48x36 Poster Template

1
Eliminating non-specific interactions for
accurate single-molecule force measurements with
magnetic tweezers Noah Johnson,, Gokul
Upadhyayula, Dr. Sharad Gupta, Dr. Valentine
Vullev University of California, Riverside, CA
92521, The Pennsylvania State University,
University Park, PA 16801
Results
Abstract
Eliminating Non-specific Interactions
Once bead velocity is calculated, we can assume
the magnetic force on the bead is equal to the
drag force of suspension fluid on the bead plus
the gravitational force on the bead.
The surfaces and beads were successfully
derivatized with PEG 3000, resulting in a contact
angle of 72.40.79 for the coated glass surface
compared to 26.70.24. We found the 1.33mM
TWEEN 20 to work very well in keeping the beads
separated.
To accurately study protein-ligand dissociation
kinetics we must minimize the non-specific
interactions between the beads and the substrate.
The effects of Van der Waals forces are
drastically reduced by separating beads and
surfaces (Figure 3).
Magnetic tweezers (MT) are widely used for
investigating nanometer-sized molecular complex
interactions by applying forces to
micrometer-sized superparamagnetic beads. In
contrast to other force measurement techniques
such as optical tweezers and atomic force
microscopy, magnetic tweezers offer a few key
advantages (1) MT allow for the recording of
hundreds of single-molecule events in parallel
with a single measurement (2) Magnetic forces
are orthogonal to most biological interactions
which eliminates the risk of altering sample
properties during MT measurements (3) MT require
relatively low energy, significantly reducing the
risk of sample overheating (4) MT are able to
conduct measurements at a constant force,
eliminating the need to take loading rates into
consideration. Due to significant measurement
error caused by non-specific interactions between
probe interfaces at nanometer separation, the
utilization of single-molecule force measurements
remains largely unexplored. By employing
surface-engineering methodologies developed in
our laboratory, we aim to suppress the
non-specific interactions between the slide
surface and the beads, greatly increasing the
accuracy of our measurements. Once both surfaces
are derivatized, we will use MT for dissociation
studies of protein-ligand complexes, and
furthermore to study the directionality of
dissociation by strategically attaching the bead
and substrate at various positions.
FM
FM FdFg
Fd
Fg
Using Stokes equation we can calculate the drag
force.
Figure 6 (A) Significant bead clumping is seen
at 40x in distilled water. (B) Single beads are
seen in TWEEN 20 suspension solution at 10x.
The gravitational force can be calculated
theoretically.
Fg mg ?beadsVfluidg
We applied a tiny force of 0.357pN with the MT
(Figure 7) for 30s to test the effectiveness of
PEGylation in minimizing non-specific
interactions.
Using surface engineering, we derivatized the
glass and beads with PEG (Figures 4 5
respectively).


Calibration Results
We controlled the distance between the magnet and
the center of the field of view as well as the
electromagnet voltage. We compiled a calibration
matrix with distances between 1 and 5mm and
magnet voltages between 2 and 12V.
Figure 7 (A) Non-PEGylated beads and surface
before and after the force is applied. (B)
PEGylated beads and surface.
Force Calibration
MT is an inverted microscope with a magnet.
However, in the calibration setup, the objective
is on the side to observe the movement of beads
in the presence of an external magnetic field
(Figure 1). Through this setup, we can calculate
the terminal velocity of beads at specific
voltages.
References

• Wan, J., Thomas, M., Guthrie, S., Vullev, V.,
  Surface-Bound Proteins with Preserved
  Functionality,Annals of Biomed Eng, 2009, 6,
  1190-1205.

Figure 2 The calibration was performed with 3µm
diameter polypropylene beads in a 1.33mM TWEEN 20
surfactant solution, used to keep the beads from
sticking together.
Acknowledgements
Each PEG layer adds 11nm of separation to the
surface. Entropic repulsion by PEGylation reduces
Van der Waals forces by nearly five orders of
magnitude (Figure 3).
We would like to thank Sean Guthrie who helped
with derivatization procedures and Stephen Bishop
who helped take calibration data. Also Jun Wang
for organizing the 2009 UCR BRITE REU program,
and the National Science Foundation for funding.
Thus far we have observed forces ranging from
0.357 to 2.44pN.