Article pubs.acs.org/accounts
Recent Progress in Molecular Recognition Imaging Using Atomic Force Microscopy Subhadip Senapati†,‡,∥ and Stuart Lindsay*,†,‡,§ †
Biodesign Institute, ‡Department of Chemistry and Biochemistry, and §Department of Physics, Arizona State University, Tempe, Arizona 85287, United States CONSPECTUS: Atomic force microscopy (AFM) is an extremely powerful tool in the field of bionanotechnology because of its ability to image single molecules and make measurements of molecular interaction forces with piconewton sensitivity. It works in aqueous media, enabling studies of molecular phenomenon taking place under physiological conditions. Samples can be imaged in their near-native state without any further modifications such as staining or tagging. The combination of AFM imaging with the force measurement added a new feature to the AFM technique, that is, molecular recognition imaging. Molecular recognition imaging enables mapping of specific interactions between two molecules (one attached to the AFM tip and the other to the imaging substrate) by generating simultaneous topography and recognition images (TREC). Since its discovery, the recognition imaging technique has been successfully applied to different systems such as antibody−protein, aptamer−protein, peptide−protein, chromatin, antigen− antibody, cells, and so forth. Because the technique is based on specific binding between the ligand and receptor, it has the ability to detect a particular protein in a mixture of proteins or monitor a biological phenomenon in the native physiological state. One key step for recognition imaging technique is the functionalization of the AFM tips (generally, silicon, silicon nitrides, gold, etc.). Several different functionalization methods have been reported in the literature depending on the molecules of interest and the material of the tip. Polyethylene glycol is routinely used to provide flexibility needed for proper binding as a part of the linker that carries the affinity molecule. Recently, a heterofunctional triarm linker has been synthesized and successfully attached with two different affinity molecules. This novel linker, when attached to AFM tip, helped to detect two different proteins simultaneously from a mixture of proteins using a so-called “two-color” recognition image. Biological phenomena in nature often involve multimolecular interactions, and this new linker could be ideal for studying them using AFM recognition imaging. It also has the potential to be used extensively in the diagnostics technique. This Account includes fundamentals behind AFM recognition imaging, a brief discussion on tip functionalization, recent advancements, and future directions and possibilities.
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INTRODUCTION Colocalization of proteins with a resolution close to the smallest linear dimensions of the proteins themselves (1−10 nm) is an important biophysical goal, one that is close to being realized with super-resolution optical microscopy. However, it requires dye or fluorescent protein labels, and attaching these may not be desirable or even possible in certain cases. Some super-resolution techniques can achieve a resolution of 25 nm. However, shortage of conventional probes and experimental challenges in live cell imaging leave scope for improvement for this technique.1,2 Here, we review a new colocalization technique based on atomic force microscopy, which has a resolution of ∼20 nm and does not require fixed labels. In the last few decades, atomic force microscopy (AFM) has become a powerful tool in field of nanoscience because of its ability to scan a surface with nanometer resolution in aqueous liquids.3 Remarkable advances have been made in the past few years in understanding the dynamics of the interaction forces between biomolecules at the single molecule level. Spatial mapping of these interaction helps to localize and visualize the events taking place in real time. This mapping technique is known as © XXXX American Chemical Society
recognition imaging and gives us critical insights about the interaction process and binding sites. Since its development in 1985,4 AFM has become an integral part of nanoscale analysis in the field of material science and biology. In the first few years after its discovery, AFM was only operated in air or vacuum. A new door was opened for biophysicists when AFM operation was demonstrated in fluids,5 which gave it significant advantages compared to the other similar imaging techniques such as SEM (scanning electron microscopy) or TEM (transmission electron microscopy). Operation in fluid medium made it possible to image the biomolecules of interest under near native and physiological conditions. Moreover, this can be achieved without any further modification of the substance such as tagging, staining, coating, crystallization, and so forth. Another application of AFM is force spectroscopy, which can provide information about the ligand−receptor interaction,6 elasticity and stiffness,7 protein folding and unfolding,8,9 adhesion,10 and so forth. The force of Received: December 9, 2015
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To solve the issues associated with force−volume imaging, recognition imaging was developed.15−17 Recognition imaging is a technique that allows interaction data to be acquired simultaneously with the normal AC mode of topographic image acquisition. In AC modes (Tapping Mode, MacMode), the probe is oscillated sinusoidally so that the height of the probe above the surface, h, changes according to
interaction between the ligand and receptor (order of piconewtons) can be measured by single molecule force spectroscopy by probing the immobilized biomolecules on the surface with an AFM tip functionalized with the cognate molecule.11 Force spectroscopy can be demonstrated by the approach-retraction curves in Figure 1. When the tip is far away
h = h0 sin 2πft where f is the modulation frequency and h0 is the amplitude. This is an oversimplification, because the interaction of the probe with the surface is highly nonlinear, as surface forces increase very rapidly as the probe tries to compress the underlying stiff surface. If the probe were truly a harmonic oscillator (i.e., moving in a sinusoidal pattern), any damping of amplitude would cause a reduction in both the amount of the upswing and the downswing (i.e., it would be equivalent to reducing the value of h0). However, in water, the motion of the probe is highly overdamped. Thus, if the probe is driven continuously (as it is with magnetic drive or “MacMode”),18 interactions at the bottom of the swing do not affect the amplitude at the top of the swing. In recognition imaging, the sample is scanned in the same way as in the normal imaging mode, but with a cognate ligand attached to the tip by a flexible linker that is about as long as the amplitude of oscillation of the AFM probe. Thus, reductions of the amplitude of the downward swing amplitude reflect interaction with surface topography. Reductions of the upward swing amplitude reflect stretching of the flexible linker if the cognate ligand sticks to the surface molecule so as to cause this stretching. In this way, with a correctly chosen oscillation amplitude and linker length (i.e., equal to about 2h0), fluctuations in the top and bottom amplitudes report topography and interactions separately. Importantly, because the probe is far from the surface when the linker stretching becomes significant, signals from nonspecific interactions between the probe and the surface are greatly reduced. Monitoring the values of top and bottom amplitudes of a functionalized tip over a surface using an electronic circuit generates simultaneous topography and recognition images (TREC imaging).16 Comparing the topography and recognition images, specific binding events can be mapped in real time (i.e., at normal AFM scanning rates, typically tens of seconds to minutes to scan an area of a micrometer squared). Since the molecule on the tip identifies and hence recognizes the molecule on the surface, this is termed as “recognition imaging” where high resolution mapping image is achievable within normal imaging time period (Figure 2). Force spectroscopy provides critical insights about the dynamics of the binding events, whereas recognition imaging maps and localizes these binding events. So far, recognition imaging has been performed on different biomolecular systems, i.e. antigen−antibody,16 aptamer-protein,19 peptide−protein,20 antibody-protein,21 chromatin,17,22,23 cells,24,25 and other ligand−receptor such as avidin−biotin and so forth.26 These early studies illustrate the potential of recognition imaging and it is our hope that this overview will encourage further applications. To this end, proper functionalization of the AFM tips and execution of the key steps for this technique are critical to a successful recognition experiment. Recognition imaging has been discussed and explained in details in some excellent review articles and book chapters previously.27−29 In this Account, brief notes on the fundamental principles, conventional AFM tip functionalization will be
Figure 1. Representative force−distance curves for functionalized AFM tip probing a substrate modified with the cognate molecules. Blue and red lines represent the approach and retraction curves, respectively.
from the surface, no interaction occurs between the surface molecule and cognate tip molecule (approach curve, blue line). As the tip goes closer, at one point, binding takes place. During the retraction (red line), first the tip goes up (with the molecules still bound to each other) followed by stretching of the linker. As a result of this, the cantilever bends and the retraction curve goes downward. Then unbinding takes place and the retraction curves suddenly jumps back to zero force. In a further development, the spatial location of specific interaction was determined by a process of taking force curves at every point on an image, so-called force-volume imaging. When the tip is functionalized with a cognate ligand, specific chemical interactions can be mapped.12−14 Combining the topography with the force measurement experiments helped to map the various interactions taking place on biomolecular surfaces, in principle, at a single molecule level. In these experiments, force−distance curves are collected and analyzed at each predefined pixel of the corresponding topographic image. Force−volume technique has been used in many ligand−receptor systems and cells. Even though it is possible to get information about both topography and interactive force using this experiment, it suffers from two major drawbacks: (i) low lateral resolution and (ii) longer experiment time compared to normal AFM imaging. Normal AFM imaging can be done in contact mode, noncontact mode, or tapping mode. Contact mode produces images with better resolution, but both the tip and sample become damaged due to direct contact during scanning. In both tapping and noncontact mode, damage to the tip and sample is minimal, but tapping mode produces better resolution images compared to noncontact mode. Force spectroscopy and force−volume imaging are done in contact mode. B
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(bare tip) and recognition imaging (functionalized tips). The compressed oscillations exhibit distinctly different oscillation maxima for imaging with bare and functionalized tips whereas the minima remained similar (Figure 3). From the minima, it
Figure 2. Interactions during recognition imaging. (A) The ligand on the tip cannot bind with the receptor on the surface, and hence, no recognition occurs. (B) The ligand binds with the receptor on the surface, and recognition takes place. Adapted with permission from ref 17, Copyright 2004 National Academy of Sciences, U.S.A.
mentioned followed by the discussion on recent progress in tip modification methods, expansion of the application range of recognition imaging, and possible future directions and scope.
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FUNDAMENTAL PRINCIPLES In the recognition imaging experiments, the biomolecule of interest is immobilized on the surface and the tip is functionalized with the corresponding cognate molecule, attached via a flexible linker of typically 5−10 nm full length. The tip raster scans the surface and the molecule on the tip periodically binds and unbinds from the molecules on the surface (Figure 2). Polyethylene glycol (PEG) is commonly used as a linker for attaching the probing molecule. PEG provides the conformational flexibility needed for proper binding. A relatively long (several nm) linker is required so that the probe tip itself is well out of the range of nonspecific interactions with the substrate when the recognition signal is generated. This may appear to present a severe limitation to the resolution of recognition imaging. However, the binding and unbinding of the cognate ligand is a stochastic event, and is repeated many times as the tip sweeps over a target molecule to obtain a high-resolution image. As a result, the “blurring” caused by the long tether is distributed in a Gaussian manner around the center of the interaction. In consequence, recognition “spots” are only a few nanometers in diameter when, for example, a 10 nm linker is used. In some cases, the specific site of recognition can be located on the surface of a large complex.30 By using a special electronic circuit box (sold as the TREC box, Keysight Technologies, Santa Clara, CA; earlier sold by Agilent Technologies), recognition signals are generated. When the functionalized tip is far away from the substrate (no interaction), the oscillation amplitude remains uniform. As the tip approaches the surface, the amplitude gradually decreases and reaches zero when in contact. When there is no binding interaction between the tip molecule and the surface molecule, the cantilever oscillates similarly to the imaging with a bare tip. But when the tip molecule is bound to the surface molecule via the stretchable PEG linker, the oscillating cantilever cannot return to its original position. As a result of this, the upper part of the deflection signal is altered and shows distinct characteristics. The lower half of the deflection signal generates a signal essentially identical to that of a bare tip, and is fed into the AFM controller to generate the topographic image. As the scanning continues, at one point, dissociation of ligand−receptor binding occurs and oscillation amplitude regains its initial state (same as when the tip is far away). When plotted against an extended period of time, the oscillation amplitude of the cantilever becomes highly compressed and shows different characteristics for normal imaging
Figure 3. Change in oscillation amplitude during the binding event. (A) Oscillation amplitudes plotted against 1 s for imaging with a bare tip. (B) Same plot with a functionalized tip. Adapted with permission from ref 16. Copyright 2004 Elsevier.
was possible to get topographic information with the functionalized tip. The decrease in the amplitude for the maxima in certain places is due to the inability of the cantilever to oscillate freely when binding takes place and binding information can be extracted from this.16 The Pico TREC box separates the upper part (Umax) of the oscillation amplitude from the lower part (Umin). Umin gives the topographic image and Umax gives the recognition image (Figure 4).26
Figure 4. Special electronic circuit in the TREC box separates the maxima and minima of the oscillation amplitude during binding and generates the recognition and topographic image from them, respectively. Reproduced with permission from ref 26. Copyright 2005 John Wiley and Sons.
Typically, the contrast in the recognition image is set to correspond to the upper level of the deflection signal so that the maximum amplitude is bright. Recognition events will then appear as dark spots (typically reductions in voltage of a few tens to a hundred mV depending on the gain of the system and the thermal noise generated by cantilever fluctuations). Thus, C
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Accounts of Chemical Research the substrate and unrecognized molecules appear as a uniformly bright background with dark spots representing the sites of interaction. Since topography and recognition signals are simultaneously acquired, the two images can be overlaid to identify the molecules that generated the recognition signals. To confirm the specificity of the binding, the molecules on the tip are made inactive by passing a free solution of corresponding cognate molecules that bind to the tip molecules. Once the molecule on the tip is blocked, it cannot bind to the molecule on the surface any more and hence recognition stops. For example, in Figure 5, recognition of
Figure 6. (A) Topographic image of the receptor IgG, (B) corresponding recognition image, and (C) zoomed-in recognition spot exhibiting scan lines with internal unbinding (circled) or without (pointed with arrows). The circled unbinding events within a scan line were used to obtain ton and toff (and then kon and koff). (D) Interpretation of ton and toff in terms of binding and unbinding events. (E) Histograms generated from voltage changes during recognition events; bound (black), unbound (white), gray (background). Reproduced with permission from ref 21. Copyright 2011 Elsevier.
Figure 5. (A) Topographic image of thrombin proteins on mica. (B) Corresponding recognition image of (A). (C) Recognition image from using a thrombin solution to block the TBA tip. Adapted with permission from ref 20. Copyright 2013 American Chemical Society.
human α-thrombin was done using anti-thrombin aptamer as the probing molecule attached to the tip.20 The bright spots in the topographic image represent thrombin molecules (Figure 5A). Corresponding dark spots in the recognition image indicate binding of thrombin molecules with antithrombin aptamer (Figure 5B). Successful recognition events depend on efficient binding, which in turn, depends on the exposure and orientation of the binding spot on the biomolecules. So, in general, not all the biomolecules are recognized, but majority of them are. Recognition efficiency denotes the percentage of molecules recognized out of all possible molecules. For example, the circled spots are the thrombin molecules, which have been recognized whereas the molecule inside the square has not been recognized. In this case, free thrombin solution was passed through the flow cell to block the aptamer molecule on the tip. Once the aptamer is blocked, the tip becomes inactive and cannot interact with the surface molecules anymore. So when the same area is scanned again, recognition spot vanishes (Figure 5C). One other intriguing aspect of recognition imaging lies in the ability to collect transient binding and unbinding data from the recognition scans. If the off rate, koff, is rapid compared to the time the probe spends within a linker length of a target molecule, transient unbinding events can be seen as “on−off” switching of the recognition signal during each line scan, and analysis of this data can give koff directly (Figure 6).21 Antibody tends to unfold in their metastable state at higher loading rates (used for recognition imaging) and that results in a weak binding interaction between the cognate molecules.21
Figure 7. Some representative methods for functionalizing the AFM tip. (A) Functionalization of silicon or silicon nitride tips, (B) functionalization of gold-coated tip, and (C) functionalization using click Chemistry.
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FUNCTIONALIZATION OF AFM TIPS Functionalization of AFM tips is a key step for recognition imaging. Nowadays, two of the most popular chemistries for modifying the silicon (or, silicon nitride) and gold tips are silanization11,31 and gold−thiol chemistry32,33 respectively (Figure 7A and B). The “new material” probes (sold by NanoWorld) also contain Si−O groups on their surface and can be modified in the same way as conventional silicon, though
they do require brief activation in an oxygen plasma. Silicon or silicon nitride tips are aminated using silane chemistry. Most common reagent for silanization has been (3-aminopropyl)triethoxysilane (APTES),21 even though, in the past few years 1-(3-aminopropyl) silatrane (APS),34 has become increasingly popular. APTES is susceptible to hydrolysis and polymerization D
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Accounts of Chemical Research and is better to use in vapor phase deposition technique. On the other hand, APS is more stable and can be used in aqueous solution making the technique simple and user-friendly. A PEG linker with NHS group is commonly used for the attachment to the amine groups on the tip. Other end of the PEG linker carries different groups depending on the reactive moiety on the biomolecules (NHS for aminated biomolecules and maleimide or vinyl sulfone for thiolated biomolecules etc.). Azide functionalized molecules can be attached to alkyne or cyclo-alkyne functionalized tip (and vice versa) by coppercatalyzed35 or copper-free Click Chemistry20 (Figure 7C). The latter method made it possible to functionalize AFM tips without any catalyst, where all the steps were carried out in aqueous medium. It should be noted these are some general methods for tip functionalization. Depending on the molecules and experimental design, many functionalization techniques are available.36
Many biological processes in nature occur with simultaneous involvement of multiple units. Wang et al. tried to address it by functionalizing the tips with two affinity molecules using an equimolar mixture of the two recognition or affinity molecules (Figure 8A).54 In a single AFM image, they identified two
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APPLICATIONS OF RECOGNITION IMAGING The very first recognition imaging technique was applied on lysozyme molecules immobilized on mica and the magnetically coated tip was functionalized with antilysozyme antibody in MAC mode operation.15 But the recognition image was not accompanied by simultaneous topographic image. For that purpose, topographic image was recorded with a bare tip before the recognition imaging. Nevertheless, this discovery paved the way for a new and critical application of AFM. This technique was then extended to generate a simultaneous topography and recognition image in the same experiment (with time and resolution being comparable to the standard AFM imaging mode).16 A home-built circuit was used, known as the TREC box. Here also lysozyme molecules were detected using AFM tip functionalized with antilysozyme antibody but in this case it was possible to attribute the binding to the corresponding molecules in topographic image generated side-by-side. This invention was followed by a number of applications. Stroh et al. later used PicoTREC system to detect antibodychromatin binding for the first time.17 The tip was functionalized with antihistone H3 antibody and the reconstituted nucleosomal arrays carrying the mouse mammary tumor virus (MMTV) were immobilized on mica. This process was further extended for monitoring biological processes in situ. Another class of affinity molecules for recognition was introduced in 2006 when human IgE was recognized using an aptamer molecule on the tip.19 Aptamers are single stranded DNA or RNA molecule, which are more stable, easy to modify and can be more specifically attached to the AFM tips compared to the antibodies. Aptamers were later used in other AFM recognition studies as well.20,37−40 An important innovation was application of AFM based recognition imaging on cells for the first time. Binding sites of vascular endothelial (VE)-cadherin on endothelial cells from mouse myocardium (MyEnd) were detected using VEcadherin-Fc cis-dimer functionalized tip.24 MyEnd cells were fixed on gelatin-coated glass slides using 0.5% glutaraldehyde. This led to many other studies where different binding sites on cells were investigated using properly functionalized AFM tips.25,41−44 Later, human tissues were investigated in their near-native state by TREC imaging, a development which might have a tremendous impact in understanding the difference in tissue structures in the diseased state.45,46 In the past few years, recognition imaging has been successfully applied to many other systems as well.47−53
Figure 8. Detection of two molecules during the same experiment. (A) Functionalization of AFM tips using equimolar mixture of two linear linkers carrying the affinity molecules. (B) Functionalization the tip using a triarm linker that carries two affinity molecules that bind to different proteins. (C) representative picture depicting the recognition of two molecules using a triarm linker.
interactions simultaneously in the multicomponent systems hSwi-Snf complex and chromatin. But this technique is difficult because of the “chance factor” involved in the functionalization process. To make sure that the tip is functionalized with both the affinity molecules, a novel three-arm linker was developed carrying two affinity molecules (Figure 8B and C).37 In this study, two different triarm linkers were synthesized, one with antithrombin aptamer and RGD peptide and another with antiVEGF and anti-TNFα aptamers. First linker was used to detect human α-thrombin and α5β1-integrin from a mixture and the second one for the detection of VEGF and TNF-α. Molecules of VEGF and TNF-α can be seen in Figure 9A. Scanning with a tip functionalized with linker carrying both anti-VEGF and antiTNF-α produced the recognition image (B) where both proteins were detected. In order to identify the individual proteins and check the specificity, a sequential blocking technique was adopted. First, VEGF solution was passed through the flow cell to block the anti-VEGF aptamer on tip and the same area was scanned. The number of recognition spots decreased, and these recognition spots should correspond to aptamer−TNF-α binding (Figure 9C). In the next step, TNF-α solution was passed through the flow cell and the same area was scanned again. Since both the affinity molecules on the tip were blocked at this point, no recognition spots were detected (Figure 9D). This conclusively proved the specificity of the interaction. Superimposing both recognition images (B) and (C), it was possible to identify individual VEGF and TNFα molecules in Figure 9E where VEGF proteins were represented by the black spots and TNF-α proteins by the black-topped-by-green spots. Due to high lateral resolution of AFM multiplex recognition, it was possible to characterize two different proteins from the recognition image where the topographic image was showing just one big spot (spot with green boundary in Figure 9A). Recognition image showed two well-separated spots (Figure 9B). Blocking the anti-VEGF aptamer on the tip and subsequent recognition experiment produced just one spot E
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Figure 9. Topographic (A) and recognition (B) image of a VEGF and TNF-α mixture; (C) anti-VEGF aptamer-blocked recognition image; (D) both aptamers-blocked recognition image. (E) Superimposition of recogntion images (B) and (C) to identify the two proteins. Black topped-bygreen spot: TNF-α protein and Black: VEGF protein. Adapted with permission from ref 37. Copyright 2015 American Chemical Society.
Halfwidth at half height (HWHH) of each Gaussian is −ln(2)k , where k is m4 or m7 giving HWHH for first Gaussian is 3.38 nm, for second Gaussian 1.98 nm, so we could not resolve them at 6 nm separation or less. Panel (c) shows the same Gaussians, but now moved to be just 20 nm apart. There is a clear difference between a 2 Gaussian fit (Black line completely overlaps the data) and a 1 Gaussian fit (red line). Thus, a realistic resolution is 20 nm. Using this technique, multimolecular changes in biological process can be monitored in near-physiological conditions, important for finding factors that require colocalization for their function.
(TNF-α) in Figure 9C. The vanished spot was attributed to VEGF protein. Subsequent blocking of the anti-TNF-α produced no recognition spot as described before. This technology has the potential to facilitate the analysis of simultaneous phenomenon in multisubunit complexes such as nucleosomes (histone octamer wrapped with DNA), extent of DNA-methylation and histone acetylation in cancer progression, clinical diagnosis of certain diseases, and so forth.37 To estimate the resolution limit for colocalization technique, the encircled region in Figure 9B was zoomed-in (Figure 10a) and this change in recognition voltage was fit to the following equation:
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m1 − m2 × exp − [(x − m3)2 /m4] − m5
CONCLUDING REMARKS AFM has become an extremely popular instrument for imaging molecules in solution with nanometer to subnanometer resolution. Functionalization of the tip with suitable molecules led to the measurement of unbinding force with piconewton sensitivity using force spectroscopy. Combining the topographic information with force spectroscopic data introduced “force−volume” experiment, which was able to map specific interactions between two biomolecules. The resolution and the experiment time of force−volume experiments were later improved when recognition imaging technique was introduced. Recognition imaging is relatively new compared to other popular AFM techniques such as imaging, force spectroscopy, or indentation. Since TREC’s first application in 2004, recognition imaging has come a long way and many biological phenomena have been investigated. But still it has not been utilized to its full potential so far, partly because it requires a combination of specialized electronics and competence in the required modification chemistry. Quality of the tips (initially and during scanning) and proper functionalization are extremely critical to carry out recognition experiments. Moreover, strong expertise is needed for experimental design, analysis, and data interpretation. Despite these challenges, however, it is almost unique in being able to locate factors with chemical sensitivity to nanometer precision. The recently developed multiplex recognition imaging should further enhance its appeal for studying multiple changes occurring simultaneously in a single biological process.
× exp −[(x − m6)2 /m7]
The black line in Figure 10b represents the actual change in voltage for the recognition events and the red line is the corresponding Gaussian fit. Centers of Gaussians are determined to 0.025 and 0.058 nm with Gaussians well separated. But this is not the actual resolution limit, as the peaks will overlap more when close.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Present Address
Figure 10. (a) Blow-up of the encircled region from Figure 9B. (b) Gaussian fitting to the change in voltage profile along the red line in recognition image. (c) Same Gaussian, but shifted to be 20 nm apart.
∥ S.S.: Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio, USA.
F
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Resolution by Force-Volume AFM. Angew. Chem., Int. Ed. 2011, 50, 12103−12108. (15) Hinterdorfer, P.; Raab, A.; Han, W.; Badt, D.; Smith-Gill, S. J.; Lindsay, S. M.; Schindler, H. Antibody Recognition Imaging by Force Microscopy. Nat. Biotechnol. 1999, 17, 901−905. (16) Stroh, C. M.; Ebner, A.; Geretschläger, M.; Freudenthaler, G.; Kienberger, F.; Kamruzzahan, A. S. M.; Smith-Gill, S. J.; Gruber, H. J.; Hinterdorfer, P. Simultaneous Topography and Recognition Imaging Using Force Microscopy. Biophys. J. 2004, 87, 1981−1990. (17) Stroh, C.; Wang, H.; Bash, R.; Ashcroft, B.; Nelson, J.; Gruber, H.; Lohr, D.; Lindsay, S. M.; Hinterdorfer, P. Single-Molecule Recognition Imaging Microscopy. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 12503−12507. (18) Han, W.; Lindsay, S. M.; Jing, T. A Magnetically Driven Oscillating Probe Microscope for Operation in Liquids. Appl. Phys. Lett. 1996, 69, 4111. (19) Lin, L.; Wang, H.; Liu, Y.; Yan, H.; Lindsay, S. Recognition Imaging with a DNA Aptamer. Biophys. J. 2006, 90, 4236−4238. (20) Senapati, S.; Manna, S.; Lindsay, S.; Zhang, P. Application of Catalyst-Free Click Reactions in Attaching Affinity Molecules to Tips of Atomic Force Microscopy for Detection of Protein Biomarkers. Langmuir 2013, 29, 14622−14630. (21) Kaur, P.; Qiang-Fu; Fuhrmann, A.; Ros, R.; Kutner, L. O.; Schneeweis, L. A.; Navoa, R.; Steger, K.; Xie, L.; Yonan, C.; Abraham, R.; Grace, M. J.; Lindsay, S. Antibody-Unfolding and Metastable-State Binding in Force Spectroscopy and Recognition Imaging. Biophys. J. 2011, 100, 243−250. (22) Wang, H.; Bash, R.; Lindsay, S. M.; Lohr, D. Solution AFM Studies of Human Swi-Snf and Its Interactions with MMTV DNA and Chromatin. Biophys. J. 2005, 89, 3386−3398. (23) Wang, H.; Dalal, Y.; Henikoff, S.; Lindsay, S. Single-Epitope Recognition Imaging of Native Chromatin. Epigenet. Chromatin 2008, 1, 10. (24) Chtcheglova, L. A.; Waschke, J.; Wildling, L.; Drenckhahn, D.; Hinterdorfer, P. Nano-Scale Dynamic Recognition Imaging on Vascular Endothelial Cells. Biophys. J. 2007, 93, L11−L13. (25) Chtcheglova, L. A.; Wildling, L.; Waschke, J.; Drenckhahn, D.; Hinterdorfer, P. AFM Functional Imaging on Vascular Endothelial Cells. J. Mol. Recognit. 2010, 23, 589−596. (26) Ebner, A.; Kienberger, F.; Kada, G.; Stroh, C. M.; Geretschläger, M.; Kamruzzahan, A. S. M.; Wildling, L.; Johnson, W. T.; Ashcroft, B.; Nelson, J.; Lindsay, S. M.; Gruber, H. J.; Hinterdorfer, P. Localization of Single Avidin-Biotin Interactions Using Simultaneous Topography and Molecular Recognition Imaging. ChemPhysChem 2005, 6, 897− 900. (27) Hinterdorfer, P.; Dufrene, Y. F. Detection and localization of single molecular recognition events using atomic force microscopy. Nat. Methods 2006, 3, 347−355. (28) Kienberger, F.; Ebner, A.; Gruber, H. J.; Hinterdorfer, P. Molecular recognition imaging and force spectroscopy of single biomolecules. Acc. Chem. Res. 2006, 39, 29−36. (29) Ebner, A.; Chtcheglova, L.; Tang, J.; Alsteens, D.; Dupres, V.; Dufrene, Y. F.; Hinterdorfer, P. Recognition Imaging Using Atomic Force Microscopy. Handbook of Single-Molecule Biophysics 2009, 525− 554. (30) Bash, R.; Wang, H.; Anderson, C.; Yodh, J.; Hager, G.; Lindsay, S. M.; Lohr, D. AFM Imaging of Protein Movements: Histone H2AH2B Release during Nucleosome Remodeling. FEBS Lett. 2006, 580, 4757−4761. (31) Riener, C. K.; Stroh, C. M.; Ebner, A.; Klampfl, C.; Gall, A. A.; Romanin, C.; Lyubchenko, Y. L.; Hinterdorfer, P.; Gruber, H. J. Simple Test System for Single Molecule Recognition Force Microscopy. Anal. Chim. Acta 2003, 479, 59−75. (32) Martines, E.; Zhong, J.; Muzard, J.; Lee, A. C.; Akhremitchev, B. B.; Suter, D. M.; Lee, G. U. Single-Molecule Force Spectroscopy of the Aplysia Cell Adhesion Molecule Reveals Two Homophilic Bonds. Biophys. J. 2012, 103, 649−657. (33) Klein, D. C.; Ovrebo, K. M.; Latz, E.; Espevik, T.; Stokke, B. T. Direct measurement of the interaction force between immunostimu-
The authors declare no competing financial interest. Biographies Subhadip Senapati Subhadip Senapati received his B.Sc. in 2007 from Jadavpur University (Kolkata, India) and M.Sc. in 2009 from Indian Institute of Technology Madras (Chennai, India). He got his Ph.D. in Chemistry under the supervision of Prof. Stuart Lindsay at the Arizona State University. At present, he is a postdoctoral research fellow in the Department of Ophthalmology and Visual Sciences at Case Western Reserve University. Stuart Lindsay Stuart Lindsay received his Ph.D. in physics from the University of Manchester in 1976. Presently, he is Regents’ Professor of Physics and Chemistry at Arizona State University and Director of the Center for Single Molecule Biophysics in the Biodesign Institute. His current research focuses on nanoscale and single molecule biophysics.
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ACKNOWLEDGMENTS We are grateful to all co-workers, who have contributed to some of the studies described above. A Grant (U54CA143862) from National Cancer Institute (NCI) supported this research.
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