Comm. Appl. Biol. Sci, XX/X, 2013
1
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 throughthe specific recognition and subsequentbinding 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 thusthe importance of studying and quantifying those features.
In this work we implementeda recently publishedtechnology (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, Arah1 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 isneeded forimprovingthe 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).
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, beingthe 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. Themaximum current used in this experiments was 0.8A. Because at this current substantial amount of heat is generated in the coil, the system iscooled using a water pump. This enablescontinuous operation below 45°C and preventsthe 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.
Figure 1. Scheme of the instrumental set-up consisting of a microscope-camera 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 RedLakeMotionPro 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.
Results
We first optimized the concentration of Ara h 1 protein immobilized on the polystyrene slides.If thedensity 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 100nM, were immobilized on the polystyrene slides (Figure 2) with1 nM of protein estimatedto be the optimal one for the assay. To test for unspecific adsorptions, modified particles were also incubated on a 1% BSA coated polystyrene slide.
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 interactionsbetween 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).
Figure 3. Average force-induced dissociation curves of Ara h 1 antibody coupled magnetic particles from an Ara h 1 protein coated surface.
Discussion
From Figure 3 it is evident thatthe curves havetwo distinguishable components. This suggests that the population of bound particles consists oftwo 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. Thiscan be explained with the random distribution of both antibodies and protein on the surface during their immobilization. It has beenalready 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, originatingfrom 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.
Theproposed bi-exponential model has its origin inBell 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 forextrapolatingthe dissociation constant atzero force,koff(0), by varying the applied force:
lnkoff(F)= lnkoff(0) +Fxb/Kb*T
The obtained dissociation curves were fitted withthe 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 recentmagnetic bead based technology in studyingthe force inducedkinetics and quantifying the strength of the bond formed between the Ara h 1 protein and its monoclonal antibody. In the on-going research we arefurther challengingthis technologyfor 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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