Biomedical Engineering Reference
In-Depth Information
silanized to allow for covalent attachment of the antibodies to the fiber tips. Covalent
attachment of the antibodies was performed by first activating the silanized fiber tip with
a solution of carbonyl diimidazole, followed by incubation of the activated fiber with the
antibody of interest at 4
C for 3 days. After antibody attachment, the biosensors were
tested for retention of antibody activity as well as their sensitivity and absolute detection
limits. From these analyses, it was found that the antibodies had retained greater than
95% of their original binding affinity for BPT and that the absolute detection limit for BPT
was approximately 300 zmol (zepto = 10
21
).
Shortly after the development of this first antibody-based fiber-optic nano-biosensor,
they were applied to in vitro measurements within individual living cells.
3,34,49-51
In one
such study, nano-biosensors for BPT were prepared and used to quantitatively measure
intracellular concentrations of BPT in the cytosol of two different cell lines: (1) rat liver
epithelial cells and (2) human mammary carcinoma cells.
34
Unlike previous intracellular
analyses that employed relatively large cells (i.e., mouse oocytes and neurons) the cells
analyzed in this study were approximately 10 µm in diameter, demonstrating that fiber-
optic nanosensors and nano-biosensors could be used to analyze cells, the size of typical
mammalian somatic cells (i.e., 10-15 µm), without destroying them (see Figure 3.5). This
study also demonstrated that the insertion of a fiber-optic nano-biosensor into a cell and
the subsequent spectroscopic measurement had little effect on the cell's routine function.
This was demonstrated by inserting the fiber-optic nano-biosensor into the cytoplasm of a
cell beginning to undergo mitosis and monitoring division of the cell into two identical
daughter cells over the next 2 h.
°
3.2.1.2.2 Protein-Based Fiber-Optic Nano-Biosensors
Since the development and application of these first antibody-based fiber-optic nano-biosen-
sors, several other nano-biosensors, employing different biological receptor molecules, have
been fabricated and applied to the analysis of various biologically relevant species in indi-
vidual cells.
52,53
These have included nano-biosensors for the detection of nitric oxide,
employing the nonenzymatic protein cytochrome
c
and fluorescently labeled cytochrome
c
,
52
as well as enzymatic-based nano-biosensors for the indirect detection of glucose and gluta-
mate. By employing enzymes as bioreceptors, not only is a high degree of specificity
achieved, but also the catalytic activity of the enzymes can amplify the species being meas-
ured, providing extremely sensitive analyses. One such enzymatic fiber-optic nano-biosensor
FIGURE 3.5
Microscopic image of a fiber-optic
nanosensor being used to probe the chem-
ical cytosol of a rat liver epithelial cell.
