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may seem small, they’re too big, at least for portability. Miniaturizing them
further is difficult because they consist of many sensing spots. The individual
spots are small, but most arrays manufactured today are millimeters or centimeters
on a side and require expensive scanners for reading.
Researchers have developed ways to shrink the
spots a thousandfold, which could greatly decrease microarray dimensions or increase
the number of spots in a given area. But a fluorescence signal from such small spots
would be a million times fainter than that from current ones. Now researchers at
Ben-Gurion University of the Negev in Beersheba, Israel, have come up with a solution:
microlenses that magnify the fluorescence signal and can be placed where needed.
According to Levi A. Gheber, current
technology produces single spots of 50 to 200 μm, with an equivalent space
between them. Features on a microarray have a pitch — the distance from one
repeating feature to the next — of 100 to 400 μm. Thus, an array that
is a centimeter on a side can hold only roughly 50 x 50 spots.
Some years ago, a research team that
included Gheber used a nanopipette as the probe at the end of an atomic force microscope
to print protein dots 200 nm in diameter. Another group, of which he also was a
member, showed that nanochannels and wells could be carved into a protein layer
using small amounts of the enzyme trypsin to do the chopping. These two investigations
showed that it was possible, from a basic research point of view, to manufacture
microarrays with spot dimensions 1000 times smaller than current ones. Fine tuning
is still needed (and is under way) to transform these concepts into commercial reality,
but even so, the detection of these nanospots is a big challenge.
These 3-D representations
of polymer microlenses, based upon atomic force microscopy data, show how various
physical parameters and the resulting focal length can be controlled by varying
the deposition time (1 s on the left and 5 s on the right). Images courtesy of Levi
Gheber and Ben-Gurion University of the Negev.
In microarrays, fluorescent labels
are attached to target molecules, those that will bind to the arrayed material.
Mapping the fluorescence pattern against the known distribution of spots on an array
shows what specific targets are present.
However, reducing the linear dimensions
of a spot by three orders of magnitude decreases the number of molecules by roughly
six orders of magnitude, and consequently the fluorescence signal from such a spot
will be a million times weaker, Gheber said.
The Ben-Gurion research team demonstrated
a solution for this by constructing lenses using the same nanopipette employed to
deposit the nanospots.
The technique uses a nanopipette, which
is a hollow glass or quartz capillary with a tapered tip and an opening of a few
hundred nanometers. Solution loaded into the nanopipette is drawn toward the tip
by capillary forces. The solution will flow from the tip only when it is in contact
with a material for which it has a greater affinity than it does for the nanopipette.
Constructed from a hollow glass or quartz
capillary, a nanopipette, seen here end on, is mounted as the probe of an atomic
force microscope. When filled with a UV-curable polymer, the nanopipette can deliver
it in precise locations to create microlenses, which could be useful in the readout
of miniaturized microarrays.
As reported in the April issue of Nano
Letters, the researchers mounted the nanopipette on the end of a cantilever
of an atomic force microscope from Nanonics of Jerusalem. They loaded the nanopipette
with a monomer mixture that would polymerize when exposed to ultraviolet radiation.
They deposited the monomer on glass coverslips, varying the contact time, and
then exposed the droplets to 254-nm radiation from a UV lamp until the molecules
cross-linked into a polymer, which was several minutes.
The result, verified by atomic force
and near-field scanning optical microscopy, was smooth spherical droplets, with
surface roughness of 1.6 nm. The diameter of the lenses was between 4 and 9 μm
for a deposition time of between 1 and 20 s, with smaller lenses produced by shorter
deposition times. The lens diameter didn’t increase linearly with contact
time. Rather, it increased rapidly at first, and the growth slowed as the diameter
neared a maximum value.
The contact angle between the droplet
and the surface was determined by the properties of the two, and the refractive
index of the materials was fixed. Therefore, the deposition time governed the optical
performance of the lenses.
The researchers characterized the lenses
using bright-field microscopy. For more precise characterization, they mapped the
point spread function of the lenses as well.
Finally, to see how much the lenses
could improve fluorescence measurement, they placed a microlens and a spot of fluorescein
on the opposite sides of a coverglass. They imaged fluorescence from the spots through
the lenses, and the measured intensity profile indicated that a detector placed
at the right location would have collected 50 percent more light as a result of
Although the nanolens technique works,
Gheber pointed out several areas where ongoing research aims to improve performance.
Achieving this will involve a balancing act between competing demands.
For example, the lenses have a low
numerical aperture because of the relative flatness of the drops and the refractive
index of the polymer. Increasing the numerical aperture requires changing one or
both of those parameters, which implies the use of a different material. On the
other hand, any material employed still has to flow out of the nanopipette on demand,
which favors higher weta-bility, a smaller contact angle and a flatter lens.
“We would like to have drops
with high contact angle, but then they will be less likely to leave the pipette,”
Contact: Levi A. Gheber, Ben-Gurion
University of the Negev, Beersheba, Israel; e-mail: firstname.lastname@example.org.