Non-destructive force measurement in liquid usingatomic force microscope

Hiroshi Sekiguchi, Hideo Arakawa, Takaharu Okajima, Atsushi Ikai*

Laboratory of Biodynamics, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology,

4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Kanagawa, Japan

Received 2 September 2001; accepted 21 September 2001

Abstract

Atomic force microscopy (AFM) has been applied to measure inter- or intra-molecular forces acting to hold biological

molecules and structures. For these measurements, it is important to keep the target molecules biologically active on a solid

surface. Besides the strategy for immobilizing them on the surface keeping their biological activities intact, it is crucial to reduce

the force applied to them through the AFM tip to avoid mechanical inactivation of the sample. In this paper, we propose a new

procedure to minimize the effect of contact force. The first step of the procedure is to bring the cantilever tip close to the sample

surface within less than 3 mm, but short of contact with the sample surface. The approximate distance of the tip from the sample

stage is measured using the thermal fluctuation of the cantilever. The second step is a ‘‘compression-free’’ force spectroscopy for

the measurement of protein–protein interactions only, which is possible when the piezo scanner was retracted before the

cantilever starts upward deflection. The interaction force can be measured in the retraction period provided a physical contact is

established between the proteins on the tip and the substrate. This procedure allowed to measure interaction forces between

GroEL and a denatured protein without mechanical deformation. # 2002 Elsevier Science B.V. All rights reserved.

Keywords: Force spectroscopy; Bio-molecule; Thermal fluctuation; Compression-free

1. Introduction

Atomic force microscopy (AFM), invented in 1986

by Binnig et al. [1], has been increasingly used in

biological science not only for imaging but also for

measuring forces [2]. One important precaution to be

considered in the force measurement is how to fix bio-

molecules on a substrate and a probe, securely enough

for measuring force but flexible enough to keep the

molecules biologically active. Various methods for

this purpose have been reported using chemical

cross-linkers, flexible spacer molecules [3], inactive

proteins as cushions [4] and/or self-assembled mono-

layers [5]. Another issue to be addressed in such

experiments is the effect of pushing the molecules

on the substrate and AFM probe in the course of force

measurements. As the AFM probe is pushed onto the

molecule, there is a possibility of denaturing the mole-

cule artificially or adsorbing it to the probe physically.

In reality, however, it has been difficult to apply an

AFM in force measurements without pressing the

sample with the probe for two reasons. First, it is

difficult for us to know how far the probe is from the

sample surface before the cantilever senses the repul-

sive force, and secondly, the probe has to be in contact

with the sample molecule on the substrate for a certain

Applied Surface Science 188 (2002) 489–492

* Corresponding author. Tel.: þ81-45-924-5828;

fax: þ81-45-924-5806.

E-mail address: aikai@bio.titech.ac.jp (A. Ikai).

0169-4332/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved.

PII: S 0 1 6 9 - 4 3 3 2 ( 0 1 ) 0 0 9 7 7 - 1

period to establish interactions or bond formation. To

keep them in contact, the force curve must include

some period of repulsive, i.e. compressing region.

In this paper, we propose a new method to overcome

these problems. The method consists of two steps. The

first step is to bring a cantilever tip close to the sample

surface within the range of piezo-electric’s movement.

We estimate the distance between the tip and the surface

without prior contact between them by detecting the

power spectrum of the cantilever’s thermal fluctuation

in liquid. We could bring the tip within less than 3 mm to

the surface in this step. The second step of the procedure

is the ‘‘compression-free’’ force spectroscopy measure-

ment where no repulsive force is exerted during the

approaching process to immobilized molecules, but the

interaction force is measured in the retraction process.

The results for a protein pair, GroEL on the substrate

and denatured pepsin on the probe, showed difference

depending on compression-free or not.

By using these procedures, force measurements can

be done almost without touching the sample surface

with the AFM probe.

2. Materials and methods

2.1. AFM

A Multimode Nanoscope IIIa (Digital Instruments,

Santa Barbara, CA) equipped with a K-scanner and

a Signal Access Module, and an SPM-9500-J2

(SHIMADZU, Japan) were used. The deflection signal

was recorded by an A/D converter (ADM-670PCI,

Microscience, Japan) mounted on a commercial PC.

The AFM was operated with a standard software or

with a special software for force spectroscopy mea-

surements, ‘‘Force Curve Software, Ver. 2.54 (SHI-

MADZU, Japan)’’. The latter program enabled us to

control the holding time of the piezo-tube at its

approach end, which was equated with the reaction

time for bio-molecular pairs at the nearest position to

each other.

2.2. Preparation of functionalized substrate and tip

Gold-coated AFM tips (OMCL-TR400-PB, Olym-

pus, Japan) were functionalized with porcine pepsin

(SIGMA, Missouri, USA) through the cross-linker,

sulfo-LC-SPDP (sulfosuccinimidyl-6-[30-(2-pyridyl-

dithio)-propionamido]hexanoate, PIERCE, Illinois,

USA) according to the method in [6]. This protein

is known to lose its native conformation [7] and

interact with GroEL at neutral pH [8].

A drop of a GroEL solution (0.5 mg/ml, 100 ml) in

HEPES buffer (20 mM HEPES, 100 mM KCl, 5 mM

MgCl2, pH ¼ 7:2) was deposited on a freshly cleaved

mica surface and protein molecules were adsorbed on

it for 1 h at room temperature. Then the mica surface

was rinsed with HEPES buffer and kept in the buffer

until use.

2.3. Cantilever’s thermal fluctuation in liquid

The frequency and amplitude of thermal fluctuation

of the cantilever (OMCL-TR400-PS, Olympus, Japan)

in HEPES buffer was measured at various distances

between the tip and the mica surface. The distance was

controlled by a piezo-tube when the distance was less

than 6 mm, and by a step motor at larger distances (i.e.

the maximum movement of piezo was about 6 mm).

Deflection data, sampled at 125 kHz, were Fourier-

transformed into power spectra in order to obtain the

frequency response. The resonance frequency of the

cantilevers used was around 10 kHz in air.

3. Results and discussion

3.1. Spectral shift according to the distance

from surface

To start force spectroscopy measurements, the tip

must be brought close enough to the surface within the

range of the piezo movement. We found that the

distance of the probe from the surface can be estimated

from the spectral shift of the thermal fluctuation of the

cantilever in HEPES buffer.

The thermal fluctuation of the cantilever was mea-

sured without externally vibrating it. Fig. 1 shows the

power spectra of a cantilever at several distances from

the substrate. At larger distances than 10 mm, the

power spectrum of the cantilever fluctuation has a

peak at around 1.5–2.0 kHz (Fig. 1, circles). When the

probe was brought closer to the surface, the peak

shifted to around 1.0 kHz at 3 mm (Fig. 1, squares),

and the peak was hardly observable at 300 nm in

490 H. Sekiguchi et al. / Applied Surface Science 188 (2002) 489–492

distance (Fig. 1, triangles). The spectra were then

analyzed by curve fitting to a Lorentzian equation

[9] given by the expression

PðoÞ ¼ f

ðo2 � o20Þ

2 þ 4g2o2

where P is the power density, o the frequency, o0 the

resonant frequency of the cantilever, f the force

applied from the surrounding particles and g the

damping parameter.

The lines in Fig. 1 represent fitted data and the

values of the parameter are shown in Table 1. It is clear

that when the cantilever is near to the surface, the

resonant frequency (o0) is lower and the damping

parameter (g) is higher. It is noted that at distance of

about 3 mm, these values change drastically. Therefore

it is possible to bring a tip close to the substrate within

3 mm by monitoring the spectrum.

3.2. Compression-free force spectroscopy

Fig. 2 is a schematic drawing of the compression-

free force measurement which is composed of

approaching, holding and releasing processes. The

sample stage piezo starts the approaching process

(Fig. 2a) after the cantilever was brought close to

the surface within 3 mm by the method described

above. For the compression-free measurement, the

stage movement should stop before any indication

of repulsive force. The holding process at the nearest

position to the substrate (Fig. 2b) is important because

Fig. 1. Power spectra of cantilever’s thermal fluctuation at three

different distances from the substrate: 300 nm for triangles, 3 mm

for squares and 10 mm (or more) for circles. Spectra were fitted to a

Lorentzian function for each curve: 0.3 mm for solid, 3 mm for

dotted and 10 mm (or more) for dashed lines. As the distance was

controlled with step motor for 10 mm, the accuracy of the distance

was lower than that of other distances; however, the spectrum did

not change in effect when the distance was longer than 10 mm.

Table 1

Values of the parameter for the Lorentzian equation of the

cantilever’s thermal fluctuation in HEPES buffer

Distance from surface (mm) o0 (kHz) g (kHz)

0.3 1.30 1.31

1 1.52 1.30

3 1.81 1.20

6 2.01 0.93

10 (or more) 2.11 0.90

Fig. 2. Schematic drawing of a compression-free force spectro-

scopy. This method is composed of approaching (a), holding (b)

and releasing (c) processes. Figures on the left represent bio-

molecular pairs and AFM probe.

H. Sekiguchi et al. / Applied Surface Science 188 (2002) 489–492 491

it constitutes the reaction time for the establishment of

biological interactions. When the nearest position of

probe to the sample happens to be within the range for

the pairwise interaction to occur, a positive indication

of interaction is to be obtained (Fig. 2c).

This method was applied to the GroEL system, a

protein machinery that interacts with misfolded pro-

teins and assists them to re-fold correctly [10]. Fig. 3

shows the examples of recorded force curves in a

normal and a compression-free method. Interestingly

the magnitude of force observed in the compression-

free method was less than that observed in the normal

method, suggesting that multiple points or molecules

were reacted when the probe was pushed. The force

curves were also rather reproducible in compression-

free method while the normal method made variety in

profile and the force. This might be caused by the

different kinds and number of interactions happened

in normal method. We believe the compression-free

method gives much better results to study single

molecular event on soft material like bio-molecules

both in quality and reproducibility.

Occasionally we found the cantilever-sensed repul-

sive force during the holding time caused by the drift

of the equipment, but this seemed not damaging the

sample on the probe and the substrate, and not affected

the next compression-free experiment done subse-

quently. This kind of drift is of course not desired

in these experiments and the AFM with less drift is

hoped to be developed.

4. Conclusion

By using the methods of force curve measurement

described above, sample damages as well as tip con-

taminations were reduced so that the force curves

become stable and reliable. Since the contact area

between the tip surface and the substrate was mini-

mized, chances of multiple interactions were also

reduced.

Acknowledgements

This work was supported in part by Grant-in-Aid

to AI from the Japan Society for the Promotion

of Science (Research for the Future Program

#99R167019) and from the Japanese Ministry of

Education, Science, Culture and Sports (Scientific

Research on Priority Areas (B) #11226202).

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Fig. 3. Examples of force curves measured by a normal (a) and a

compression-free (b) method. Three typical force curves were

reproduced for each method applied to measure a biological

interaction. Denatured protein (pepsin) attached to the tip was

brought into contact with GroEL on the substrate and binding force

was measured during the retraction period.

492 H. Sekiguchi et al. / Applied Surface Science 188 (2002) 489–492