Comm. Appl. Biol. Sci, XX/X, 2013
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STUDY OF THE FORCE INDUCED DISSOCIATION OF
MOLECULAR BONDS
E. PÉREZ-RUIZ*, D. SPASIC*, L.J. VAN IJZENDOORN**, M.W.J. PRINS***,
J. LAMMERTYN*
*Department of Biosystems, MeBioS - Biosensors group, KU Leuven - University of Leuven,
Willem de Croylaan 42, 3001 Leuven, Belgium.
** Department of Applied Physics, Eindhoven University of Technology, The Netherlands.
*** Philips Research, Eindhoven, The Netherlands.
INTRODUCTION
Most of the biological processes are controlled through the specific
recognition and subsequent binding of a ligand to a receptor. Therefore, the
quantification and characterization of biomolecular interactions is essential
for completely understanding these processes. Moreover, these interactions
have been successfully employed for designing different bioassays
implemented in various biotechnological applications. The sensitivity and
specificity of these assays rely on the affinity and strength of the molecular
interactions, emphasizing thus the importance of studying and quantifying
those features.
In this work we implemented a recently published technology (Jacob et al.,
2012), based on magnetic beads and application of magnetic forces in the
picoNewton
range,
for
studying
intermolecular
interactions.
Superparamagnetic beads, functionalized with a receptor, are incubated on
polymer substrates coated with the respective ligand. Different magnetic
pulling forces are subsequently applied to the ligand-receptor bond and the
number of beads detaching from the surfaces is quantified. From these
experiments we could determine the dissociation rate constant at zero force
as well as the transition state distance in antibody-protein complexes.
As a model system we used main peanut allergen, Ara h 1 protein, and its
complementary monoclonal antibody. Peanut allergy is a common and often
severe condition with no medical treatment available so far and the only
existing therapy being avoidance of allergen-containing food. Because
rigorous labelling of products is required, it is essential to improve the
performance of current bio-assays for detecting potential allergens in food
samples. To date, several immunoassay techniques (Mills et al., 1997, Pomes
et al., 2004, Wen et al., 2005) and immunosensors (Huang et al., 2008;
Pollet et al., 2011) for detection of Ara h 1 have been described in literature.
Because all of them are based on antibodies, characterizing their affinity
towards Ara h 1 protein is needed for improving the analysis.
MATERIAL AND METHODS
Reagents and biomolecules
Carboxilic acid coated superparamagnetic particles with a diameter of 2.8
µm (Dynabeads™ M270) from Life Technologies (Norway) were used in these
experiments. Monoclonal antibody against Ara h 1 purchased from Indoor
Biotechnologies Limited (UK) was bound to the particles using EDC/NHS
chemistry. Modified particles were stored in 150 mM phosphate buffered
saline (PBS) solution (pH 7.4) containing 0.5% BSA and 0.01% Tween 20.
An open cell of 9 mm diameter and 0.12 mm depth was created on
polystyrene substrates (Agar Scientific, UK) using a double sided adhesive
cell-dots (Secure-Seal™ imaging spacer, Sigma-Aldrich, The Netherlands).
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Ara h 1 protein was immobilized by physical adsorption (1 hour at room
temperature) on the polystyrene surface enclosed within the cell. All modified
polystyrene surfaces were finally blocked with 1% BSA in 150 mM PBS
buffer.
All buffer reagents were supplied by Sigma-Aldrich (Belgium) and all
solutions were prepared using deionized water purified with a Milli-Q
Simplicity 185 system (Millipore, USA)
Bead-based magnetic detection technology
As shown in Figure 1, the bead-based magnetic detection technology setup
contains three different components: a sample holder, a microscope-camera
system and an electromagnet. A fluid cell for incubation of functionalized
magnetic particles is placed on the sample holder on top of an
electromagnet. The electromagnet consists of a copper wire coil around a soft
iron core with a tip that has been flattened off, being the diameter of the
flattened area 1 mm. Due to this large tip radius, the magnetic force applied
to the particles is uniform across the binding surface. The distance between
the tip of the magnet and the binding surface of the sample is 300 µm. The
current going through the coil controls the magnetic field generated. The
maximum current used in this experiments was 0.8 A. Because at this
current substantial amount of heat is generated in the coil, the system is
cooled using a water pump. This enables continuous operation below 45°C
and prevents the coil from melting. In addition, a push-pull current
controller that temporarily applies a higher voltage up to a maximum of 25 V
has been developed to reduce the rise-time of the current and to allow the
application of time-dependent currents for demagnetization of the magnet
core after every force application.
camera
microscope
fluid cell
sample
holder
synchronized external
triggering
computer
magnetic system
(electromagnet)
Figure 1. Scheme of the instrumental set-up consisting of a microscopecamera system, an electromagnet and a sample holder.
The particles on the polystyrene surface were imaged with a Leica DM6000
microscope and images were acquired at 30 Hz frame rate by a RedLake
MotionPro HS-3 speed camera. The camera is triggered to the electromagnet
through a function generator (Agilent, 33250A). The first frame at t=0
captures the total number of particles bound on the surface before the
application of the force. To measure the dissociation rate, the microscope
was focused on the polystyrene surface and the number of beads remaining
was counted on the consecutive image frames using a home-written MatLab
(The MathWorks, Inc., US) program.
Comm. Appl. Biol. Sci, XX/X, 2013
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RESULTS
We first optimized the concentration of Ara h 1 protein immobilized on the
polystyrene slides. If the density of protein present on the surface is too
high, multiple bonds may occur between particles coated with the Ara h 1
antibody and the surface. On the other hand, if the concentration is too low,
the number of single-bonds within the analysed surface area will not be
statistically significant. Therefore, different concentrations of Ara h 1
protein, ranging from 0.1 nM to 100 nM, were immobilized on the
polystyrene slides (Figure 2) with 1 nM of protein estimated to be the optimal
one for the assay. To test for unspecific adsorptions, modified particles were
also incubated on a 1% BSA coated polystyrene slide.
1% BSA
0,1 nM
1 nM
100 nM
Figure 2. Optimization of the concentration of Ara h 1 protein on the
polystyrene slide surface. Images of particles bound to the surface as
recorded by the camera.
As it can be seen from Figure 2, functionalized magnetic particles bind to the
polystyrene surfaces only when Ara h 1 protein is present. This suggests
that there are no unspecific adsorptions of magnetic particles and that their
binding to the surface is only due to the specific interactions between
antibody and its ligand.
Subsequently, different magnetic pulling forces, from 10 to 50 pN, were
applied to the beads bound to the functionalized polystyrene surfaces. At
least two different samples were studied for each applied force and an
average dissociation curve for each experiment was obtained (Figure 3).
1,0
10 pN
Bound fraction
0,8
30 pN
0,6
0,4
50 pN
0,2
0
10
20
30
40
time/ s
Figure 3. Average force-induced dissociation curves of Ara h 1 antibody
coupled magnetic particles from an Ara h 1 protein coated surface.
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DISCUSSION
From Figure 3 it is evident that the curves have two distinguishable
components. This suggests that the population of bound particles consists of
two different fractions:
- a weakly bound fraction with faster dissociation rate due to non-specific
interactions between Ara h 1 antibody and Ara h 1 protein. This can be
explained with the random distribution of both antibodies and protein on the
surface during their immobilization. It has been already described that
passive adsorption of antibodies on hydrophobic surface leads to a mixture
of orientations with different efficiency of capture (Tajima et al., 2011,
Wiseman et al., 2012).
- a stronger bound fraction with slow dissociation rate, originating from the
specific interactions between both biomolecules when they are favourably
oriented.
Based on the existence of these two fractions of bound particles, we propose
a bi-exponential model with three independent parameters for fitting the
dissociation curves:
P = Ps*exp[-koff(F)s*t] + (1-Ps)*exp[-koff(F)ns*t)]
Using this equation, both dissociation rates for the non-specific (koff(F)ns)
and for the specific interactions (koff(F)s), as well as the population of
particles specifically bound for each experiment (Ps), can be obtained.
The proposed bi-exponential model has its origin in Bell and Evan’s
equations to describe the force dependent dissociation of molecular bonds
(Bell et al. 1978, Evans et al., 2001). According to their model, the
dissociation rate constant of a molecular bond at zero force, koff(0), is related
to the change in free energy to the transition state, Eb(0), by the following
expression:
koff(0) = ʋ*exp[-Eb(0)/Kb*T]
The energy needed to overcome the transition state is lowered by the work
done on the bond when a force, F, is applied: W= -Fxb, where xb is the
distance between the minimum and the maximum of the energy barrier. This
denotes that there is an exponential relationship between the off rate of a
molecular bond changes and the applied force:
koff(F) = ʋ*exp[-(Eb(0)-Fxb )/Kb*T] = koff(0)*exp[-Fxb/Kb*T],
allowing for extrapolating the dissociation constant at zero force, koff(0), by
varying the applied force:
ln koff(F) = ln koff(0) + Fxb/Kb*T
The obtained dissociation curves were fitted with the biexponential model
described above using Origin® software (OriginLab Corporation, US) and the
extracted dissociation rates of the specific bonds were plotted as a function
of the force on a logarithmic scale. From this plot the dissociation constant
to zero force of the bond formed between the Arah1 protein and its
monoclonal antibody was obtained.
CONCLUSION
We showed a successful application of a very recent magnetic bead based
technology in studying the force induced kinetics and quantifying the
strength of the bond formed between the Ara h 1 protein and its monoclonal
antibody. In the on-going research we are further challenging this technology
Comm. Appl. Biol. Sci, XX/X, 2013
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for investigating the affinity of a new receptor against Ara h 1 protein, a
recently selected aptamer in our group (Tran et al., 2012).
ACKNOWLEDGEMENTS
This work was financially supported by EU-FP7 ITN BioMaX and EFRO INTERREG - NanosensEU.
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