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Label-Free Detection of Lectins on Carbohydrate-Modified Boron-Doped Diamond Surfaces Sabine Szunerits,*,† Joanna Niedzioˇłka-Jo¨nsson,†,| Rabah Boukherroub,† Patrice Woisel,‡ Jean-Se´bastien Baumann,§ and Aloysius Siriwardena§ Institut de Recherche Interdisciplinaire (IRI, USR 3078), Universite´ Lille Nord de France, Parc de la Haute Borne, 50 Avenue de Halley, BP 70478, 59658 Villeneuve d’Ascq, France, Unite´ des Mate´riaux Et Transformations (UMET, UMR 8207), Team “Inge´nierie des Syste`mes Polyme`res” (ISP), Universite´ Lille Nord de France, 59650 Villeneuve d’Ascq Cedex, France, Laboratoire des Glucides (UMR 6219), Universite´ de Picardie Jules Vernes, 33 rue saint Leu, 80039 Amiens, France, and Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland This paper describes the label-free detection of carbohydrate-lectin interactions. The sensor consists of a boron-doped diamond (BDD) electrode terminated with alkynyl surface groups, which have been functionalized via the CuACC (copper(I)-catalyzed azide-alkyne cycloaddition) “click” reaction with carbohydrate analogues bearing an azido-terminating arm. In this work, electrochemical impedance spectroscopy (EIS) was used as an effective technique to probe the specific interactions of the surfacebound carbohydrates with their complementary lectin partners, and the response was found to be dependent on the relative density of sugar units immobilized on the BDD surface. A BDD interface with 60% surface-bound mannose showed a detection limit of ∼5 ( 0.5 nM for Lens culinaris lectin, with an affinity constant of KA ) (2.63 ( 0.5) × 106 M-1. Advances in biology continue to reveal that cell-surface oligosaccharides play an essential role in the development and maintenance of all living systems and that they confer an exquisite level of structural and functional diversity characteristic of higher organisms.1-3 Establishing the details of specific carbohydrateprotein interactions is, therefore, essential in developing a clear understanding of the roles of surface carbohydrates in biology and, moreover, how their often intrinsically low-affinity association with proteins are transformed into those of biological significance.4,5 It is not surprising then that the development of carbohydrate * To whom correspondence should be addressed. Tel: +33 3 62 53 17 25. Fax: +33 3 62 53 17 01. E-mail:
[email protected]. † Institut de Recherche Interdisciplinaire, Universite´ Lille Nord de France. ‡ Unite´ des Mate´riaux Et Transformations, Universite´ Lille Nord de France. § Universite´ de Picardie Jules Vernes. | Polish Academy of Sciences. (1) Ambrosi, M.; Cameron, N. R.; Davis, B. G. Org. Biomol. Chem. 2005, 3, 1593. (2) Rudd, P. M.; Wormald, M. R.; Dwek, R. A. Trends Biotechnol. 2004, 22, 524. (3) Munoz, F. J.; Rumbero, A.; Sinisterra, J. V.; Santos, J. I.; Andre´, S.; Gabius, H.-J.; Jime´nez-Barbero, J.; He´rnaiz, M. J. Glycoconjugate J. 2008, 25, 633. (4) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 2754. (5) Sharon, N.; Lis, H. Science 1989, 246, 227. 10.1021/ac1016387 2010 American Chemical Society Published on Web 09/09/2010
microarrays has received sustained attention over the past 10 years.3,6-11 Most of the sensors thus far developed rely on an end point and thus indirect detection of a labeled protein (fluorescence tag, biotin motif). The field would undoubtedly benefit greatly from additional bioanalytical tools that allow glycosignatures to be scrutinized rapidly and in a label-free fashion. Among the label-free methods available, plasmonic-based techniques such as surface plasmon resonance (SPR), SPR-imaging, and localized surface plasmon resonance (LSPR) have received the greatest attention to date in high-throughput glycan profiling.7,11–14 Electrochemical impedance spectroscopy (EIS) has received little consideration.15,16 Although similar to SPR in several respects, EIS would enable rapid label-free assays, through an analysis of the changes in the interfacial properties of a sensor interface expected to be induced upon analyte binding. EIS measures the response (current and its phase) of an electrochemical system to an applied oscillating potential as a function of frequency. Electrochemical systems offer, in addition, further attractive advantages such as low cost and the ability to perform EIS on conductive, metallic, semiconductor interfaces and heterostructures.17 It is thus an elegant way to interface biorecognition events and signal transduction. EIS detection has been intensively used in immunosensors, enzymatic, and DNA sensors (6) Jelinek, R.; Kolusheva, S. Chem. Rev. 2004, 104, 5987. (7) Mercey, E.; Sadir, R.; Maillart, E.; Roget, A.; Baleux, F.; Lortat-Jacob, H.; Livache, T. Anal. Chem. 2008, 80, 3476. (8) Park, S.-M.; Shin, I. Angew. Chem., Int. Ed. 2002, 41, 3180. (9) Wang, D.; Liu, S. W.; Trummer, B. J.; Deng, C.; Wang, A. Nat. Biotechnol. 2002, 20, 275. (10) Housemann, B. t.; Mrksich, M. Chem. Biol. 2002, 9, 443. (11) Smith, E. A.; Thomas, W. D.; Kiesslinger, L. L.; Corn, R. M. J. Am. Chem. Soc. 2003, 125, 6140. (12) Linman, M. J.; Tauylor, J.; Yu, H.; Cheng, Q. Anal. Chem. 2008, 80, 4007. (13) Yonzon, C. R.; Jeoung, E.; Zhou, S.; Schatz, G. C.; Mrksich, M.; Van Duyne, R. P. J. Am. Chem. Soc. 2004, 126, 12669. (14) Mizukami, K.; Takakura, H.; Matsunaga, T.; Kitano, H. Colloids Surf., B: 2008, 66, 110. (15) Pjcic, B.; De Marco, R. PEJCIC 2006, 6217. (16) Dubois, M.-P.; Gondran, C.; Renaudet, O.; Dumy, P.; Driguez, H.; Fort, S.; Cosnier, S. Chem. Commun. 2005, 4318. (17) Manesse, M.; Stambouli, V.; Boukherroub, R.; Szunerits, S. Analyst 2006, 133, 1097.
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to study the strong monovalent recognition events by measuring the changes induced upon a binding event. Recognition of proteins by their monovalent carbohydrate ligands is, however, usually weak. This might account for the fact that label-free electrochemical approaches have, until now, been rarely applied in the carbohydrate area.16,18,19 In this work, we investigate, for the first time, the possibility to exploit EIS as a label-free method for the detection of carbohydrate-protein recognition events on boron-doped diamond (BDD) electrodes. BDD has emerged as an alternative electrode material due to its undeniably good electrical conductivity and low double-layer capacitance and as a consequence of its low background current density, wide electrochemical potential window, and biocompatibility.20-22 High chemical and morphological stabilities under extreme conditions characteristic of BDD electrodes make it an extremely attractive interface for electrochemical studies. Despite this, such an interface has only been reported for the EIS sensing of DNA hybridization events.23-25 Hamers et al. showed that DNA hybridization leads to changes in the conductivity of the interfaces via a molecular field effect in which the negative charge on the DNA strand alters the conductivity of moderately doped (NA ) 1018 B cm-3) diamond thin films.24,25 Loh et al. investigated DNA hybridization on polyaniline/polyacrylate-modified BDD electrodes with a detection limit of 2 × 10-8 M.23 In the current work we aimed to extend the range of “clickable” interfaces26-30 to also include carbohydrate-modified BDD interfaces and, further, to demonstrate that these were effective for the electrochemical detection of carbohydrate-protein binding events. Indeed, the key to the success of such a biosensor is the choice of chemistry for linking glycans to the sensor surface. Selfassembled monolayers of thiol-functionalized carbohydrates have found wide use to decorate gold interfaces.31,32 However, the prerequisite thiol-functionalized glycan analogues are not straightforward to synthesize, and furthermore the packing of such molecules to form structurally well-defined monolayers is not guaranteed.10 Alternative surface attachment scenarios have thus (18) La Belle, J. T.; Gerlach, J. Q.; Svarovsky, S.; Joshi, L. Anal. Chem. 2007, 79, 6959. (19) Oliveira, M. D. L.; Correia, M. T. S.; Coelho, L. C. B. B.; Diniz, F. B. Colloids Surf., B: 2008, 66, 13. (20) Szunerits, S.; Boukherroub, R. J. Solid-State Electrochem. 2008, 12, 1205. (21) Granger, M. C.; Swain, G. M. J. Electrochem. Soc. 1999, 146, 4551–4558. (22) Chen, Q.; Granger, M. C.; Lieser, T. E.; Swain, G. M. J. Electrochem. Soc. 1997, 144, 3806. (23) Gu, H.; di Su, X.; Loh, K. P. J. Phys. Chem. B 2005, 109, 13611. (24) Hamers, R. J.; Butler, J. E.; Lassetera, T.; Nicholsa, B. M.; Russell, J. N.; Tsea, K.-Y.; Yanga, W. Diam. Relat. Mater. 2005, 14, 661–668. (25) Tse, K.-Y.; Nichols, B. M.; Yang, W.; Butler, J. E.; Russell, J. N.; Hamers, R. J. J. Phys. Chem. B 2005, 109, 8523. (26) Das, M. R.; Wang, M.; Szunerits, S.; Gengembre, L.; Boukherroub, R. Chem. Commun. 2009, 2753. (27) Wang, M.; Das, M. R.; Li, M.; Boukherroub, R.; Szunerits, S. J. Phys. Chem. C 2009, 113, 17082. (28) Collman, J. P.; Devaraj, N. K.; Eberspacher, T. P. A.; Chidsey, C. E. D. Langmuir 2006, 22, 2457. (29) Devaraj, N. K.; Miller, G. P.; Ebina, W.; Kakaradov, B.; Collman, J. P.; Kool, E. T.; Chidsey, C. E. D. J. Am. Chem. Soc. 2005, 127, 8600–8601. (30) Collman, J. P.; Devaraj, N. K.; Chidsey, C. E. D. Langmuir 2004, 20, 1051– 1053. (31) Mann, D. A.; Kanai, M.; Maly, d. J.; Kissling, L. L. J. Am. Chem. Soc. 1998, 120, 10575. (32) Grant, C. F.; Kanda, V.; Yu, H.; Bundle, D. R.; McDermott, M. T. Langmuir 2008, 24, 14125.
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been explored in recent years.3,16,33,34 Among these are “click” methods which are appealing in that, by definition, they tolerate a wide variety of reaction conditions and functionalities and occur quantitatively with high selectivity.35 Such click-based approaches have, for example, already been used in classical glycoarray formats for carbohydrate-lectin detection.36-41 This paper reports on the fabrication of carbohydrate chips and the use of EIS to monitor their binding with selected lectins. Two relevant carbohydrates, mannose, a monosaccharide, and lactose, a disaccharide, as well as three commercial plant lectins from Lens culinaris, Arachis hypogaea, and Maackia amurensis have been selected to demonstrate the feasibility of the approach in this model study. The covalent immobilization of properly derivatized mannose and lactose analogues on alkynyl-terminated BDD surfaces was achieved by the Cu(I)-catalyzed Huisgen 1,3dipolar cycloaddition of alkynes and azides, one of the most popular “click” reactions to date.42 EXPERIMENTAL SECTION Materials. Details of the syntheses of 5-oxo-5-(prop-2-ynyloxy)pentanoic acid (1), per-O-acetyl lactose (2), per-O-acetyl-(3bromopropyl)-β-D-lactoside (3), per-O-acetyl-(3-azidopropyl)-β-Dlactoside (4); 3-azidopropyl-β-D-lactoside (D), azidopropanol (6), and 3-azidopropyl-β-D-mannose (7) are reported in the Supporting Information. Lectins from Lens culinaris were obtained from Aldrich and used without further purification. Lectins from Arachis hypogaea and Maackia amurensis were obtained from EY Laboratories, Inc. Boron-Doped Diamond Films. Polycrystalline boron-doped diamond (BDD) films (1.5-2 µm thick) (CSEM, Neuchatel, Switzerland) were deposited on silicon substrates in a hot filamentassisted chemical vapor deposition reactor supplied with diborane and methane in hydrogen. The doping level of boron was determined to be NA ∼3 × 1019 B cm-3 by SIMS measurements. Functionalization of BDD Surfaces. Photochemical Oxidation of Hydrogen-Terminated Diamond Surface. A low pressure mercury arc lamp (UVO cleaner, Nr. 42-220, Jelight, Irvine, CA, P ) 1.6 mW cm2, distance from sample: 3 mm, t ) 60 min) was used to photochemically oxidize as-received BDD samples, as reported previously.43 Preparation of Alkynyl-Terminated BDD Diamond Surfaces. 5-Oxo-5-(prop-2-ynyloxy)pentanoic acid (2 mmol) and dicyclohexylcarbodiimide (2 mmol) were dissolved in CH2Cl2 (10 mL). (33) Jaipuri, F. A.; Collet, B. y. M.; Pohl, N. L. Angew. Chem., Int. Ed. 2008, 47, 1707. (34) Zhi, Z.-L.; Powell, A. K.; Turnbull, J. E. Anal. Chem. 2006, 787, 4786. (35) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004. (36) Zhang, Y.; Luo, S.; Tang, Y.; Hou, K.-Y.; Cheng, J.-P.; Zeng, X.; Wang, P. G. Anal. Chem. 2006, 78, 2001. (37) Bryan, M. C.; Fazio, F.; Lee, H.-K.; Huang, C.-Y.; Chang, A.; Best, M. D.; Calarese, D. A.; Blix, O.; Paulson, J. C.; Burton, D.; Wilson, I. A.; Wong, C.-H. J. Am. Chem. Soc. 2004, 126, 8640. (38) Fazio, F.; Bryan, M. C.; Blix, O.; Paulson, J. C.; Burton, D. J. Am. Chem. Soc. 2002, 124, 14397. (39) Michel, O.; Ravoo, B. J. Langmuir 2008, 24, 12116. (40) Santoyo-Gonzalez, F.; Hernandez-Mateo, F. Chem. Soc. Rev. 2009, 38, 3449. (41) Norberg, O.; Deng, L.; Yan, M.; Ramstrom, O. Bioconjugate Chem. 2009, 20, 2364–2370. (42) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 2596. (43) Boukherroub, R.; Wallart, X.; Szunerits, S.; Marcus, B.; Bouvier, P.; Mermoux, M. Electrochem. Commun. 2005, 7, 937–940.
Figure 1. Description of the BDD surface functionalization strategy using a “click” reaction as the key step for the introduction of carbohydrate molecules.
Next, dimethylaminopyridine (0.66 mmol) was added and stirred for 15 min. The oxidized diamond surface was placed into the resulting mixture and stirred at room temperature for 48 h. The resulting surface was washed with CH2Cl2 and water and dried under a stream of nitrogen. Clicking of N3-Molecules. The alkynyl-terminated BDD surface was immersed into 10 mL of aqueous solution of the corresponding azido derivative (2 mmol), CuSO4 · 5H2O (100 µmol), and sodium ascorbate (150 µmol) and stirred at ambient temperature for 24 h. The resulting surface was washed with water and dried under a stream of nitrogen. Surface Characterization. X-ray Photoelectron Spectroscopy. X-ray photoelectron spectroscopy (XPS) measurements were performed with an ESCALAB 220 XL spectrometer from Vacuum Generators. A monochromatic Al KR X-ray source (1486.6 eV) was operated in the CAE (constant analyzer energy) mode (CAE ) 100 eV for survey spectra and CAE ) 40 eV for high-resolution spectra), using the electromagnetic lens mode. No flood gun source was needed due to conducting character of the substrates. The angle between the incident X-rays and the analyzer is 58°. The detection angle of the photoelectrons is 90°, as referenced to the sample surface. Contact Angle Measurements. Water contact angles were measured using deionized water. We used a remote-computercontrolled goniometer system (DIGIDROP by GBX, France) for measuring the contact angles. The accuracy is ±2°. All measurements were made in ambient atmosphere at room temperature. Electrochemical Impedance Spectroscopy (EIS). Electrochemical impedance measurements were performed using an Autolab 20
potentiostat (Eco Chimie, Utrecht, The Netherlands). EIS experiments were carried out in an aqueous solution of a mixture of 10 mM Fe(CN)64-/10 mM Fe(CN)63- in HEPES (0.1 M) and 50 mM NaCl using the following parameters: amplitude of 10 mV at open circuit potential with a frequency range of 100 kHz to 0.1 Hz. Impedance data were modeled using ZView2 software. For EIS studies with carbohydrate-derivatized BDD surfaces, 10 µL of a 100 µg/mL solution of lectin in HEPES (0.1 M) with 50 mM NaCl was added to 1 mL of electrolyte. RESULTS AND DISCUSSION Preparation of Carbohydrate-Terminated BDD Surfaces. Figure 1 depicts schematically the stepwise assembly of the carbohydrate-modified BDD interfaces investigated in this work. Photochemical oxidation of the as-grown diamond interface constitutes the first step of the process. The treatment generates surface hydroxyl functions that can be easily coupled with 5-oxo5-(prop-2-ynyloxy)pentanoic acid (1) through ester bond formation. The terminal alkynyl groups on the BDD interface are then reacted with any chosen azido-terminated molecule depicted in Figure 2. X-ray photoelectron spectroscopy (XPS) is a valuable tool to evaluate modifications in surface chemical composition and binding at each stage of surface derivatization (see Supporting Information, Figure S1). Deconvolution of the C1s multiplex spectrum of the oxidized BDD surface indicates the presence of bulk diamond at 284.1 eV, C-O at 285.2 eV, CdO at 287.1 eV, and a small contribution of sp2 carbon at 283.1 eV. Upon reaction of the surface hydroxyl groups with 5-oxo-5-(prop-2-ynyloxy)pentanoic acid (1), the C1s spectrum broadens and can be Analytical Chemistry, Vol. 82, No. 19, October 1, 2010
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Figure 2. (A) Structure of the various synthesized molecules used in this study. (B) Synthetic approach used for the preparation of an azideterminated lactose: (i) Ac2O, pyridine; (ii) BF3 · Et2O, HO(CH2)3Br, CH2Cl2; (iii) NaN3, DMF; (iv) MeONa, MeOH.
deconvoluted into four components at 283.5 (Csp2), 284.5 (bulk diamond), 285.3 (C-O), 287.1 (CdO), and 288.0 eV (HO-CdO), respectively. The band at 287.1 eV is particularly intense and reflects that a pair of ester functions is simultaneously introduced on the surface. In addition, the band ascribed to the C-O function increases in accordance with the chemical composition of the monolayer. The acylation of the BDD is further confirmed by the change of the water contact angle value from that characteristic of a hydrophilic oxidized interface (θ < 10°) to that of an alkynyl-terminated interface θ ) 60°. “Clicking” Azido Substrates onto Alkynyl-Terminated BDD Interfaces. Various azide-terminated molecules (5-7) have been “clicked” onto the alkynyl-terminated BDD interface (Figure 2A). Carbohydrates carrying a three-carbon spacer arm bearing a terminal azido group were selected, because such sugar derivative is typically prepared in a straightforward manner using 8206
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a classical approach (Figure 2B).44-46 Furthermore, direct introduction of an azido function on unprotected oligosaccharides has recently been described and promises to render this particular “click” format all the more amenable for the fabrication of future sensors using natural glycans. Each of the azido precursors in this work could be “clicked” onto the alkynyl-terminated BDD surface under identical conditions as outlined in the Experimental Section. In brief, Cu(I) was generated in situ by the reduction of catalytic Cu(II) sulfate by ascorbic acid in a reaction solution containing both the azido precursor and the immersed alkynylderivatized BDD surface. Thus “clicking” 3-azidopropyl-β-D-lacto(44) Yang, Z.-Q.; Puffer, E. B.; Pontrello, J. K.; Kiessling, L. L. Carbohydr. Res. 2002, 337, 1605. (45) Yu, H.; Chokhawala, H.; Karpel, R.; Yu, H.; Wu, B.; Zhang, J.; Zhang, Y.; Jia, Q.; Chen, X. J. Am. Chem. Soc. 2005, 127, 17618. (46) Arranz-Plaza, E.; Tracy, A. S.; Siriwardena, A.; Pierce, J. M.; Boons, G.-J. J. Am. Chem. Soc. 2002, 124, 1305.
Table 1. Static Water Contact Angle of the Investigated BDD Surfaces BDD interface
θ (deg)
as-grown oxidized alkynyl-terminated mannose-terminated lactose-terminated
92 ± 2
