2. • Many applications in medicine, environmental analysis, and
the chemical Industry require sensitive methods for sensing
small organic molecules.
• Our sense of smell and taste are designed to perform exactly
this task, and the immune system recognizes millions of
different molecules.
• Recognition of small molecules is a specialty of biomolecules,
so they provide an attractive approach to the creation of
specific sensors.
• Two components are needed:
• the recognition element and some mechanism for readout once
the recognition element has found its target.
3. Antibodies Are Widely Used as
Biosensors
• Antibodies are nature’s premier biosensors, so it comes as no
surprise that the development of diagnostic tests using
antibodies has been one of the major successes of
biotechnology.
• Perhaps the most familiar example is the simple test used to
determine blood type.
• This is the simplest possible form of immunotesting, taking
advantage of two properties of antibodies:
• their specificity and their ability to cross-link targets.
4. • The blood type test is composed of a collection of antibodies
that recognize specific sugars on the surfaces of red blood
cells.
• The antibody is added to the blood, and if the particular blood
type is present on the cells, the antibodies bind to the surface,
sticking many cells together.
• The result is a clumping of cells that is easily seen with the
naked eye.
5. • Antibodies may be labeled with radioactive iodine or tritium
and the presence of radiation used to quantify the amount of
bound antibody.
• Powerful biosensors are created by linking antibodies to
specific enzymes such as -D-galactosidase or alkaline
phosphatase.
• These enzymes then convert dye molecules to colored forms
that can be detected.
• The most sensitive methods employ the detection of
luminescent or fluorescent molecules, either connected to the
antibodies or created by antibody-linked enzymes.
6. • Pregnancy tests provide an example of how these tests can be
streamlined for use in the home.
• Many variations are available from different providers using
monoclonal antibodies that detect the presence of chorionic
gonadotropin (CG), a small protein in the urine.
• A test from Abbott Laboratories uses a clever one-step process.
• The sample of urine is applied at one end, and it soaks
through a fiber pad from one end to the other.
• First, sample encounters a section with antibodies that have
colloidal selenium particles attached, which are bright red. If
the sample contains CG, it binds to the antibody.
7. • Then, as the sample continues through the pad, it drags the
colored antibody and the bound CG with it.
• They next encounter two stripes of antibodies that are
attached to the pad.
• One stripe is horizontal and contains specific antibodies that
attach to the test antibodies.
• Some of the red antibodies stick here, creating at least a
“minus” sign in all tests.
• The other stripe is vertical, and contains more antibodies that
are specific for CG.
• If the sample contains CG, the CG-antibody complexes bind to
these, forming a vertical stripe and completing a “plus” sign.
8. Biosensors Detect Glucose Levels for
Management
of Diabetes
• Glucose biosensors are some of the most successful biosensors
on the market today.
• People living with diabetes require convenient methods for
monitoring glucose levels.
• Implanted sensors and noninvasive sensors are under
development, but currently the most accessible approach is a
handheld biosensor that analyzes a drop of blood.
9. • The biosensor relies on the fungal enzyme glucose oxidase,
which combines glucose and oxygen to form gluconic acid and
hydrogen peroxide.
• A sensor can be designed to detect the amount of hydrogen
peroxide formed.
• In the 1960s, Leland C. Clark had the clever idea to hold the
enzyme very close to a platinum electrode with a membrane,
so that the chemical changes could be followed by watching
changes in the current at the electrode.
• This idea proved effective, and a series of laboratory-sized
instruments were developed based on the sensing of peroxide.
10. • To make a portable, consistent glucose biosensor for the home,
however, a change in methodology was needed.
• The oxygen-to-peroxide change is hard to standardize, because
of the need for consistent oxygen levels and interference by
other molecules in the blood.
• Instead of oxygen, a mediator molecule is used to deliver the
signal to the electrode.
• Ferrocene, a small molecule with an iron ion trapped between
two cyclopentadiene rings, was found to be an effective
mediator.
• Handheld meters that use disposable electrodes with enzyme
and mediator are available commercially.
• Now, in a matter of seconds, glucose levels may be measured
in a small drop of blood.
11. • A small modification can change a biosensor into a biofuel
cell.
• In biofuel cells, specific enzymes are tethered to two
electrodes, performing reactions that strip electrons from the
fuel at one electrode and replace them on oxygen at the other
electrode.
• Adam Heller has created a biofuel cell that uses glucose as its
fuel.
• On one electrode, glucose oxidase extracts electrons from
glucose, converting it to glucolactone.
• Then a second electrode is added with an enzyme that
replaces the electrons, forming a closed circuit.
• For instance, the enzyme laccase may be used, which adds
electrons to oxygen, forming water.
12. Engineered Nanopores Detect Specific
DNA Sequences
• Researchers at Texas A&M University have designed a
biosensor that can detect short strands of DNA, about seven
nucleotides in length (Figure).
• The sensor is based on the bacterial protein hemolysin.
Hemolysin is composed of seven protein chains that form a
pore through lipid bilayers.
• In nature, this is used as a toxin. As a biosensor, hemolysin is
embedded in a membrane separating two chambers.
• An electrical potential is applied across the membrane, which
draws ions through the pore from one chamber to the other.
• The current through this pore is monitored, and when the
nanopores are blocked an abrupt change in the current is easily
detected.
13. A DNA detector is created by tethering a short single strand of
DNA (shown in pink) inside the
pore of hemolysin (shown in gray). When the complementary
strand is added to the solution, it binds to the
tethered strand, blocking the pore.
14. • Hemolysin has a large chamber at one end, 3–4 nm in
diameter, and a narrow tube that crosses the membrane, about
1.4 nm in diameter.
• To create the sensor, the researchers tethered a short DNA
strand to one protein subunit inside the large chamber.
• This single strand does not block the pore, so ions are free to
pass.
• The DNA strands to be tested are added to the solution, where
they are drawn into the pore by the electrical potential.
15. • If a DNA strand does not match the DNA tethered inside, it
passes quickly through the pore, reducing the current for about
a tenth of a microsecond.
• If a DNA strand is complementary, however, it binds to the
tethered strand and partially blocks the entry to the pore,
causing a reduction in the current that lasts about 45 s.
• Eventually, the strand dissociates and passes through the pore,
restoring the current.
• By monitoring the time that DNA strands remained bound to
the sensor, they could discriminate perfect matches from
matches with a single mismatched nucleotide.
