Langmuir 2003, 19, 1745-1751
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Probing Specific Lectin-Carbohydrate Interactions Using Atomic Force Microscopy Imaging and Force Measurements† Ahmed Touhami,‡ Barbara Hoffmann,§ Andrea Vasella,§ Fre´de´ric A. Denis,‡ and Yves F. Dufreˆne*,‡ Unite´ de chimie des interfaces, Universite´ catholique de Louvain, Croix du Sud 2/18, B-1348 Louvain-la-Neuve, Belgium, and Laboratorium fu¨ r Organische Chemie, HCI H 317, ETH-Ho¨ nggerberg, CH-8093 Zu¨ rich, Switzerland Received June 28, 2002. In Final Form: August 8, 2002 Knowledge of the molecular interactions between lectins and carbohydrates is a key to understand cellular interactions and to develop new bioanalytical applications. We have used atomic force microscopy (AFM) imaging and force measurements to probe the specific interactions between the lectin concanavalin A (Con A) and oligoglucose saccharides. To this end, gold-coated substrates were first functionalized with Con A and thiol-terminated hexasaccharide molecules. The functionalization procedures were validated by means of X-ray photoelectron spectroscopy and AFM. AFM images recorded in aqueous solution revealed that the hexasaccharide-terminated substrates interact specifically with the lectin. Force-distance curves were then recorded between hexasaccharide-terminated AFM probes and Con A-terminated substrates. About half of the retraction curves showed unbinding forces of 96 ( 55 pN (n ) 100), along with elongation forces and rupture lengths ranging from 0 to 200 nm. These features were not observed when the measurements were performed in the presence of mannose or with a hydroxyl-terminated probe. These results, together with the AFM images, indicate that the measured unbinding forces originate from specific lectin-carbohydrate interactions. The carbohydrate AFM probes designed here offer promising prospects for mapping lectin receptors at cell surfaces.
Introduction Molecular recognition between ligand and receptor molecules is a central event in a variety of biological phenomena. Examples of specific interactions between ligands and receptors are those between complementary strands of DNA, enzyme-substrate, antigen-antibody, and lectin-carbohydrate molecules. Because lectins mediate cell-cell interactions by combining with complementary carbohydrates on opposing cells, they play a role in the control of various processes in living organisms such as initiation of infection, promotion of symbiosis, control of differentiation, and organ formation.1-4 Understanding and exploiting the selectivity of lectincarbohydrate interactions is also of great relevance for developing new bioanalytical (detection) and biomedical (diagnostic) applications.1-4 Therefore, direct measurement of these interactions at the molecular level is of considerable interest from both the basic and applied points of view. Because atomic force microscopy (AFM) is capable of measuring forces in the piconewton range under physiological conditions, it has emerged as a powerful tool for probing the interaction forces between individual ligand* Corresponding author. Phone: (32) 10 47 36 00. Fax: (32) 10 47 20 05. E-mail:
[email protected]. † Part of the Langmuir special issue entitled The Biomolecular Interface. ‡ Universite ´ catholique de Louvain. § Laboratorium fu ¨ r Organische Chemie. (1) Sharon, N.; Lis, H. Science 1989, 246, 227. (2) Sharon, N., Lis, H., Eds.; Lectins; Chapman and Hall: New York, 1989. (3) Liener, I. E., Sharon, N., Goldstein, I. J., Eds.; The Lectins. Properties, Functions, and Applications in Biology and Medicine; Academic Press: Orlando, 1986. (4) Singh, R. S.; Tiwary, A. K.; Kennedy J. F. Crit. Rev. Biotechnol. 1999, 19, 145.
receptor complexes. In these measurements, ligands are bound to the AFM probe and receptors to a solid substrate, or vice versa. The modified probe and substrate are brought in contact so that ligand-receptor complexes can form and the unbinding forces are then measured by pulling the probe away from the substrate. Successful experiments require that the binding of the biomolecules to the probe and substrate is much stronger than the intermolecular force being studied. This is typically achieved by using the chemisorption of alkanethiols on gold or the covalent binding of silanes on silicon oxide. Using this technique, a variety of intermolecular ligand-receptor forces have been measured in recent years, among which are the forces between biotin-avidin,5-8 antibody-antigen,9,10 complementary strands of DNA,11,12 carbohydrate-carbohydrate,13 and lectin-carbohydrate.14 The measured unbinding forces were typically in the range 50-400 pN, depending on the nature of the molecules and on the loading rate. Concanavalin A (Con A), the most widely investigated plant lectin, exhibits a series of remarkable biological properties, including the ability to agglutinate erythro(5) Lee, G. U.; Kidwell, D. A.; Colton, R. J. Langmuir 1994, 10, 354. (6) Florin, E.-L.; Moy, V. T.; Gaub, H. E. Science 1994, 264, 415. (7) Lo, Y. S.; Huefner, N. D.; Chan, W. S.; Stevens, F.; Harris, J. M.; Beebe, T. P. Langmuir 1999, 15, 1373. (8) Lo, Y. S.; Zhu, Y. J.; Beebe, T. P. Langmuir 2001, 17, 3741. (9) Hinterdorfer, P.; Baumgartner, W.; Gruber, H. J.; Schilcher, K.; Schindler, H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 3477. (10) Ros, R.; Schwesinger, F.; Anselmetti, D.; Kubon, M.; Scha¨fer, R.; Plu¨ckthun, A.; Tiefenauer, L. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 7402. (11) Lee, G. U.; Chrisey, L. A.; Colton, R. J. Science 1994, 266, 771. (12) Schumakovitch, I.; Grange, W.; Strunz, T.; Bertoncini, P.; Gu¨ntherodt, H. J.; Hegner, M. Biophys. J. 2002, 82, 517. (13) Dammer, U.; Popescu, O.; Wagner, P.; Anselmetti, D.; Gu¨ntherodt, H. J.; Misevic, G. N. Science 1995, 267, 1173. (14) Grandbois, M.; Dettmann, W.; Benoit, M.; Gaub, H. E. J. Histochem. Cytochem. 2000, 48, 719.
10.1021/la026145w CCC: $25.00 © 2003 American Chemical Society Published on Web 10/24/2002
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Figure 1. Schematic presentation of the strategy used for immobilizing the lectin Con A on gold surfaces.
Figure 2. Structure of the thiol-terminated hexasaccharide used in this study. Table 1. Surface Chemical Composition of Solid Substrates Determined by XPS after the Various Steps of the Con A Immobilization Procedure mole fraction (%)a sample Au Au/NH2 Au/NH2/CM-amylose Au/NH2/CM-amylose/Con A
Au 4f
C 1s
48.4 50.2 40.3 38.4 24.3 22.7 14.2 13.8
43.7 42.3 42.5 43.5 54.5 57.4 56 58
S 2p
O 1s
N 1s
4.7 4.3 1.5 1.7 1.3 1.1
7.9 7.5 7.0 8.7 16.5 15.3 18.3 17.5
5.5 5.1 3.2 2.9 10.2 9.6
a Mole fraction of elements excluding hydrogen; values of two independent determinations.
cytes, to clump certain bacteria, and to precipitate glycogen and starch from solution. These properties result from the specific binding between the lectin and R-mannosyl and R-glucosyl groups. Yet, the nature of this specific interaction remains unclear. In this paper, AFM is used to probe the specific interaction between Con A and oligoglucose molecules. In a first step, solid substrates are functionalized with Con A lectins and thiol-terminated hexasaccharides. The functionalization strategies are validated with X-ray photoelectron spectroscopy (surface chemical composition) and AFM imaging (surface morphology in aqueous solution). In a second step, forcedistance curves are recorded between hexasaccharideterminated AFM probes and Con A-terminated substrates. Two control experiments are performed to demonstrate the specificity of the measured interaction forces, i.e., blocking of the binding sites of Con A with mannose and the use of hydroxyl-terminated AFM probes. Materials and Methods Immobilization of Con A. The lectin concanavalin A (Con A) was covalently immobilized onto gold substrates by using a procedure similar to that developed by others.14,15 The main steps of the immobilization protocol are drawn in Figure 1. Silicon wafers (Siltronix, France) were coated by electron beam thermal evaporation with a 4-nm-thick Ti layer followed by a 30-nmthick Au layer. To create surfaces bearing amine groups (step 1), the coated samples were cleaned for 5 min by UV/ozone treatment (UVO-Cleaner, Jelight, CA), rinsed in ethanol, immersed for 16 h in a 1 mM solution of HS(CH2)2NH2 (Aldrich; used as received) (15) Johnsson, B.; Lo¨fas, S.; Lindquist, G. Analytical Biochemistry 1991, 30, 268.
Figure 3. AFM topographic images (3 × 3 µm; z-ranges, 10 nm) recorded in aqueous solution for a native gold substrate (A) and for gold substrates after functionalization with Con A (B). Characterization of the thickness of the grafted film (C): a 4 × 4 µm image was first recorded at large forces and high rates on a Con A-functionalized substrate, followed by imaging a 7 × 7 µm image of the same area under small forces. Similar results were obtained in different spots and when using independent preparations. and then rinsed with ethanol. During rinsing, sonication was briefly applied to remove alkanethiol aggregates that may be adsorbed. In the second step, a phosphate-buffered saline solution (PBS; pH 7.2) of 10 mg/mL carboxymethylamylose (CM-amylose) (Sigma) was activated with 20 mg/mL N-hydroxysuccinimide (NHS) (Aldrich) and 50 mg/mL 1-ethyl- 3-(3-dimethylamino-
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Figure 4. AFM topographic images (3 × 3 µm; z-range, 10 nm) recorded in aqueous solution for a hexasaccharide-terminated substrate (A), a hexasaccharide-terminated substrate after adsorption of Con A (B), a hexasaccharide-terminated substrate after adsorption of Con A in the presence of 100 mM of D-mannose (C), and a hydroxyl-terminated substrate after adsorption of Con A (D). Similar results were obtained in different spots and when using independent preparations. propyl)carbodiimide (EDC) (Sigma) for 5 min in a 1:1:1 proportion (vol/vol/vol). The amino-functionalized surface was then incubated with the NHS-activated amylose for 10 min and rinsed 3 times in PBS. In the third step, the modified surface was incubated with 0.5 mg/mL Con A (Sigma) in PBS, pH 7.2, for 30 min and intensively rinsed with water to remove the unbound proteins. Water used in our experiments was HPLC grade produced by a MilliQ plus system from Millipore (MilliQ water). Preparation of Hexasaccharide and Hydroxyl-Terminated Surfaces. Self-assembled monolayers (SAMs) of thiolterminated hexaamylose molecules on gold were used to produce carbohydrate-terminated surfaces (Figure 2).16 Silicon wafers (Siltronix, France) and Si3N4 AFM probes (ThermoMicroscopes, Sunnyvale, CA) were coated by electron beam thermal evaporation with a 5-nm-thick Cr layer followed by a 30-nm-thick Au layer. They were cleaned during 5 min by UV/ozone treatment, rinsed in ethanol, and immersed for 3 h in a 0.05 mM solution of the thiol-terminated hexasaccharide in a 1:1 solution of methylene chloride and ethanol. The functionalized surfaces were then rinsed in three baths of methylene chloride, sonication being briefly applied during the rinsing step to remove loosely bound aggregates. (16) Fritz, M. C.; Ha¨hner, G.; Spencer, N. D.; Bu¨rli, R.; Vasella, A. Langmuir 1996, 12, 6074.
For control experiments, hydroxyl-terminated surfaces were prepared by immersing cleaned gold-coated silicon wafers and AFM probes for 16 h in 1mM solutions of HS(CH2)11OH (Aldrich; used as received) in ethanol, and rinsing with ethanol. Adsorption of Con A. Hexasaccharide and hydroxylterminated substrates were deposited on the bottom of wells of tissue culture plates (Falcon, Becton Dickinson, Belgium) and incubated for 30 min at 20 °C with 2 mL of buffered solution of Con A (0.5 mg/mL; PBS; pH 7.2). Rinsing was accomplished by 10 successive dilutions without exposure of the sample to the air-water interface: after adding 2 mL of water, 3 mL of the liquid was aspired; then, 3 mL of water was added and the procedure was repeated. Blocking experiments were performed by adding D-(+)-mannose (100 mM) to the PBS solution. Surface Characterization. X-ray photoelectron spectroscopy (XPS) analyses were carried out with an SSI X-Probe (SSX-100/ 206) spectrometer from Fisons. Samples were dried by flushing with a gentle nitrogen flow for about 20 s and then immediately introduced in the XPS vacuum chamber. The pressure during analysis was between 2.5 × 10-6 and 2.5 × 10-7 Pa. The X-ray beam from the Al source was monochromated (10 kV, 20 mA). The angle between the normal to the sample and the direction of photoelectron collection was 55°. The irradiated zone was an elliptic spot, with a shorter axis of 1000 µm. The constant pass
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Figure 5. Force measurements between hexasaccharide-terminated AFM probes and Con A-terminated substrates: (A) typical retraction force-distance curves and schematic representation of the experimental system; (B) histogram of the largest unbinding forces; (C) histogram of rupture lengths corresponding to the largest unbinding forces. Different probes and samples prepared independently yielded similar results. energy in the hemispherical analyzer was 150 eV (all elements and survey spectrum). Electron binding energies were calibrated by using the Au 4f (84.0 eV). A linear background was used. Mole fractions were calculated by using the peak areas normalized on the basis of acquisition parameters and of sensitivity factors proposed by the manufacturer (mean free path varying according to the 0.7th power of the photoelectron kinetic energy; Scofield cross sections;17 constant transmission function). Water contact angles were measured under ambient atmosphere at 20 °C, using the sessile drop method and an image analysis of the drop profile. The water droplet volume was 0.3 µL. AFM Measurements. AFM images and force-distance curves were obtained at room temperature, with a commercial microscope (Nanoscope III, Digital Instruments, Santa Barbara, CA). The bottom side of hydrated substrates was quickly dried by using precision wipes (Kimwipes, Kimberlay-Clarck) and the substrates were then immobilized on a steel sample puck using a small piece of adhesive tape. The mounted samples were immediately transferred into the AFM liquid cell, while avoiding dewetting. Imaging was performed in aqueous solutions with Si3N4 cantilevers (ThermoMicroscopes, Sunnyvale, CA) using an applied force well below 1 nN. Force measurements were performed in aqueous solutions containing 1 mM CaCl2 and 1mM MnCl2, using hexasaccharide and hydroxyl-terminated probes prepared as described above. The spring constants of these functionalized cantilevers were found to be 8 ( 0.4 mN/m, as determined by measuring the free resonance (17) Scofield, J. H. J. Electron Spectrosc. 1976, 8, 129.
frequency in air.18 Retraction force curves were recorded at a rate of 0.5 µm/s. Blocking control experiments were performed by injecting 100 mM D-(+)-mannose solutions into the liquid cell.
Results and Discussion Design and Validation of Lectin and Carbohydrate-Terminated Surfaces. A key prerequisite for successful investigation of molecular recognition forces by AFM is that receptor and ligand molecules are firmly anchored to solid surfaces while keeping sufficient mobility.9 In this study, the lectin Con A was immobilized on gold surfaces by using carboxymethylamylose (CM-amylose) as a spacer to provide mobility to the protein and minimize nonspecific adsorption (Figure 1). Carbohydrate surfaces were created by assembling monolayers of a thiolterminated hexaamylose on gold (Figure 2). Compared to monosaccharides, hexasaccharides offer the advantage of providing enhanced accessibility to the Con A binding sites. Because of their ability to form monolayers on gold, thiol-terminated saccharides make it possible to create carbohydrate surfaces that are much better defined than what would be obtained by using classical grafting of polysaccharide macromolecules. (18) Cleveland, J. P.; Manne, S.; Bocek, D.; Hansma, P. K. Rev. Sci. Instrum. 1993, 64, 403.
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Figure 6. Force measurements between hexasaccharide-terminated AFM probes and Con A-terminated substrates blocked with 100 mM mannose: (A) typical retraction force-distance curve and schematic representation of the experimental system; (B) histogram of the largest unbinding forces; (C) histogram of rupture lengths corresponding to the largest unbinding forces. Different probes and samples prepared independently yielded similar results.
Substrates functionalized with Con A were thoroughly characterized by using surface analysis by XPS and AFM imaging. Table 1 presents the surface chemical composition determined by XPS for substrates after the various steps of the immobilization procedure. Several observations can be made. First, after treatment with HS(CH2)2NH2 the gold samples showed significant sulfur and nitrogen concentrations, which reflect the occurrence of amino-terminated alkanethiols. Second, substrates functionalized with CM-amylose showed much smaller gold, sulfur, and nitrogen concentrations and a significant increase of the carbon and oxygen concentrations. This is consistent with the presence of a thin layer of polysaccharide, i.e., CM-amylose, at the substrate surface. Third, following incubation with Con A, the contributions of gold and sulfur were even smaller and a large increase of the nitrogen concentration was noted (from 3 to 10%), indicating the presence of a significant amount of Con A proteins at the surface. The surface morphology of functionalized substrates was investigated by AFM. Figure 3 shows AFM height images recorded in aqueous solution for the surface of gold substrates, as such, or after functionalization with Con A. After functionalization, the surface roughness of the underlying gold substrate was attenuated and dotlike features, of 9 ( 1 nm height, were distributed accross the surface. Interestingly, this height is about twice the size of a Con A monomer, indicating that dotlike features may consist of tetramers, which is expected for Con A at neutral pH.3 To evaluate the thickness of the grafted layer, a 4 × 4 µm image was first recorded at large forces for short period of times, followed by imaging a 7 × 7 µm image of the same area under normal load. Figure 3C shows that imaging at high forces resulted in pushing the grafted
material along the scanning direction, thereby revealing the underlying substrate. The thickness of the layer removed from the central area was found to be 3 ( 1 nm. From these results, the following picture emerges: the surface of substrates functionalized with Con A consists of a thin, continuous layer, presumably made of a mixture of CM-amylose and Con A monomers, from which tetramers or supramolecular assemblages are protruding. Hexasaccharide-terminated substrates were also analyzed by XPS. The results (not shown) were similar to those reported earlier16 and consistent with the presence of a monolayer of hexasaccharide molecules at the surface. This was further confirmed by the water contact angle, found to be
