Using indicator-displacement assays in test strips and to follow reaction kinetics

Article abstract of DOI:10.1021/ol0493599

(Matrix Presented) An indicator-displacement assay was used to study the kinetics of reactions that produce tartrate. The same assay was also found to be useful in developing tartrate test strips, by coating filter paper with the same receptor and indicator.

Full text of DOI:10.1021/ol0493599

ORGANIC
LETTERS
2004
Vol. 6, No. 15
2499-2501
Using Indicator-Displacement Assays in
Test Strips and To Follow Reaction
Kinetics
Binh T. Nguyen, Sheryl L. Wiskur, and Eric V. Anslyn*
The Department of Chemistry and Biochemistry, The UniVersity of Texas,
1 UniVersity Station A5300, Austin, Texas 78712
anslyn@ccwf.cc.utexas.edu
Received April 8, 2004
ABSTRACT
An indicator-displacement assay was used to study the kinetics of reactions that produce tartrate. The same assay was also found to be
useful in developing tartrate test strips, by coating filter paper with the same receptor and indicator.
Indicator-displacement assays (IDAs) have been used widely
in analytical sciences to detect various analytes. In an IDA,
an indicator is first allowed to bind to a host. Then, an analyte
is introduced into the system causing the displacement of
the indicator from the host, which in turn modulates an
optical signal.1 The IDA offers many advantages over
traditional sensing routines.2 First, it gives a color change
for a guest or host lacking a chromophore. Second, the
method does not require the indicator to be covalently
attached to the host. Third, because there are no covalent
bonds between the host and the indicator, one can use many
different indicators with the same host, and fourth, the assay
works well in both organic and aqueous media. Early work
using this method was successful in measuring DNA helicase
activity,3 characterizing binding of RecA protein to double-
stranded DNA,4 sensing of phosphate,5 halides,6 and histi-
dine,7 and the detection of cocaine.8 Our group has developed
a series of chemosensors that utilize an IDA to determine
analytes such as gallic acid,9 aspartate,10 inorganic phos-
phate,11 nitrate,12 citrate,2 glucose-6-phosphate,2 inositol-
1,4,5-triphosphate,2 2,3-bisphospho glycerate,13 heparin,14 and
tartrate.15
Given the utility and advantages of an IDA in sensing
sciences, we set out to extend the method to tracking reaction
kinetics and creating test strips for “naked eye detection”.
Our goal was to show that an IDA can be used in these
analyses by studying the kinetics of reactions that produce
diastereomeric tartrates, and to qualitatively detect tartrates
on test strips. Herein, we report kinetic studies of the
(6) Gale, P. A.; Twyman, L. J.; Handlin, C. I.; Sessler, J. L. Chem.
Commun. 1999, 1851
(7) Hortala, M. A.; Fabbrizzi, L.; Marcotte, N.; Stomeo, F.; Taglietti,
A. J. Am. Chem. Soc. 2003, 125, 20.
(8) Stojanovic, M. N.; Landry, D. W. U.S Patent Appl. Publ. 2003.
(9) Wiskur, S. L.; Anslyn, E. V. J. Am. Chem. Soc. 2001, 123, 10109.
(10) Ait-Haddou, H.; Wiskur, S. L.; Lynch, V. M.; Anslyn, E. V. J. Am.
Chem. Soc. 2001, 123, 11296.
(11) Tobey, S. L.; Anslyn, E. V. Org. Lett. 2003, 5, 2029.
(12) Niikura, K.; Bisson, A. P.; Anslyn, E. V. J. Chem. Soc., Perkin
Trans. 1999, 2, 1111.
(1) Martinez-Manez, R.; Sancenon, F. Chem. ReV. 2003, 103, 4419.
(2) Wiskur, S. L.; Ait-Haddou, H.; Lavinge, J. J.; Anslyn, E. V. Acc.
Chem. Res. 2001, 34, 963.
(3) Eggleston, A. K.; Rahim, N. A.; Kowalczykowsky, S. C. Nucleic
Acids Res. 1996, 24, 1179.
(4) Zaitsev, E. N.; Kowalczykowsky, S. C. Nucleic Acids Res. 1998,
26, 650.
(5) Han, M. S.; Kim, D. H. Angew. Chem., Int. Ed. 2002, 41, 3809.
(13) Best, M. D.; Anslyn, E. V. Chem. Eur. J. 2003, 9, 51.
(14) Zhong, Z.; Anslyn, E. V. J. Am. Chem. Soc. 2002, 124, 9014.
(15) Lavinge, J. J.; Anslyn, E. V. Angew. Chem., Int. Ed. 1999, 38, 3666.
10.1021/ol0493599 CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/26/2004

hydroxylation of maleic acid and fumaric acid in water to
create tartrate, as well as the hydrolysis of D-dimethyl tartrate.
We further show that an IDA is easily adapted to test strips.
Scheme 1. Formation of Tartratea
The design of host 1 has been reported.9 Previous studies
showed that boronic acids form reversible bonds with 1,2-
diols and R-hydroxy carboxylate,16 and guanidinium groups
bind carboxylates through H-bonds or charge-pairing interac-
tions.17,18 Further, on the basis of our previous studies, we
knew that upon addition of 1 to a solution of 2 (0.05 mM,
25% water in methanol (v/v), 20 mM HEPES, pH 7.4) the
λmax of the indicator shifts from 441 nm to that of the
indicator-host complex. Addition of 3 to the solution of the
1:2 complex causes the λmax to shift back to 441 nm. The
binding constants between 1 and 2 and between 1 and d,l-3
are known to be KHI ) 6.1 × 104 M-1 and KHT ) 1.4 × 105
M-1, respectively.9,19
To follow the kinetics of tartrate formation, one must relate
the free indicator concentration to tartrate concentration. In
an IDA, the change of absorbance of the indicator is not
directly proportional to tartrate formation because of the
equilibria in eq 1, where H is the host, I is the indicator, and
T is tartrate. We solved for the relationship between indicator
concentration and tartrate concentration (eq 2). [HI] and [I]
can be related to absorbance changes via eqs 3 and 4, where
HI is host-indicator complex, HT is the host-tartrate
complex, [H]0 is the total concentration of the host, [I]0 is
the total concentration of the indicator, AI is the absorbance
of free indicator, A is measured absorbance, and AHI is
absorbance of the host-indicator complex. Last, [T] is used
in eq 5 to create the standard first order kinetics plot, where
[S]0 is the total concentration of the reactant, t is time, and
k is the reaction rate constant.
a Conditions: (a, b) 0.5 M, NaOH (2 equiv), NaClO3 (1.6 equiv),
water, 4% w/w OsO4 in water (0.4% mol equiv), 50 °C; (c) 0.2 M,
water, pH 2, 100 °C.
acid, 2 equiv of NaOH were used to bring the pH of the
solution near 7 so that when adding an aliquot (2 µL) of the
reaction mixture to the solution of the 1:2 complex in buffer
the overall pH of the solution would not be perturbed.
Typically, the reaction kinetics were followed by adding 2
µL of the reaction mixture into 1 mL of a solution of the
1:2 complex (0.25 mM 1, 0.05 mM 2, 25% water in methanol
(v/v), 20 mM HEPES, pH 7.4) at each time interval, and
the absorbance was measured. For each interval of time, a
fresh 1 mL of the solution of the 1:2 complex was used
because for this particular reaction, the OsO4 would dihy-
droxylate the indicator after several minutes. The UV-vis
spectra and plots of kinetics are shown in Figure 1. Least-
squares fitting of the data to eq 5 gives rate constants for
the dihydroxylation of maleic acid and fumaric acid catalyzed
by osmium tetraoxide in water as 3 × 10-4 h-1 and 6 ×
10-4 min-1 respectively. The rate constant for the hydrolysis
of D-dimethyl tartrate was 3 × 10-4 h-1.
Besides applying the IDA to reaction kinetics, we also
developed a tartrate test strip that produced colors with
different intensities upon applying different tartrate concen-
trations. The test strip was prepared by adding 1-2 mL of
buffer (20 mM HEPES, 25% water in methanol (v/v), pH
7.4) to a 3 cm × 1 cm filter paper (Fisher, Q8, cat. no. 09-
790F). After drying in air, 1 mL of the 1:2 complex (0.2
mM 1, 0.2 mM 2) was allowed to coat the paper, which
was placed on a watch glass. The solution was absorbed by
the paper, and the paper was dried in air. Using capillary
tubes, four different solutions of tartaric acids (1, 0.5, 0.1,
and 0.01 M at pH 7.2) were spotted on the paper. The spots
turned dark blue and the intensity of the color decreased as
the concentration of tartrate decreased (Figure 2).
HI + T h HT + I
[H]0KHI[I] - [HI] - KHI[I][HI]
KHT[HI]
(1)
(2)
[T]
AI - A
[HI] ) [I]
0AI - AHI
(3)
(4)
To exclude the possibility of residual metal in the paper
matrix, which may influence the color change, a control test
strip was made by pretreating the paper with a solution of
20 mM EDTA and washing with the same buffer, and the
[I]0 ) [I] + [HI]
[S]o - [T]
-ln
) kt
(5)
[S]o
(16) Wulff, G. Pure Appl. Chem. 1982, 54, 2093.
(17) Linton, B. R.; Goodman, M. S.; Fan, E.; Van Arman, S. A.;
Hamilton, A. D. J. Org. Chem. 2001, 66, 7313.
(18) Berger, M.; Schmidtchen, F. P. J. Am. Chem. Soc. 1999, 121, 9986.
(19) Wiskur, S. L. Ph.D. Thesis. The University of Texas, Austin, Texas
2003.
We applied the mathematic derivation above to study the
kinetics of the dihydroxylation of maleic acid and fumaric
acid in water, and the hydrolysis of D-dimethyl tartrate
(Scheme 1). In the hydroxylation of maleic acid and fumaric
2500
Org. Lett., Vol. 6, No. 15, 2004

Figure 1. UV-vis spectrum for the IDA and kinetic plot for the
analysis of the dihydroxylation of maleic acid.
Figure 2. Test strips tested with 1.0, 0.5, 0.1, and 0.01 M tartrate
at pH 7.2.
procedure above was repeated. The same color change was
observed. When using the same coating conditions with the
addition of buffer or water, no color change was observed.
Also, a test strip coated with the indicator alone (0.2 mM,
20 mM HEPES, 25% water in methanol (v/v), pH 7.4) tested
with the same tartrate solutions produced no color change.
These results suggest that the IDA is suitable for making
test strips for visual detection of specific chemicals that have
no chromophore of their own.
In conclusion, we have found two new applications for
indicator-displacement assays. It is now possible to study
reaction kinetics, and we specifically followed reactions that
create tartrate. Further, the first test strip based upon an IDA
was reported. The results of this research open a window of
opportunity for the future of IDAs. This very simple method
could be used to follow the kinetics of simple reactions and
detect other natural or synthetic products using colorimetric
test strips.
Acknowledgment. We gratefully acknowledge support
for this project from the Welch Foundation and the Beckman
Center for array sensors.
Supporting Information Available: Full derivation of
equations. This material is available free of charge via the
Internet at http://pubs.acs.org.
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