Biomedical Engineering Reference
In-Depth Information
sequence of the molecular beacon, the loop sequence binds to the complementary strand,
causing the stem to unzip and creating a spatial separation between the fluorophore and
the quencher. This spatial separation in turn generates a dramatic increase in fluorescence
intensity of the molecular beacon, with some reports of enhancement exceeding 200-fold.
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Owing to their inherent sensitivity and specificity, molecular beacons have recently been
used to fabricate fiber-optic nano-biosensors for single-stranded DNA and RNA.
55,57
In
this work, molecular beacons were attached to the silica core of a tapered fiber-optic nano-
biosensor through biotin-avidin linkages. By binding the molecular beacons to the
tapered tip of the fiber optic in this method, it was shown that a strong attachment could
be achieved while not degrading the activity of the beacon.
3.2.1.3 Fiber-Optic Nano-Imaging Probes
While fiber-optic nanosensors and nano-biosensors have proven to be useful tools for the
spatially localized, quantitative analysis of chemical species in individual living cells, they
are limited in the number of locations that can be probed simultaneously. In most cases
only one sensor can be inserted into a typical mammalian somatic cell at a time, without
causing significant damage or altering the normal cellular function of the cell.
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This
inability to monitor multiple locations on or within an individual living cell simultane-
ously can dramatically limit the applicability of fiber-optic nanosensors and nano-biosen-
sors for many types of cellular analyses. To overcome this limitation, two different types
of fiber-optic nano-imaging probes, comprised of thousands of individual fibers arranged
in a coherent bundle, have recently been developed by a couple of research groups and
used for chemical imaging.
59,60
These nano-imaging probes represent the latest advance in
fiber-optic-based nanosensors, and are capable of obtaining measurements at thousands
of different locations simultaneously with nanometer-scale spatial resolution.
Fabrication of these fiber-optic nano-imaging probes is performed through either a
chemical etching process or a heated pulling process, similar to single-fiber-based
nanosensors and nano-biosensors. In the first of these fabrication methods, chemical etch-
ing is performed on commercially available coherent imaging bundles having as many as
30,000 individual fibers. The individual fiber elements comprising the imaging bundle are
etched in HF to form an array of silica tips, with the core of each fiber element correspon-
ding to a nanotip.
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After the array of silica tips is formed from this etching process, a layer
of gold is sputtered over the entire surface to prevent light from escaping from the tapered
sides of each fiber element. Once the gold overlayer is deposited on the fiber tips a poly-
mer coating is electrochemically deposited over the gold and heat-cured to expose the
gold only at the tips of the individual fiber elements. Lastly, the exposed gold on the tips
of the coated fiber elements is dissolved in aqua regia to produce free silica surfaces for
spectroscopic analyses.
As with the single-core fiber-optic nanosensors and nano-biosensors, nano-imaging
probes have also been fabricated via a heated pulling process.
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When fiber-optic nano-
imaging probes are fabricated via this process, the resulting fiber probe has a flat tip, with
each of the individual fiber elements comprising the bundle having been tapered equally.
This can be seen from the microscopic image shown in Figure 3.7a, which shows the side-
on view of a tapered fiber-optic nano-imaging probe produced on a CO
2
laser-based
micropipette puller that has been specially optimized for producing nano-imaging probes.
To produce the flat surface on these nano-imaging probes, which is necessary for imaging,
the fiber is allowed to cool slightly before the final hard pull of the micropipette puller
cleaves the bundle into two individual pieces. In addition to providing a flat surface, this
tapering process also ensures that each fiber element in the bundle is tapered equally. By
changing the heating temperature and the initial pull strength of the micropipette puller,
