Biomedical Engineering Reference
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physiologically relevant glucose levels (30-300 mg/dL), were incubated with the
iDIOL-functionalized surface. Detection of free, labeled DBA indicated loss of
fluorescent signal from the iDIOL environment following exposure to glucose,
confirming successful competition. A plot of the fluorescence signal in response
to increasing glucose concentrations produced a response curve that defined the
glucose sensitivity of the candidate DBA relative to the iDIOL. Response profiles
of DBAs that showed a significant, competitive response to increasing glucose
concentration were considered to have a desirable binding equilibrium between
glucose and the iDIOL. On the one hand, the DBA needed to bind to the iDIOL with
sufficient affinity to produce a useful signal. On the other hand, the DBA needed to
bind to the iDIOL weakly enough relative to the DBA:glucose affinity so that
glucose could compete to produce a signal. The slope and IC
50
values of each
response curve were the parameters used to compare the binding sensitivity of each
DBA:glucose:iDIOL detection system.
6.1 Examples of Different Component Sensitivity and Selectivity
In one representative study, multiple candidate DBAs were used to generate
glucose response curves using a reference iDIOL, over a broad glucose concentra-
tion range. Figure
9a
shows the glucose response curves, which are the inverse of
the free solution fluorescence intensity measured during the assay. Upon addition of
glucose, the fluorescence intensity of DBA not bound to the iDIOL increased. This
was due to the competitive binding of glucose to the boronic acid receptors of the
DBA, which prevented the fluorescently labeled DBA from binding to the iDIOL.
Previously determined binding constants for DBA:glucose and DBA:diol were
correlated with the glucose response curves of each DBA:iDIOL system (Fig.
9b
).
These binding curves illustrate that the candidate DBAs (Fig.
10a-c
) respond
differently to changing levels of glucose when exposed to a particular iDIOL
(Fig.
10d
), as would be expected from their DBA:diol Keq values. DBA 2 and
DBA 3 are on or below the DBA:glucose versus DBA:diol 1:1 line, indicating that
glucose has equal or greater affinity for DBA 2 and DBA 3 than the diol. The
opposite is true for DBA 1, which has minimal DBA:glucose affinity relative to the
DBA:diol. These data correlate with the observed glucose response curves where
DBA 1 produced a minimally responsive curve and DBA 2 and DBA 3 showed
typical competitive assay curves. Furthermore, the greater I
50
sensitivity of DBA
2 relative to DBA 3 (Fig.
11
) is in agreement with the difference in DBA 2:glucose
affinity versus DBA 3:glucose affinity.
In a second representative study, multiple candidate iDIOL conjugates
(Fig.
13a-c
) were used to generate glucose response curves (Fig.
12a
) using a
reference DBA (Fig.
13d
) over a broad glucose concentration range. As in the
previous example, upon addition of glucose, the fluorescence intensity of unbound
DBA increased due to the competitive binding of glucose to the boronic acid
receptors of the DBA, which prevented further binding of the DBA to the iDIOL
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