quantitative measurement of the mechanical properties of ... · quantitative measurement of the...

Nano Res

1

Quantitative measurement of the mechanical properties

of human antibodies with sub-10-nm resolution in a

liquid environment

Agnieszka Voss1,2, Christian Dietz1,2 (), Annika Stocker1,2, and Robert W. Stark1,2 ()

Nano Res., Just Accepted Manuscript • DOI 10.1007/s12274-015-0710-5

http://www.thenanoresearch.com on January 6, 2015

© Tsinghua University Press 2015

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Nano Research

DOI 10.1007/s12274-015-0710-5

Quantitative measurement of the mechanical

properties of human antibodies with sub-10-nm

resolution in a liquid environment

Agnieszka Voss, Christian Dietz*, Annika Stocker,

Robert W. Stark*

Technische Universität Darmstadt, Germany

An atomic force microscope in the Peak-Force tapping mode

can be used to simultaneously obtain structural images with

sub-10-nm resolution of human antibodies and a diversity of

maps of quantitative mechanical properties, such as elasticity,

adhesion, and deformation in a liquid environment.

http://www.pos.tu-darmstadt.de/pos/index.en.jsp



liquid environment


Received: day month year

Revised: day month year

Accepted: day month year

(automatically inserted by

the publisher)

© Tsinghua University Press

and Springer-Verlag Berlin

Heidelberg 2014

KEYWORDS

atomic force microscopy,

structure and physical

properties, piconewton

forces, flexibility,

antibodies,

immunoglobulin

ABSTRACT

The nanomechanical properties of single human immunoglobulin G and M

antibodies were measured in a liquid environment using a fast force-volume

technique with sub-10-nm spatial resolution. Ultrastructural details of these

molecules could be resolved in the images. Simultaneously, important physical

properties, such as elasticity, adhesion and deformation, were measured.

Considering their dimensions and adsorption onto the substrate, the

immunoglobulin M antibodies were highly flexible, with a low elastic stiffness

(34 ± 10) MPa and high deformability (1.5 ± 0.5) nm.

1 Introduction

Structural, physical, and chemical properties of

biological macromolecules govern their functionality

on the nano- and microscale. In particular,

mechanical properties affect the biological function

of proteins in processes such as cell signaling,

membrane signal transduction [1] and immune

responses [2, 3]. For example, antibodies are often

highly flexible, allowing them to adopt

conformations to bind to structurally diverse

antigens [4]. To better understand the basic principles

of such molecular processes, sophisticated methods

are necessary to determine the relationship between

Nano Research

DOI (automatically inserted by the publisher)

Address correspondence to Christian Dietz, [email protected]; Robert W. Stark, [email protected]

Research Article

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2 Nano Res.

the ultrastructure and the mechanical properties of

molecules in physiological conditions.

There are various methodologies to visualize the

physical properties of individual macromolecules.

Most common approaches are based on one of the

following techniques: (1) fluorescence microscopy, (2)

electron microscopy, or (3) atomic force microscopy.

Atomic force microscopy allows the measurement of

the mechanical properties of protein crystals or

individual molecules [5-15]. Dynamic atomic force

microscopy methods are suitable for high-resolution

imaging in a liquid environment and can provide

information on the sample mechanics [16-18]. Most

commonly, force-distance curves are used to

simultaneously record information about the

structural, physical and chemical properties of the

sample [19] because this information can be directly

derived from the cantilever deflection data.

Here, we exploit an imaging technique for

quantitative nanomechanical mapping which is

based on the so-called Peak-Force tapping mode

(PeakForce QNM) [20]. In this mode, the tip moves

pixel-wise across the sample surface recording a

force-distance curve at each point, thus measuring

the sample morphology together with mechanical

sample properties. To obtain quantitative data, the

cantilever is driven at a few kHz, which is far below

the mechanical resonance but is fast enough to

achieve high data acquisition rates. Thus, the

topographical imaging and simultaneous mapping of

mechanical properties extracted from each

force-distance curve can be achieved at a high

resolution and at reasonable imaging rates in an air

or liquid environment.

This study focuses on two macromolecules,

immunoglobulin M (IgM) and immunoglobulin G

(IgG), which play a key role in the mammalian

immune response [21]. Both human antibodies (Ig*)

can be found in large quantities in our blood. They

are produced in various types with different amino

acid sequences and antigen-binding sites, and these

antibodies bind specifically to viruses or microbial

toxins. The biochemical function and structural

models of Ig* obtained by X-ray scattering have been

investigated in depth [22, 23]. IgM macromolecules

are secreted by B cells in two functional polymeric

forms consisting of heavy (H) and light (L) chains.

The predominant form of these H2L2 polymers

exhibits a joining (J-) chain. High J-chain

concentrations during polymerization result in high

levels of pentameric IgM antibodies while in their

absence, mostly hexametric IgM molecules are

expressed. However, a significant amount of J-chain

free pentameric IgM molecules was also found

[24-26]. This study focuses on the pentameric IgM

form that can be observed with and without J-chain

positioned in the macromolecule center [27]. In

addition to the control of the polymeric formation,

the J-chain was found to have a major influence on

the binding to the hepatic polymeric

immunoglobulin receptor of Ig* molecules [28].

The protein structure of the antibodies permits a

large amount of flexibility. Due to dynamic changes

in the macromolecular substructure, Ig* can adapt to

a large number of antigens of various sizes and

shapes [2, 29]. As noted by Pumphrey et al. [30],

mechanical properties are crucial for functionality.

Several groups have succeeded in resolving the

structure of Ig* with an atomic force microscope

[31-41], providing complementary information about

the molecules, such as the elasticity and flexibility [36,

42]. Although these measurements provide

information about the surface properties, there is a

close relationship between the surface elasticity and

the flexibility of thin samples. Here, we discuss an

approach to obtain a whole package of physical maps,

including the structural, elastic, adhesive and

deformation properties of these immunoglobulins,

which allows us to understand their flexibility.

2 Experimental

2.1 Materials

Human IgM serum (product number I8260) and IgG

(product number A6029) were purchased from Sigma

Aldrich (St Louis, USA). The stock solution, with an

initial concentration of 0.2 – 1.0 mg/µl, was diluted

1:60 with ultrapure deionized Milli-Q water. A 100 µl

droplet of the diluted IgM solution was deposited on

freshly cleaved muscovite mica for approximately 60

s before being rinsed off with distilled water. We

used the residual water droplet (pH = 5.4) on the

mica surface as our imaging medium. Constant pH

conditions are essential for the reproducibility of the

experiments with Ig* antibodies because the stability

as well as the molecular conformation are dependent

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

3 Nano Res.

on the surrounding medium [43]. For this reason, the

pH value of the Milli-Q water was determined by a

standard pH meter (pH 314i, WTW GmbH, Weilheim,

Germany) prior to each experiment.

2.2 Atomic force microscopy

All measurements were performed using the

PeakForce QNM mode with a Dimension Icon AFM

from Bruker AXS (Santa Barbara, USA). This mode

was applied to measure surface topography and to

quantify the various mechanical properties of the

specimens. PeakForce QNM is an extension of the

pulsed-force mode [44], with an improved force

resolution of 10-10 N, which allows one to map

adhesion, elastic modulus, deformation, or

dissipation simultaneously at line rates of

approximately 1 Hz. In the PeakForce QNM mode,

force-separation curves are measured and analyzed

pixel-wise. The maximum force (peak force) is

controlled precisely each time the tip touches the

surface. The elastic modulus of the sample was

extracted by the Derjaguin-Muller-Toporov (DMT)

model [45], which was proposed for force distance

measurements that involve relatively weak adhesion

forces and small tip-radii, and hence applies for the

experimental conditions of this study. By definition,

the DMT model fits the equation

(1)

to force-versus-separation data obtained when the tip

is retracted from the surface after the approach. This

equation describes the relationship between the

difference in the applied load and the adhesion force,

( ), and the sample deformation, ( ).

Furthermore, is the radius of the tip, and is

the reduced modulus given by the formula:

, (2)

where and are the sample’s and tip’s Poisson

ratios and Es and Et the elastic moduli of the sample

and tip, respectively. For biomolecules, the stiffness

of the tip is much larger than that of the sample and

hence the last term in formula (2) can be neglected.

This technique covers a broad range of elastic moduli

between 700 kPa and 70 GPa [20]. Nonetheless,

quantitative measurements require calibration, either

with a reference sample (relative method) or by

measuring the cantilever tip radius and its spring

constant (absolute method) [20]. Several additional

mechanical properties can be obtained. The adhesion

force at each contact point can be extracted from the

vertical difference of the minimum force during

retraction and the zero force in a force-distance plot.

The deformation of the sample is assigned to the

horizontal difference between the “peak force”

tip-sample separation and the tip-sample separation

corresponding to the onset of the overall repulsive

force during an approach. A detailed description of

the data quantification can be found elsewhere [46].

Cantilevers of the type “ScanAsyst – Fluid” and SNL

were purchased from Bruker AFM probes (Camarillo,

USA) and used without further modification. Typical

force constants were k = 1.0 N/m with a nominal

resonant frequency f0 = 150 kHz and k = 0.4 N/m with

a nominal resonant frequency f0 = 65 kHz in air,

respectively. The individual cantilevers were

calibrated with the thermal noise spectrum method

[47]. Tip radii were estimated from reference

measurements on a sharp-edged TiO2 sample (RM15,

Bruker AFM probes). The tip curvatures were

obtained with the blind tip reconstruction method

provided by the AFM operation system. We obtained

radii of 7 nm and 9 nm for the “ScanAsyst – Fluid”

tips and SNL tips, respectively.

The acquisition rate was set to 2 kHz (1 Hz

corresponds to 1 force curve per second), and the

peak forces were varied between 300 pN and 800 pN.

The lower force limit was defined by the necessity of

a certain indentation depth to successfully extract the

elastic modulus of the sample. Peak forces smaller

than 300 pN also led to false triggering due to the

hydrodynamic damping in the surrounding medium.

Forces larger than 800 pN either caused structural

damage of the antibodies or contamination of the tip

with biological material. The force trigger limited the

mechanical stress in both tip and sample and limited

the indentation depth to 0.7 nm in mica. In repeated

measurements with forces in this range, we did not

experience topographical alterations indicating that

the deformation of the molecules during

characterization was reversible.

2.3 Image processing

The topography images shown in this work were

subject to a line-wise 1st order flattening and

low-pass filtering. We used the low-pass command of


4 Nano Res.

the NanoScope software (Bruker AXS, Santa Barbara,

USA) to suppress high spatial frequency components

within the topography images by averaging a 3 x 3

pixel region around each pixel [48]. Adhesion, elastic

modulus, and deformation maps were rendered from

the raw data as provided by the NanoScope software

without further treatment.

3 Results and discussion

As shown in Fig. 1, PeakForce QNM allows one to

resolve the morphology of tiny biological samples

with a high resolution in a liquid environment.

Figure 1(a) depicts a homogeneous distribution of

human IgM molecules adsorbed on mica. Figure 1(b)

illustrates the overall surface charge as a function of

the pH value for mica and IgM antibodies. Mica is

negatively charged at all pH values, whereas IgM has

an isoelectric point at 5.5 ± 1.0 [49]. The

measurements were performed in ultrapure

deionized water at pH = 5.4, where the molecules are

nearly uncharged. Although the molecules are

trapped at the surface by van der Waals forces or

counterions shared with the mica substrate, the

molecules retain their flexibility [50, 51]. Most of the

antibodies reveal a typical pentameric structure,

which is shown in Fig. 1(c). The five subunits

(Y-shaped branches - IgG) are held together by

disulfide bonds in the center region of the

biomolecule (yellow), where the J-chain connects two

of these subunits via heavy chains (blue). Each

subunit consists of two identical heavy chains and

two identical light chains. Disulfide bonds bridge the

heavy and the light chains (as shown in the lower

part of Fig. (1(c))). Fab and Fc fragments are

designated by braces [22]. Interestingly, the IgM

molecules apparent in Fig. 1(a) differ slightly in

shape and size, most likely due to the different

positions of the Fab domains with respect to the

central body of the molecule. This suggests a high

mechanical flexibility of the antibodies during the

adsorption process [23].

Additionally, we imaged single IgG molecules

adsorbed on mica. The Y-shaped morphology of the

molecule is apparent in the high-resolution

topographical PeakForce QNM image of Fig. 1 (d).

The observed molecular structures and their total

sizes in the lateral dimensions are in good agreement

with other studies [34, 35]. A similar resolution was

achieved with a different type of cantilever (Bruker

SNL, Fig. S1 in the Electronic Supplementary

Material (ESM)). These cantilevers were slightly

softer and they were specified with a smaller

nominal tip radius. In general, we observed an

enhanced probability of picking up biological

material from the sample with these tips.

For a more detailed analysis of the morphology, we

focused on two single IgM pentamers and recorded

highly resolved images (Fig. 2). From these images

(Fig. 2(a) and (b) top) and the respective

cross-sectional profiles (bottom) drawn through the

horizontal positions (red arrows), we estimated the

lateral and vertical dimensions of the IgM. The

average height of the IgM molecules was

approximately 2.2 nm. The obvious difference in the

height (4 – 10 nm) reported by Munn et al. [52],

judged from edge-on views with electron microscopy,

can be explained by the elastic deformation of the

sample caused by the pressure of the tip on the

sample (see the differences in the deformation

between IgM and mica, Fig. 3(h)) and the different

electrostatic interactions. As described by Knoll et al.

[53], this can cause an overcompensation of the

z-piezo that introduces an additional height

Figure 1 (a) Topographical image of IgM antibodies adsorbed on

mica in a liquid environment obtained in peak-force tapping

mode. (b) Illustration of the overall surface charges of Si3N4, IgM

and mica at different pH values [49, 59]. (c) Schematic structure

of IgM (top) and IgG (bottom) antibodies depicting their

sub-domains. (d) High-resolution topographical image of a single

IgG human antibody adsorbed on mica in distilled water. The Fab

domains are indicated by the green arrows. Cantilevers: Bruker

“ScanAsyst-Fluid”.


5 Nano Res.

Figure 2 Highly magnified topography peak-force tapping mode

images of single IgM human antibodies, revealing two possible

orientations. (a) The center region (J-chain) shows a protrusion

from the main body (top). The horizontal profile taken at the

position indicated by the red arrows reveals a 0.5 nm higher tip

region (blue arrow) than the surrounding area (bottom). (b)

Missing J-chain configuration exhibiting a hollow in the center

region (top). The valley (blue arrow) has a depth of

approximately 0.5 nm, as depicted in the horizontal

crosssectional profile (bottom).

difference between soft materials and stiff substrates.

Additionally, force differences between tip-sample

and tip-substrate of attractive nature (electrostatic or

van-der-Waals) can lead to the same effect. PeakForce

QNM measures the topography at the peak force, i.e.

at the point of maximum indentation. In contrast,

frequency and amplitude modulation atomic force

microscopy rely on weighted forces. Thus, the

compensation of attractive forces between tip and

sample by the z-piezo can cause differences in the

apparent height of the biomolecules obtained with

amplitude [37] or frequency modulation atomic force

microscopy [36].

The lateral size of the molecules can also be

estimated from the height profiles. Considering the

finite size of the tip (R = 7 nm) and the geometric

relationship between the tip shape and the sample

topology [54], the apparent width of the molecules in

the cross-sectional profile of approximately 42 nm

can be reduced to a lateral size of approximately 28

nm. Strikingly, in Fig. 2(b), the Fab domains (reduced

to a length of 7 nm) clearly stick out of the center

region, and in some cases, two single neighboring Fab

domains within the IgG substructure can be

unambiguously distinguished (e.g., two fragments

pointing to the left). The main body consists of five Fc

domains, including the J-chain, with an apparent

diameter of 18 nm. These values are consistent with

the dimensions of IgG and IgM found in earlier

studies [31, 36, 42]. Comparing Fig. 2(a) with 2(b), the

antibody in the left panel exhibits a clear protrusion

inside the main body, whereas the antibody in the

right panel possesses a hollow (see the blue arrows in

the cross-sectional profile). We assume that the

protrusion of the molecule apparent in Fig. 2(a)

corresponds to a J-chain that can be incorporated in

the pentagonal molecular structure at a relatively late

stage of the intracellular assembly process [27]. The

fact that the majority of the macromolecules observed

in this study has a pentagonal structure indicates that

J-chains were present in a significant amount during

polymerization. In contrast, the presence of a hollow

of a particular molecule (topography and height

profile, Fig. 2(b)) indicates the absence of a J-chain of

this individual antibody molecule. J-chain free

pentagonal IgM molecules were reported in several

studies [24-26]. However, it cannot be excluded that

this molecule adsorbed upside-down on the mica

substrate, i.e., the protrusion points downward.

In addition to the topographical images, PeakForce

QNM provides maps of several mechanical surface

properties, such as adhesion, elastic modulus

(DMT-modulus), and deformation. Figure 3(a) shows

a (332 x 138) nm2 topographical image accomplished

with PeakForce QNM, including five single IgM

molecules. The peak force set point was set to a force

of 800 pN, and the standard deviation of the

respective feedback loop error was 16 pN (Fig. 3(b)).

The DMT modulus map (Fig. 3(c)) reveals two

dominant regions of constant elasticity: one region

for the mica surface and another for the IgM

antibodies. We set the lower value of the color bar to

-100 MPa for better visibility, although no values

below zero were measured. For analysis purposes,

we drew averaged cross-sectional profiles (120 nm x

12 nm) through the dashed red line indicated in Fig.

3(a) of a single molecule to exhibit particular values

of different mechanical properties of IgM compared

to the mica surface. Figure 3(d) represents the profile

of the elasticity fitted with the DMT modulus. The

average DMT modulus of mica was estimated to (1.3

± 0.4) GPa and of IgM antibodies to (34 ±10) MPa


6 Nano Res.

Figure 3 Mechanical properties of human IgM antibodies as obtained from quantitative nanomechanical mapping measurements in a

liquid environment. (a) Topography and (b) peak-force error. (c) Map of the elastic modulus (DMT fitted) and (d) averaged profile (120

nm x 13 nm) corresponding to a cross-section drawn through the dashed red line indicated in (a). Note that the ordinate is split for better

visibility. (e) Adhesion force image and (f) respective averaged cross-section. (g) Image of the deformation generated by the pressure

applied through the tip on the sample and (h) averaged cross-section.

(note the split y-axis). Figure 3(e) shows the adhesion

map, and Fig. 3(f) shows the respective profile with

an adhesion force of (174 ± 22) pN on mica and

approximately zero on IgM. The deformation map

(Fig. 3(g)) and its profile (Fig. 3(h)) provided values

of (0.7 ± 0.1 nm) on mica and (1.5 ± 0.5 nm) on IgM.

The results show that mechanical properties, such as

the elasticity, can be imaged with high resolution.

Strikingly, the exact contour of single molecules

becomes apparent. However, the substructure of the

IgM subdomains is barely observable in the

nanomechanical property maps in contrast to

topographical images. On the basis of the smallest

structures observable in these images, the achieved

lateral resolution of the technique can be

approximated to 7 nm. The elastic modulus

measured on IgM antibodies is very close to the

value recently reported by Martinez et al. [36].

Control experiments performed in a phosphate

buffered saline solution (Fig. S2 in the Electronic

Supplementary Material (ESM)) resulted in elasticity

values of the same range of the IgM antibodies. The

lateral resolution of the topography images and the

physical maps, however, was significantly reduced

because of the high salt concentration.

The measured values of mica, however, deviated

slightly from the nominal elastic modulus (10 GPa).


7 Nano Res.

One reason for this difference could be the

insufficient indentation depth of the tip into the mica

surface while sensing the necessary mechanical

response. The inset of Fig. 4 displays single

force-versus-separation curves we obtained on an

IgM molecule (blue) and on mica (red). The curve

measured on mica clearly reveals that most of the

sample deformation occurs directly after the

minimum in force curve. After a fraction of

nanometers in deformation, the tip-mica force

depicts an almost vertical trend. The indentation of a

sharp tip into the surface of phyllosilicates is a

complex physical process on the nano- and atomic

scale which is out of scope of this study.

These results show that the method allows one to

quantify the mechanical properties of heterogeneous

samples even if the elasticity varies over orders of

magnitude. However, sensing the mechanical

properties of soft biological systems adsorbed on

hard substrates by nanoindentation has to be carried

out with care. The sharp tip can induce local strains

within the soft material which can exceed the linear

material regime and hence, lead to a damage of the

sample surface [55]. To address this problem, we

limited the maximum load during force curve

acquisition to avoid damage to the biomolecules and

to minimize mechanical stress. Repeated probing of

the same individual molecule did not lead to a

measurable degradation of topography or local

stiffness.

Additionally, the hard mica substrate might affect the

measured elasticity of the IgM molecules due to their

small dimensions [56]. A comprehensive

nanomechanical study of amyloid fibrils with similar

height but larger mechanical stiffness as compared to

IgM, demonstrated that the various AFM methods

such as nanoindentation, peak-force-tapping, image

analysis, and force spectroscopy reliably result in

similar elasticity values [56, 57]. Assuming that the

total height of the molecule on the substrate is the

measured height (typically 2.2 nm) plus deformation

(typically 1.5 nm), the relative compression is up to

40 %. However, the measured value still is a valid

upper estimate for the ‘true’ bulk modulus [56]. More

importantly, quantitative nanomechanical imaging

gives an insight into the local variations of the

stiffness and thus the local flexibility with high

spatial resolution.

The large adhesion measured on mica compared to

the values measured on IgM antibodies is surprising

at first glance. Taking into account the overall

charges of mica (negative) and Si3N4 (material of the

tip, positively charged) at pH = 5.4, an attractive force

between both parties is expected, leading to high

adhesion forces (compare Fig. 1(b)). Christenson

extensively studied the adhesion forces between two

mica surfaces in dry air and liquid with the surface

force apparatus and found that shielding of ionic

contributions takes place in water at the interface [58].

Similar studies for the Si3N4 mica interface have to be

performed to find the physical chemistry behind the

origin of the interaction as London dispersion alone

cannot explain the amount of adhesion. On the other

hand, the pH value of 5.4 is close to the isoelectric

point of IgM antibodies, which explains the neutral

character of the adhesion results found on IgM. We

note that there are regions of different adhesion

values apparent on the mica substrate within the

adhesion map (Fig. 3(e)). A continuous

attachment/detachment of biomolecules to/from the

Figure 4 Force-distance curves captured on mica (red curve – top)

and IgM (blue curve – bottom). Crosses in the topographical

image mark the positions where force-distance curves were

manually exported during imaging. The inset magnifies the area

of interest. The z-position of the piezo was converted into

tip-sample separation, and the curves were fit by the DMT model

(black lines, extracted elastic modulus: Emica = 0.52 GPa; EIgM =

2.2 MPa).


8 Nano Res.

tip during scanning can lead to different adhesive

forces at the interface. The detected DMT modulus,

however, remains unaffected by this very thin layer

of molecules covering the end of the tip. The mean

deformation of the molecules (1.5 nm) is sufficient to

determine the elastic modulus using the DMT-model.

Under these conditions, the molecules are squeezed

by the tip, which leads to an apparent reduced height

in the topographical images. The morphology and

mechanical response of IgM molecules remained

unchanged even after several scans over the same

area with a peak force of approximately 800 pN;

however, some of the molecules were slightly

distorted in such a way that the Fab domains were

marginally twisted. Because of a 7-nm-tip-radius, the

deformation process into the mica surface can be

considered as a global elastic deformation rather than

an actual surface-atom displacement.

In Fig. 4, we compare a single force-distance curve

extracted manually from the software during

imaging. These data were obtained on mica with a

force distance curve measured on IgM to verify the

elastic modulus, adhesion, and deformation. This

analysis serves as a verification of the quantitative

DMT maps obtained after data reduction. To assess

elasticity, the plots were converted into force vs.

separation curves, and the DMT model was

externally fit using Eq. (1) (see experimental section)

over a range that is similar to the range adjusted by

the microscope software (see the black lines of the

inset; fitting parameter: R = 7 nm, υ = 0.3, obtained

elasticity Emica = 0.52 GPa; EIgM = 2.2 MPa). These

results are close to the values offered by the

microscope software. Measuring the minimum force

provides adhesion values of -400 pN on mica. The

force-distance curve taken on IgM shows almost no

adhesion. Sample deformations range from

approximately 1.5 nm on mica to 4 nm on IgM. The

manually extracted values of elasticity, adhesion and

deformation were in the range of the data provided

by the AFM software.

4 Conclusions

We present a straightforward approach to

simultaneously image and generate maps of the

mechanical properties of the human IgG and IgM

antibodies adsorbed on mica. Quantitative

nanomechanical mapping is a method that provides

high spatial resolution with complementary

information on adhesion, elasticity, or deformation.

All of this information is extracted from

force-distance curves taken at a kHz acquisition rate.

This approach is non-destructive to soft samples

under the operating parameters that were applied in

this study. The IgM sub-structure could be clearly

resolved in water, suggesting that the molecules lie in

different positions on mica, where the Fab domains

can be oriented in various angles to each other. The

analyses of the topographical data of IgM molecules

revealed the relatively large size of the Fab domains in

comparison with its central portion, where the j-chain

is located. Strikingly, we were able to simultaneously

measure the elastic modulus of a stiff material, such

as muscovite mica (1.3 ± 0.4) GPa, and a soft

biomolecule, such as IgM (34 ±10) MPa. The low

stiffness found on IgM together with the high

deformability (1.5 ± 0.5 nm) in comparison with the

dimensions of the molecule (nominal height: 7 nm)

corroborates the high flexibility of the antibodies.

This flexibility may be the key property that these

antibodies are able to adopt confirmations to bind to

a large number of antigens with varying sizes and

shapes and leads to a high mobility in the organism.

Acknowledgements

The authors thank the Center of Smart Interface for

financial support.

Electronic Supplementary Material: Supplementary

material (control experiments performed in a

phosphate buffered saline solution) is available in the

online version of this article at

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Nano Res.

Electronic Supplementary Material



liquid environment


Supporting information to DOI 10.1007/s12274-****-****-* (automatically inserted by the publisher)

Address correspondence to Christian Dietz, [email protected]; Robert W. Stark, [email protected]


Nano Res.

Figure S1 (a) PeakForce QNM topographical image of IgM antibodies adsorbed on mica in a liquid

environment. (b, c) Two high-resolution topographical images of single IgM human antibodies adsorbed on

mica taken in distilled water using PeakForce QNM. Cantilevers: Bruker SNL.


Nano Res.

Figure S2 PeakForce QNM image of IgM antibodies adsorbed on mica measured in a phosphate buffered saline

solution. (a) Topography, (b) elasticity map, (c) adhesion map and (d) local deformation. Cantilevers: Bruker

SNL.

quantitative measurement of the mechanical properties of ... · quantitative measurement of the...

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