Int. J. Mol. Sci. 2010, 11, 2134-2151; doi:10.3390/ijms11052134
OPEN ACCESSInternational Journal of

Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
ArticleReconsideration of Dynamic Force Spectroscopy Analysis ofStreptavidin-Biotin Interactions
Atsushi Taninaka, Osamu Takeuchi and Hidemi Shigekawa *Institute of Applied Physics, CREST-JST, University of Tsukuba/1-1-1 Tennodai, Tsukuba 305-8573,
Japan; E-Mails: jun_t@bk.tsukuba.ac.jp (A.T.); osamu@big.or.jp (O.T.)* Author to whom correspondence should be addressed; E-Mail: hidemi@ims.tsukuba.ac.jp;
Website: http://dora.bk.tsukuba.ac.jp; Tel.: +81-29-853-5276; Fax: +81-29-853-5276.
Received: 9 April 2010; in revised form: 3 May 2010 / Accepted: 6 May 2010 /Published: 13 May 2010

Abstract: To understand and design molecular functions on the basis of molecular
recognition processes, the microscopic probing of the energy landscapes of individualinteractions in a molecular complex and their dependence on the surrounding conditions isof great importance. Dynamic force spectroscopy (DFS) is a technique that enables us to
study the interaction between molecules at the single-molecule level. However, the obtained
results differ among previous studies, which is considered to be caused by the differences in
the measurement conditions. We have developed an atomic force microscopy technique thatenables the precise analysis of molecular interactions on the basis of DFS. After verifying
the performance of this technique, we carried out measurements to determine the landscapesof streptavidin-biotin interactions. The obtained results showed good agreement withtheoretical predictions. Lifetimes were also well analyzed. Using a combination of crosslinkers and the atomic force microscope that we developed, site-selective measurement was
carried out, and the steps involved in bonding due to microscopic interactions are discussed
using
the
results
obtainedbysite-selective analysis.
Keywords: dynamic force spectroscopy; atomic force microscopy; site-selective analysis;
streptavidin-biotin interaction

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1. Introduction
The variety and selectivity of interactions between a pair of functional molecules, for example, DNA,ligand-receptor and antigen-antibody systems, play essential roles in biological processes and moleculardevices based on molecular recognition properties [1-6]. However, such interactions include various
effects, for example, those of solvent parameters and the manifold structures of functional molecules,
which prevent the design of detailed functions in predetermined structures [1-6]. Despite theirimportance, local variations between two molecules during their chemical reaction may not have beenprobed by a thermodynamic method and remain hidden behind the averaged function. Therefore, themicroscopic probing of the energy landscapes of individual interactions in a molecular complex and theirdependence on the surrounding conditions is strongly desired to achieve further advances in biophysics
and chemistry and their applications.
Dynamic force spectroscopy (DFS) is a technique that enables us to study the interaction between
molecules at the single-molecule level [7-26]. In DFS, the unbinding force applied to a pair of molecules
is increased at a constant rate, and the force required to rupture the bond is measured. By analyzing therelationship between the modal rupture force and the logarithm of the loading rate, microscopic
potential barrier landscapes (the distance from the potential minimum) and the lifetimes of bonds can beobtained [7-26]. However, the obtained results for potential barriers and bond lifetimes differed among
the previous studies [9-13], and some results were inconsistent with theoretical predictions. Table 1
shows previously obtained results for the interactions of streptavidin/biotin complexes.
The different measurement conditions, such as the types of cross-linkers, substrates andmeasurement systems shown in Table 1, may affect the results. For example, streptavidin is directly
fixed to a cantilever in the case of [13]. The length of the molecular chains used for linkers is not thesame for each method as noted in Table 1. However, in a conventional DFS analysis, since the sampling
rate is not sufficiently high at a high loading rate and it is difficult to maintain a constant loading rate ata low loading rate, even though the same measurement conditions are used it is difficult to performmeasurements to acquire accurate rupture forces [27], resulting in the difference in the slope of therelationship between the modal rupture force and the logarithm of the loading rate, which provides
potential barrier positions and lifetimes. In addition, the existence of the cross-linker molecule used to
fix the target molecules to the cantilever and substrate affects force measurement [19-24]. These points
must be considered before discussing the details of molecular interactions.
We have developed an atomic force microscopy (AFM) technique that enables the precise analysis ofmolecular interactions on the basis of DFS [15,16]. In this paper, after examining the performance of
this spectroscopy technique in detail, we discuss the results obtained for determining the landscapes of
streptavidin-biotin interactions. The results obtained by this spectroscopy technique showed good
agreement with theoretical predictions. Furthermore, using the combination of cross-linkers and theatomic force microscope that we developed, site-selective measurement was carried out. The steps
involved in bonding due to microscopic interactions will be discussed using the results obtained by siteselective analysis.

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Table 1. Bell model parameters obtained from previous studies: potential barrier positions(xb) and dissociation rates (k).
Surface spacer
molecule andTip crosslinker andlengthlength

BFP

PEG(30 nm)PEG(30 nm)AFM

Agarosepolymer(-)

AFM

MethodAFM

Potential
barrierPotential
barrier

xb1/nmxb2/nmPBS(pH 6.8)

0.5

BSA(3 nm~)PBS(pH 7.2)

BSA(3 nm~)

BSA(3 nm~)-

poly-L-lysine

Glutaraldehyde

PBS

(-)

(1 nm)(pH 7.2)

Solution

k1/s-1

k2/s-1

Reference

0.12

6.14  10-5

2.9

[9]

0.49

0.05

1.67  10-5

2.09

[11]

0.6

0.14

0.2

54

[12]

0.081

0.024

0.56

2.98

[13]

2. Developed Techniques and Experimental Methods
First, we explain the methodology we developed and the details of sample preparation.
2.1. For Precise and Deeper Analysis
In DFS, the unbinding force applied to a pair of molecules is increased at a constant rate, and theforce required to rupture the bond is measured. The lifetime of a molecular bond depends on thepotential barrier height. When a tensile force is applied to a bond, the potential landscape is deformed;
thus, the barrier height decreases. When the tensile force is applied at a constant loading rate, theprobability distribution of the rupture force can be expressed as( f  f * ) xb
( f  f * ) xb
P( f )  C exp
exp
1

exp



k BT 
k B T 




(1)

where P(f), f, f*, xb, kB, T, and C are the probability distribution of the rupture force, the rupture force,the modal rupture force, the distance of the potential barrier position from the potential bottom, theBoltzmann constant, the temperature and a constant, respectively [9,10,15,16]. According toEquation 1, f* linearly depends on the logarithm of the loading rate r0 (df/dt), as shown byf *  k BT t (0) xb

ln r0  ln  offxb 
k B T 
(2)where toff(0) is the lifetime of the molecular bond. Equation 2 indicates that the potential barrier position
can be obtained from the slope of the linear relationship. When using Equations 1 or 2, it is essential tomaintain a constant loading rate [15-20]. However, when the DFS measurement is carried out by AFM
with a cross-linker molecule, a constant retraction velocity does not result in a constant loading rate
because of the stretching of the cross-linker molecule [19,20]. Therefore, we introduce a mechanism forcontrolling the applied force to maintain a constant loading rate. To satisfy this requirement, we haveInt. J. Mol. Sci. 2010, 11

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developed an AFM system with a force feedback system that enables precise analysis by DFS [15-18],
which features (1) fine control of the loading rate to reduce the effect of the soft cross-linker connecting
a sample molecule to the tip or substrate [15-18] and (2) a high sampling rate (100 kHz) to obtain asufficient amount of data at a high loading rate [15,16]. A high sampling rate (at least 1 kHz) is required
to resolve the subtle processes underlying the interactions between the pair of molecules [27].
Furthermore, since AFM measurement is stable under various pHs, different buffer solutions withdifferent pHs can be used.
2.2. Structure of Streptavidin Molecule
We used the streptavidin-biotin complex, a typical ligand-receptor system, as a sample [28,29].
Streptavidin is a tetrameric protein that has a high affinity for biotin molecules. Each monomer of
streptavidin bonds with one biotin molecule. The binding pocket of streptavidin has several reaction
sites with a hydrogen-bonding network, which can be classified into the following three groups
depending on the distance from the bottom of the binding pocket (Figure 1) [28,29]: (1) the innerbinding sites of amino acid residues SER27, ASN23, TYR43 and ASP128, (2) the middle binding sites
SER45 and THR90 and (3) the outer bonding sites ASN49 and SER88. Since streptavidin has acomplex structure when considering its chemical reactions, various processes are expected to occur inthis system depending on the operating conditions used. Therefore, probing to obtain a deeper
understanding of the energy landscapes of individual interaction sites of this system will provide afoundation for designing and controlling the mechanism of chemical reactions between two more
complex functional molecules.
Figure 1. Structure of streptavidin-biotin complex (reproduced from [28,29]).

2.3. Cantilever and Sample Preparation
A rectangular gold-coated cantilever (Bio-Lever, Olympus, 6 pN/nm and 30 pN/nm) was immersed
in a solution of 8-amino, 1-octanethiol hydrochloride molecules (Sigma-Aldrich, 1 mM in ethanol) for
48 h to form a closely packed SAM with amino groups on the surface. After rinsing with ethanol, thecantilever was immersed in a solution of biotin-PEG3400-COO-NHS (Shearwater Polymers, 0.1 mM in
ethanol) for 20 h to fix a biotin (biotin-PEG) molecule onto the probe apex (Figure 2(a)). Finally, theInt. J. Mol. Sci. 2010, 112138

cantilever was rinsed with ethanol. The formation of closely packed SAMs on the probe apex andsubstrate was necessary to decrease the strong interaction between the gold layers on both surfaces.
Figure 2. Schematic illustrations of the apex of a modified cantilever (a) and the three typesof cross-linker for site-selective analysis; streptavidin is fixed to a SAM of (b)1,10-decanedithiol/1-octanethiol (1/100 ratio) solution on a Au-coated substrate via a
streptavidin-maleimide structure (SM condition), (c) 8-amino, 1-octanethiol molecule on a
Au-coated substrate via a biotin-PEG-COO-NHS molecule (B-PEG condition) and (d)HS-(PEG)-SH/1-octanethiol (1/100 ratio) mixed solution on a Au-coated substrate via a
streptavidin-maleimide structure (SM-PEG condition).

For the microscopic analysis of the energy landscapes and lifetimes of streptavidin-biotin interactions,which will be discussed in Sections 3-3 and 3-4 in detail, we prepared three types of cross-linker, as
schematically illustrated in Figure 2. Namely, streptavidin was fixed to a SAM of (b)1,10-decanedithiol/1-octanethiol (1/100 ratio) mixed solution on a Au-coated substrate via a
streptavidin-maleimide structure (SM), (c) an 8-amino, 1-octanethiol molecule on an Au-coatedsubstrate via a biotin-PEG-COO-NHS molecule (B-PEG) and (d) HS-(PEG)-SH/1-octanethiol(1/100 ratio) mixed solution on a Au-coated substrate via a streptavidin-maleimide structure(SM-PEG). To avoid multiple-bonding events, the density of target molecules in the SAM was reduced
by adjusting the ratios of 1,10-decanedithiol/1-octanethiol and HS-(PEG)-SH/1-octanethiol molecules
to 1/100. The distances between the streptavidin molecule and the substrate are ~30 nm for the B-PEG
and SM-PEG conditions, and ~1.5 nm for the SM condition, as shown in Figure 2.
First, a thin gold film (100 nm) was evaporated as a substrate onto a freshly cleaved mica surface in ahigh vacuum at 400 °C and flame-annealed using a hydrogen gas burner for 30 s. A flat Au surface wasobtained, as shown by the AFM image in Figure 3(a). Then, for the SM condition, the substrate was
immersed in a solution of 1,10-decanedithiol/1-octanethiol (1/100 ratio) (Sigma-Aldrich, 1 mM in
ethanol) for 48 h to form a closely packed SAM with thiol groups on the surface. The formation of a

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flat closely packed SAM with etch pits was confirmed by AFM, as shown in Figure 3(b),(c). Afterrinsing with ethanol, the substrate was immersed in a solution of streptavidin-maleimide (Sigma-Aldrich,
10 mg/l in phosphate-buffered saline (PBS), pH 7.4: N,N-dimethylformamide = 99:1) for 5 h to fix
streptavidin molecules to the substrate. Finally, the substrate was rinsed with PBS (Figure 2(b)). As
shown in Figure 3(d),(e), isolated streptavidin molecules of 2.3-4.4 nm height were observed by AFM.
The density of streptavidin molecules is ~5/100  100 nm2, which is sufficiently low to enable themeasurement of a single bond.
Figure 3. Tapping-mode AFM images of a sample substrate: (a) bare Au, (b) with
1,10-decanedithiol/1-octanethiol SAM, (c) magnified image of an etch pit in (b), (d)substrate immersed in 10 mg/l streptavidin-maleimide solution and (e) line profile ofstreptavidin-maleimide molecules. The spring constant of the cantilever used for all
measurements was 2 N/m.

For the B-PEG condition, the substrate was immersed in a solution of 8-amino, 1-octanethiol(Sigma-Aldrich, 1 mM in ethanol) for 48 h to form a closely packed SAM with amino groups on thesurface. After rinsing with ethanol, the substrate was immersed in a solution of biotin-PEG-COO-NHS(0.1 mM in ethanol) for 20 h to fix biotin onto the substrate. Finally, the substrate was rinsed withethanol (Figure 2(c)).
For the SM-PEG condition, the substrate was immersed in a solution of HS-(PEG)-SH/
1-octanethiol (1/100 ratio) (SUNBRIGHT, NOF Corporation, 1 mM in ethanol) for 48 h to form aclosely packed SAM with thiol groups on the surface. After rinsing with ethanol, the substrate was
immersed in a solution of streptavidin-maleimide (Sigma-Aldrich, 10 mg/l in phosphate-PBS, pH 7.4:
N,N-dimethylformamide = 99:1) for 5 h to fix streptavidin molecules to the substrate. Finally, thesubstrate was rinsed with PBS (Figure 2(d)).
2.4. Measurement Procedures
In DFS, the unbinding force applied to a pair of molecules is increased at a constant rate, and theforce required to rupture the bond is measured [15-20]. In our experiment, the loading rate wascontrolled between 10 and 105 pN/s, and 5,000-10,000 approach and retract cycles were carried out toform a histogram for each loading rate measurement. To avoid multiple-bonding events, as mentioned
above, the density of target molecules in the SAM was reduced so that the probability of bonding

Int. J. Mol. Sci. 2010, 112140

became 5-10% for each tip-sample approach. Furthermore, only single ruptures were counted toremove the errors caused by the effects of multiple-rupture events on the analysis results. All DFS
measurements were performed at room temperature.
In general, for a selected loading rate, many rupture forces are measured from force curves(Figure 4(a)). The measured rupture forces are shown in a histogram, from which the modal rupture
force is obtained for each loading rate measurement (Figure 4(b)). In the analysis, the modal rupture
forces are plotted as a function of the logarithm of the loading rate (Figure 4(c)), thereby, information
concerning the energy landscape of the interaction is derived from the relationship between the modal
rupture force obtained from the histogram and the loading rate of the unbinding force. In addition, thewidth of the histogram also provides the potential barrier position by analysis using Equation 1(Figure 4(b)) [15,16], which can be used to examine the validity of the results obtained from the slope
shown in Figure 4(c).
Figure 4. Schematic diagram representing the method of DFS analysis.

3. Results and Discussion
3.1. Confirmation of Measurement Conditions
We first confirmed that no rupture event occurred when streptavidin molecules were not prepared on
the substrate. This is because we covered the gold surfaces of the probe and substrate with closely
packed SAMs of spacer molecules to prevent direct bonding between their bare surfaces.
Secondly, the stretching motion of a PEG molecule was observed for each rupture event.
Figure 5(a),(b) show two different forms of the typical force curve obtained at a loading rate of
39 pN/s under the SM condition (Figure 2(b)) as functions of (a) distance and (b) time. The distance
represents the length of the cantilever retraction measured from the zero-force position. Since thecantilever was first moved inward until the force reached 50 pN, a region of negative distance exists.
Although the force does not exhibit a linear change in (a) owing to the existence of a PEG molecule, alinear change is shown in (b). This result indicates that a constant loading rate is achieved during thestretching of the PEG molecule. The gradient of the force-distance curve in Figure 5(a) after reaching
the zero-force position is smaller than that in the negative-distance region because of the existence of
the PEG molecule. The rupture force is 11.5 pN, which is the value expected for the measurement at
the loading rate.

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When there is a direct interaction between the cantilever and the substrate, no stretching of the PEG
molecule is observed and the rupture force should be much larger than 11.5 pN (Figure 5(c),(d)). The
distance between the zero-force position and the rupture point includes the amount of bending of thecantilever, which is obtained by dividing the rupture force by the spring constant of the cantilever(6 pN/nm). Therefore, the actual length of the PEG molecule can be estimated by subtracting theamount of bending of the cantilever from the distance of 30.4 nm between the zero-force and rupture
positions obtained from Figure 5(a). The obtained length is 28 nm, which is in good agreement with thelength expected from the PEG molecules that we used (molecular weight, ~3400: length, ~30 nm).
Figure 5(e) shows a typical force curve obtained for the B-PEG condition (Figure 2(c)). The length of
the PEG molecule, obtained from the PEG-PEG distance (61.4 nm), the rupture force (18.9 pN) and thespring constant of the cantilever (6 pN), is about 58 nm, which is in good agreement with twice thelength of a single PEG molecule, as expected.
Figure 5. (a) and (b): Two different forms of the typical force curve obtained using oursystem [15,16] as functions of (a) distance and (b) time. (c) and (d) show the data obtained
for a direct interaction between the cantilever and the substrate, where no stretching of the
PEG molecule is observed. (e) shows a typical force curve obtained for the B-PEG
condition. (f) Schematic diagram of the approach and retract cycle.

Next, we examined the effect of the viscous drag on the cantilever. Figure 6 shows a force curve
obtained at a high loading rate of 104 pN/s for the B-PEG condition. The observed force includes theviscous drag on the cantilever. In fact, although the cantilever (Bio-lever) we used was smaller than the
cantilever typically used for AFM, an increase in force was observed after the rupture event, where theretraction of the cantilever was accelerated to realize a constant loading rate without the formation of amolecular bond. To remove such undesirable effects, we estimated the rupture force by subtracting theforce immediately after the rupture event. We examined the validity of this treatment by plotting 23
histograms obtained using three cantilevers with different spring constants and shapes (6 pN/nm,
rectangular, Au-coated; 30 pN/nm, rectangular, Au-coated; and 20 pN/nm, triangular, noncoated Si2N3).
No marked dependence on the type of cantilever was observed [15].

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Thirdly, we confirmed the formation of a streptavidin-biotin structure from the reduction in bonding
probability following the introduction of free biotin molecules into the solution. Figure 7 shows thechange in binding rate, obtained under the SM condition, induced by the introduction of free biotin
molecules. When free biotin molecules were added, the peak signal, indicated by an arrow, gradually
decreased in intensity and eventually almost disappeared, whereas the peak position did not shift, as
expected (Figure 7). The histograms in Figure 7 appear to show distributions with different shapes. Thisis due to the reduction in the amount of data obtained resulting from the introduction of free biotin
molecules, which greatly reduced the probability of forming the streptavidin-biotin structure. In fact,
each histogram in Figure 7, for which a sufficient amount of data was obtained, has the expected shape.
This shows the importance of collecting a sufficient amount of data. In addition, the phosphate buffer
ensures that the pH remains constant even when biotin molecules are added.
From these results, the observed force curves are considered to originate from the biotin-streptavidin
interaction.
Figure 6. Schematic illustration of the force measurement and a typical force curve
obtained at a high loading rate of 104 pN/s to analyze the effect of the viscous drag on
the cantilever.

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Figure 7. Change in binding rate caused by the introduction of free biotin molecules. The
loading rate was set at 2.25  102 pN/s for all measurements. In the measurements, theratios of the number of detected streptavidin-biotin bonds to the total number ofmeasurements were (a) 8.1%, (b) 4.5% and (c) 2.2%.

3.2. Rupture Force Distribution and Potential Barrier Position
Figure 8(a)-(c) shows typical histograms of the rupture forces obtained for the SM condition in PBS(pH 7.4) at the loading rates of (a) 1.63  104 pN/s, (b) 3.45  103 pN/s and (c) 2.95  102 pN/s, where
the modal rupture forces are (a) 45.1 pN, (b) 38.5 pN and (c) 31.0 pN. The modal rupture force
decreases with the loading rate, as expected [7-26]. As mentioned in Section 2, the potential barrier
position can be estimated by analyzing the shape of the rupture force distribution [15,16]. The solid
green lines in Figure 8 show theoretical fitting curves (Equation 1), and the potential barrier position xb
obtained from each distribution in Figure 8 using the method in [15] and [16] is determined to be (a)0.51 nm, (b) 0.52 nm and (c) 0.69 nm. The barrier position obtained by this method is estimated to besmaller than the actual value, when there is a broadening of the histogram. The results are consistent
with the barrier position of 0.68 nm obtained from the slope in Figure 8, which is the best-fit value,
because of the many data points used for estimation. The analysis based on the Bell model [9-26] isvalid when the change in the potential shape of the system is small [9-26], and the obtained results
validate the results of measurements performed in this study and indicate the stability of the potential
barrier determined in each experiment.
On the other hand, Figure 8(d)-(f) shows typical histograms of the rupture forces obtained for the BPEG condition in PBS (pH 7.4). Similarly to the results obtained for the SM condition, the modal
rupture force decreases with the loading rate. From the theoretical fitting curves, the potential barrier
position xb is estimated to be (d) 0.15 nm, (e) 0.40 nm and (f) 0.50 nm. Although these estimatedpotential barrier positions are similar to those for the SM condition, the potential barrier position
obtained from the fitting in Figure 8(d) is smaller than those obtained from the fitting in Figure 8(e),(f).
When the Bell model can be applied with the assumption of the shape of potential landscapes
described in previous studies [9,10,25], as in the present case, the deformation of the histogram isconsidered to be small at high and low loading rates. However, the shape of the histogram is broadened

Int. J. Mol. Sci. 2010, 112144

at a loading rate where two potential barriers are probed, as shown in Figure 8(d). In fact, two potential
barriers are observed as will be discussed later for the results shown in Figure 9. There are othermethods for the analysis of more complex cases [23,24].
Figure 8. Typical histograms of the rupture forces obtained for the (a-c) SM and (d-f) BPEG conditions in PBS (pH 7.4) at various loading rates.

3.3. Analysis of Molecular Interactions–Potential Barrier Position
Figure 9 shows the relationships between the modal rupture force and the logarithm of the loading
rate obtained in 0.01 M PBS (pH 7.4) for the (a) SM, (b) B-PEG and (c) SM-PEG conditions shown in
Figure 2. According to the DFS theory (Equation 2), each slope provides the potential barrier of one
unbinding process, and the distance of the barrier position from the potential bottom, xb, can be deduced
from the reciprocal of the gradient [7-26].
For the B-PEG and SM-PEG conditions, the gradient of the slopes exhibits a marked increase at
2  103 pN/s. The potential barrier positions were estimated from the two slopes to be 0.13 ± 0.01 nm
and 0.63 ± 0.09 nm for the B-PEG condition and 0.11 ± 0.05 nm and 0.61 ± 0.27 nm for the SM-PEG
condition, In pioneering studies on the DFS of streptavidin-biotin complex, a potential landscape withtwo barrier widths (0.12 nm and 0.5 nm) was observed [9,10]. On the basis of the results obtained by
X-ray diffraction and MD calculations, the small (0.12 nm) and large (0.5 nm) potential barrier positions
were attributed to the direct hydrogen bonding of the inner amino acid residues, such as ASP128,
TYR43 and ASN23, with the biotin molecule, and the indirect interaction of the middle sites, such as
SER45, with the biotin molecule via water molecules, respectively [29-31]. The results obtained under
B-PEG and SM-PEG conditions are consistent with that obtained in previous studies.

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Figure 9. Relationships between the modal rupture force and the logarithm of the loading
rate obtained for (a) SM (Figure 2(b)), (b) B-PEG [Figure 2(c)] and (c) SM-PEG(Figure 2(d)) conditions in 0.01 M PBS (pH 7.4). The barrier positions obtained from theseresults are shown in the figure. A similar experimental result obtained in 0.05 M sodium
nitrate solution under the SM condition is also shown. The result obtained for the 0.01 M
PBS (pH 7.4) solution shown in (b) is also indicated for comparison (blue dashed line).

For the SM condition, in contrast, only one slope was observed and the potential barrier position
estimated from this slope was 0.68 ± 0.05 nm. Namely, only the bridged bonding at the middle reaction
sites was observed. To confirm the validity of this result, we plotted the values from several histograms
obtained using three cantilevers with different spring constants and shapes (6 pN/nm, rectangular, Aucoated; 30 pN/nm, rectangular, Au-coated; and 20 pN/nm, triangular, noncoated Si2N3). No marked
dependence on the type of cantilever was observed. Since the waiting time (30 ms or 300 ms), i.e., thetime before the cantilever was retracted after coming into contact with the sample, had no significant
effect on the results, the bonds are considered to be fully formed [15].
Although a more detailed study is necessary to determine the mechanism, it is considered that thebiotin molecule attached to the cantilever can enter deep into the binding pocket and conjugate evenwith the inner amino acid residues in streptavidin under the B-PEG or SM-PEG condition(Figure 10(a)). Under the SM condition (Figure 10(b)), in contrast, this process is prevented. This may
be due to the lack of flexibility or to the effect of the hydrophobicity of the substrate surface on theformation of a hydrogen bond. Since the SAM of octanethiol molecules formed on the Au substrate has
a hydrophobic characteristic, the reduction in the distance between the streptavidin molecule and theInt. J. Mol. Sci. 2010, 11

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substrate may increase the effect of the hydrophobicity of the substrate. The potential barriers obtained
for the 3 nm linkers in [12] (in Table 1) are similar to those obtained for the B-PEG condition,suggesting that the effect of the molecular chain is negligible in the case of 3 nm linkers, enabling
precise measurements. Since the linker length for the SM condition is 1.5 nm, the effect of the SM
condition may occur between linker lengths of 1 nm and 3 nm.
Figure 10. Schematic illustration of the site-selective analysis using two types of crosslinker. (a) B-PEG condition and (b) SM condition.

With this method, bonding at the middle reaction sites can be selectively probed. To analyze theorigins of the potential barriers of 0.68 nm and 0.63 nm in more detail, we changed the buffer solution
from phosphate (pH 7.4) to sodium nitrate (pH 7) [15]. The measurements were performed under the
SM condition. For the sodium nitrate, the slope changed at a loading rate of about 10 2-103 pN/s, andthe two potential barrier positions were estimated to be 0.26 nm and 1.6 nm.
Since no bond corresponding to the potential barriers of 0.68 nm and 0.63 nm exists in the 0.05 M
sodium nitrate solution, the bond related to the potential barrier positions of 0.68 nm and 0.63 nm issuggested to be formed by molecular bridging between the amino acid residues at the middle reaction
sites and the biotin molecule [15]. Namely, the potential barrier positions of 0.68 nm and 0.63 nm areformed by the phosphate molecules in the buffer solution [15] instead of the water molecules, incontrast to the mechanism predicted in previous studies [9,10].
The bonding at the inner or middle sites was distinguished and separately analyzed for the SM,
B-PEG and SM-PEG conditions. To the best of our knowledge, this is the first demonstration ofsite-selective analysis by DFS. Using a combination of cross-linkers and the atomic force microscopethat we developed for precise analysis by DFS, direct and bridging interactions at each reaction site in aligand-receptor system were distinguished and individually analyzed.

Int. J. Mol. Sci. 2010, 112147

3.4. Analysis of Molecular Interactions–Lifetime
The lifetime of bonds can be estimated from the intercept obtained by extrapolating the linear
relationship between the modal rupture force and the logarithm of loading rate (Equation 2), as shownin Figure 9 [7-26]. The rupture forces obtained for the B-PEG condition were larger than those for the
SM condition at all loading rates, as shown in Figure 9, and the lifetimes obtained for the SM, B-PEG
and SM-PEG conditions were 1.0 s, 6.4 s and 13 s, respectively.
Since two rupture points exist for the B-PEG condition (Figure 11(a)) and one rupture point exists
for the SM-PEG condition (Figure 11(b)), the rupture probability for the B-PEG condition is considered
to be two times larger than that for the SM-PEG condition. Namely, the lifetime for the
B-PEG condition is expected to be half that for the SM-PEG condition. From the result of the B-PEG(PEG-biotin-streptavidin-biotin-PEG) condition (6.4 s), the lifetime for streptavidin-biotin-PEG isestimated to be 12.8 s [12]. This value is in good agreement with the lifetime obtained for the SM-PEG
condition (13 s). The obtained values are ten times larger than that obtained for the SM condition. Thisis caused by the fact that the direct bonds with the inner amino acid residues, such as ASP128, TYR43
and ASN23, are stable under the B-PEG and SM-PEG conditions because the effect of thehydrophobicity of the substrate, for example, is less than that under the SM condition. Similarly to thepotential barriers mentioned in Section 3.3, the lifetime obtained for the 3 nm linkers in the previous
experiment [12] (in Table 1) is similar to that obtained for the B-PEG condition, suggesting that theeffect of the SM condition is negligible at 3 nm from the substrate in the case of steric hindrance.
Figure 11. Schematic illustrations of the rupture types for (a) B-PEG and (b) SM-PEG
conditions.

The lifetimes obtained from DFS measurements are, in general, much shorter than that expected fromthe dissociation constant (Kd ~ 10-15) of the streptavidin-biotin complex [28,29]. This is because thedissociation constant is measured under an equilibrium condition where biotin molecules form bonds
with all three group sites in streptavidin. However, the lifetime of each bond is evaluated using theresults obtained by DFS measurement instead of the total strength, resulting in the observed shorteningof the lifetime.

Int. J. Mol. Sci. 2010, 11

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The lifetime, and thereby the observed rupture force, changes with the experimental conditions.
Despite this fact, different conditions were used in previous experiments. Namely, some data wereobtained using a cantilever without a PEG molecule, and other data were obtained using a PEG
molecule at different buffer concentrations. In addition, the loading rate was not kept constant.
Therefore, the difference in lifetime between our study and previous studies (for example, that indicatedin [9] may be caused by the differences in pH and ion concentration. Such variations among thereported values must be due to important effects yet to be understood in detail, which, therefore, must
be clarified in the future. In any case, a precise DFS measurement technique will be essential for the
analysis.
3.5. Analysis of Molecular Interactions – Reaction Processes
The results obtained in this study are shown in Table 2. As shown in Figure 1, the binding pocket of
streptavidin has several reaction sites with a hydrogen-bonding network, which are classified into thefollowing three groups depending on the distance from the bottom of the binding pocket: (1) the innerbinding sites of amino acid residues SER27, ASN23, TYR43 and ASP128, (2) the middle binding sites
SER45 and THR90, and (3) the outer binding sites ASN49 and SER88 [28,29]. The barrier positions
observed correspond to these three types: two direct bonds at inner and middle sites, and one indirectbond via solvent molecules at middle sites. Since the –COOH of the biotin molecule was attached to the
PEG molecule, the interactions between the –COOH of the biotin molecule and the outer binding sites
ASN 49 and SER88 are considered to be very weak, resulting in the absence of potential barriers
between a biotin-PEG molecule and the outer binding sites.
Table 2. Potential barrier positions obtained for various conditions.
pH
pH 7.4
pH 7
pH 7.4pH 7.4pH 6.8

condition
SM
SM [15]
B-PEG [15]SM-PEG
R. Merkel et al.[9,10]

Buffer solution
Phosphate
NaNO3
Phosphate
Phosphate
Phosphate

Bond type
Amino acid residue
Barrier positions by MD [31]Reaction area

0.13 nm0.11 nm0.12 nmDirect bond
ASP128,TYR43,
ASN23
0.10 nm
inner

Potential barrier position (xb)0.68 nm0.26 nm0.63 nm0.61 nm0.50 nm

1.6 nm-Direct bondSalt bridge-

SER27,SER45

SER27,
SER45-

0.26 nmmiddle--These results obtained by site-selective analysis enable us to discuss the step-by-step processes forvarious conditions: For the B-PEG and SM-PEG conditions in PBS, the biotin molecule is trapped at
the inner sites due to direct bonding and at the middle sites via the bridging of buffer molecules (Figure12(a)). For the SM condition in PBS, the biotin molecule is not trapped at the inner sites but only at the
middle sites via the bridging of buffer molecules (Figure 12(b)). For the SM condition in sodium nitrate

Int. J. Mol. Sci. 2010, 112149

solution, the biotin molecule is trapped at the middle sites via the hydrogen bonding of amino acidresidues (Figure 12(c)).
Figure 12. Schematics of the bonding structures of streptavidin-biotin complex.

4. Conclusions
We have developed an atomic force microscopy technique that enables precise analysis of themolecular interactions on the basis of DFS. The landscapes of streptavidin-biotin interactions obtainedby this technique are consistent with theoretical predictions, indicating the importance of the precise
measurement conditions realized by this microscopy technique. Lifetimes were also well analyzed.
Furthermore, using a combination of cross-linkers and the atomic force microscope that we developed,
site-selective measurement was carried out. Direct and bridging interactions at each reaction site in a
typical ligand-receptor system, i.e., the streptavidin-biotin complex, were clearly distinguished andindividually analyzed. This methodology will provide a foundation for further advances in biophysics
and chemistry and their applications, such as designing and controlling the mechanism of chemical
reactions between functional molecules.
Acknowledgements
This work was supported in part by a Grant-in-Aid for Scientific research from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan (Yong Scientists B).
References and Notes
1.
2.
3.

Badjic´, J.D.; Balzani, V.; Credi, A.; Silvi, S.; Stoddart, J.F. A molecular elevator. Science 2004,
303, 1845-1849.
Kay, E.R.; Leigh, D.A.; Zerbetto, F. Synthetic molecular motors and mechanical machines. Angew.
Chem.: Int. Ed. 2007, 46, 72-192.
Ariga, K.; Hill, J.P.; Endo, H. Developments in molecular recognition and sensing at interfaces. Int.
J. Mol. Sci. 2007, 8, 864-883.

Int. J. Mol. Sci. 2010, 11
4.

2150

Vestergaard, M.; Kerman, K.; Tamiya, E. An overview of label-free electrochemical protein.
Sensors 2007, 7, 3442-3458.
5. Yasuda, S.; Okutsu, Y.; Suzuki, I.; Shinohara, K.; Komiyama, M.; Takeuchi O.; Shigekawa, H.
Single molecular anatomy of host-guest chemistry based on atomic force microscopy study ofcyclodextrin-ferrocene molecular interaction. Jpn. J. Appl. Phys. 2007, 46, 5614-5616.
6. Yasuda, S.; Suzuki, I.; Shinohara, K.; Shigekawa, H. Single molecular anatomy of solvophobic
effects in host-guest interactions based on surface tension using atomic force microscopy. Phys.
Rev. Lett. 2006, 96, 228303:1-228303:4.
7. Sulchek, T.A.; Friddle, R.W.; Langry, K.; Lau, E.Y.; Albrecht, H.; Ratto, T.V.; DeNardo, S.J.;
Colvin, M.E.; Noy, A. Dynamic force spectroscopy of parallel individual Mucin1–antibody bonds
Proc. Natl. Acad. Sci. 2005, 102, 16638-16643.
8. Neuert, G.; Albrecht, C.; Pamir, E.; Gaub, H.E. Dynamic force spectroscopy of the digoxigenin–
antibody complex. FEBS Lett. 2006, 580, 505-509.
9. Merkel, R.; Nassoy, P.; Leung, A.; Ritchie, K.; Evans, E. Energy landscapes of receptor–ligand
bonds explored with dynamic force spectroscopy. Nature 1999, 397, 50-53.
10. Evans, E. Introductory Lecture Energy landscapes of biomolecular adhesion and receptor
anchoring at interfaces explored with dynamic force spectroscopy. Faraday Discuss. 1998, 111, 1.
11. Yuan, C.; Chen, A.; Kolb, R.; Moy, V.T. Energy landscape of streptavidin−biotin complexes
measured by atomic force microscopy. Biochemistry 2000, 39, 10219-10223.
12. Patel, A.B.; Allen, S.; Davies, M.C.; Roberts, C.J.; Tendler, S.J.B.; Williams, P. M. Influence ofarchitecture on the kinetic stability of molecular assemblies. J. Am. Chem. Soc. 2004, 126,
1318-1319.
13. Piramowicz, M.O.; Czuba, P.; Targosz, M.; Burda, K.; Szymoński, M. Dynamic force
measurements of avidin–biotin and streptavdin–biotin interactions using AFM. Acta Biochim.
Polon. 2006, 53, 93-100.
14. Wong, J.; Chilkoti, A.; Moy. V.T. Direct force measurements of the streptavidin–biotin interaction.
Biomol. Eng. 1999, 16, 45-55.
15. Taninaka, A.; Takeuchi, O.; Shigekawa, H. Site-selective anatomy of step-by-step reactions inligand–receptor bonding processes using dynamic force spectroscopy. Appl. Phys. Express 2009, 2,085002:1-085002:3.
16. Takeuchi, O.; Miyakoshi, T.; Taninaka, A.; Tanaka, K.; Cho, D.; Fujita, M.; Yasuda, S.; Jarvis,
S.P.; Shigekawa, H. Dynamic-force spectroscopy measurement with precise force control using
atomic-force microscopy probe. J. Appl. Phys. 2006, 100, 074315:1-074315:6.
17. Oberhauser, A.F.; Hansma, P.K.; Vazquez, M.C.; Fernandez, J.M. Stepwise unfolding of titin
under force-clamp atomic force microscopy. Proc. Natl. Acad. Sci. 2001, 98, 468-472.
18. Marszalek, P.E.; Li, H.; Oberhauser, A.F.; Fernandez J.M. Chair-boat transitions in single
polysaccharide molecules observed with force-ramp AFM. Proc. Natl. Acad. Sci. 2002, 99,
4278-4283.
19. Evans, E.; Ritchie, K. Dynamic strength of molecular adhesion bonds. Biophys. J. 1999, 72,
1541-1555.

Int. J. Mol. Sci. 2010, 112151

20. Evans, E.; Ritchie, K. Strength of a weak bond connecting flexible polymer chains. Biophys. J.
1999, 76, 2439-2447.
21. Pierres, A.; Touchard, D.; Benoliel, A.-M.; Bongrand, P. Dissecting streptavidin-biotin interaction
with a laminar flow chamber. Biophys. J. 2002, 82, 3214-3223.
22. Friedsam, C.; Wehle, A.K.; Kühner, F.; Gaub, H.E. Dynamic single-molecule force spectroscopy:
bond rupture analysis with variable spacer length. J. Phys: Condens. Matter 2003, 15,
S1709-S1723.
23. Pincet, F.; Husson, J. The solution to the streptavidin-biotin paradox: The influence of history on
the strength of single molecular bonds. Biophys. J. 2005, 89, 4374-4381.
24. Husson, J.; Pincet, F. Analyzing single-bond experiments: Influence of the shape of the energy
landscape and universal law between the width, depth, and force spectrum of the bond. Phys. Rev.
E 2008, 77, 026108:1-026108:14.
25. Strunz, T.; Oroszlan, K.; Schumakovitch, I.; Güntherodt, H.-J.; Hegner, M. Model energy
landscapes and the force-induced dissociation of ligand-receptor bonds. Biophys. J. 2000, 79,
1206-1212.
26. Björnham, O.; Schedin, S. Methods and estimations of uncertainties in single-molecule dynamic
force spectroscopy. Eur. Biophys. J. 2009, 38, 911-922.
27. We investigated the sampling rate dependence of the DFS analysis. Particularly for avidin-biotin
bonding, the shape of the histogram of rupture force was severely deformed at the sampling rate of
1 kHz in comparison with that at the sampling rate of 100 kHz.
28. Weber, P.C.; Ohlendorf, D.H.; Wendoloski, J.J.; Salemme, F.R. Structural origins of high-affinity
biotin binding to streptavidin. Science 1989, 243, 85-88.
29. Katz, B.A. Binding of biotin to streptavidin stabilizes intersubunit salt bridges between Asp61 and
His87 at low pH. J. Mol. Biol. 1997, 274, 776-800.
30. Grubmüller, H.; Heymann, B.; Tavan, P. Ligand binding: Molecular mechanics calculation of thestreptavidin-biotin rupture force. Science 1996, 271, 997-999.
31. Zhou, J.; Zhang, L.; Yongsheng, L.; Tsao, H.-K.; Sheng, Y.-J.; Jiang, S. Unbinding of thestreptavidin-biotin complex by atomic force microscopy: A hybrid simulation study. J. Chem. Phys.
2006, 125, 104905:1-104905:7.
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