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Sensing Discrete Streptavidin-Bio tin Interactions with Atomic Force Microscopy Gil U Lee,* David A. Kidwell, and Richard J. Colton Surface Chemistry Branch, Naval Research Laboratory, Code 6177, Washington, DC 20375-5342 Received September 13, 1993. I n Final Form: November 12, 199P Molecular recognition forms the basis for assembly and regulation in living organisms. We have used the atomic force microscope (AFM)to study the interaction of a model receptor streptavidin with its ligand biotin under physiological conditions. Surfaces functionalized with biotin and streptavidin exhibited adhesive forces 3-8 times greater than the nonspecific interactions observed between blocked streptavidin and biotinylated surfaces. The magnitude and distribution of the observed adhesive forces suggest they result from individualstreptavidin-biotin interactions. This technique provides a means to directly study molecular recognition interactions at the molecular level. Nature has developed the unique ability for molecular recognitionthrough the use of multiple noncovalent bonds (Le., van der Waals, hydrogen, ionic, and hydrophobic interactions) which possess a high degree of spatial and orientational specificity. Molecular recognition plays a central role in cellular behavior1 and the immunological response,2and has also become the basis for a wide range of bioanalytical technique^.^ Although the structure and binding properties of molecular recognition systems can be measured, the forces involved in intermolecular interactions remain largely unknown. Molecular recognition interactions have been characterized through observations of the behavior of cells on which ligands and receptors naturally occur or have been a t t a ~ h e d . Although ~?~ these studies have largely been qualitative, the application of micropipet techniques to the study of cellular interactions5 has provided the meansfor micromanipulation with a force sensitivity of N. The surface force apparatus has also recently been used to characterize quantitatively the surface forces between model molecular recognition systems.6 The control of surface properties and the very small forces and distances involved in intermolecular interactions continue to limit the characterization of discrete molecular recognition interactions. The AFM has several properties that make it an ideal tool for measuring intermolecular forces: theoretical force sensitivity on the order of N, displacement sensitivity of 0.01 nm, contact areas as small as 10 nm2, and ability to operate under physiological condition^.^ In fact, the measurement of interactions as small as asingle hydrogen bond has recently been reported.8 The bond energies for specific molecular interactions fall between those typical
* To whom correspondence should be addressed. a Abstract published in Aduance ACS Abstracts, January 1,1994.
(1)Bongrand, P. Physical Basis of Cell-Cell Adhesion; CRC Press: Boca Raton, FL, 1988. (2)Eisen, H. N. Immunology, 3rd ed.; Harper and Row Publishers: New York, 1990. (3)Tijssen, P. Practice and Theory of Enzyme Immunoassays; Elsevier: Amsterdam, 1985. (4)Tha, S.P.; Goldsmith, H. L. Biophys. J. 1986,50,1117. Simoon, S. I.; Chambers, J. D.; Sklar, L. A. J. Cell Biol. 1990,111, 2747. (5)Evans, E.;Berk, D.; Leung, A. Biophys. J. 1991,59,838. (6)Leckband,D.E.; Israelachvili,J. N.;Scmitt, F.-J.; Knoll, W. Science 1992,255,1419-1421. Helm, C. A.; Knoll, W.; Israelachvili, J. N. Proc. Natl. Acad. Sci. U.S.A. 1991, 88,8169. (7)Binnig, G.; Quate, C. F.; Gerber, Ch. Phys. Reu. Lett. 1986,56,930. Meyer, G.; Amer, N. M. Appl. Phys. Lett. 1988,53,1045.Burnham, N. A.; Colton,R. J. In Scanning Tunneling Microscopy: Theory, Techniques, and Applications; Bonnell, D. A., Ed.; VCH Publishers Inc.: New York, 1993;pp 191-250. (8)Hoh, J. H.; Cleveland, J. P.; Pratter, C. B.; Revel, J.-P.; Hansma, P. K. J. Am. Chem. SOC.1993,114, 4917.
Table 1. Comparison of the Bond Energy, Rupture Force, and Bond Length of Several Noncovalent Bonds and the StreDtavidin-Biotin Interaction ~~
bond energy
U interaction
(kJ/mol)
ionic (NaC1) streptavidin-biotin van der Waals (Ar-Ar)
520b 88c
Id
~
rupture force’
F(N)
~
~~~~
effective bond rupture length length ro(nm) Y (nm)
2.1 X lkgb 0.228b 1 X lkg varies 1.2 X 1k1ld 0.382d
0.46 0.15 0.14
‘ F = dUIdr calculated a t the point at which d2U/dr2 = 0. Calculated with the Madelung potential for a single NaClinteraction at room temperature and atmospheric presswe.2’ c Reference 28. Calculated with the Lennard-Jones potential for two Ar atoms at 0 K and zero pressure.%
of ionic and vander Waals bonds, Table 1. The magnitude of the rupture force calculated for the ionic and van der Waals bonds is well within the theoretical force sensitivity of the AFM. In order to measure the interaction between biotin and streptavidin, glass microspheres and mica surfaces were functionalized with biotin and streptavidin, and the forces between two biotinylated surfaces, a biotin and blocked streptavidin surface and a biotin and streptavidin surface, were measured. Microsphere and muscovite mica surfaces were functionalized with biotin and streptavidin using the nonspecific adsorption of bovine serum albumin (BSA), which is known to spontaneously and “irreversibly”adsorb to glass and mica surfaces? BSA to which 7-9 biotin groups were covalently attached using biotin-eaminocaproic acid N-hydroxysuccinimide ester (hereafter called BBSA) was obtained from Sigma Chemical Co. (St. Louis, MO). Freshly prepared microspheres and mica surfaces were incubated at room temperature in 250 pg/mL BBSA in pH 5.6 phosphate buffer saline (PBS) (10 mM phosphate buffer, 6 mM KCl, 120 mM NaC1) for 2 h, and rinsed with pH 7.0 PBS. Streptavidin-functionalizedmica surfaces were formed by exposing BBSA-mica films to 30 pg/mL streptavidin (Sigma Chemical Co.) in pH 7.0 PBS for 2 h followed by thorough rinsing with pH 7.0 PBS. Blocked streptavidin surfaces were prepared by exposing streptavidin-functionalized surfaces to 10 mM biotin for -20 min. Figure 1A is an AFM image of a BBSA film that has been partially removed by applying a high (>3 nN) imaging force in a 1pm2 area. Cross-sections of BBSA films, such (9)Andrade, J. D. Surface and Interfacial Aspects of Biopolymers; Plenum Press: New York, 1985;Vol. 2,Chapters 1, 2, 7,and 8.
This article not subject to U.S. Copyright. Published 1994 by the American Chemical Society
* Langmuir, Vol. 10, No.2, 1994 355
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as the one shown in Figure lB, give a film thickness of 3.5 f 0.7 nm. The similarity in the thickness of the BBSA film to the globular domain size of human serum albuminlo suggests that the protein adsorbs in a monolayer with a surface coverage of 44 nm2/molecule. The streptavidin surface coverage and number of active sites per streptavidin molecule were determined with 3H-labeled biotin and 1251-labeledavidin (New England Nuclear, Boston, MA)." For each BBSA molecule on the surface, 0.5 f 0.2 avidin molecule was adsorbed, with each avidin molecule having 2.6 i 1 biotin binding sites that were free. The AFM (Nanoscope 111, Digital Instruments, Santa Barbara, CA) has a conventional optical-beam-deflection detector scheme with a liquid cell. The sensitivity of this instrument was -0.01 nN.12 The instrumentwas operated in a variable-force mode in which the surface was ramped toward the probe at frequencies of 0.1-0.001Hz while the (10) He, X. M.; Carter, D. C. Science 1992,358,209. (11) Avidinandstreptavidinhaveahighdegreeof similarityinstructure and binding characteristics. Avidin, however, tends to adsorb in a nonspecificmannerwhich may accountfor the relativelyhigh uncertainty in the number of free biotin binding sites. (12) The theoretical force Sensitivity of the optid-beam-deflection detection scheme is *0.002 nN for a 0.048 N/m cantilever: Putman, C. A. J.; De Grooth, B. D.; Van Hulst, N. F.; Greve, J. J. Appl. Phys. 1992, 72, 6. The remaining sources of noise (i.e., thermal and mechanical excitationof the cantileverand variationsin opticalproperties)determine the force sensitivity of the instrument.
Figure 2. Measured forcesmbetween BBSA monolayers in pH 7.0 PBS and the theoretical DLVO force curve. The repulsive double-layerforcewas calculatedfor surfacesof constant potential (-35 mV39 using the weak overlap approximation, and the van der Waals force has been calculated with a Hamaker constant (A=A-~~,-~ ofA4) X J. The retarded A ~ A - ~ B has A been calculated by NiP2 to vary between 7.6 X and 8.7 X J. Ionic screening of the zero-frequency contribution of the Hamaker constant, however, results in a -50% decrease in the ~ A16, p 396). The primary minimum of value of A ~ A - P B S -(ref the DLVO force curve, not shown in the figure, occurs a t 2 nm. The asymptote of the measured force curves has been set a t a surfaceseparation of 4 nm to facilitatecomparison with the DLVO force curve. The diamondson the measured force curverepresent every 10th data point. The maximum repulsive force applied to the surfaces, which was not recorded due to the limited dynamic range of the digital oscilloscopeand detector, is -5.0 nN. This force did not exceed the yield stress of the BBSA film due to the large contact area of the spherefilm junction.20
piezowaveform and the response of the cantilever were acquired with a digital storage oscilloscope. Microfabricated Si3Na cantilevers with spring constants of 0.048 f 0.005 and 0.120 f 0.010 N/m were obtained from a commercial supplier (Park Scientific Instruments, Mountain View, CA).13 Glass microspheres (MoSci, Rolla, MO) of 15-pm radius were epoxied to the cantilevers14 and subsequently cleaned with an oxygen plasma. The microspheres were imaged with AF'M, and their root-meansquare roughness varied between 2 and 10 nm.15 The force measured between two BBSA monolayers is plotted asa function of relative surfaceseparation in Figure 2. The interaction of BBSA monolayers is characterized by the absence of long-range (>6 nm) forces. The theoretical Derjaguin-Landau-Very-Overbeek (DLVO) force curve (dashed line in Figure 2),16 which is the sum of the repulsive double-layer force and attractive van der Waals force, reveals that the long-rangeforces are strongly attenuated by the high ionic strength of the PBS buffer. At short range the measured force curve exhibits a net repulsiveforce that is indicative of a short-rangerepulsive force (not accounted for by the DLVO theory). Shortrange repulsive forces may originate from steric-hydration forces" and/or the effects of the surface roughness of the (13)The spring comtanta of the microfabricated cantilevers were measured using a 12.5-pm-diameter gold wire teat spring: Lee, G. U, Ph.D. Thesis, University of Minnesota, 1992. (14) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Nature 1991,359, 239. (15) The quality of the commercial microapheres was found to vary significantly from lot to lot. Some microspheres were coated with 0.1pm-size particles which can be removed with a 0.1 M KOH etch. (16) Israelachvili, J. Intermolecular and Surface Forces; Academic Press: San Diego, 1991; p 246.
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microsphere.14 The lack of a substantial attractive force makes the BBSA films ideal systems for observingspecific interactions. The force measured between a BBSA monolayer and blocked streptavidin surface is plotted as a function of the relative surfaceseparationin Figure 3. The BBSA-blocked streptavidin interaction exhibits an adhesive force of 0.08nN magnitude, in contrast to the interaction between BBSA monolayers. These adhesive forces may originate from an increase in the magnitude of the van der Waals force or a decrease in the magnitude of the short-range repulsive forces. A histogram of the nonspecific adhesive forces, Figure 4, showsa skewed distribution that decreases with increasingforce. The mean nonspecificadhesiveforce was 0.06 f 0.04 nN (n = 30). The interaction between the BSA monolayer and streptavidin surface in Figure 5a is dominated by an adhesive force of 0.29 nN. Strong adhesive forces were observed in d O % of the measurements, and the mean magnitude of these forces was 0.34 f 0.12 nN (n = 42). The remaining measurements exhibited adhesive forces 0.6 nN reveal an unperturbed BBSA film. This suggests that the streptavidin-biotin bond was ruptured before the BBSA film was perturbed. The maximum number of streptavidin-biotin interactions may be calculated from the Hertzian contact area of the microsphere and surface.lg For a sphere of 15-pm radius under a 15-nN load20the contact area is 880 nm2. From the surfacedensity, at most 10streptavidin molecules (18) Streptavidin-labeled gold colloids (Sigma Chemical, St. Louie, MO) were incubated with BBSA-mica films at a concentrationof 3.7 X 1013 particles/mL in pH 7.0 PBS. The colloid surface density was determined to be 1 colloid/lO BBSA molecules by optical density measurements. (19) The Hertzian model rather than the JKRS or DMT models was used tocalculatethe areaof contactdue tothesmall surfaceforceeohrved between the BBSA and blocked streptavidin-BBSA surfaces: Weihe, P.; Nawas, Z.; Jarvis, S. P.; Pethica, J. B. Appl. Phys. Lett. 1991; 59,3536; Pethica, J. B.; Oliver, W. C. Phys. Scr. 1987, T19,61. The JKRS model would predict a contact area that is only 15% larger than that calculated with the Hertzian model.
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Langmuir, Vol. 10, No. 2, 1994 357
are within this area. Steric limitations imposed by the immobilization of streptavidin and biotin, and the location of the biotin binding site deep within the streptavidin molecule,2l however, are expected to significantly reduce the viable number of biotin-streptavidin interactions. In fact, 30% of the measurements did not result in specific interactions. Likewise, when the contact area was decreased by using a probe of radius =0.3 pm, no specific interactions were observed. Both these observations lead us to believe that the observed adhesive forces result from only a few specific interactions. The force necessary to rupture a bond may be calculated if the form of the potential is known. However, potentials have not been determined for complex systems such as the streptavidin-biotin interaction which involve at least seven hydrogen bonds and a similar number of hydrophobic interactions.21 Rupture forces for complex interactions have commonly been estimated by dividing experimentally determined bond energies (0by a length (y) that is thought to be representative of the range of the force, F = -U/y.Z2 Using the ionic and van der Waals interactions as models (Table l), it appears that the effective rupture length, y, increases with the range of the interaction potential or the inverse of the bond length. Given the nature of the bonding interactions in the streptavidin-biotin system, we would expect the effective rupture length to be relatively short-ranged and similar to that of a van der Waals bond, i.e., y 0.15 nm. This would give an estimated rupture force of N, a factor of 3 larger than the measured adhesive force. Bell23 has refined this simple model by taking into account the reversibility of specific interactions and the finite time over which the bond is ruptured. The external force applied to a reversible bond is related to the period, T , over which the bond will rupture throQghthe equation
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F=
U - kT ln(7/T0) h
(1)
where kT is the thermal energy, TOis the reciprocal of the natural frequency of oscillation (=10-l2 s for biotin), and h is a parameter approximately equal to the effective rupture length, y.5 The average rupture period and adhesive force of the streptavidin-biotin interactionwere found to be dependent on the spring constant (k) of the cantilever^:^^ for k = 0.048 N/m, Fav = 0.3nN, Tgy = 0.34 S; for k 0.12 N/m, Fa" = 0.4 nN, T~~ = 0.04 s. The decrease in the rupture force with the increase in period is consistent with Bell's ~
(20) A 10-GPa elastic modulus and a Poisson ratio of 0.4 (typical of amorphous materiels) have been used to calculate the area of contact: Gavieh, B.;Gratton, E.; Hardy, C. J. Proc. Natl. Acad. Sci. U.S.A. 1983, 80,750. (21) Hendrickson, W. A.; Pahler, A.; Smith, J. L.; Satow, Y.; Merritt, E. A.; Philzackerley,R. P. Roc. Natl. Acad. Sci. U S A . 1989,86,2190. (22) Tabor, D. Rep. Prog. Appl. Chem. 1951,36,621. (23) Bell, G. I. Science 1978,200,618. (24) For a given probe-surface velocity and rupture force, a stiffer spring resulted in a shorter rupture period.
mode1.25 In fact, for h = 0.15 nm the calculated rupture forces, F(7 = 0.30 s) = 0.25 nN and F(T = 0.04 a) = 0.30 nN, are equivalentto the average measured adhesive forces (if the nonspecific adhesive force is taken into account). Evans and co-workers5 have related the number of adhesive interactions to the probability of observing a specific adhesive force using a generalized form of eq 1 and treating the intervals between the rupture of the ligand-receptor interactions as a random process. The resulting probability distribution of adhesive forces,which is a function of both the applied force and rupture period, shifts to larger forces with wider distributions as the number of ligand-receptor interactions increases. The narrow distribution of adhesive forces (Figure 4)and the skewed distribution of rupture times26 observed in this study are characteristic of single molecular interactions (ref 5,Figure A l ) . Therefore, analysis of the magnitude and distribution of the adhesive forces again leads us to conclude that the observed adhesive forces are predominantly the result of a single streptavidin-biotin interaction. This study has demonstrated that the elements of AFM may be used to directly measure a molecular recognition interaction while controlling the surface properties, area of contact, and environmental conditions. We believe this technique is versatile enough to be applied to synthetic and living molecular recognition systems. Potential applications include the simultaneous mapping of surface topography and receptor location on living cells, as well as chemical sensors, based on the measurement of intermolecular interactions.
Acknowledgment. This research has been funded by the Office of Naval Research (ONR), and G. U Lee acknowledges the support of the ASEE on an ONR Postdoctoral Fellowship. We would also like to recognize the useful conversations and assistance we have received from S. Brandow, B. Farrell, D. DiLella, B. Spargo, and A. McGill. (25) Equation 1 does not take into account the fact that the force applied to the streptavidin-biotin bond increases as a linear function of time. However, the constant-forceapproximation is reasonable as the rupture force is proportional to the logarithm of time (i.e., the constant force rupture period is less than 10% greater than the bond survival periods calculated with a linearly increasing force). For a thorough theoretical discussion, see Appendix 1 of ref 5. (26) For k = 0.12 N/m, T- = 0.02 8, and mi. = 0.20 8. For k = 0.048 N/m, r, = 0.27 s and T& = 0.47 8. (27) Tosi, M. P. SoZid State Phys. 1964, 16, 1. (28) Chiet, L.; Wolf, F. J. Arch. Biochem. Biophys. 1964,106, 1. (29) Kittel, C.Introduction to Solid State Physics, 6th ed.; J. Wiley and Sons: New York, 1986. (30) The sensitivity of the AFM detector was determined using the slope of the loading curve. The relative displacementof the surface and probe was calculated by subtracting the displacement of the cantilever from the piezo displacement. The force curves have been corrected for thermal drift, low-frequency optical interference, and piezo hyetemie. (31) Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1978, 66, 285. (32) Nir, S. Prog. Surf. Sci. 1976,8, 1.
