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Applied Biosystems, Eagle Research and
Development Double Up for Single-Molecule Detection
By
Laurie Sullivan
Applied Biosystems and
Eagle are co-developing a device for single-molecule detection. The device,
invented by Eagle, identifies and quantifies molecules based on their unique
electronic charge signatures. Notably, the device has the potential to
perform low-cost, high-throughput DNA sequencing. Currently in prototype
stage, Applied Biosystems will provide development support for a two-year
period, initially focusing on applications in protein identification and
detection of protein-binding events.
Pharma
DD talked to Timothy G. Geiser, Director of Strategy & Business
Development for Applied Biosystems Group, and Jon Sauer, Founder of Eagle
Research and Development, about the technology’s potential applications,
its unique advantages, and next steps in its development.
PDD:
What is the significance of the deal for both companies—Eagle and Applied
Biosystems?
Jon:
For Eagle, it’s a simple answer—we’re a small company. The agreement
with Applied Biosystems (ABI) provides financial support and biochemical
expertise such that the device’s development will not be constrained, at
least by these factors.
Tim:
Current analytical techniques for determining the presence and concentration
of biomolecules (e.g., proteins, DNA, RNA) in biological samples require
modification of the biomolecule being measured. Typically, a fluorescent or
chemiluminescent moiety is attached to the target molecules via PCR or
antibody association, enabling detection by a laser-based fluorimeter or
luminometer, respectively. Not only are such modifications expensive and
perturbing to the biomolecule under study, such assays usually cannot be
done at high levels of multiplex where, particularly for proteins, detection
and quantification in the same sample volume of multiple biomolecules is
desired. While DNA-chip analysis can achieve a high degree of multiplexing,
it requires expensive and onerous labeling procedures.
ABI’s interest in
Eagle’s technology is driven by its potential to identify and quantify a
wide variety of biomolecules using a single device—without the need for
labeling.
PDD:
What are potential applications for the device?
Tim:
ABI envisions a number of application areas.
First, the device’s
label-free analysis capability would be of supreme interest to scientists
studying fundamental molecular biology. It’s interesting to study RNA
expression, but proteins more directly mediate the real business of biology.
A technology such as this, which could identify and quantify specific,
multiple proteins in real time, identify specific protein-protein and
protein-DNA interactions would be a powerful tool for understanding biology.
Second, if the technology
can identify individual proteins, it may also be able to detect viral and
bacterial pathogens. The device could thus be utilized for high-throughput,
ultra-sensitive pathogen detection.
Third, the device could be
of interest to clinical researchers interested in real-time changes in
protein concentrations, and their interactions, as a patient’s disease
progresses. In particular, as drugs are administered, it’s important to
understand an individual’s specific biological response to them. In such a
scenario, this tool could provide highly resolved, individual-based data on
the effect a drug treatment has on protein profiles. For example: Perhaps
you have tissue biopsies collected over time from a patient undergoing
cancer therapy, and you want to map the drug treatment’s effect on
biomarkers characterizing that particular cancer. In principle, Eagle’s
technology would allow one to utilize very small amounts of sample that are
minimally processed (as it doesn’t require amplification or fluorescent
labeling of the sample) and measure multiple specific proteins on the
chip—not only to identify them but also to determine their concentration
relative to other proteins of interest in the sample.
In the same vein,
researchers could also do high-throughput drug testing (using either in
vivo or in vitro systems) to
see how protein panels change over time after exposure to drugs hypothesized
to target a particular protein. Any protein subject to chemical inhibition
is likely to have ripple effects—affecting other proteins’
functions—so this device could be a good way to study off-target drug
effects.
Finally, a fourth potential
area is high-throughput, low-cost DNA sequencing.
And there’s the crux of
why Applied Biosystems was originally interested in this technology—it has
the potential to perform high-throughput DNA sequencing at a very low cost.
However, that application has been relegated as a secondary interest on
ABI’s part, as it will be more challenging than applying the technology to
protein analysis. Exploring the potential for protein analysis and DNA
sequencing is probably beyond the scope of the current two-year agreement.
From a sequencing point of view—and even from a protein-detection point of
view—this is really next- (or next-next) generation technology. Many basic
questions must be answered before we’ll have a good practical sense for
when certain products could materialize.
Jon:
Attempting sequencing pushes the edge of the engineering limit, and that
will take at least a couple of years. In the near future (i.e., within the
next two years), it will be possible to use the technology in extremely
useful ways involving proteins. So, during the two-year period during which
ABI provides funding, Eagle will seek to develop effective chip-device
fabrication processes to enable evaluation of the devices to achieve those
nearer-term goals.
PDD:
What advantages does Eagle’s single-molecule detection device offer?
Tim:
In addition to label-free detection, it offers high-density detection as
these devices can be fabricated with a large number of pores—they’re
highly scalable. It’s possible to have thousands of nanopores, each of
which can identify and count a particular biomolecule as it passes through a
pore. It doesn’t matter where a pore is located or which molecule of
interest passes through a particular nanopore, because they’re all
individually addressable.
Jon:
Furthermore, because it’s comprised of a silicon chip, the device is
fairly inexpensive and easy to manufacture in volume. While the speed of
analysis by microprocessor standards is not very fast, it surely is by
biochemical standards. DNA has been demonstrated elsewhere to pass through
nanopores at a rate of approximately 1 million bases per second. Thus, 1,000
pores, running in parallel, equates to one gigabase per second.
But perhaps the biggest
point is the minimal amount of sample preparation involved. For all
practical purposes, the DNA or protein is processed raw, requiring no
complex biochemistry (e.g., PCR, labeling, or extensive purification steps)
to prepare it.
Tim:
Sample prep is the bane of biological assays. PCR is a fairly sophisticated
technique for amplifying DNA or RNA. But because it’s quite sensitive to
biological contaminants that could inhibit the PCR reaction, it requires
extensive sample preparation to eliminate them. We believe Eagle’s
approach would, in principle, be much less sensitive to such contaminants.
PDD:
Last but certainly not least, please describe the underlying technology of
the single-molecule detection device.
Jon:
Consider a vertical wall, or one side of a funnel-shaped nanopore with a
rectangular cross section. On one side is a semiconductor and on the other
is a charged solution. The wall’s surface is covered by a thin insulator
to prevent charges from crossing it. On the solution side, a charge is
present, e.g. due to a biomolecule,—close to the wall surface (within a
couple hundred nanometers). This charge, due to the electrostatic potential
it creates, pulls mobile charges in the semiconductor that pile up on the
insulator (as near as possible to [and opposite]) the charge in the
solution.
This “image charge”
distribution is controlled by the potential, which depends on the magnitude
of the charge, distance from the wall, and the semiconductor mobile charge
densities. If there is also a vertical field in the semiconductor producing
a small background current, these image charges will be rapidly pulled away
toward the drain in the semiconductor, causing an additional source-drain
current in the semiconductor. As soon as the image charges are pulled away,
they are replaced by new charges that come in the semiconductor to the
insulator opposite the external charge, which in turn are also pulled away,
sustaining the changed semiconductor current.
The region where this
happens within the semiconductor is the “gate region.” When the
relatively slow-moving charge in the solution finally moves away from the
region near the gate pulled by an external field through the pore, the added
current ceases and leaves only the background current. The changing current
in the semiconductor then measures the size and position of the solution
charge as a function of time as it passes by the gate. Since all
biomolecules have non-spherical charge distributions (with both positive and
negative regions on their surfaces), biomolecules passing by the gate region
produce changing currents in the semiconductor.
Since the device has multiple gate regions surrounding the solution
channel, the device effectively maps those charge distributions (within the
boundaries of its resolution). With sufficient time and space resolution,
the device can unambiguously identify the biomolecule from this signature
and provide information about its properties.
Tim:
Because a protein’s conformation is based on different amino-acid
sequences held in three-dimensional configurations, we expect that
individual proteins will have a unique three-dimensional charge signature
measurable by the semiconductor, creating an ability to uniquely identify
proteins based on their charge signature. That’s the first fundamental
objective of studying these devices.
PDD:
With that premise, what’s the technology’s current stage of development,
and what are next steps?
Jon:
ABI’s support for continued development was predicated on Eagle’s proven
ability to fabricate the vertical embedded transistor structures and employ
them for detection of biomolecules. At this time, the device dimensions are
not quite small enough, and the silicon-processing sequence is not
sufficiently developed for high-yield manufacturing, to field an initial
product.
As such, ABI’s support is
primarily to fund development through production of a viable prototype
device. Hopefully it will involve fairly straightforward size-reduction
techniques, fine-tuning of the silicon processing, and device
characterization using appropriate biomolecules provided by ABI.
Copyright 2007, Cambridge Healthtech Institute. All Rights Reserved.
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