Biomedical Engineering Reference
In-Depth Information
was developed for the indirect detection of glucose. 54 In this work, Kopelman and coworkers
photopolymerized the enzyme glucose oxidase and the oxygen-sensitive indicator tris(1,10-
phenanthroline)ruthenium chloride in an acrylamide polymer onto the tapered end of a
nanofiber. When this nano-biosensor is in the presence of glucose, the enzyme catalyzes the
oxidation of glucose into gluconic acid, thereby consuming oxygen. The resulting changes in
oxygen levels are then measured by monitoring the fluorescence of the oxygen-sensitive indi-
cator dye. Using glucose oxidase as an enzymatic bioreceptor, these nano-biosensors were
capable of absolute detection limits of approximately 10 15 mol and a sensitivity 5 to 6 orders
of magnitude greater than the previous glucose optodes. 54 In another example of enzymatic-
based nano-biosensors, the enzyme glutamate dehydrogenase was attached to the end of a
tapered fiber optic by Tan et al . 53 When glutamate was present, the glutamate dehydrogenase
reduced NAD to NADH and the fluorescence of the NADH was measured. Employing such
an enzymatic-based nano-biosensor, the continuous monitoring of glutamate levels released
from an individual cell were monitored, providing a powerful new tool for the field of neu-
rophysiology.
3.2.1.2.3 Molecular Beacon-Based Fiber-Optic Nano-Biosensors
To detect the presence of oligonucleotides (i.e., ribonucleic acid (RNA) and DNA), in intra-
cellular environments, a class of fiber-optic nano-biosensors has been developed that
employs a bioreceptor molecule known as a molecular beacon. 55 Molecular beacons are
hairpin-shaped oligonucleotide probes that rely on the complementarity of nucleic acids
(i.e., adenine:thymine, cytosine:guanine, etc.) to provide the molecular recognition for a
specific oligonucleotide sequence. 56 In their native form molecular beacons form a stem-
loop structure, in which a fluorophore (e.g., fluorescein isothiocyanate (FITC)) on one end
of the stem is in close proximity to a fluorescence-quenching moiety (e.g., dimethy-
laminophenylazobenzoic acid (DABCYL)) on the other end (see Figure 3.6).
In this state, when the fluorophore on the beacon is excited, energy transfer takes place
between the excited fluorophore and the quencher, by either direct energy transfer or
fluorescence resonance energy transfer (FRET), resulting in minimal fluorescence emis-
sion. However, in the presence of an oligonucleotide sequence complementary to the loop
Target/analyte
oligonucleotide
sequence
Molecular beacon
Loop
Fluorophore
Quencher
Stem
Fluorophore
Quencher
FIGURE 3.6
Schematic depiction of a molecular beacon. In its native form, the molecular beacon exists in a hairpin structure
in which a quenching species minimizes any fluorescent emission. After binding the complementary oligonu-
cleotide sequence, the quencher and fluorophores are separated resulting in fluorescence emission.
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