Effect of Mechanical Contact on the Molecular Recognition of

Several developing technologies rely upon the activity of surface-bound biomolecules to control the functionality of an interface. The specificity of ...
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Langmuir 1999, 15, 238-243

Effect of Mechanical Contact on the Molecular Recognition of Biomolecules John-Bruce D. Green,† Alexey Novoradovsky,‡ and Gil U Lee*,† Chemistry Division, Code 6177, Naval Research Laboratory, Washington, D.C. 20375-5342, and Stratagene, Inc., La Jolla, California 92037 Received July 24, 1998. In Final Form: October 14, 1998

Several developing technologies rely upon the activity of surface-bound biomolecules to control the functionality of an interface. The specificity of these biomolecules can be compromised by mechanical stresses present during their preparation or use. Moreover, many new biomechanical devices are also sensitive to mechanical stress. This paper is concerned with the direct assessment of the mechanical limits beyond which biomolecules are rendered inactive. These mechanical limits are evaluated by a combined approach involving an atomic force microscope (AFM), an optical microscope, and standard colorimetric techniques. The AFM is used to systematically stress micrometer-sized domains of biomolecule-laden surfaces; the results of which can be directly observed with an optical microscope following histochemical staining of the surface with biologically specific dyes. Using this method, we examined the limiting conditions of two benchmark systems, i.e., streptavidin and DNA. Because of the breadth of histochemical staining combined with the nature of the study, our approach is equally applicable to a wide range of biomolecular systems, perhaps even including the surfaces of living cells.

Introduction Force is being used in an increasing number of techniques to probe the structure and function of individual biological macromolecules. In the atomic force microscope (AFM), force is applied to surface-bound molecules through a microfabricated probe to produce molecular resolution images.1-3 Optical tweezers, micropipet techniques, and AFM have been used to measure the interaction forces and mechanical properties of individual macromolecules. Intermolecular force measurements have produced detailed force-distance profiles of single molecule interactions, producing insight into the mechanisms of molecular recognition,4-6 replication,7 and motion.8,9 Intramolecular force studies have focused on the structure of individual molecules.10-12 The range of forces that are being applied to and measured between these molecules varies over at least 6 orders of magnitude. The AFM can easily apply tensile * To whom the correspondence should be addressed. E-mail: [email protected]. † Naval Research Laboratory. ‡ Stratagene, Inc. (1) Mueller, D. J.; Bueldt, G.; Engle, A. J. Mol. Biol. 1995, 249, 239243. (2) Weisenhorn, A. L.; Hansma, P. K.; Albrecht, T. R.; Quate, C. F. Appl. Phys. Lett. 1989, 54, 2651-2653. (3) Binnig, G.; Quate, C. F.; Gerber, C. Phys. Rev. Lett. 1986, 56, 930-933. (4) Evans, E.; Ritchie, K.; Merkel, R. Biophys. J. 1995, 68, 25802587. (5) Lee, G. U; Kidwell, D. A.; Colton, R. J. Langmuir 1994, 10, 354357. (6) Florin, E.-L.; Moy, V. T.; Gaub, H. E. Science 1994, 264, 415-417. (7) Yin, H.; Wang, M.; Svoboda, K.; Landick, R.; Block, S.; Gelles, J. Science 1995, 270, 1653-1657. (8) Finer, J. T.; Simmons, R. M.; Spudich, J. A. Nature 1994, 368, 113-118. (9) Svoboda, K.; Schmidt, C. F.; Schnapp, B. J.; Block, S. M. Nature 1993, 365, 721-727. (10) Smith, S. B.; Cui, Y.; Bustamante, C. Science 1996, 271, 795799. (11) Lee, G. U; Chrisey, L. A.; Colton, R. J. Science 1994, 266, 771774. (12) Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E. Science 1997, 276, 1109-1112.

and shear forces as large as 10 nN while forces as small as 0.01 pN can be measured with optical tweezers. At high AFM imaging forces, macromolecules that are adsorbed on surfaces such as mica are clearly displaced. While lower imaging forces decrease the amount of molecular displacement, the force may still produce subtle changes in molecular structure that impact function. The effect of mechanical load on single molecules has been reported in a few systems. AFM probes have been used to dissect surface-confined DNA plasmids at loads in the ∼6 nN regime.13 However, the mechanical strength of biological molecules appears to be highly system dependent, as the micropipet measurements of the tensile strength of F-actin have revealed the intermolecular interactions between actin monomers fail at 108 pN.14 In the lower limit, forces smaller than 10 pN have not been associated with irreversible conformational changes but may produce significant reversible changes.8-10 AFM studies of friction,15 elasticity,16,17 surface forces,18,19 and intermolecular forces5,6,11,12 commonly apply forces greater than 0.1 nN to surface-bound biomolecules. The ability of a biological macromolecule to retain molecular specificity and activity following the application of mechanical stress is a parameter of fundamental and practical importance. Fundamentally, the irreversible deformation of a biological macromolecule is a property that stems from its structure and intramolecular energy. Practically, this ability to functionally survive mechanical (13) Hansma, H. G.; Vesenka, J.; Siegerist, C.; Kelderman, G.; Morrett, H.; Sinsheimer, R. L.; Elings, V.; Bustamante, C.; Hansma, P. K. Science 1992, 1180-1184. (14) Kishino, A.; Yanagida, T. Nature 1988, 334, 74-76. (15) Mathew Mate, C.; McClelland, G. M.; Erlandsson, R.; Chiang, S. Phys. Rev. Lett. 1987, 59, 1942-1945. (16) Meyer, E.; Heinzelmann, H.; Gru¨tter, P.; Jung, T.; Hidber, H.R.; Rudin, H.; Gu¨ntherodt, H.-J. Thin Solid Films 1989, 181, 527-544. (17) Overney, R. M.; Takano, H.; Fujihira, M. Europhys. Lett. 1994, 26, 443-447. (18) Burnham, N. A.; Colton, R. J. J. Vac. Sci. Technol., A 1989, 7, 2906-2913. (19) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Nature 1991, 359, 239.

10.1021/la980934p CCC: $18.00 © 1999 American Chemical Society Published on Web 12/05/1998

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Figure 1. Representation of the key elements of this procedure. (A) A monolayer of biomolecules was covalently immobilized on a glass substrate. (B) A load was applied to the surface with the AFM tip while imaging. (C) Incubation with the HRP molecular recognition conjugate and development resulted in staining of the biologically active areas.

disturbance is of extreme importance to the fabrication of biomechanical devices, of both large and small dimensions. This paper describes a new technique for detecting the functional activity of biological molecules that have been mechanically perturbed. In this approach, the AFM is used to deliver a controlled load to a surface and a highly specific histochemical staining reaction is used to detect their biological activity. A schematic diagram of our technique is presented in Figure 1. Using this technique, we have established a scale over which force modifies the activity of the protein streptavidin and a derivatized oligonucleotide. The approach developed here has applications to molecular mechanics, biomaterials development, and cell/tissue engineering. Materials and Methods The specific steps used for the biochemical immobilization and the subsequent staining are illustrated schematically in Figure 2. First a transparent glass substrate is functionalized with maleimide groups, which react with thiol-containing biomolecules. These biomolecular films are then scanned with the AFM, as illustrated in Figure 1. Finally, the surfaces are exposed to a highly specific staining chemistry. The following is a detailed description of these steps. Activation of Glass Substrates. Glass substrates (1 × 1 cm) were cut from premium-grade microscope slides (Fisher, Pittsburgh, PA) and cleaned in a piranha solution (1:3 30% H2O2/ concentrated H2SO4) for 1 h. Caution: the H2O2/H2SO4 solution reacts violently with organic compounds and should be handled with extreme care. Unless otherwise noted, all reactions were

Figure 2. Detailed schematic diagram of the experimental approach. A clean glass substrate is exposed to an amino silane (A), which is baked to drive the cross-linking reaction to completion (B). A heterobifunctional cross-linker is added to functionalize the surface with maleimide groups (C). Reaction with either thiolated streptavidin (D1) or a thiol-containing oligonucleotide (D2) covalently attaches the biomolecules to the plate. After the surface is processed with the AFM, it is exposed to either biotinylated horseradish peroxidase (E1) or a streptavidin-conjugated horseradish peroxidase (E2). The HRP then catalytically precipitates a stain from solution (F1 and F2). performed at ambient temperature. Substrates were thoroughly rinsed in triply distilled water and absolute methanol and then dried with filtered nitrogen (Figure 2A). Cleaned substrates were silanized in a methanolic solution of 0.1% (v/v) N-(2-aminoethyl)3-aminopropyltrimethoxysilane (Gelest, Tullytown, PA) and 1 mM glacial acetic acid (Fisher) for 20 min (Figure 2B). These substrates were rinsed with copious amounts of methanol to remove excess silane and then dried with nitrogen. Condensation of the silane monolayer on the glass surface was driven to completion by baking the substrates in air for 10 min at 120 °C (Figure 2C). Subsequent treatment with 1 mM succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB; Pierce, Rockford, IL) in a solution of anhydrous dimethyl sulfoxide for 1 h produced transparent maleimidebearing glass plates, which react preferentially with molecules containing free thiol groups (Figure 2D).20 Immobilization of Streptavidin. Streptavidin was covalently linked to the substrates by converting its free surface (20) Lee, G. U; Chrisey, L. A.; O’Ferrall, C. E.; Pilloff, D. E.; Turner, N. H.; Colton, R. J. Isr. J. Chem. 1996, 36, 81.

240 Langmuir, Vol. 15, No. 1, 1999 amines to thiols. The streptavidin was thiolated by reacting 6 µM immuno-pure streptavidin (Pierce) with 50 mM Traut’s reagent (2-iminothiolane-HCl, Pierce) in a triethanolamine (TEA; Sigma Chemical, St. Louis, MO) buffer at pH 8.0 for 30 min. Streptavidin surfaces were prepared by reacting fresh 0.25 mM thiolated streptavidin with the maleimide-bearing glass plates for 1-2 h in a deaerated 10 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES; Sigma) and 5 mM ethylenediaminetetraacetic acid (EDTA) buffer at pH 6.6 (Figure 2E1). The activity of the streptavidin monolayers was characterized colorimetrically as described below. Immobilization of Oligonucleotides. A 5′-biotin-GCAGCCACAGGCACCTACCAGCTTA-(CH2)3S-S(CH2)3OH 3′-oligonucleotide was synthesized using a DNA synthesizer (model 394, PE-ABS, Foster City, CA) with conventional phosphoramidite chemistry. The oligomer was created with a 3′-terminal thiol using a thiol-modifier DNA synthesis support C3-S-S CPG (Glen Research, Sterling, VA). An oxidizer, 0.02 M I2, was used to preserve the dithiol group, which functions as an anchor to the support. 5′-Biotin was achieved using a bioTeg phosphoramidite (Glen Research). The concentration of each oligonucleotide was determined spectroscopically using an optical adsorption at 260 nm. Prior to immobilization the disulfide group was deprotected by incubating 5 µM oligonucleotide with 100 mM dithiothreitol (DTT; Sigma) in a 0.17 M phosphate buffer of pH 8.0 for 0.5 h. Excess DTT was removed by repeated extraction with equal volumes of ethyl acetate (MTM Research Chemicals, Windham, NH). Covalent 3′ immobilization of the oligonucleotides was achieved by immersing the maleimide-bearing glass plates with 0.1 µM thiolated oligonucleotide for 1-2 h in a deaerated HEPES/ EDTA buffer (Figure 2E2). After covalent immobilization the surfaces were immersed in an agitated solution of 1 N NaCl for 1 h to remove noncovalently bound oligonucleotide.21 The activity of the DNA monolayers was characterized colorimetrically as described below. AFM. AFM measurements were made on a commercial AFM equipped with a liquid cell (Digital Instruments, Santa Barbara, CA). Contact and friction images were taken in the constantdeflection mode at a scan rate of 100 µm/s. A single oxidesharpened microfabricated Si3N4 cantilever22 (Park Scientific Instruments, Sunnyvale, CA) was used. This cantilever had a nominal force constant of ∼30 pN/nm, as determined by spectral evaluation of the thermal noise,23 and a tip radius of 30 nm, as determined from SrTiO3 calibration standards.24,25 The sensitivities of the optical lever detector and the applied load were determined using the loading portion of the force curves. Drift in the force baseline occurred during imaging, which in turn caused the applied load to vary from the beginning to the end of the image. This drift was monitored and took the form of a linearly increasing force baseline with approximately 120 pN/ min slope. To account for the effect of drift, the loading force was set before each image and recorded after each image. The initial load was set at the following values from the minimum tracking load (∼0.01-0.05 nN) to 1.5, 3, 4.5, 6, 9, 12, and 24 nN. Colorimetry and Staining of Surfaces. The presence or absence of the biological activity of the surface-immobilized biomolecules was determined with specific molecular receptors conjugated to the enzyme horseradish peroxidase (HRP). HRP decomposes two molecules of hydrogen peroxide, its natural substrate, into water and oxygen. However, the specificity of HRP for the second molecule of hydrogen peroxide is low, and other electron donors can be substituted, which has led to the development of several colorimetric detection schemes.26 Prior to addition of the HRP conjugate, the surfaces were incubated with 10 mg/mL of bovine serum albumin (BSA; fraction V, Sigma) in a phosphate buffer saline solution (PBS: 5 mM (21) Chrisey, L. A.; O’Ferrall, C. E.; Lee, G. U Nucl. Acids Res. 1996, 24, 3031. (22) Albrecht, T. R.; Quate, C. F. J. Vac. Sci. Technol., A 1988, 6, 271. (23) Hutter, J. L.; Bechhoefer, J. Rev. Sci. Instrum. 1993, 64, 1868. (24) Mesoscale standard supplied by M. Mo¨ller, Organische Chemie III, Universita¨t Ulm, D89069 Ulm, Germany. (25) Sheiko, S. S.; Moeller, M.; Reuvekamp, E. M. C. M.; Zandbergen, H. W. Phys. Rev. B 1993, 48, 5675-5678. (26) Hosoda, H.; Takasaki, W.; Oe, T.; Tsukamoto, R.; Nambara, T. Chem. Pharm. Bull. 1986, 34, 4177.

Green et al. Na2HPO4, 5 mM NaH2PO4, 5.4 mM KCl, 0.12 M NaCl) at pH 7.0 for 1 h. Streptavidin-coated surfaces were immersed for 1 h in a 0.3 µM solution of biotinylated horseradish peroxidase (Pierce) in citrate buffer (0.1 M Na3C6H5O7, pH 6.0) (Figure 2F1). The 5′-biotinylated oligonucleotide surfaces were incubated with 0.01 µM streptavidin-HRP conjugate (Pierce) in PBS buffer with 1 mg/mL of BSA for 1 h (Figure 2F2). HRP activity was detected with 1.3 mM 4-chloro-1-naphthol (4-CN; Sigma), 0.23 mM N,N′diethylphenylenediamine dihydrochloride (DEPDA; Sigma), and 4.4 µM H2O2 in a 10 mM phosphate buffer at pH 7.0. The 4-CN and DEPDA substrates react to form a water-insoluble dye (see below), resulting in highly specific staining of the surface.27

The HRP reaction was run until a uniform coat of blue dye could be detected by eye. The stained surfaces were imaged using a transmission optical microscope (Zeiss Axiovert 100, Thornwood, NY) with a video camera (DAGE-MTI, Michigan City, MI) and frame grabber (Data Translation, Marlboro, MA). Care was taken to center and focus the light source and condenser of the microscope to minimize optical intensity variations within the field of view.

Results A schematic of the technique used to perturb a biomolecule monolayer and to detect its state is presented in Figures 1 and 2. A composite image of the results from a streptavidin monolayer is presented in Figure 3. At low loading forces no difference in optical transmission was detected between the area exposed to load and the unperturbed area (Figure 3A). The first detected attenuation in activity occurred at 1.5 nN (Figure 3B). In this image a 50 × 50 µm square is visible. The lighter optical intensity is a measure of the lower density of the stain, which suggests a lower density of the HRP conjugate and, in turn, implies a lower density of active streptavidin. As the level of the force was increased, the relative optical intensity inside the imaged squares also clearly increased. The arrows to the right of each image indicate the slow scan direction for the AFM scanning, and for the 6.0 nN scan shown in Figure 3E, the scan direction was allowed to reverse and the top portion of the scanned area was imaged twice. This will be addressed in more detail below. Figure 3 contains many bright circular features, which we suspect are due to surface-bound gas bubbles formed during the staining procedure. The level of optical attenuation due to load was directly computed from the digitized images. The optical intensity of specific areas was measured inside the imaged area, outside the imaged area, and in an area in which the monolayer had been completely destroyed by the excessive uncontrolled loads of a neighboring cantilever. The rectangular areas in Figure 3G contain 19 × 229 pixels, and the intensities of these 4351 pixels were tabulated as a histogram, which is shown in Figure 3H. The optical (27) Conyers, S. M.; Kidwell, D. A. Anal. Biochem. 1991, 192, 207211.

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Figure 4. Optical intensity of areas of the streptavidin (solid symbols) and DNA (open symbols) surfaces that experienced AFM imaging. The solid lines are the Hertzian fits of these data. The optical intensity was normalized, with the intensity of the most severely damaged portion of the surface assigned a value of 1 and the unmodified intensity given a value of 0. The lines drawn through the data are best fits in which E* and pth were used as fitting parameters. Those fits suggest that for streptavidin E* ) 20 MPa and pth ) 4.5 MPa, while for the biotinylated oligonucleotide E* ) 2.5 GPa and pth ) 0.12 GPa.

Figure 3. Optical micrograph of a controlled loading experiment on a streptavidin monolayer. Fast scan axis is from left to right. (A) Area in which the applied load was initially set at the minimum required to track to the surface (bottom) but was increased approximately 0.5 nN by the end of the scan (top). (B) The initial applied load was 1.5 nN. The light area represents an area in which the streptavidin activity has been attenuated. The remaining parts were imaged at increasing initial applied loads of (C) 3, (D) 4.5, (E) 6, (F) 9, and (G) 12 nN. (H) Histogram of optical intensities from inside (solid line) and outside (dashed line) of the affected area in G, as illustrated by the rectangles.

intensity was determined from the mean intensity for a given histogram. These measured values allow us to calculate a normalized optical intensity (IN)

IN )

IO - I IO - I D

where I is the mean intensity inside the affected area, IO is the mean intensity just outside that same affected area, and ID is the mean intensity in one of the completely destroyed areas. The images of the DNA surfaces are similar to those of the streptavidin shown in Figure 3, with the only notable difference being that the normalized intensities are consistently smaller. The load dependence of the normalized optical intensity of the streptavidin and DNA monolayers is shown in Figure 4. There was an initial region in which no attenuation in the optical intensity of the monolayers could be detected. This regime extends from ∼0.05 to 1.5 nN. Beyond this range it is clear that the optical intensity, and thus the biological activity, is a function of load. These

experiments were repeated several times, and the results were very similar, with the exception that the absolute intensity depended upon the extent of staining, which was controlled by visual inspection. The glass substrates used for these experiments were not conducive to high-quality images; however, a similar experiment on oriented Au substrates (data not shown) suggested that streptavidin films are not physically disturbed at loads below ∼2 nN. Above this load, some contrast could be observed in the frictional images but not in topography. Topographic images of the streptavidin monolayers indicate that the molecules were not removed from the surface up to loads of 10 nN. AFM images of the DNA monolayers did not reveal changes in topographic or frictional images in this range of load. Ideally, the molecular activity will be a linear function of IN. Activity is related to intensity through the fidelity of the optics and functional form of the staining chemistry. The influence of illumination-based optical intensity variations was minimized by optimizing the microscope optics. Proportional staining is dependent on the level of HRP development, and the monotonic form of the 4-CN/ DEPDA response requires that the staining reaction be either under- or overdeveloped. By developing the films to the point where there is both a light blue background and a transparent fully destroyed square clearly visible, we ensured that the staining was within the proportional regime. Discussion In the case of AFM imaging, and likely in many other biomechanical devices, the kinds of mechanical stresses that may be applied to immobilized biomolecules are quite complex. Further, the corresponding molecular response, its strain, is expected to be highly nonlinear and anisotropic. Nonetheless, we can use simple models to understand mechanisms leading to loss of biological activity. Figure 5 illustrates how a film of spherical molecules might distort under the vertically applied pressure of an AFM

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Green et al.

Figure 5. (A) Contact of the probe with a hypothetical monolayer. (B) The Hertz model illustrating the pressure distribution along with a threshold pressure and radius.

probe. The load will be distributed over the molecules within the contact area, and the stress on an individual molecule will be related to its location. Molecules under the apex of the probe will be under more normal stress (compressive) than molecules at the periphery. The actual functional dependence of this stress and the resulting strain will be related to the geometry and chemistry of the probe, as well as to the chemical structure of the molecules (hydrophobic domains, etc.). Although the stress under a laterally translating tip is a complicated four-dimensional vector field, to a first approximation it can be correlated with the pressure. If the film is densely packed, the pressure can be approximated by Hertzian contact mechanics.28 For our model we assume that at some threshold Hertzian pressure, pth, the stress experienced by the molecules will produce a strain necessary to inactivate the molecule. Under this assumption the equation for this threshold radius, rth, within which inactivation occurs is given by

rth )

( )[ 3RF 4E*

1/3

1 - pth2

( )] π3R2 6FE*2

2/3 1/2

where E* is the reduced elastic modulus, F is the net applied load, and R is the probe radius. Thus for molecules where 0 < r < rth, the pressure will exceed pth and the molecules will be irreversibly deactivated. Molecules located in the range rth < r < a, where a is the Hertzian contact radius, may experience reversible deactivation or even no affect at all. Finally, molecules located outside of the contact, a < r, will be relatively unaffected. During imaging the AFM probe apex is rastered with respect to the sample surface in the xy plane by two orthogonal triangular functions. The geometry of this path and the contact radius may be used to calculate the fractional area contacted by the probe:

(nrL) - (nrL)

f)2

2

where r is the contact area radius, L is the scan size of the AFM image, and n is the number of passes the probe makes across the surface. In this case, the AFM probe made 1024 linear passes across the 50 × 50 µm test area. When we combine this path with the threshold radius, we can

calculate the fractional surface area within the scanned square that experienced pressures in excess of pth. Using this model, with the reduced elastic modulus and the threshold pressure as fitting parameters, we produced good fits to the data including both the functional form of the data at moderate forces and the threshold behavior. This model produces a convincing fit of the DNA results and to a lesser extent the streptavidin results, as seen in Figure 4. The reduced elastic moduli of the monolayers (E*strep ) 20 MPa and E*oligo ) 2.5 GPa) were also consistent with previous measurements for proteins and polymers, which range from 10 MPa to 10 GPa. The relative quality of the fits suggests that the moderate load behavior is more a function of damaged area as a function of load instead of an intrinsic force dependent property of the molecule. Nonetheless, the numerical values for the elastic constants should be treated as qualitative at best, since the Hertz model is not specifically designed to describe a thin compliant film between two rigid substrates. Similar results were obtained with the Winkler elastic foundation model,28 which is not surprising since it is commonly used as an approximation of the Hertz model. This model does, however, explain two features of the activity attenuation behavior observed in Figure 3. First, the fact that the total attenuation of the streptavidin activity was higher than that of DNA can be interpreted in terms of contact area; that is, the streptavidin monolayer was thicker than the single-stranded DNA, resulting in a larger geometric contact area and a higher level of activity attenuation. More important is the realization that the form of the data at loads greater than 1.5 nN was determined by the contact area of the probe rather than the inherent strength of the molecules. This is also supported by the observation in Figure 3E, where the region scanned twice at the same load was significantly more deactivated because the fraction of the affected area was roughly doubled. The exact nature of the contact is critical to the amount of deactivation. A sudden change in the contact will produce a change in the amount of deactivation. For example, temporary contamination of the tip might produce a larger contact area, which might deactivate a wider swath of the substrate than the corresponding clean tip. This is one possible mechanism for the creation of the occasional bands of increased deactivation in Figure 3. The loss of activity of the streptavidin and DNA films at 1.5 nN represents an upper limit of the ability of these molecules to retain activity, but this does not mean that the molecules have not been modified at lower loads. We have observed that lipid bilayers in the liquid-crystalline phase are also penetrated by AFM probes at loads of approximately 1-2 nN. This represents a potential mode of failure for the protein, which may be the disruption of its hydrophobic core. At forces of 10 nN, we have observed displacement of streptavidin molecules from the substrate; thus, at that load the stresses of imaging must be high enough to cleave the covalent bonds with the surface. This is the same order of magnitude as the DNA dissecting measurements performed earlier;13 however, we observe no such effects in our oligonucleotide system, probably because of its size and mode of attachment, which allows significant flexibility. This study demonstrates a novel combined approach to the characterization of load-induced deactivation of biological macromolecules. Using this technique, we were able to assess the mechanical limits associated with AFM (28) Johnson, K. L. Contact Mechanics; Cambridge University Press: New York, 1985.

Molecular Recognition of Biomolecules

imaging of immobilized streptavidin and DNA. It was found that the streptavidin was significantly more susceptible to tip-induced deactivation than DNA. In addition to its direct nature, a fundamental value of this approach is its broad applicability, as many of the biomolecular systems of interest are associated with colorimetric agents, which are commonplace in biotechnology. The protocol is quite general: damage the film, expose the film to a reactive group, and determine if a probe molecule has reacted with the damaged portion. This combined approach can be modified by replacing the stain with another molecular recognition marker (e.g., fluorescently labeled isotopes) or replacing optical microscopy with another microscopic surface analysis tool (e.g., FTIR microscopy); in fact, large proteins could even be used and analyzed by noncontact imaging with an AFM. However, some of the most interesting future work involves the variation of environmental conditions, such as ionic strengths, buffer

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species, pH, loading conditions, etc. We anticipate that force resolution on the order of tens of piconewtons will be readily achieved, and as the maximum applied load is decreased, as in noncontact and intermittent contact imaging modes, it will be possible to apply this technique to membrane-supported protein assemblies, perhaps even in vitro. Acknowledgment. This research was supported by the Office of Naval Research and Stratagene under Navy CRADA 96-111. J.-B.D.G. acknowledges the support of the American Society for Engineering Education on an ONR Postdoctoral Fellowship. We also thank Linda Chrisey for use of the DNA synthesizer and Larry Bottomley and S. A. Syed Asif for valuable discussions. LA980934P