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DURHAM, N.C. - Biochemists have developed a technology that
will enable proteins to be engineered as sensitive, specific
"bioelectronic" sensors for a vast array of chemicals. These
engineered proteins, when attached to electrodes, can detect a
specific chemical in a complex mixture and produce an electric
signal reflecting its identity and concentration.

The researchers already have demonstrated that they can
engineer proteins to detect glucose in blood serum and the
sugar maltose in beer - showing that such proteins can pick
specific molecules out of complex mixtures without interference
or fouling from other constituents.

The scientists, led by Duke University Medical Center
biochemist Homme Hellinga, reported the new approach to
biosensors in the Aug. 31, 2001 issue of Science. Their research is
sponsored by the Office of Naval Research and the National
Institutes of Health. Besides Hellinga, other authors are David
Benson, currently at Wayne State University; David Conrad,
Duke; Robert de Lorimier, Duke; and Scott Trammell, now with
the Naval Research Laboratory in Washington.

"Since these engineered proteins are robust and potentially
miniaturizable, we believe they will provide a basis for a vast
array of chemical sensors," Hellinga said.

"For medical applications, you could imagine a multitude of
sensors on a tiny chip that physicians could use at the
patient's bedside to immediately determine from a drop of blood
the concentrations of drugs, or metabolites such as glucose.
Anesthesiologists could use such biosensors to instantly
measure during surgery the concentration of anesthetic or key
metabolites such as epinephrine in a patient's body, rather
than having to rely on the less accurate monitoring of vital
signs. Thus, with these biosensors, in many cases you would no
longer need expensive chemical laboratories and time-consuming
clinical analysis."

Also, said Hellinga, an implantable glucose sensor would
enable constant monitoring of blood glucose in people with
diabetes and could also provide a long-term sensor as the basis
for an artificial pancreas.

In other applications, Hellinga foresees use of the
biosensors to monitor pollutants and chemical and bio-warfare
agents. He emphasized the adaptability of the system.

"These engineered proteins are based on proteins that
bacteria use to sense their chemical environment, and since
there are perhaps hundreds or thousands that exist, they
provide a basis for a vast array of chemical sensors," Hellinga
said. "Using powerful computational design tools that we have
developed, it is possible to engineer these candidates to
dramatically alter their specificity and sensitivity."

In contrast, said Hellinga, other approaches to biosensors
are more complex and less robust, depending on enzymatic
reactions that involve measuring the output of chemical
reactions and replenishing consumed chemicals. Also, such
biosensors have largely depended on natural proteins, limiting
their adaptability.

"Our engineered proteins can be thought of as solid-state
entities that include both a biological component and an
electronic component," he said

In the Science
paper, Hellinga and his colleagues described how they started
with natural bacterial proteins called "bacterial periplasmic
binding proteins." These proteins constitute a large
"superfamily" of proteins on the bacterial surface that the
organisms use to sense food sources such as sugars and to avoid
toxic chemicals.

Besides their broad variability, the major advantage of such
proteins is that the protein's chemical-sensing active site is
"allosterically" coupled to the domain that sends a signal to
the bacterial metabolic pathways. Such internal signals trigger
actions such as moving toward a food source. Allosteric
coupling means that the two domains are separated on the
protein, and thus one can be altered even drastically without
affecting the other. In particular, the bacterial protein acts
like a hinge, such that when a chemical plugs into the active
site, the hinge closes, switching on the distant metabolic
signal.

"Since hinge-bending motions are easy to understand, they
are easy to manipulate," said Hellinga.

In one experiment, for example, the scientists altered a
bacterial maltose binding protein, tethering to it a metal
ruthenium group that would produce a voltage when its
conformation was altered. Thus, when they coated a gold
electrode with the proteins and added maltose, that sugar
altered the proteins' conformation, producing an electric
current proportionate to the maltose concentration.

To demonstrate the generality of their "hinge-bending"
mechanism, the scientists created additional bioelectronic
sensors using other proteins that responded specifically to
glucose and glutamine.

And as a demonstration of the extreme adaptability of their
system, they radically redesigned the maltose binding protein
active site so that it acted as a zinc detector.

Finally, to show that their biosensors could function in
complex mixtures without being "confused" by related chemicals
or fouled by contaminants, the scientists showed that their
bioelectronic sensors could specifically measure maltose
concentrations in beer and glucose concentrations in human
blood serum.

"We believe that these experiments in real-life mixtures
dramatically demonstrated that the chemistry of these systems
is very robust and specific, and does not suffer fouling by the
multitude of other substances in such mixtures," Hellinga
said.