PNNL-13501 - Pacific Northwest National Laboratory

Study Control Number: PN99005/1333
Biomolecular Interaction Assay System
Jay W. Grate, Cynthia Bruckner-Lea
Understanding protein-protein and protein-DNA interactions is integral to understanding the function of living organisms
and how organisms respond to insults such as environmental exposure to chemicals and radiation. Methods are being
developed on this project to rapidly measure the interactions between these biomolecules.
Project Description
The purpose of this project was to build and test a device
for rapidly measuring the interactions between
biomolecules (proteins and DNA) and multiple protein
complexes. We constructed two different renewable
microcolumn devices with on-column optical detection,
and wrote computer control programs to automate the
experimental systems. The renewable microcolumn
systems were then used to monitor the binding of several
model proteins (antibodies) and measure the interaction
between proteins and DNA fragments. This project
advances our ability to rapidly measure biomolecular
interactions and therefore understand biological systems.
Introduction
A number of sensor technologies have been adapted to
monitoring biomolecular interactions. Acoustic wave
devices such as flexural plate wave devices, surface
transverse waves, and quartz crystal microbalances detect
the mass increase observed upon binding of a solution
biomolecule to a surface bound biomolecule. However,
these devices also respond to changes in viscosity,
temperature, liquid density, and viscoelastic effects,
which may confound the interpretation of observed
signals. In addition, nonspecific binding is often
indistinguishable from specific binding. Several
techniques for refractive index sensing, such as planar
wave guides and surface plasmon resonance, can also be
used to observe biomolecular interactions localized at a
surface. Again, nonspecific binding is indistinguishable
from specific binding, and the derivatized surface must be
very thin and uniform to obtain adequate sensitivity and
reproducibility. All of these techniques use planar
surfaces that must be derivatized with the first
biomolecule. These surfaces are difficult to prepare and
characterize, and must be prepared fresh for each assay.
In addition, these techniques cannot measure the binding
of multiple proteins to form large protein complexes.
Therefore, there is the need for the development of new
techniques for conducting many rapid, automated
measurements of biomolecular binding events, including
the formation of multiple protein complexes.
Approach
This project will develop renewable surface sensing
techniques for monitoring biomolecule binding events. In
this approach, a suspension of surface derivatized beads is
introduced into a flow system and then automatically
trapped by a barrier that stops the beads but allows the
fluid to proceed (Bruckner-Lea et al. 1999, 2000;
Chandler et al. 1999, 2000; Ruzicka and Hansen 2000;
Ruzicka and Scampavia 1999; Ruzicka 1998). This
produces a small microcolumn of beads (only about a
microliter in volume) in a location for observation. The
beads can then be automatically perfused with reagent or
sample solutions to perform separations or surface
reactions. Detection methods can be used to observe
optical changes (absorbance or fluorescence) on the bead
surfaces. At the completion of the observation, the beads
can be flushed from the observation area and disposed. A
new bed of beads can then be packed for the next assay.
This new bead bed has a fresh surface, hence the name
renewable surface sensing.
The renewable surface approach has a number of
advantages over planar formats, and over the surface
plasmon resonance method. The flow injection system is
far more flexible for delivering solutions to the
observation area. This feature may become particularly
important when attempting to create and observe
assemblies of more than two components. The spectral
detection, as opposed to a single refractive index change,
provides more selective detection by providing more
information for observing particular species and zeroing
out undesirable contributions to signals. In addition, the
column format is compatible with typical measurements
by affinity chromatography for obtaining thermodynamic
Biosciences and Biotechnology 41