem. Soc. 2004, 126, 4812–4819. (b) Brun, M. A.; Disney, M. D.; Seeberger, P. H. ChemBioChem 2006, 7, 421–424.
Letters
In conclusion, we have developed a straightforward fabrication method for carbohydrate arrays by microcontact chemistry which can be readily extended to more complex carbohydrates. The layout and dimensions of the array can be designed according to the stamp. Although alkynes are readily introduced in carbohydrates, bio-orthogonal reactions other than azide-plusalkyne “click” chemistry may also be valuable for carbohydrate immobilization. We believe that the methods proposed here will be of general use in glycomics. Acknowledgment. We are grateful to Michael Hirtz and Prof. Lifeng Chi for the AFM measurements, to Matthias Rinke and Prof. Hellmut Eckert for XPS measurements, and to the Federal State of North Rhine-Westphalia for financial support. Supporting Information Available: Synthesis and analysis of carbohydrates 1-4. Preparation of azide SAMs, microcontact printing, and surface analysis. This material is available free of charge via the Internet at http://pubs.acs.org. LA802304W
