Langmuir 2002, 18, 6659-6665
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The Development, Characterization, and Demonstration of a Versatile Immobilization Strategy for Biomolecular Force Measurements Molly M. Stevens,†,‡ Stephanie Allen,† Martyn C. Davies,† Clive J. Roberts,† Etienne Schacht,§ Saul J. B. Tendler,*,† Stefan VanSteenkiste,§ and Philip M. Williams† Laboratory of Biophysics and Surface Analysis, School of Pharmaceutical Sciences, The University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom, and Department of Organic Chemistry, University of Ghent, Krijgslaan 281, B-9000 Ghent, Belgium Received February 25, 2002. In Final Form: May 28, 2002 Biomolecular force measurements, obtained for example using atomic force microscopy (AFM), can provide valuable molecular-level information on the interactions between biological receptor-ligand pairs. However, in such experiments, a significant level of care must be taken to ensure that adhesion events due to these specific biomolecular interactions are not confused with unwanted interactions that can originate from the underlying surfaces. To this end, we have developed a versatile immobilization strategy of general applicability for biomolecular force measurements. Specifically, dextrans were modified by O-succinoylation to produce a range of partially succinoylated dextrans of different molecular weights and degrees of esterification. The introduced monosuccinate group, upon chemical activation, is able to form covalent bonds with a wide range of functional groups on substrate surfaces and biomolecular species of interest. X-ray photoelectron spectroscopy measurements and AFM tapping-mode images were employed to demonstrate that modified dextran molecules were able to form a surface-immobilized layer that could be hydrated under aqueous conditions. The general utility of this immobilization method for the investigation of a range of receptor-ligand complexes was then demonstrated through force measurements recorded for the streptavidin-biotin system and an antigen-antibody system. Importantly, our results show that specific biomolecular recognition was retained in these systems, indicating that the dextran allows preservation of the native structure of the biomolecules.
Introduction In atomic force microscopy (AFM) investigations of the forces involved in the unbinding of receptor-ligand complexes,1-5 it is essential that these specific biomolecular interactions are appropriately detected. The importance of excluding the background forces that might arise from underlying substrates has been highlighted on numerous occasions.1 Such interactions can lead to large nonspecific adhesional forces that can mask or be indistinguishable from the biomolecular interaction under study. Here, we report the development, characterization, and experimental demonstration of a novel immobilization strategy for AFM force measurement applications that offers a versatile approach to the immobilization of receptor-ligand complexes. In this study, a range of dextrans were chemically modified by O-succinoylation (see Figure 1) to introduce varying percentages of monosuccinate groups. Such succinoylated dextrans can be covalently tethered to aminefunctionalized surfaces to produce a monolayer of hydro* To whom correspondence should be addressed. E-mail:
[email protected]. † The University of Nottingham. ‡ Current address: Department of Chemical Engineering, Massachusetts Institute of Technology, 45 Carleton Street, Room E25342, Cambridge, MA 02139. E-mail:
[email protected]. § University of Ghent. (1) Willemsen, O. H.; Snel, M. M. E.; Kuipers, L.; Figdor, C. G.; Greve, J.; de Grooth, B. G. Biophys. J. 1999, 76, 716. (2) Leckband, D. E.; Schmitt, F.-J.; Israelachvili, J. N.; Knoll, W. Science 1992, 255, 1419. (3) Leckband, D. E.; Schmitt, F.-J.; Israelachvili, J. N.; Knoll, W. Biochemistry 1994, 33, 1614-1624. (4) Florin, E.-L.; Moy, V. T.; Gaub, H. E. Science 1994, 264, 415. (5) Lee, G. U.; Kidwell, D. A.; Colton, R. J. Langmuir 1994, 10, 354.
philic dextran, which is known to form a neutral proteinresistant hydrated barrier at the surface under aqueous conditions.6-11 Any remaining succinoyl groups can then be utilized to covalently immobilize virtually any biomolecule of interest via native amine, thiol, aldehyde, or carboxylic acid functionalities.6,7 It was envisaged that this type of immobilization approach would enable proteins, subsequently immobilized to the dextran, to be shielded from surface forces generated by the underlying substrates, thus preventing the detection of nonspecific interactions during force measurements. Furthermore, it was anticipated that the hydrophilic nature of the dextran would allow the preservation of the native state of attached biomolecules, while the flexible conformational structure of its chains helped to ensure that steric constraints on the system were minimized. Similar immobilization approaches have, in addition, been utilized for several years for the attachment of proteins to SPR sensors, in particular in those employed by BIAcore systems.12,13 Utilizing standard active ester chemistry, the applicability of this approach to the immobilization of (6) Hermanson, G. T. In Bioconjugate Techniques; Hermanson, G. T., Ed.; Academic Press: London, 1995. (7) Bruneel, D.; Schacht, E. Polymer 1994, 35, 2656. (8) Petro, M.; Gemeiner, P.; Berek, D. J. Chromatogr. A. 1994, 665, 37. (9) Matthijs, G.; Schacht, E. J. Chromatogr. A. 1996, 755, 1. (10) O ¨ sterberg, E.; Bergstro¨m, K.; Holmberg, K.; Riggs, J. A.; Van Alstine, J. M.; Schuman, T. P.; Burns, N. L.; Harris, J. M. Colloids Surf. A: Physicochem. Eng. Aspects 1993, 77, 159. (11) O ¨ sterberg E.; Bergstro¨m, K.; Holmberg, K.; Schuman, T. P.; Riggs, J. A.; Burns, N. L.; Van Alstine, J. M.; Harris, J. M. J. Biomed. Mater. Res. 1995, 29, 741. (12) Lo¨fås, S. Pure Appl. Chem. 1995, 67, 829. (13) Lo¨fås, S.; Johnsson, B.; Edstro¨m, A° .; Hansson, A.; Lindquist, G.; Hillgren, R.-M. M.; Stigh, L. Biosens. Bioelectron. 1995, 10, 813.
10.1021/la0202024 CCC: $22.00 © 2002 American Chemical Society Published on Web 07/24/2002
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Figure 1. Scheme for the chemical modification of dextran by O-succinoylation. The percentage succinoylation is controlled by changing the ratio of succinic anhydride or 4-(dimethylamino)pyridine (4-DMAP) to dextran.
proteins via lysine residues is demonstrated. Specifically, we utilize this approach to record force measurements for the model receptor-ligand interaction streptavidinbiotin3-5,14,15 and an antigen-antibody system, namely, that of human chorionic gonadotrophin (hCG) and antihCG antibody (anti-hCG). Although this coupling strategy can involve a diversity of potential attachment points in proteins, because of the high prevalence of lysine, it does provide a generally applicable coupling route.6 Furthermore, in previous studies, it has been demonstrated that specific biomolecular recognition is retained in antigenantibody systems and in the streptavidin-biotin system when these molecules are immobilized via amine groups.15,16 Experimental Section Materials. Dextrans of molecular weights 10 000 and 70 000 (Dextran T-10 and Dextran T-70, respectively) were obtained from Pharmacia Biotech AB (Uppsala, Sweden) and were dried over phosphorus pentoxide before use. Succinic anhydride was (14) Wilchek, M.; Bayer, E. A. In Avidin-Biotin Technology; Wilcheck, M., Bayer, E. A., Eds.; Methods in Enzymology Series; Academic Press: New York, 1983; No. 184, pp 5-13 and 49-67. (15) Allen, S.; Davies, J.; Dawkes, A. C.; Davies, M. C.; Edwards, J. C.; Parker, M. C.; Roberts, C. J.; Sefton, J.; Tendler S. J. B.; Williams, P. M. FEBS Lett. 1996, 390, 161. (16) Allen, S.; Chen, X.; Davies, J.; Davies, M. C.; Dawkes, A. C.; Edwards, J. C.; Roberts, C. J.; Sefton, J.; Tendler S. J. B.; Williams, P. M. Biochemistry 1997, 36, 7457.
Stevens et al. obtained from Fluka Chemie AG (Sigma-Aldrich Co. Ltd., Gillingham, Dorset, U.K.) and was purified prior to use by refluxing with redistilled acetic anhydride (J. T. Baker Chemicals, Deventer, Holland) for 1 h 20 min (0.5:1 mol/mol). 4-(Dimethylamino)pyridine (4-DMAP) (Janssen Chemical, Beekse, Belgium) was used without further purification. Dimethyl sulfoxide (DMSO) was obtained from Allied Signal (Riedel-de-Hae¨n AG, Seeize, Germany) and was dried and distilled before use. Monoclonal mouse IgG directed against hCG (anti-hCG) was obtained from Ortho-Clinical Diagnostics (Chalfont St. Giles, Buckinghamshire, U.K.). N-Ethyl-N′-(dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), biotinylated bovine serum albumin (BBSA), and hCG were all obtained from Sigma-Aldrich Co. Ltd. (Gillingham, Dorset, U.K.). 3-Aminopropyldimethylethoxysilane (3-APDES) was obtained from ACBR (Manchester, U.K.). Spherical silica particles to be attached to the ends of cantilevers were obtained from Supelco (Supelco Inc., Bellefonte, PA) and had a nominal diameter of 12 µm. O-Succinoylation of Dextrans. Partially succinoylated dextrans were prepared with different degrees of acylation from dextran with a Mw of 10 000 (dextran T-10) or 70 000 (dextran T-70). Two partially succinoylated dextrans with a lower degree of acylation were prepared by adding 1 mmol of succinic anhydride in 10 mL of DMSO to a solution of 1 g of dextran T-10 or T-70 in 15 mL of DMSO. The reaction mixture was warmed to 40 °C, and 0.1 mmol of 4-DMAP was added as a catalyst with stirring. The reaction mixture was kept at 40 °C for 24 h. The same procedure was employed to prepare two partially succinoylated dextrans with a higher degree of acylation by using 4 and 0.4 mmol of succinic anhydride and 4-DMAP, respectively. All four partially succinoylated dextrans were isolated from the DMSO by precipitation in a 5-fold volume of ethanol/ether (1:1, v/v). The dried precipitate was dissolved in 5 mL of highpurity water (purified on an ELGA water system, Maxima HPLC, Elga Ltd., Bucks, U.K.; resistivity of approximately 15 MΩ cm) and injected into a preparative gel filtration column (Sephadex G-25, eluent water (distilled and deionized), flow rate 2 mL min-1, detection by Sicon Analytic differential refractometer LCD 201 apparatus). The purified polymer fraction was collected and freeze-dried. The number- and weight-average molar masses and the polydispersity of the purified polymers were determined by gel permeation chromatography utilizing a Tessek 40-100-300-1000 4 (4 × 25 cm) column (Millennium 2000, Waters) and a calibration curve constructed utilizing dextran standards of Mw ) 9890, 21 400, and 43 500. The samples were dissolved in 0.1 M citric acid buffer (pH 6.0). The degree of esterification was determined for the purified polymers by 1H NMR spectroscopy performed on an AM500 Bruker spectrometer (Bruker Analystische, Karlsruhe, Germany). Deuterium oxide (Aldrich, Milwaukee, WI) was used as the solvent. Preparation of Succinoylated Dextran-Coated Substrates and Spherical Particles. Amine-functionalized (3APDES functionalized) silicon substrates and spherical particles (nominal diameter of 12 µm) were prepared via a covalent immobilization strategy as previously described.16 These materials were then incubated in a 10 mg/mL solution of the succinoylated dextran containing 400 mM EDC and 115 mM NHS in sodium phosphate buffer (10 mM, pH 8). This resulted in the activation of the carboxy groups on the dextran to form N-hydroxysuccinimide esters, which spontaneously react with amine groups to allow covalent attachment of the dextran to the surface. After 1 h of incubation, the substrates and spherical particles were rinsed thoroughly with high-purity water to remove any reagents or noncovalently bound dextran. X-ray Photoelectron Spectroscopy (XPS) Analysis. XPS spectra were acquired on a VG Scientific ESCALAB Mark II instrument (VG Scientific Ltd., East Grinstead, Sussex, U.K.). Survey spectra were recorded for each sample (0-1100 eV) at a take-off angle of 45°. AFM Imaging. AFM tapping-mode images were obtained with a multimode AFM with Nanoscope IIIa Controller (Digital Instruments, Veeco Metrology Group, Santa Barbara, CA). Images were obtained in either high purity water or propanol. V-shaped silicon nitride AFM cantilevers with integrated py-
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Table 1. Degree of Substitution (DS) for the Partially Succinoylated Dextrans as Determined by 1H NMR Spectroscopy, and the Weight Average Molar Mass (Mw), Number-Average Molar Mass (Mn), and Polydispersity (d) as Determined by Gel Permeation Chromatography sample
DSa (%)
partially succinoylated dextran T-10 partially succinoylated dextran T-10 partially succinoylated dextran T-70 partially succinoylated dextran T-70
13 62 17 65
Mw
Mn
Table 2. Surface Elemental Atomic Percentages Determined Using XPS for an Amine-Functionalized Silicon Wafer and Succinoylated Dextran-Coated Silicon Wafers elemental atomic percentage
d
12 793 9170 1.4 17 801 11 832 1.5 70 753 41 644 1.7 111 020 69 323 1.6
a Degree of substitution (DS) is defined as the amount of substitution per 100 dextran glucosidic rings.
ramidal probes (Nanoprobe, Digital Instruments, Veeco Metrology Group, Santa Barbara, CA) were employed. AFM Force Measurements. AFM force measurements were obtained using instrumentation constructed in the Laboratory of Biophysics and Surface Analysis (School of Pharmaceutical Sciences, The University of Nottingham, Nottingham, U.K.). AFM spherical probes with controlled geometry were utilized for force measurements. The probes were prepared by fixing an individual dextran-coated spherical particle to the apex of a V-shaped silicon nitride cantilever (Nanoprobe, Digital Instruments, Santa Barbara, CA) with Araldite rapid set epoxy (Ciba-Geigy Plastics, Duxford, U.K.). The spherical particles were placed at the end of the cantilever using a micromanipulator (Research Instruments Ltd., Cornwall, U.K.) while being viewed under an optical microscope. Measurements of sphere diameters were obtained using an eyepiece micrometer in the optical microscope, and found to be within the range of 10-12 µm. Cantilever spring constants were determined using the resonant frequency method17 and were found to be within the range of 0.097-0.224 N m-1. AFM force measurements were obtained in 100 mM sodium phosphate buffer (pH 7.4) at a piezo approach-retract speed of 1 µms-1. Biomolecules were immobilized to the dextran-coated spherical probes and substrates by incubating the probes and substrates for 1 h in a 1.5 mM solution of the protein containing 400 mM EDC and 115 mM NHS in sodium phosphate buffer (10 mM, pH 8). These samples were then rinsed thoroughly with high purity water to remove any reagents or unbound protein prior to the AFM force measurements. For each experiment, control force measurements were first performed between the dextran-coated substrates and spherical probes to ensure that no nonspecific adhesion was detected prior to covalent immobilization of the proteins of interest. To confirm that the subsequently observed adhesion events were due to specific receptor-ligand binding events, a control blocking step was also performed at the end of each experiment. This involved flooding the experimental system with an excess of the ligands biotin or hCG (0.1 mg/mL in 100 mM sodium phosphate buffer, pH 7.4) for 1 h, for force measurements performed between streptavidin and biotinylated bovine serum albumin (BBSA) or hCG and anti-hCG, respectively. After 1 h, additional force measurements were obtained between the ligands and the blocked receptors.
Results and Discussion Purified succinoylated dextrans (presented in Table 1) were synthesized with a range of degrees of esterification (13-65%), as quantitatively determined by 1H NMR spectroscopy analysis.18 The number-average molar mass (Mn), weight-average molar mass (Mw), and polydispersity (d) of the polymers, as determined by gel permeation chromatography (GPC), are also reported in Table 1. Mw for the unmodified dextrans T-10 and T-70 lies in the ranges 9000-11 000 and 63 000-77 000, respectively.19 The greater determined values for Mw for the partially succinoylated dextrans therefore reflect the increase in (17) Cleveland, J. P.; Manne, S.; Bocek, D.; Hansma, P. K. Rev. Sci. Instrum. 1993, 64, 403. (18) Bruneel, D; Schacht, E.; Debruyn, A. J. Carbohydr. Chem. 1993, 12, 769. (19) Pharmacia Biotech AB (Uppsala, Sweden).
sample 3-APDES-coated silicon wafer partially succinoylated dextran (T-10, DS ) 13%) partially succinoylated dextran (T-10, DS ) 62%) partially succinoylated dextran (T-70, DS ) 17%) partially succinoylated dextran (T-70, DS ) 65%)
silicon (Si 2p)
carbon (C 1s)
oxygen (O 1s)
52 23
13 39
35 38
27
36
37
39
23
38
37
27
36
mass from the monosuccinate groups, with higher values being observed for higher degrees of acylation. The polydispersity values for the modified dextrans lie between 1.4 and 1.7, reflecting a reasonably uniform distribution of the modified dextran chains (typically Mw/Mn is in the range 1.5-2.020). XPS Analysis. Dextran-coated substrates were prepared by utilizing a mixture of EDC and NHS to form N-hydroxysuccinimide esters, which subsequently reacted with 3-APDES functionalized (amine-functionalized) substrates. XPS was employed to confirm the immobilization of the succinoylated dextrans to the aminated silicon substrates. Table 2 details the peak areas observed for the carbon (C 1s), oxygen (O 1s), and silicon (Si 2p) regions of the prepared substrates. Analysis of 3-APDES functionalized silicon substrates prior to immobilization of the succinoylated dextran revealed a large signal in the Si 2p region. XPS spectra recorded for partially succinoylated dextran-coated substrates had relatively larger signals in the C 1s regions, confirming the presence of the modified dextran on the substrate surface. In such spectra, signals in the Si 2p region were significantly reduced, particularly for the dextran T-10 functionalized substrates, indicating that these substrates had the greatest surface coverage. Imaging of Dextran-Coated Substrates. To further investigate surface coverage, silicon substrates functionalized with the partially succinoylated T-10 dextran (13% degree of esterification) were subsequently imaged in liquid environments utilizing tapping-mode AFM. Imaging was undertaken in both water and propanol environments with reference to work by Frazier et al.,21 who imaged gold substrates functionalized with thiolated dextrans. In that study reversible dehydration of the immobilized dextran layer was demonstrated upon replacement of the water with propanol, enabling a resolution of single dextran molecules. Figure 2 displays representative topography [a(i)] and phase-contrast [a(ii)] AFM images of a 3-APDES-functionalized silicon substrate prior to dextran immobilization, recorded in water. Also displayed are topography [b(i)] and phase-contrast [b(ii)] images recorded for this substrate following the covalent attachment of dextran. A comparison of the images reveals a marked change in the phase contrast, together with an apparent decrease in image resolution upon dextran immobilization. For the dextran-coated surface [b(ii)], the phase contrast has a uniform appearance, suggesting the presence of a continuous layer of dextran molecules with very little of the (20) Young, R. J.; Lovell, P. A. In Introduction to Polymers, 2nd ed.; Young, R. J., Lovell, P. A., Eds.; Chapman and Hall: London, 1991. (21) Frazier, R. A.; Davies, M. C.; Matthijs, G.; Roberts, C. J.; Schacht, E.; Tendler, S. J. B.; Williams, P. M. Langmuir 1997, 13, 4795.
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Figure 2. Typical AFM images of (i) surface topography (size, 500 nm × 500 nm; z scale, 6 nm) and (ii) phase contrast (size, 500 nm × 500 nm; z scale, 4.77°) of silicon substrates functionalized with (a) 3-APDES, (b + c) Partially succinoylated dextran (T-10, DS ) 13%). AFM images a and b were collected in water and image c was obtained following replacement of water with propanol. The surface roughness values for a(i), b(i), and c(i) are 0.25, 0.49, and 0.62 nm, respectively.
underlying silicon substrate apparent. The low image resolution observed in the topography (b(i)) is likely to be due to the hydration of the discrete dextran molecules in the water environment. In this state, the molecules will be swollen and able to coalesce to form a continuous film. The low elastic modulus of this hydrated dextran film allows a greater indentation of the sample by the imaging AFM probe and thus creates a larger probe-sample
contact area and, hence, a decrease in image resolution.21-23 To test this hypothesis the same dextran-coated substrate was subsequently imaged in a propanol environment to investigate whether dehydration of the dextran (22) Weihs, T. P.; Nawaz, Z.; Jarvis, S. P.; Pethica, J. P. Appl. Phys. Lett. 1991, 59, 3536. (23) Shao, Z.; Yang. J. Q. Rev. Biophys. 1995, 28, 195.
Strategy for Biomolecular Force Measurements
Figure 3. Control force measurements performed between a spherical AFM probe and a substrate both functionalized with succinoylated dextran (T-10, DS ) 13%). (a) Schematic of the experimental setup. (b) Typical probe-sample separation curve for a force measurement performed between these two dextrancoated surfaces.
molecules could be observed (see Figure 2c). The emergence of discrete globular features with an average diameter of 16 nm ((2 nm, n ) 10) is observed in both the topography [c(i)] and the phase contrast [c(ii)]. These globules are most likely attributable to dehydrated dextran aggregates, and a full surface coverage of dextran is observed on the silicon substrate. AFM Force Measurements. Prior to the covalent immobilization of biomolecules to the dextran, control force measurements were performed between substrates and spherical probes functionalized with each of the partially succinoylated dextrans (see Figure 3a for a schematic diagram of the experimental setup). Figure 3b displays a typical force versus extension trace collected after a period of about 15 min (sufficient to allow the dextran layer to hydrate), demonstrating that nonspecific interactions between underlying substrates were prevented by the dextran coating. Loss of linearity is observed in the contact region as the approaching spherical probe deforms the flexible dextran chains. The succinoylated T-10 dextran (13% degree of esterification) was utilized for all AFM force measurements of specific biomolecular interactions as it was found to afford the best surface coverage (from XPS measurements and AFM images), together with excellent screening of nonspecific interactions from underlying substrates (occurrence of nonspecific adhesion events of
