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Specific Interactions between Biotin and Avidin Studied by Atomic Force Microscopy Using the Poisson Statistical Analysis Method Yu-Shiu Lo, Neil D. Huefner, Winter S. Chan, Forrest Stevens, Joel M. Harris, and Thomas P. Beebe, Jr.* Department of Chemistry and Center for Biopolymers at Interfaces, University of Utah, Salt Lake City, Utah 84112 Received August 10, 1998. In Final Form: November 30, 1998 The interactions between biotin and avidin or streptavidin, a prototypical example of a specific biological ligand-receptor system, were studied by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and time-of-flight secondary ion mass spectrometry (TOF-SIMS). Although this and other ligandreceptor systems have been studied by several techniques, including AFM, in this paper, a statistical analysis method which makes use of the properties of the Poisson distribution was applied, and the rupture strength of an individual interaction was obtained from the total adhesion forces measured by the AFM. Tip- and surface-modification chemistries were investigated by XPS and TOF-SIMS. The magnitudes of the interactions between biotin-avidin and biotin-streptavidin pairs, as determined by the Poisson method, were found to be 173 ( 19 and 326 ( 33 pN, respectively, for loading rates between 2 × 105 and 8 × 105 pN‚s-1. These values are comparable to the values reported by other groups for the same systems. The statistical method used in this work has several advantages. It requires no assumptions about the surface energies or contact area between the AFM tip and the substrate, it is not limited by the force resolution of the instrument, and the number of measurements required to extract the individual unbinding force is significantly lower than that required by other methods.
Introduction Highly specific interactions between biomolecules, such as antigen-antibody, ligand-receptor, and complementary DNA-DNA interactions, play important roles in governing molecular recognition processes in numerous biological functions. The expression of genes, the functionality of an enzyme, and the protection of an organism’s cells are a few examples of processes that are dependent upon such interactions. The direct measurement of the forces involved in these interactions has become possible with the invention of sensitive force transducers such as the biomembrane force probe with micropipet suction,1-4 optical tweezers,5 and the surface forces apparatus (SFA).6-8 Although some of these techniques are very sensitive, some lack the spatial or force resolution necessary to measure discrete molecule-molecule interactions. Atomic force microscopy (AFM), which uses a minute tip as a force probe and a soft cantilever as a force transducer, combines moderately high force resolution and high spatial resolution, as well as the capability of operating under physiological conditions. This makes AFM well suited for investigations of biological interactions with modified AFM tips and substrates. (1) Evans, E.; Berk, D.; Leung, A Biophys. J. 1991, 59, 838-848. (2) Evans, E.; Berk, D.; Leung, A.; Mohandas, N. Biophys. J. 1991, 59, 849-860. (3) Berk, D.; Evans, E. Biophys. J. 1991, 59, 861-872. (4) Evans, E.; Ritchie, K.; Merkel, R. Biophys. J. 1995, 68, 25802587. (5) Kuo, S. C.; Sheetz, M. P. Science 1993, 260, 232-234. (6) Israelachvili, J. N. J. Colloid Interface Sci. 1973, 44, 259-272. (7) Horn, R. G.; Israelachvili, J. N. J. Chem. Phys. 1981, 75, 14001411. (8) Helm, C. A.; Knoll, W.; Israelachvili, J. N. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 8169-8173.
The forces between several ligands and receptors,9-14 antibodies and antigens,15-18 and single strands of DNA19,20 have now been studied with AFM. Biotin-avidin and biotin-streptavidin interactions are prototypical systems for ligand-receptor recognition studies because of their high specificity, high affinity, well-known solution-phase thermodynamic properties, ease of preparation of functionalized substrates, and wide applications in bioanalytical techniques. Biotin (MW ) 244.3), also known as vitamin H, is a coenzyme which plays an important role in many carboxylation reactions of metabolism.21 Avidin and the homologous protein streptavidin (MW ) 66 000 and 60 000, respectively) are tetrameric proteins. Each can bind up to four molecules of biotin with high affinity (9) Moy, V. T.; Florin, E.-L.; Gaub, H. E. Colloids Surf. A 1994, 93, 343-8. (10) Moy, V. T.; Florin, E.-L.; Gaub, H. E. Science 1994, 266, 257259. (11) Florin, E. L.; Moy, V. T.; Gaub, H. E. Science 1994, 264, 415-17. (12) Lee, G. U.; Kidwell, D. A.; Colton, R. J. Langmuir 1994, 10, 354-7. (13) 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, 161-164. (14) Chilkoti, A.; Boland, T.; Ratner, B. D.; Stayton, P. S. Biophys. J. 1995, 69, 2125-2130. (15) 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, 7457-7463. (16) Hinterdorfer, P.; Baumgartner, W.; Gruber, H. J.; Schilcher, K.; Schindler, H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 3477-3481. (17) Dammer, U.; Hegner, M.; Anselmetti, D.; Wagner, P.; Dreier, M.; Huber, W.; Guntherodt, H. J. Biophys. J. 1996, 70, 2437-2441. (18) Stuart, J. K.; Hlady, V. Langmuir 1995, 11, 1368-74. (19) Moy, V. T.; Florin, E.-L.; Rief, M.; Lehmann, H.; Ludwig, M.; Gaub, H. E.; Dornmair, K. Proc. SPIE Int. Soc. Opt. Eng. 1995, 2384, 2-12. (20) Lee, G. U.; Chrisey, L. A.; Colton, R. J. Science 1994, 266, 7713. (21) Lehninger, A. L.; Nelson, D. L.; Cox, M. M. Principles of Biochemistry, 2nd ed.; Worth Publishers: New York, 1993; pp 465467.
10.1021/la981003g CCC: $18.00 © 1999 American Chemical Society Published on Web 01/08/1999
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(dissociation constant Kd ) 10-15 M, which is among the strongest known protein-ligand interactions22,23). Such strong and specific binding between avidin or streptavidin and biotin has led to the general applicability of these biomolecules to form essentially irreversible and specific linkages between biological macromolecules in various immunochemical and diagnostic assays.24-26 Investigations of this strong recognition process have been carried out by several techniques8,22,23,27,28 including AFM,9-14 although none using the Poisson method employed here. In this paper, biotin-avidin and biotin-streptavidin interactions were studied by AFM using a novel Poisson statistical analysis method developed in our group. There are several advantages associated with the Poisson method: (1) it does not require any assumptions about surface energies or tip-surface contact areas, nor does it suffer from the large errors associated with attempting to measure the effective contact area; (2) it does not require a large number of force measurements, thus minimizing sample damage; and (3) it is not limited by the force resolution of the AFM instrument. The results to be presented here for the biotin-avidin and biotin-streptavidin interactions using the Poisson method are comparable to the values reported by groups for the same systems using other analysis methods. Biotin-modified surfaces were prepared via the essentially irreversible adsorption of a protein “glue”, bovine serum albumin (BSA; which had been previously modified by attaching biotin), onto the AFM cantilever tips and the glass substrates. Avidin-modified surfaces were prepared by adsorbing avidin onto the biotinylated surface or tip. Although this modification approach has been used in previous studies,9,12,13 no surface analytical characterization of the protein-modified surfaces has been performed to our knowledge. Therefore, a considerable portion of this work (see the Results section) is devoted to the surface analytical characterization of the substrates and AFM tips employed. It is the opinion of the authors that such a characterization, although often lacking in AFM measurements, is an essential “background” experiment necessary for a full understanding of tip-surface interaction forces. In this work, in order to confirm the immobilization of proteins onto the surfaces, the proteincoatedAFMcantilevertipsandsubstrateswerecharacterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (TOF-SIMS), and contact-angle measurement. Poisson Statistical Analysis. The statistical method of force curve analysis used in this study is described in detail in previously published papers from our group.29-32 Like any other processing technique, certain assumptions (22) Chilkoti, A.; Stayton, P. S. J. Am. Chem. Soc. 1995, 117, 1062210628. (23) Weber, P. C.; Ohlendorf, D. H.; Wendoloski, J. J.; Salemme, F. R. Science 1989, 243, 85-88. (24) Diamandis, E. P.; Christopoulos, T. K. In Immunoassay; Diamandis, E. P., Christopoulos, T. K., Eds.; Academic Press: San Diego, 1996. (25) Deshpande, S. S. Enzyme Immunoassays: From Concept to Product Development; Chapman & Hall: New York, 1996. (26) Wild, D. In The Immunoassay Handbook; Wild, D., Ed.; Stockton Press: New York, 1994. (27) Miyamoto, S.; Kollman, P. A. Proteins 1993, 16, 226-245. (28) Grubmu¨ller, H.; Heymann, B.; Tavan, P. Science 1996, 271, 997999. (29) Wenzler, L. A.; Moyes, G. L.; Raiker, G. N.; Hansen, R. L.; Harris, J. M.; Beebe, T. P., Jr. Langmuir 1997, 13, 3761-3768. (30) Wenzler, L. A.; Moyes, G. L.; Harris, J. M.; Beebe, T. P., Jr. Anal. Chem. 1997, 69, 2855-2861. (31) Williams, J. M.; Taejoon, H.; Beebe, T. P., Jr. Langmuir 1996, 12, 1291-1295. (32) Han, T.; Williams, J. M.; Beebe, T. P., Jr. Anal. Chim. Acta 1995, 307, 365-76.
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regarding the system must be made before the technique can justifiably be applied to a set of data. Assumptions made in the Poisson method are that the total adhesive force in an AFM pull-off event is composed of a finite number of discrete interacting bonds and that the distribution of the number of interacting bonds formed at the pull-off point from multiple measurements within a fixed contact area follows a Poisson distribution. Although the distribution is strictly binomial, a low probability for the occurrence of a given event (i.e., bond formation) can be approximated by a Poisson distribution.33 Factors such as steric hindrance and direction of approach can play a significant role in the interactions between biological systems. Thus, it is a reasonable assumption that the bondformation probability is low with the biotin-avidin system. This assumption is further supported by the close fitting of Poisson distribution curves to the histograms of AFM force measurements taken in the biotin-avidin and biotin-streptavidin systems. As defined by the Poisson distribution, the mean and the variance of the number n of interacting bonds formed will be the same, i.e., µn ) σn2. The adhesive force m measured in one force-distance curve is related to the number of bonds ruptured during a pull-off event by
m ) nFi
(1)
where Fi represents the average individual bond-rupture force in the system, the value that we hope to determine. A sampling of many of these events produces a mean measured pull-off force µm and a pull-off force variance, σm2. On the basis of the relationship between the measured force and the number of bonds ruptured, we can derive the following equations:
µm ) µnFi
(2)
σm2 ) σn2Fi2
(3)
Because µn ) σn2 for Poisson, the magnitude of Fi can be determined from easily measured quantities as shown below.
Fi ) σm2/µm
(4)
When any possible nonspecific and long-range interactions, F0, are taken into account, eqs 2 and 3 become:
µm ) µnFi + F0
(5)
σm2 ) σn2Fi2 ) µmFi - FiF0
(6)
A linear regression curve of the variance (σm2) versus the mean (µm) of the pull-off force from several sets of measurements taken at different locations and with different tips and surfaces will give the magnitude of the individual bond-rupture force Fi and the product -FiF0 as the slope and intercept, respectively. The latter quantity can be used to estimate any nonspecific “background” forces that may be present. It is important to note that, in most biological systems, several different chemical forces are involved in the interactions between biomolecules such as protein-ligand and antibody-antigen complexes. From X-ray crystallography studies, it is known that multiple hydrogen bonds and van der Waals interactions are associated with the (33) Barlow, R. J. Statistics: A guide to the use of statistical methods in the physical sciences; John Wiley & Sons: New York, 1989; p 204.
Specific Interactions between Biotin and Avidin
biotin-avidin or biotin-streptavidin complex.23 In the Poisson analysis described above, the interaction between one biotin-avidin or biotin-streptavidin pair is considered as an individual bond and Poisson statistics is used to describe the distribution of a discrete number of interacting biotin-avidin or biotin-streptavidin pairs during a set of AFM force measurements. Thus, it is the average “overall” interactions of individual biotin-avidin or biotinstreptavidin pairs, not the specific chemical interactions involved (hydrogen bonds or van der Waals forces), that are extracted with the Poisson statistical analysis in this work. A computer modeling study of AFM bond-rupture force measurements was recently carried out in our group with the bond-formation probability, individual bond strength, and nonspecific background force as variable parameters.34 The computer modeling work provides a much more detailed discussion about the feasibility and applicability of our statistical analysis method for different systems, and the interested reader is referred to this and other studies from our group.29-32,34 Experimental Section Preparation of Modified Tips and Substrates. Glass microscope slides (Fisher Scientific) and as-received commercial Si3N4 AFM cantilever tips (Park Scientific) were soaked in a mixture of concentrated H2SO4 and 30% H2O2 (70:30, v/v) (“piranha solution”) for 30 min. This mixture has very strong oxidizing power and is extremely dangerous to handle in the lab; goggles, face shields, and gloves are needed for protection.35 It was found from TOFSIMS analysis that commercial AFM cantilever tips shipped and stored on silicone or poly(dimethylsiloxane) (PDMS) pads are contaminated with silicone oils which can be effectively removed only by piranha solution.36 Glass substrates were washed five times by ultrasonication in 18 MΩ‚cm Milli-Q water (Millipore) and then dried at 150 °C for 1 h. The AFM cantilever tips were rinsed with copious amounts of 18 MΩ‚cm Milli-Q water and then dried at 150 °C for 1 h. To create biotin-functionalized tips and surfaces, the clean glass substrates and Si3N4 AFM cantilever tips were incubated in 1 mg/mL biotinylated bovine serum albumin (BBSA; Sigma) solution in phosphate-buffered saline (PBS; 20 mM Na2HPO4, 150 mM NaCl, Milli-Q water, pH 7.0) at room temperature overnight. Avidin- and streptavidin-functionalized tips and substrates were prepared freshly from biotin-modified surfaces via 30 min incubation in 100 µg/mL avidin or streptavidin (ICN) in PBS before force measurement. Force Measurements. A commercial AFM system (Topometrix Explorer) with a typical optical beam deflection detection system was used to obtain force-distance curves. The noise level of this instrument was ∼30 pN.37 Commercial AFM cantilevers (Park Scientific) were modified as described above. The force constants of the (34) Stevens, F.; Lo, Y.-S.; Harris, J. M.; Beebe, T. P., Jr. Computer Modeling of Atomic Force Microscopy Force Measurements: Comparisons of Poisson, Histogram, and Continuum Methods. Langmuir, in press. (35) Kern, W. In Handbook of Semiconductor Wafer Cleaning Technology; Kern, Werner, Ed.; Noyes Publications: Park Ridge, NJ, 1993. (36) Lo, Y.-S.; Huefner, N. D.; Chan, W. S.; Beebe, T. P., Jr. Organic and Inorganic Contamination on As-Received AFM Cantilevers. In preparation. (37) The noise level was estimated from the standard deviation of the flat portions of sampled force-distance curves before jump-in during the advancing process and after pull-off during the retracting process. Sources of noise that affect the force sensitivity of the instrument include thermal and mechanical vibrations and intensity fluctuations of the laser used in the optical detection system.
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cantilevers were determined from their individually measured resonance frequencies with a series of added masses.38 The average force constants for the two types of cantilevers employed here were 0.039 ( 0.001 and 0.150 ( 0.011 N/m. All force measurements were made with individually calibrated cantilever force constants, not an average or nominal manufacturer’s value. The forcedistance curves were obtained under a PBS medium in a home-built liquid cell. All force-distance measurements were performed at a vertical scan rate of 5 µm/s, which is less than the scan rate reported by Hoh39 to cause oscillations at the pull-off point. As a result, loading rates ranged from 2 × 105 to 8 × 105 pN‚s-1. XPS. XPS analysis in this study was performed using an ESCALab 220i-XL (Fisons, U.K.) with a monochromatic Al KR (1486.7 eV) X-ray source. Survey spectra were acquired using a 100 eV pass energy while high-resolution, multiplex spectra of the individual elements were acquired using a 20 eV pass energy, and these spectra were signal averaged for 50 scans. A low-energy electron flood gun was used to stabilize and compensate for sample charging. Peak positions were assigned by referencing the methylene component of the C 1s peak to a binding energy of 284.6 eV and linearly shifting all other peaks by an equal amount, as is customary. Contact-Angle Measurement. A standard contactangle goniometer (model A-100, Rame-Hart) and microscope were used to make contact-angle measurements on modified surfaces. Water (18 MΩ‚cm) was used as the sessile drop for all samples. Advancing contact-angle measurements were made visually within 30 s after application of the drop to the sample. Each value was reported as an average with standard deviation from at least 12 measurements. TOF-SIMS. Static TOF-SIMS was performed with the TOF SIMS IV by Cameca/ION-TOF (Paris/Mu¨nster). Spectra were acquired using a 30 keV Ga+ primary ion pulse with a current of 1.2 nA and a pulse width of 15 ns and an irradiated area of approximately (250 µm)2 for positive-ion spectra and (120 µm)2 for negative-ion spectra, resulting in a mass resolving power M/∆M greater than 6000. To obtain spatially resolved ion maps, Ga+ was used as the primary ion with a kinetic energy of 30 keV and a current of 1.2 nA. The images were obtained with a pulse width of 70 ns, and image sizes are given in the figures. In all cases, charge compensation was achieved by applying low-energy electrons (∼30 eV) from a pulsed flood gun. All measurements were made below the static flux limit of 1 × 1012 primary ions/cm2. Results XPS Surface Characterization. Before the AFM force measurements were obtained, XPS, TOF-SIMS, and contact-angle measurements were performed to determine the optimal conditions of protein adsorption. After being incubated in different concentrations of bovine serum albumin (BSA) solutions in phosphate-buffered saline (PBS) overnight, BSA-coated glass substrates were rinsed with deionized water for about 1 min to remove ionic species before surface characterization was carried out. Figure 1 shows the survey XPS spectra of a clean glass substrate and a substrate incubated overnight in a 1 mg/ mL BSA solution. High-resolution elemental narrow scans of C 1s and N 1s regions on the sample incubated in a 1 mg/mL BSA solution are shown in Figure 2. Three different (38) Cleveland, J. P.; Manne, S.; Bocek, D.; Hansma, P. K. Rev. Sci. Instrum. 1993, 64, 403-405. (39) Hoh, J. H.; Engel, A. Langmuir 1993, 9, 3310-12.
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Figure 1. XPS survey spectra of (A) a clean glass substrate and (B) a glass substrate after incubation in a 1 mg/mL BSA protein solution overnight. Arrows shown in B indicate the major peaks of C and N due to the adsorption of BSA protein onto the substrate.
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Figure 2. High-resolution XPS spectra of a BSA-coated glass substrate. (A) High-resolution scan of the C 1s region. (B) Highresolution scan of the N 1s region. Dotted peaks represent spectral decomposition of the data. Three types of C and two types of N expected in the protein BSA are shown in these XPS elemental narrow scans.
types of carbon are present in proteins in significant amounts: alkyl, carbonyl, and ether. Peaks for all three are clearly resolved and visible in the fitted C 1s spectrum of the BSA-incubated glass substrate. The presence of the N 1s peak in the spectrum of a BSA-incubated substrate (which is not seen on the clean glass substrate) also indicates the adsorption of BSA on the glass substrates. High-resolution XPS spectra in the N 1s region exhibited primarily the amide species (399.6 eV) and to a lesser extent the protonated amine species (400.9 eV). Peak areas of the photoelectron signals from N atoms in XPS survey spectra were used to quantify the amount of BSA adsorbed on the glass substrates and will be discussed below.40 XPS spectra were also acquired from BSA-modified AFM cantilevers. Due to the fact that the AFM cantilevers were made of Si3N4, as well as the ubiquitous hydrocarbon contamination present on almost any surface, no unambiguous evidence of protein adsorption on AFM cantilevers could be proved by the appearance of N and C peaks in the XPS survey spectra. However, elemental small-spot spectra of C 1s and N 1s regions on the cantilevers (data not shown) incubated in 1 mg/mL BSA did exhibit separate peaks for the several types of carbon and nitrogen present in the BSA protein. These peaks, similar to those depicted in Figure 2, were not seen in the spectra of the same regions on unmodified control cantilevers. This is indicative of the adsorption of protein onto the cantilevers.
Contact-Angle Measurements. Advancing contactangle measurements were made on glass substrates incubated, for a fixed amount of time overnight (24 h), in a range of protein concentrations. The glass substrates underwent an obvious increase in hydrophobicity as the concentration of incubation protein solutions increased. A plot of the contact angles and the atomic percentages of different elements from XPS versus BSA concentration is shown in Figure 3. Both curves reach a plateau at a point corresponding to a concentration of ∼1 mg/mL and indicate that a 1 mg/mL BSA solution is suitable for preparation of BSA-covered substrates under our experimental conditions. TOF-SIMS Surface Characterization. TOF-SIMS analysis was performed on both an unmodified control AFM cantilever tip and an AFM cantilever incubated in 1 mg/mL BSA solution. Peaks present only for the BSAmodified AFM cantilever sample in the positive-ion spectrum that are characteristic of amino acids included peaks at m/z 30 (CH4N+; Gly), 70 (C4H8N+; Pro), and 84 (C4H6NO+ and C5H10N+; Glu and Lys) and many others.41,42 Several alkyl fragments were also present in the positiveion spectrum. These hydrocarbon fragments are an expected part of any spectrum of a biological specimen such as a random-sequence protein, or they could result from ubiquitous hydrocarbon contamination. Protein
(40) The equation Cx ) (Ix/Sx)/(∑Ii/Si) was used for the calculation of the atomic concentration ratio of the species on surfaces. C, I, and S represent the atomic concentration, XPS intensity, and XPS sensitivity factor, respectively.
(41) Lhoest, L.-B.; Castner, D. G. Time-of-Flight SIMS studies of adsorbed protein mixtures. 11th Annual SIMS Workshop, Austin, TX, 1998; p 41. (42) Mantus, D. S.; Ratner, B. D. Anal. Chem. 1993, 65, 1431-1438.
Specific Interactions between Biotin and Avidin
Figure 3. Plot of (A) contact angle and (B) atomic percentage of different elements calculated from XPS vs BSA solution concentration. The curves of contact angle and N atomic percentage, a characteristic element of proteins, reach a plateau at about 1 mg/mL and indicate that a 1 mg/mL solution of BSA is suitable for the preparation of protein-covered substrates under these experimental conditions. Error bars were calculated from replicate measurements.
peaks in the high-mass region provided additional evidence of the adsorption of BSA proteins on the surfaces. CN- and CNO- peaks in the negative-ion spectrum (data not shown here) also indicated the presence of protein on the AFM cantilever tips. A portion of a positive-ion spectrum as well as ion maps of some characteristic amino acid fragments obtained by TOF-SIMS is shown in Figure 4. While hydrogen and oxygen exist in substantial amounts in proteins, they are also found in many common contaminants. As such, the first two images (B and C) serve primarily to define the shape, position, and condition of the AFM cantilever. The vertical feature covering the farright quarter of the image is not part of the AFM cantilever chip. It is a glass cover slip used to establish a more uniform extraction field conducive to ion extraction and collection. The two subsequent images (D and E) depict ion maps of two negative ion fragments common to proteins, CN- and CNO-. Arrows and labels in the positive-ion spectrum point out the position of several peaks indicative of protein adsorption. These signals indicate the presence of proteins on the cantilever, although neither the TOF-SIMS nor the XPS data can be used to infer conformational or functional integrity of the adsorbed proteins. Force-Distance Measurement. Figure 5 shows representative examples of the force-distance curves acquired in these systems. No noticeable pull-off force was observed in the BBSA-BBSA control system (Figure 5A). In the BBSA-avidin (Figure 5B) and BBSAstreptavidin (Figure 5C) systems, however, a clear force hysteresis was present as a result of the adhesion force between the biotin-avidin and biotin-streptavidin pairs. This suggests that the force measured in the BBSA-avidin
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Figure 4. (A) TOF-SIMS positive-ion spectrum of a BSA-coated AFM cantilever. Peaks typical of proteins are labeled. (B-E) Spatially resolved TOF-SIMS ion maps of a BSA-coated AFM cantilever. Images B and C depict ion maps of hydrogen and oxygen and serve primarily to define the shape, position, and condition of the AFM cantilever. Images D and E depict ion maps of two negative ion fragments common to proteins, CNand CNO-. These signals indicate the presence of proteins on the cantilever. It can be inferred that the protein is present in a uniform coverage (not patchy) on the scale of microns. The vertical feature covering the far-right quarter of the image is not part of the AFM cantilever chip. It is a glass cover slip used to establish a more uniform extraction field conducive to ion extraction and collection.
system can be attributed to the specific biotin-avidin interaction. Blocking Experiments. Additional evidence that the force measured is due to the binding of biotin to its complement receptor comes from “blocking” control experiments in which a small amount of free avidin or streptavidin solution was injected between BSA-avidin or biotinstreptavidin systems during force measurements. The free avidin or streptavidin molecules apparently bound to the biotin on the BBSA-modified surface. As a result, the tip and substrate no longer had an affinity for one another, and the adhesive forces detected prior to injection disappeared. Multiple Pull-off Events and Zero Pull-off Events. In some force curves, there were multiple steps in the retracting process which may be attributed to multiple or sequential breaking of interacting bonds between the probe and the substrate.11,15-17 A number of other groups have also related this experience anecdotally at recent meetings. When observed, the adhesive forces were measured and treated individually for each pull-off step in this work. In
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Lo et al. Table 1. Results for the Unbinding Forces between Biotinylated Bovine Serum Albumin (BBSA) and Avidin in pH 7.0 Phosphate-Buffered Saline data set
tip-substrate modification
mean force µm (pN)
variance σm2 (pN2)
size of set N
A B C D E F G
avidin-BBSA avidin-BBSA BBSA-avidin BBSA-avidin avidin-BBSA BBSA-avidin BBSA-avidin
822 386 752 801 631 979 760
1.14 × 105 0.55 × 105 1.21 × 105 1.32 × 105 1.00 × 105 1.63 × 105 1.12 × 105
47 55 36 50 42 50 51
Table 2. Results for the Unbinding Forces between Biotinylated Bovine Serum Albumin (BBSA) and Streptavidin in pH 7.0 Phosphate-Buffered Saline
Figure 5. Representative force-distance curves of (A) BBSABBSA control, (B) BBSA-avidin, and (C) BBSA-streptavidin systems under phosphate-buffered saline at pH 7. Part A shows no interactions, as expected for this control system with no protein receptors present. This indicates that the forces measured in B and C are due to the interactions within biotinavidin and biotin-streptavidin pairs.
addition to force curves in which multiple steps were observed, approximately two-thirds of the force curves exhibited no noticeable interaction. The Poisson method does predict some zero-force pull-off events, and the number predicted depends on the mean of the Poisson distribution. All zero-force pull-off events were neglected in our calculations, and the effect this could have on our results is discussed in subsequent sections and in the Appendix to this paper. Application of the Poisson Method. Several sets of measurements were taken and the mean and variance calculated for each set. Each set was composed of 36-74 force measurements, as summarized in Tables 1 and 2. The variance-vs-mean plots for the data collected in the BBSA-avidin and BBSA-streptavidin systems are shown in Figure 6. As discussed above, the slope of the linear regression fit of the variance-vs-mean plot represents the magnitude of a single unbinding force between the ligand and receptor. The individual unbinding forces in the
data set
tip-substrate modification
A B C D E F G
BBSA-streptavidin BBSA-streptavidin BBSA-streptavidin BBSA-streptavidin BBSA-streptavidin streptavidin-BBSA streptavidin-BBSA
mean force variance µm (pN) σm2 (pN2) size of set N 311 356 621 801 617 1010 1794
1.54 × 104 2.03 × 104 1.26 × 105 1.92 × 105 1.16 × 105 3.35 × 105 4.76 × 105
50 40 73 57 74 65 52
BBSA-avidin and BBSA-streptavidin systems were found to be 173 ( 19 and 326 ( 33 pN, respectively, from their linear regression slopes in Figure 6. These values showed good agreement with those that have been reported for the same systems by other groups.43 A comparison of these values is shown in Table 3. Thermodynamic parameters associated with the binding of biotin to avidin and to streptavidin44 are also included in Table 3. Through application of the Poisson method, the nonspecific interaction, F0, can be determined from the intercept of the linear regression curve, -FiF0 (see eq 6), in the variance-vs-mean plot. The nonspecific interactions in the biotin-avidin and biotin-streptavidin systems, calculated from the ratio of the absolute value of the intercept (FiF0) and the slope (Fi) in Figure 6, were found to be 75 ( 85 and 225 ( 96 pN, respectively. On the basis of the Student’s t test, in the biotin-avidin pair, the nonspecific interaction F0 can be considered as zero with very high confidence. In the latter system, the biotinstreptavidin pair, the nonspecific interaction F0 is not significantly different from zero at the 95% confidence level. A possible factor that results in a slightly negative intercept in the variance-vs-mean plots arises from neglect of the zero-force pull-off events in the data processing, which will be discussed in more detail below. Based on the results of the control and blocking experiments where no interaction was detected, and because of the fact that the zero-force pull-off events were neglected in data processing, both the biotin-avidin and biotin-streptavidin systems in this work are believed to exhibit no significant nonspecific interactions. A histogram of pull-off forces from one set of measurements (set F from Table 1) and the theoretical curve of the Poisson distribution with the same mean is shown in Figure 7. The curves which exhibited no adhesive force were not used in data processing and are not included in the histogram (see Appendix). The theoretical curve was (43) Evans52 and Shapiro53 have argued persuasively that the bondrupture force is not a “constant” that can be tabulated in the absence of consideration of the loading rate under which the bond was ruptured. (Conditions such as solvent, pH, etc., may also affect the measured bond force.) The measured bond-rupture forces that we have reported should therefore be considered as conditional bond-rupture forces measured under a specific set of conditions. (44) Swamy, M. J. Biochem. Mol. Biol. Int. 1995, 36, 219-225.
Specific Interactions between Biotin and Avidin
Figure 6. Plots of force variance σ2m vs mean µm for the (A) BBSA-avidin and (B) BBSA-streptavidin systems acquired under phosphate-buffered saline at pH 7. Each point represents a data set taken with a different tip and/or sample location, as shown in Tables 1 and 2. The filled-circle symbols in part A were obtained several months after the initial data sets at the suggestion of a reviewer. They fit well onto the prior data trend, supporting the applicability of the method and reproducibility of the chemistry.
constructed using the experimental mean force and individual bond-rupture force to calculate the mean number of ruptured bonds. This value was then used in the formula of the Poisson distribution to calculate the theoretical frequency of different bond-rupture events. The measurements fit the Poisson curve reasonably well, supporting the applicability of this analysis method. Two advantages of the Poisson analysis method include the fact that it can be applied to AFM data that does not have force resolution high enough to resolve weak discrete unbinding events (this is generally the case for commercial AFM instruments and noncovalent bonds), and the number of measurements required to obtain an individual bond-rupture force is much less than that required using other methods. Discussion Surface Characterization. From the results of XPS atomic percentage and contact-angle measurements on substrates incubated overnight in BSA solutions of various concentrations (Figure 3), both reached a plateau at a concentration of ∼1 mg/mL. Although the curves exhibit shapes similar to the Langmuir adsorption isotherm, the detection of a significant intensity from silicon (which came from the glass substrate and AFM cantilever) in the XPS analysis indicates that the surfaces were not completely
Langmuir, Vol. 15, No. 4, 1999 1379
covered with protein. The adsorption of protein has been addressed using the random sequential adsorption (RSA) model.45,46 This model takes the size-exclusion effects of the adsorbed molecules into account and is applicable to cases when molecules irreversibly adsorb onto surfaces and exhibit no diffusionscases for which it would be inappropriate to apply a Langmuir model.47-49 The RSA model predicts that, at the so-called jamming limit, the surface coverage of a monolayer of “disklike” molecules irreversibly adsorbed is about half. It has been found that BSA irreversibly adsorbs onto silica and silicon nitride materials,11,12,50 and it is known that most proteins have a low diffusivity.11,12,50 This suggests that the RSA model is applicable to our system. The RSA model predicts a Langmuir-like curve and explains the presence of silicon in the XPS spectra. Because the elemental quantification from XPS data could not easily be used to determine the surface coverage on an absolute scale, no thermodynamic information such as an adsorption constant could be extracted by fitting the atomic percentage curve in Figure 3B to the RSA model. Because this was not the primary concern of our work, no further effort was spent on this issue. Biotin-Avidin and Biotin-Streptavidin Interactions. In most sets of the force-distance measurements in both the biotin-avidin and biotin-streptavidin systems, approximately half to two-thirds of the curves exhibited no adhesive force. The population of these “zeroforce pull-off curves” was much higher than would be expected from the Poisson statistics of the interacting bonds between the biotin and avidin or streptavidin pairs, given the means typically observed. Such a high number of zero-force pull-off curves can be explained by the inactive engagement of the BBSA and the avidin (or streptavidin) on the tips and the substrates. When this occurs, there is no ability for the biotin on the BBSA-modified surface to reach the biotin-binding sites on the avidin- (or streptavidin-)modified surface. This is a different population from the nonbinding events attributed to the statistics of interacting bonds. In other words, more than one statistical explanation is required to account for all of the zero-force pull-off events. Because at the present time we have not been able to find a way to distinguish between zero-force pull-off curves from these different populations, we have omitted all zero-force measurements in our data processing. It has been proved analytically that the relative error of the variance-over-mean ratio caused by neglecting the zero events in a Poisson distribution depends on the mean of that population, reaches a maximum of only ≈0.37 when the mean equals 1, and exponentially decays for means greater than 1. A detailed mathematical proof is given in the Appendix. By neglecting the zero-force pull-off events, which causes a mean-dependent error in the variance-over-mean ratio as mentioned above, both the slope and intercept of the linear regression curve in the variance-vs-mean plot are affected. This affects, to a small degree, both the value obtained for the individual bond-rupture force Fi and the value obtained for the nonspecific interaction F0. Depending on the range of mean covered in the plot, the error for the slope varies only from ∼10% to ∼20%; however, a (45) Ramsden, J. J. Biosens. Bioelectron. 1996, 11, 523-528. (46) Van Tassel, P. R.; Viot, P.; Tarjus, G. J. Chem. Phys. 1997, 106, 761-770. (47) Schaaf, P.; Talbot, J. J. Chem. Phys. 1989, 91, 4401-4409. (48) Hinrichsen, E. L.; Feder, J.; Jossang, T. J. Stat. Phys. 1986, 44, 793-827. (49) Widom, B. J. Chem. Phys. 1966, 44, 3888-3894. (50) Andrade, J. D. Surface and Interfacial Aspects of Biomedical Polymers; Andrade, J. D., Ed.; Plenum Press: New York, 1985; Vol. 2.
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Table 3. Comparison of Unbinding Forces between Biotin-Avidin and Biotin-Streptavidin Pairs Measured by AFM from Different Research Groupsa thermodynamic parameters Ka ) 1.67 × 1015 M-1 ∆G ) -20.75 kcal‚mol-1 ∆H ) -23.40 kcal‚mol-1 T∆S ) -2.65 kcal‚mol-1 Ka ) 2.50 × 1013 M-1 ∆G ) -18.3 kcal‚mol-1 ∆H ) -32.0 kcal‚mol-1 T∆S ) -13.7 kcal‚mol-1
unbinding force (pN)
method
ref
Biotin-Avidin 173 ( 19 160 ( 20
Poisson statistics histogram autocorrelation
this work 11
Biotin-Streptavidin 326 ( 33 257 ( 25 340 ( 120 409 ( 166
Poisson statistics histogram autocorrelation discrete-pair measurement discrete-pair measurement
this work 11 12 13
a Thermodynamic parameters for the binding of biotin to avidin and streptavidin are included for informational purposes. These were taken from ref 43.
ments are required, and that the resolution of the instrument is less of a limiting factor when the weak noncovalent interactions between biological molecules are studied. Finally, no model-dependent assumptions about surface energies or tip-surface contact area are required. Instead, one obtains the number of individual bonds ruptured. Conclusion
Figure 7. Histogram of the unbinding forces from one set of 50 measurements (set F from Table 1) between a BBSA-coated AFM tip and an avidin-coated substrate within a fixed contact area under pH 7 phosphate-buffered saline. The data for this system fits a Poisson distribution curve PP(x) ) e-µµx/x! reasonably well, as did other data sets (not shown).
significant change in the y-intercept does result from neglect of the zero-force pull-off events. This shift in intercept can potentially mask any true nonspecific interactions in the experimental system. A more detailed discussion on this can be found in the Appendix. Force “Quantization”. Histograms of force measurement sets taken from the biotin-avidin (and biotin-streptavidin) system were generated using a bin size of 10 pN (data not shown here). These histograms did not exhibit any observable periodicity. To observe unambiguous “quantized” results, the signal-to-noise ratio must be high. The signal-to-noise ratio in our data was too low to exhibit clear periodicity. Factors contributing to the low signalto-noise ratio include the relatively small number of measurements obtained for the Poisson method when compared to the several hundreds or even thousands of measurements typically required by the histogram method to see the quantization of forces.11,17,51 Another important limitation is the force resolution of a typical commercial AFM instrument such as the one used in this study. The agreement of the present results obtained using the Poisson method and the values obtained by others using methods such as histogram quantization suggests that the Poisson method is comparable to these methods and is applicable to such biological systems. In addition, the Poisson method has some advantages over these other methods including the fact that typically fewer measure(51) Hoh, J. H.; Cleveland, J. P.; Prater, C. B.; Revel, J.-P.; Hansma, P. K. J. Am. Chem. Soc. 1992, 114, 4917-4918. (52) Evans, E.; Ritchie, K. Biophys. J. 1997, 72, 1541-1555. (53) Shapiro, B. E.; Qian, H. Biophys. Chem. 1997, 67, 211-219.
Biotin-avidin and biotin-streptavidin interactions, a widely investigated prototype of biomolecular recognition systems, have been studied by AFM with a unique statistical analysis method developed in our group. The results from this Poisson approach were comparable to the values obtained by groups who used other data processing methods for the same systems. It was found in this study that neglect of the zero-force pull-off events in the Poisson method did not introduce large errors in the analysis. As long as the criterion of low bond-forming probability is fulfilled, a variety of biological interactions can be studied using this statistical method without making assumptions regarding the tip geometry or being limited by the resolution of the instrument. In addition, the number of measurements required to apply this method is comparatively lower than that required in most other methods, such as the histogram and autocorrelation methods. More studies of other systems are being carried out and a modification of the statistical analysis method is being developed to make it a more general technique for various types of systems. Acknowledgment. The authors thank Paul Dryden for help in obtaining XPS data. Birgit Hagenhoff of Tascon/ ION-TOF and Felix Kollmer, Ralf Kamischke, and Alfred Benninghoven of University of Mu¨nster are acknowledged for collaborations involving TOF-SIMS. This work was supported by the National Science Foundation (CHE9357188), the Center for Biopolymers at Interfaces, the University of Utah Research Committee, and the UROP program at the University of Utah to N.D.H. and W.S.C.. Appendix In a Poisson distribution, the probability PP of observing x events, when the expected number of events has a mean of µ, is given by the formula
PP(x) ) e-µµx/x!
(1)
The total probability is 1. ∞
∑PP(x) ) 1 x)0
(2)
Specific Interactions between Biotin and Avidin
Langmuir, Vol. 15, No. 4, 1999 1381
Therefore, the probability of a “zero event” occurrence is
PP(x)0) ) e-µµ0/0! ) e-µ
(3)
and the probability for all other occurrences, in which one or more events are involved, is
PP(xg1) ) 1 - PP(x)0) ) 1 - e-µ The mean and variance for a system can be described by eqs 4 and 5. ∞
µ)
∑
∞
xPP(x) )
x)0
∑
∞
xPP(x) + 0PP(0) )
x)1
∞
σ2 )
∑xPP(x)
x)1
(4)
∞
∑(x - µ)2PP(x) ) x)0 ∑(x2 - 2µx + µ2)PP(x) x)0
Figure 8. Plot of the relative error of the variance-vs-mean ratio in a Poisson distribution from neglect of the zero-event occurrences. Ignoring all zero-event occurrences, the ratio of apparent variance vs apparent mean becomes 1 - e-µ. The second term, e-µ, is the relative error plotted in this figure. See text of Appendix.
∞
)
∑(x2 - 2µx + µ2)PP(x) + µ2PP(0) x)1
)
(x2 - 2µx + µ2)PP(x) + µ2e-µ ∑ x)1
∞
(5)
By neglecting of all instances for which x ) 0, an apparent mean µapp and variance σapp2 are observed and can be represented by eqs 6 and 7. ∞
µapp )
∑xPP(x) x)1 ∞
∑
µ
)
PP(x)
(6)
1 - e-µ
x)1 ∞
∑(x - µ)2PP(x)
σapp2
)
x)1
)
∞
∑
PP(x)
σ2 - µ2e-µ 1 - e-µ
x)1
)
-µ µ - µ2e-µ µ(1 - µe ) ) 1 - e-µ 1 - e-µ
(7)
One of the most important properties of the Poisson distribution is that the mean and variance of the number of events are equal. By determining the ratio of the apparent variance and mean (resulting from neglect of zero events),
σapp2 ) 1 - µe-µ µapp
(8)
we can predict a relative error of -µe-µ in the variancevs-mean ratio when all zero event occurrences are discarded. This error is dependent on the mean number of events µ. A plot of the relative error in the variance to mean ratio versus mean number of events is shown in Figure 8. The maximum relative error, ∼37%, occurs when the mean number of events equals 1. When the mean number of events is larger than 4, the relative error in the variance to mean ratio due to ignoring all zero event occurrences is less than 10%.
Figure 9. Plot of variance vs mean of a set of 10 Poisson distributions using true analysis and the apparent analysis which neglects all of the zero events. Each data set has an integral mean ranging from 1 to 10. Within this mean range, the linear regression curve from the apparent analysis has a slope ∼10% larger than that from the true analysis and a negative y intercept. As a result, in the Poisson method, neglect of zero-force pull-off events may cause a slight overestimate of individual bond-rupture force (from slope) and indicate a false nonspecific interaction (from y-intercept). These errors are estimated to be less than 10% (see Appendix).
To study how much neglect of the zero events affects the Poisson method, several true and apparent analyses were performed using Poisson statistics with integral means ranging from 1 to 10 bonds formed. This range was chosen because it included the mean number of bonds ruptured for the biotin-avidin and biotin-streptavidin data sets. The results are plotted in Figure 9. In the true analysis, the variance of each data set equaled its mean, as must be true for the Poisson distribution. For the apparent analysis, the apparent means and variances were calculated using eqs 6 and 7. Based on eq 8, the apparent analysis of neglecting the zero events always causes a negative error in the variance-over-mean ratio. This, in turn, results in the negative deviation of data points away from the true Poisson analysis in the variance-vs-mean plot, which forms a straight line with a slope of 1. As a result, the linear regression curve of the apparent analysis has a slope greater than 1 and a negative y intercept. It can be seen in Figure 9 that for the apparent analysis the slope of the linear regression curve is ∼10% higher than that of the true Poisson analysis and the negative y intercept is significantly different from zero. When this is related to the Poisson method used in this work to study
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molecular interactions between the AFM tip and the surface, neglect of the zero-force pull-off events can predict a slightly greater individual bond-rupture force Fi (∼10% in slope) and a false nonspecific interaction F0 (∼0.7Fi in y intercept), which is not actually present for control experiments in the system. The foregoing derivation and conclusion provide some confidence that by ignoring all zero-force pull-off events in the AFM data analyzed here, we have not introduced inordinately large errors in the analysis. This is fortunate,
Lo et al.
because it is not possible to distinguish between zeroforce pull-off events that result from failure of an otherwise proximal ligand-receptor pair to form a bond (a case in which we would desire to include the zero-force event) and those that result from tip-surface touches where there are no possible ligand-receptor pairs within bonding distance (a case in which we would desire to exclude the zero-force event). LA981003G
