Design and Development of In Vivo Sensor Systems: The Long and Tortured Road to a Self-Contained, Implantable Glucose Sensor for Diabetes Management - Designing Receptors for the Next Generation of Biosensors - page 218

3.1.3 DBA Construction
Boronic acid analogues were selected for inclusion in the DBA library based on
prescreening of their interactions with our target analyte (glucose) versus their
interactions with our saccharide mimic diol compounds. Utilizing ARS, a diol
selective fluorescent dye, we characterized the binding interactions of the initial
kit of boronic acids with each diol species [ 97 - 100 ]. Indicator displacement assays,
such as the ARS assay, rely on the relative affinity of two competing guests for the
receptor host. Specifically, the saccharide or diol-containing species, as the analyte
of interest, competes with and preferentially displaces the diol-containing ARS
from the boronic acid host. The displacement of the ARS reporter molecule from
the boronic acid structure causes a measurable change in fluorescence. The magni-
tude of the fluorescence change that results from increasing concentrations of
analyte provides a straightforward method to determine which boronic acid
structures bind competitively with glucose and/or the saccharide mimics under
the conditions (e.g., pH, ionic strength) of the assay.
ARS competitive assays were performed in a physiological buffer at pH 7 to
confirm that the preselected kit of boronic acid ligands, which were selected to
include a range of structural and chemical properties, bound glucose with adequate
affinity in an aqueous environment. If the observed ARS fluorescence dropped
substantially as the concentration of glucose titrated into the assay solution
increased, we could conclude that glucose was competitive with the ARS diol
relative to the boronic acid. In that case, the boronic acid was deemed to have
passed our screening guidelines. On the other hand, if there was no observed change
in fluorescence as increasing amount of glucose was titrated into the assay solution,
we could conclude that glucose could not compete for the boronic acid with
adequate affinity and, as a result, that particular boronic acid would no longer be
considered as a viable candidate.
Response curves from a representative ARS assay experiment are shown in
Fig. 4 . The observed drop in fluorescence intensity as the concentration of glucose
titrated into the solution increased demonstrated that glucose could bind to phenyl
boronic acid 1 (Fig. 5a ) and compete with ARS. In other words, the affinity of
phenyl boronic acid 1 for glucose was greater than the affinity of phenyl boronic
acid 1 for ARS, causing phenyl boronic acid 1 to preferentially bind with glucose.
In contrast, phenyl boronic acid 2 (Fig. 5b ) showed little affinity toward glucose and
was not included in construction of the DBA library. As predicted from
structure-p K a relationships, phenyl boronic acid 1 would have greater binding
affinity does glucose than does phenyl boronic acid 2. According to Hammet
equation predictions, the quantifiable difference between phenyl boronic acid 1
and phenyl boronic acid 2 is the fluoro substituent located in the para- position on
the phenylboronic acid structure. The electron withdrawing effect of the fluoro
substituent in the para- position, on phenyl boronic acid 1, combined with a less
sterically hindered boronic acid, will cause a drop in p K a and an increase in binding
affinity for glucose.