Building a Better — But Still Tiny — Sensor
DNA aptamers improve sensing ability of atomic force microscopy
For an atomic force microscope, familiarity can breed better images
— in more ways than one. When the microscope’s tip has a sensing molecule
tethered to it, the molecule enables the device to recognize targets in the sample
being scanned. That chemical familiarity also enables the microscope to perform
biological imaging of complex samples in water, something it could not accomplish
The standard choice for a sensing molecule has
been an antibody, which helps pick out proteins for which it has an affinity. Now
a group from Arizona State University in Tempe has used DNA aptamers — manufactured
single-stranded DNA molecules. Stuart Lindsay, a professor of physics and chemistry
and leader of the research team, noted that aptamers offer a significant advantage
over the typical antibody approach because their chemistry is simpler. “The
simpler chemistry seems to make everything work better,” he said. “Better
signal to noise, for example.”
A schematic, not to scale, shows an atomic force microscope
probe with attached aptamer scanning a surface. Because of its structure and chemistry,
the aptamer alters the probe’s response near particular molecules, enabling
the location of that type to be mapped. Image courtesy of Stuart Lindsay, Arizona
In atomic force microscopy, a stylus
with a very sharp point is brought close to a sample surface. The tip is mounted
on the end of a cantilever, and the interaction between the tip and the sample deflects
the cantilever. That deflection is amplified by an optical lever in which a laser
is fired at the cantilever and bounces off it; the reflected beam is detected by
a photodiode. Because the lever arms are long, they magnify the deflection by a
factor of thousands or more, making it possible to spot small movements and achieve
For biomolecular imaging, one of the
biggest problems with this technique is that it is nearly impossible to distinguish
between proteins, even if their molecular weight is very different, based on a topographical
image alone. So researchers have attached sensor molecules, such as antibodies,
to the tip of the stylus and bounced the probe up and down slightly. As the tip
moves across the surface, the oscillation changes when the sensor binds to a target
on the surface. This information is used to locate the target proteins.
The scheme is promising, but the investigators
note that one issue is the high surface concentration under the probe. Consequently,
the chemical recognition signal may appear even if the affinity between the antibody
and a molecule is small. The result is noise that can mask the true signal.
In contrast to antibodies, which are
natural, aptamers are man-made. Starting with a random sequence of DNA or RNA, they
are created through a process called systematic evolution of ligands by exponential
enrichment. Aptamers can recognize specific ligands and bind to nucleic acids, proteins
or small organic compounds. Because they consist of a single DNA strand, they are
easy to synthesize and store, and they are easily attached to the microscope’s
Aptamers also have a high affinity
for some small molecules, which might allow recognition imaging of minor chemical
changes, such as elements of an epigenetic code, information in addition to that
encoded in the DNA. “My own goal is to map epigenetic markings at the molecular
level,” Lindsay said.
For a demonstration of their approach,
which was published in the June issue of Biophysical Journal, the scientists
chose the aptamer that binds to human immunoglobin E (IgE). They did so primarily
because this aptamer produces significant adhesion in atomic force microscopy force
Immunoglobin E (IgE) was imaged with a DNA aptamer, a recognition molecule shown
in green and tethered to an atomic force microscope probe via a polyethylene glycol
linker, shown in red (a). The technique enables the depiction of the topography of the IgE molecules on a mica substrate (b) as well as simultaneous acquisition of recognition signals derived from the aptamer’s interaction with IgE (c). Adding a solution of IgE blocked further signals (d). Traces (between
the green arrows) are shown for the topography (e) and recognition (f) steps.
They functionalized the probes with
an ethylene-glycol oligomer, leaving a thiol-reactive maleimide at the end of the
molecule to act as a tether for the aptamer. Because of the tether length, the
resulting resolution was ∋5 nm. They used an atomic force microscope from
Tempe-based Molecular Imaging Corp., now part of Agilent Technologies Inc.
The researchers coated a mica substrate
with a solution containing IgE. After some additional processing, they imaged the
substrate at a scanning speed of about 2 μm/s and with an oscillation amplitude
of ∋5 nm. They found that nine out of 10 features on the surface of the right
size to be IgE molecules were picked up by the aptamer sensor, a recognition efficiency
of 90 percent. Moreover, there was a clear difference between the background signal
and that of a legitimate spot, indicating a higher signal-to-noise ratio than is
typically achieved by antibody-based sensors.
The investigators demonstrated that
the effect was specific. When IgE molecules were injected into the solution, the
recognition signal disappeared because it was blocked. When they imaged surfaces
coated with another protein, thrombin, there were no recognition events. Also, when
they imaged a surface coated with a mixture of thrombin and IgE, the recognition
events tracked to a degree the molar ratio of the mixture. The actual ratio of molecular
recognition was higher than the molar ratio, a difference that could have resulted
from preferential surface adsorption of the IgE.
The improved signal-to-noise ratio
of the aptamer approach as compared with one based on antibodies was not due to
stronger binding. Tests showed that the force required to break the aptamer bonds
was actually somewhat smaller than the force required to break the antibody bonds.
However, other tests have shown a similarly small difference, but with the aptamer
bond slightly stronger than the antibody one.
In applying the technique, it would
be necessary to have the appropriate aptamers, with the molecules designed to bind
to a given target, Lindsey noted. Therefore, work must be done developing and producing
the aptamers. That can be a challenge, but the researchers are working on it.
“We are building an aptamer factory,
but it’s not easy. On the other hand, if we get things working, we’ll
avoid the batch-to-batch variation that plagues natural products like antibodies,” Lindsay said.
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