Biomedical Engineering Reference
In-Depth Information
DBA binding environment (iDIOL). The selection of materials for this component
was governed by the need to (1) construct a glucose-competitive binding environ-
ment that would form a reversible complex with the DBA signaling component in
aqueous media and (2) select commercially available saccharide mimics with a
diversity of diol sub-structures and a suitable functional moiety for covalent
immobilization to a support.
4.1
iDIOLs as Competitive Binding Environments
for Glucose Detection
This iDIOL versus DBA strategy, as discussed in detail earlier, uses the observation
that the hydroxyl groups on saccharides, specifically 1,2- or 1,3-diols, are known to
competitively bind with boronic acids to form five- or six-membered ring structures
[
72
,
90
]. We initially selected diols, which would subsequently be immobilized to
produce the required iDIOLs, based on a comparison of their binding affinity to
DBAs versus the binding affinity of the respective DBA for glucose. Our diol
selection strategy involved exploiting the differential in relative binding affinity
that would be created when a DBA is concurrently exposed to an immobilized diol
(iDIOL) and a range of glucose concentrations. The objective was to identify DBA:
iDIOL pairs that would permit discriminatory binding of the DBA to glucose, due
to increased relative affinity over DBA binding to the iDIOL.
Selection of diols for ultimate preparation of iDIOLs, via immobilization of the
diol on the sensing system's transduction interface, was based on our evaluation of
the interactions between our kit of boronic acid-derived DBAs and various candi-
date diol species. We again used the ARS assay, as described previously, to
characterize the binding of the DBAs with the candidate diols. The magnitude of
the change in ARS fluorescence that resulted from increasing the amount of diol
titrated into the assay solution provided a straightforward method to determine
which diols, and ultimately which iDIOL structures, would competitively interact
with the various DBA species.
Response curves from an ARS assay experiment performed in a physiological
buffer at neutral pH are shown in Fig.
6
. An enhanced response of the DBA
2 (Fig.
7a
) for diol 1 (Fig.
7b
) versus diol 2 (Fig.
7c
) was observed. Phenylboronic
acids are known to have different binding affinities for diols depending on the
dihedral angle of the diol. Smaller dihedral angles often accompany higher binding
constants [
72
]. Additionally, rigid cyclic
cis
diols tend to form stronger cyclic esters
than acyclic diols [
72
,
90
]. Thus, the enhanced binding of diol 1 can be attributed to
the improved compatibility of the boronic acid recognition motif on DBA 2 with the
dihedral angle of the diol. In contrast, it can be inferred that diol 2 formed a weaker
cyclic ester with the same boronic acid of DBA 2 as a result of increased angle
strain of the larger dihedral angle structure of the acyclic diol.
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