Using an indicator displacement assay to monitor glucose oxidase activity in blood serum

Article abstract of DOI:10.1021/ol070280o

Using a boronic acid receptor that was previously found to have high affinity for gluconic acid, we created a colorimetric indicator displacement assay (IDA) that can report the concentration of the product of glucose oxidase (GOx) catalyzed glucose oxidation. The color change obtained directly reflects the concentration of glucose. Our sensing ensemble was then successfully applied to determine the glucose concentration in human serum, which offers a facile, colorimetric, sensitive, and accurate glucose test.

Full text of DOI:10.1021/ol070280o

ORGANIC
LETTERS
2007
Vol. 9, No. 9
1627-1629
Using an Indicator Displacement Assay
to Monitor Glucose Oxidase Activity in
Blood Serum
Tianzhi Zhang and Eric V. Anslyn*
Department of Chemistry and Biochemistry, 1 UniVersity Station A5300,
The UniVersity of Texas, Austin, Texas 78712
anslyn@ccwf.cc.utexas.edu
Received February 4, 2007
ABSTRACT
Using a boronic acid receptor that was previously found to have high affinity for gluconic acid, we created a colorimetric indicator displacement
assay (IDA) that can report the concentration of the product of glucose oxidase (GOx) catalyzed glucose oxidation. The color change obtained
directly reflects the concentration of glucose. Our sensing ensemble was then successfully applied to determine the glucose concentration
in human serum, which offers a facile, colorimetric, sensitive, and accurate glucose test.
Glucose sensing continues to be an active area of research
because of the increasing number of individuals diagnosed
with diabetes.1 The traditional commercial glucose test is
electrochemical, involving an amperometric electrode where
the glucose concentration is monitored by a change in current
flow caused by the glucose oxidase (GOx) catalyzed produc-
tion of hydrogen peroxide, or sometimes by the consumption
of oxygen.2 In this method, the generated hydrogen peroxide
is oxidized under a constant working potential, and the extent
of oxidation corresponds to the glucose concentration.
However, it needs careful calibration because other oxidiz-
able components may interfere at the working potential.
Acid-sensitive polymers have alternatively been used.3 In
this method, GOx generates gluconic acid which lowers the
pH and causes shrinking of the polymer. A vibrational
frequency change correlates to the glucose concentration. The
detecting process is reversible, but the sensitivity will
dramatically decrease with increasing ionic strength. Optical
methods have also been reported, along with their potential
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10.1021/ol070280o CCC: $37.00
© 2007 American Chemical Society
Published on Web 03/29/2007

Scheme 1. IDA Application in Gluconic Acid Association
applicability in vivo for noninvasive detecting and high
could monitor this product of GOx. As shown in Figure 1,
little to no displacement of the indicator occurs upon the
addition of glucose. This demonstrates that we could detect
the gluconic acid in the presence of glucose or other sugars.
sensitivity.4 Asher has reported a crystalline colloidal array
embedded polyacrylamine hydrogel that responds to low
concentrations of glucose by a Bragg diffraction change from
greenish blue to red.5 Further, the Shear group applies
horseradish peroxide to uptake the GOx product H2O2, and
the generated O2 is caught by 4-aminoantipyrine and 4-hy-
droxybenzoic acid to generate quinoneimine, a bright red
dye. Via the absorbance change, the concentration of glucose
can be determined in various drinks.6 We wanted to extend
the use of indicator displacement assays (IDA), which have
been successfully applied in various media using sensitive
colorimetric dyes. Here, we report the application of an IDA
method to glucose sensing in serum, creating an attractive
assay of potential relevance to diabetes.
We previously reported a metalated boronic acid receptor
(1) that shows excellent selectivity and high affinity to
gluconic acid with a stoichiometry of 1:1 and a Ka of 5.6 ×
106 M-1.7 We now report the use of 1 in a sensing assay. A
colorimetric response to gluconic acid was created using an
indicator displacement assay in 3:1 (v/v) MeOH/H2O at
neutral pH. Pyrocatechol violet (PV) was chosen as the
indicator (Scheme 1), and a 1:1 (molar ratio) of 1/PV solution
was prepared, giving a purplish red color. Upon introduction
of the gluconic acid, because of its stronger association to
receptor 1, PV was displaced from the receptor pocket, giving
the free indicator color, yellowish green. A large spectral
response is found for gluconic acid, demonstrating that we
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8, 279-287.
Figure 1. (A) UV/vis titration results, where [1] ) 0.13 mM and
[PV] ) 0.13 mM in MeOH-H2O 3:1 (v/v). (B) Addition of
gluconic acid to receptor 1 causes a displacement of PV from 1,
resulting in a large color change. [1] ) 0.13 mM, [PV] ) 0.13
mM, and [G] ) 0.13 mM, all in MeOH-H2O 3:1 (v/v).
Using this IDA and the fact that gluconic acid was the
only product generated with GOx, we explored the deter-
mination of gluconic acid concentrations in blood. Because
the reaction generates hydrogen peroxide, the enzyme
catalase was applied to sequester the H2O2 (Scheme 2). The
H2O2 was sequestered because boronic acid based receptors
have previously been shown to react with H2O2.8
(5) Asher, S. A.; Alexeev, V. L.; Goponenko, A. L.; Sharma, A. C.;
Lednev, I. K.; Wilcox, C. S.; Finegold, D. N. J. Am. Chem. Soc. 2003,
125, 3322-3329.
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Org. Lett., Vol. 9, No. 9, 2007

above, instead of using glucose, the human serum was
directly applied in varying amounts (50, 100, 150, 200, 250,
300 µL). We used a series of concentrations because patients
with diabetes routinely withdraw varying volumes that are
not necessarily controlled. The sample treatment procedure
was the same as that in the calibration experiment. The
absorbance was recorded and converted to glucose concen-
tration using the calibration curve (mg/mL) (Figure 2). For
each addition of serum, the glucose concentration was
determined. In each analysis, the concentration was that
quoted by Sigma-Aldrich with an average ( 7% error:
[glucose] ) 110 ( 8 mg/dL (Figure 3). Hence, this
Scheme 2. Enzyme-Catalyzed Glucose Oxidation
To optimize the reaction conditions, several variables were
examined: the [GOx], [catalase], pH, and reaction time
(Supporting Information). The optimization led us to prepare
a series of 5 mL aqueous solutions at pH ∼ 7 as standards,
each of which contained 30 µL of GOx (1 mg/mL), 200 µL
of catalase (1 mg/mL), 50 mM Tris buffer, and 0-140 µL
of a glucose solution (50.23 mM), resulting in glucose
sensitivity between 0 and 1.41 mM. This concentration range
was targeted because the procedure ultimately used with
blood involves a dilution (see below).
The oxidation commences upon the addition of different
amounts of glucose to the 5 mL aliquot containing GOx and
catalase. Aliquots of 250 µL of this solution were removed
from the reaction and mixed with a 750 µL MeOH solution
containing receptor 1 (0.13 mM) and an indicator (pyrocata-
chol violet, 0.13 mM). The solutions were analyzed by UV-
vis spectroscopy, and the absorbance was correlated to
gluconic acid concentrations, giving a calibration curve
(Figure 2). This curve was then used to analyze for glucose
in crude blood samples.
Figure 3. Glucose determination in human serum. All samples
were 110 mg/dL of glucose, and several tests were performed for
different volumes of the serum. Each test was within 7% of the
correct glucose value.
colorimetric methodology is accurate for determining glucose
concentration in human blood at a variety of different blood
volumes.
In conclusion, using a receptor that was previously found
to have high affinity to gluconic acid, we created a
colorimetric IDA that can report the concentration of the
product of GOx catalyzed glucose oxidation. The color
change obtained directly reflects the concentration of glucose.
Our sensing ensemble was then successfully applied to
determine the glucose concentration in human serum, which
offers a facile, colorimetric, sensitive, and accurate glucose
test.
Acknowledgment. We gratefully acknowledge the dis-
cussions with J. B. Shear and financial support from the NIH
(EB00549-5), and Welch Foundation.
Figure 2. Glucose calibration curves. [Glucose] ) 50.34 mM.
Supporting Information Available: A PDF file of
condition optimization. This material is available free of
charge via the Internet at http://pubs.acs.org.
Human serum (glucose 110 mg/dL, Sigma-Aldrich) was
analyzed. With the enzyme and buffer solutions described
(8) Wu, S.; Waugh, W.; Stella, V. J. J. Pharm. Sci. 2000, 89, 758-765.
OL070280O
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