Optical glucose detection across the visible spectrum using anionic fluorescent dyes and a viologen quencher in a two-component saccharide sensing system

Article abstract of DOI:10.1039/b418953a

A very general system is described in which anionic fluorescent dyes possessing a wide range of absorbance and emission wavelengths are used in combination with a boronic acid-modified viologen quencher to sense glucose at pH 7.4 in buffered aqueous solut

Full text of DOI:10.1039/b418953a

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A R T I C L E
Optical glucose detection across the visible spectrum using anionic
fluorescent dyes and a viologen quencher in a two-component
saccharide sensing system†‡
David B. Cordes, Aaron Miller, Soya Gamsey, Zach Sharrett, Praveen Thoniyot,
Ritchie Wessling and Bakthan Singaram*
Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95060,
USA. E-mail: singaram@chemistry.ucsc.edu
Received 21st December 2004, Accepted 9th March 2005
First published as an Advance Article on the web 29th March 2005
A very general system is described in which anionic fluorescent dyes possessing a wide range of absorbance and
emission wavelengths are used in combination with a boronic acid-modified viologen quencher to sense glucose at
pH 7.4 in buffered aqueous solution. The present study demonstrates this capability with the use of eleven anionic
fluorescent dyes of various structural types. Signal modulation occurs as the monosaccharide binds to the viologen
quencher and alters its efficiency in quenching the fluorescence of the anionic dyes. The degree of quenching and the
magnitude of the glucose signal were found to correlate roughly with the number of anionic groups on the dye.
Optimal quencher : dye ratios were determined for each dye to provide a fairly linear signal in response to changes in
glucose concentration across the physiological range.
Introduction
particular excitation or emission wavelengths. For example, bi-
ological and particularly in vivo applications normally preclude
the use of damaging UV wavelengths, whereas these may be
appropriate for food processing or in vitro biochemical analysis.
Previously, we have used the well-studied anionic dye pyranine
The development of fluorescent synthetic chemosensors for
the detection of carbohydrates has attracted much attraction
for over the past decade. The majority of these sensing
systems consist of a single molecule possessing both a fluo-
rophore and a boronic acid receptor. The ability of boronic
acids to reversibly bind saccharides has been well known for
1–5
(HPTS) and several of its derivatives, including a polymerizable
sulfonamide immobilized in a thin film hydrogel, to sense
9,10
monosaccharides.
The present study describes the use of
6
over forty years and has since been extensively reviewed.
various anionic dyes to sense glucose across the visible spectrum.
Typically, upon glucose binding, the resulting changes in the
electronic and/or steric states of the sensor molecule cause
modulations in the fluorescence emission of the fluorophore
that can be correlated with monosaccharide concentration. In
these single molecule systems, the difficulty associated with
synthetically combining a boronic acid receptor with the desired
fluorophore in a single moiety can be a significant drawback
since synthetic transformation can cause unpredictable changes
in the photophysical properties of the dye. In a two-component
saccharide sensing system, however, the fluorophore units can be
interchanged without any synthetic modification of the receptor.
Such a system can therefore make use of numerous commercially
available fluorescent dyes.
Results and discussion
Structurally diverse ionic dyes were selected to provide a
wide range of emission wavelengths while providing structural
diversity. All the dyes possess at least two sulfonic or carboxylic
acid groups, except fluorescein-SA which has one of each, and
all are expected to exist in their anionic forms at pH 7.4. The
eleven dyes used in this study are shown in Fig. 1.
Quenching studies
Studies of several boronic acid-modified viologen quenchers
In our two-component saccharide sensing system, boronic
acid-functionalized viologen quenchers are used as the saccha-
possessing both bipyridinium and phenanthrolinium cores have
8,10
already been reported.
The ability of these viologens to
7
–10
ride receptor unit.
Viologens have been found to quench
quench the fluorescence of anionic HPTS was demonstrated and
found to be most significant when there was a large number of
cationic groups on the quencher. Among the quenchers studied,
11–13
the luminescence of both simple dyes and macromolecular
through an ionic attraction between quencher and
14–17
systems
fluorophore to form a non-fluorescent complex. Upon saccha-
ride binding to the boronic acid of the viologen quencher, a
negatively charged boronate ester is formed. We hypothesize
that this causes a diminished electrostatic attraction between
quencher and dye resulting in a fluorescence increase. The
putative general sensing mechanism is represented in Scheme 1
4+
the 4+ charged bipyridium m-BBVBP exhibited superior
fluorescence quenching while providing a moderately strong
4+
glucose signal. In the present study, m-BBVBP was used to
quench the fluorescence of the dyes shown in Fig. 1. Based on
our previous results, we expected stronger quenching to occur for
dyes possessing a greater number of anionic groups. Generally
we found this to be the case, with highly negatively charged
4
+ 18
with the viologen quencher m-BBVBP .
Various anionic dyes were explored to demonstrate the
flexibility of the two-component system. The ability to use any
desired fluorophore in an optical sensing system is a considerable
advantage since certain applications may require the use of
HPTS, HPTS(Lys) , PTCA, MPTS and the porphyrin dyes,
3
TSPP and TCPP, providing the most significant quenching. Dyes
with fewer anionic groups quenched less effectively. A Stern–
Volmer plot of quenching of selected dyes by m-BBVBP at
4+
pH 7.4 is given in Fig. 2.
Graphical methods and UV-visible absorbance spectroscopy
were used to determine the mechanism responsible for the fluo-
rescence quenching of selected dyes. For most dyes, quenching
involves mainly a static mechanism in which association between
†
‡
Dedication: dedicated to the memory of Herbert C. Brown.
Electronic supplementary information (ESI) available: addi-
tional experimental details. See http://www.rsc.org/suppdata/ob/b4/
b418953a/.
1
7 0 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 0 8 – 1 7 1 3
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5

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Scheme 1 General sensing mechanism.
Fig. 1 Selected ionic dyes for use in saccharide sensing. All compounds are shown as purchased or prepared. Fluorescein-SA = fluo-
rescein-5-(and-6-)sulfonic acid; lucifer yellow-I = lucifer yellow iodoacetamide; SR-B = sulforhodamine-B; SR-101 = sulforhodamine-101;
MPTS = methoxypyrenetrisulfonic acid trisulfonate; CTR = carboxytetramethylrhodamine; TSPP = tetrakis(4-sulfophenyl)porphine; TCPP =
tetrakis(4-carboxyphenyl)porphine; HPTS = hydroxypyrenetrisulfonic acid trisulfonate; PTCA = perylenetetracarboxylic acid tetracarboxylate.
dye and quencher provides an essentially non-fluorescent com-
plex. An alternate quenching process, dynamic quenching, can
occur via deactivating collisions between the dye and quencher
F
o
is the initial fluorescence and F is the fluorescence
after addition of quencher. For most of the dyes studied,
we determined that the dynamic, or collisional, quenching
component contributed only a small portion of the overall
19
in solution. To determine the contribution of each of these
processes to the overall quenching we relied on a “sphere
quenching (Table 1). An exception was the HPTS(Lys) dye,
3
20
of action” model modified from earlier work by Frank and
which was observed to undergo a significant degree of dynamic
quenching. We speculate that this may occur due to the lengthy
lysine units attached to the pyrene core. This would be expected
to significantly increase the radius of gyration in solution
and, thus, the likelihood of deactivating collisions with the
21
Vavilov that provides quenching constants for both static (Ksv
and dynamic (V) processes (eqn. (1)).
)
V[Q]
20
(
F
o
/F) = (1 + Ksv[Q])e
(1)
quencher.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 0 8 – 1 7 1 3
1 7 0 9

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Table 1 Quenching data
Static quenching
Dynamic quenching
a
b
c
Dye
K
sv
K
uv
V
HPTS
50 000 ± 4000
22 000 ± 1000
16 000 ± 4000
14 400 ± 100
10 900 ± 200
4600 ± 100
(45 000 ± 5000)
(18 000 ± 1000)
(9000 ± 1000)
(22 000 ± 4000)
(15 000 ± 4000)
(3200 ± 400)
3800 ± 700
300 ± 90
4500 ± 200
0
14 000 ± 1000
100 ± 40
30 ± 10
MPTS
PTCA
TCPP
HPTS(Lys)
TSPP
3
Fluorescein-SA
1960 ± 70
(1500 ± 600)
a
b
K
sv (static Stern–Volmer quenching constant determined from fluorescence data).
K
uv (apparent binding constant for quencher–dye complexation
c
from UV-vis data). V (dynamic or collisional quenching constant derived from fluorescence data); errors are reported as standard deviations based
on three trials. All values are reported in M units.
−
1
constants (Kuv) for complexes between the strongly quenched
4
+
dyes and m-BBVBP were calculated from Benesi–Hildebrand
plots of the UV-vis absorbance data and were found to roughly
follow the same order as the Stern–Volmer quenching constants
obtained from fluorescence studies (See Table 1). When quench-
ing does occur by a static mechanism, as in the present case,
the Ksv obtained from fluorescence should match closely the
apparent quencher–dye stability constant (Kuv) derived from
UV-vis absorbance data (Ksv ≈ Kuv). The dyes showing weak
quenching showed no perturbation in their UV-vis absorbance
spectra as they were titrated with quencher; as discussed, we
believe this is due to the weak electrostatic attraction of these
dyes for the quencher. Despite the differences in quenching, all
of the dyes studied were successfully used to sense glucose.
Fig. 2 Stern–Volmer plot of fluorescence quenching of selected ionic
4+
−6
dyes by m-BBVBP at pH 7.4. All dyes were studied at [4 × 10 M]
−
7
except SR-B and SR-101 [1 × 10 M].
Glucose sensing studies
The dyes CTR, SR-B, SR-101 and LY-I were only weakly
The binding isotherm derived from the fluorescence signal was
determined by adding glucose to a quenched solution of dye.
As glucose is added to the quenched solution, the fluorescence
increases as the quenching becomes less efficient due to glucose
binding the boronic acid of the quencher. Experiments show
that the shape of the binding isotherm can be conveniently
manipulated by adjusting the ratio between quencher and dye,
an added advantage of the two-component system. Increasing
the quencher : dye ratio raises the concentration at which the
signal response saturates by lowering the apparent binding
affinity between dye and quencher. In this way, we can avoid
the common problem of rapid saturation that occurs in many
one-component saccharide sensors. This allows us to obtain
a fairly linear glucose response over the physiological range.
Similar behavior was observed for the monosaccharides fructose
and galactose, with the monosaccharide selectivity observed in
our study roughly corresponding with the 33 : 3 : 1 ratio of
binding affinities for fructose, galactose and glucose determined
4
+
quenched by m-BBVBP and we were unable to determine if
the small degree of quenching was due to a static or dynamic
mechanism. Each of these dyes bears only two anionic groups
and we speculate that the quenching efficiency of these dyes
is poor due to their relatively weak electrostatic attraction for
the cationic quencher. In all of the cases of weak quenching
we observed nearly linear Stern–Volmer plots indicating only a
single mechanism at work. Relatively, small quenching constants
−
1
ranging from 80 to 260 M were obtained from Stern–Volmer
22
analysis of the fluorescence quenching of these dyes. All
of the dyes showing strong quenching interactions with m-
4
+
BBVBP displayed changes in their UV-visible spectra as they
were titrated with quencher solution. Representative spectra are
shown in Fig. 3.
These spectra strongly indicate complex formation between
dye and quencher. The isosbestic points present in each spectra
suggest a single complex being formed from each quencher–
dye pair with what we speculate is 1 : 1 stoichiometry. Stability
23
for simple phenylboronic acid. In cases where only weak
4
+
−5
−5
Fig. 3 Changes in UV-visible absorbance spectra upon titration with m-BBVBP . a) HPTS (1 × 10 MM ) ,. b) PTCA (1 × 10 M) and c) TCPP (4 ×
−
6
4+
−5
−4
−3
1
0
M). Final concentration of m-BBVBP for a) 6.25 × 10 M, b) 1.13 × 10 M and c) 1.13 × 10
1
7 1 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 0 8 – 1 7 1 3

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fluorescence quenching was observed, we generally had to use
very high quencher : dye ratios to obtain linearity in the glucose
response signal. The effects of adjusting the quencher : dye ratio
Generally, we observed that these dyes did provide stronger
glucose signals and that the magnitude of signal response could
be roughly correlated with the number of anionic groups on the
dye (Fig. 5).
4
+
for TSPP with m-BBVBP are shown in Fig. 4.
Conclusions
In conclusion, we have demonstrated a general glucose sensing
system, operable at pH 7.4, using a broad range of anionic
fluorescent dyes in combination with a boronic acid-modified
viologen quencher. The mechanism relies on the ability of the
viologen to quench the fluorescence of the anionic dye. The
signal is produced when quenching is diminished upon addition
of glucose, which binds to the boronic acids of the viologen
quencher. The system operates at pH 7.4 and its sensitivity for
sensing glucose in the physiological range can be adjusted by
altering the ratio between quencher and dye. We found that both
the degree of quenching and the magnitude of the glucose signal
were generallygreater for dyes possessingagreater number ofan-
ionic groups. The generality of this two-component sensing sys-
tem and the ease with which desired fluorophores can be intro-
duced into the sensing scheme allows for considerable versatility
in the design of saccharide sensing systems. We are actively
investigating the use of polymerizable analogs of these dyes for
use in glucose sensing hydrogels and are exploring the appli-
cation of this two-component sensing system towards other
analytes.
Fig. 4 Glucose response curves for addition of glucose to quenched
4+
solutions of TSPP at pH 7.4 with varying m-BBVBP : TSPP ratios.
The dashed line indicates the optimal quencher : dye ratio at 188 : 1. The
physiological glucose range is boxed. F
o
= initial quenched fluorescence;
F = fluorescence after addition of glucose. Note: 10 mM [glucose] =
−
1
1
80 mg dL .
Quencher : dye ratio optimization experiments were carried
out for all of the dyes in the study to obtain a linear signal
response in the physiological range. The optimized quencher :
dye ratios and apparent binding constants for quencher binding
glucose are given in Table 2, along with percentage increases in
signals observed for increasing the glucose concentration from
Experimental
General
0
to 30 mM.
As in the quenching experiments, we expected to find a strong
charge dependence with regard to glucose sensing, with the
dyes carrying more anionic groups providing greater signals.
The following fluorescent dyes were purchased from
Molecular Probes or Aldrich and used as received: fluorescein-
SA = fluorescein-5-(and-6-)sulfonic acid (MP#F1130); lucifer
4
+
Fig. 5 Glucose response of various ionic dyes combined with m-BBVBP at pH 7.4. F
o
= initial fluorescence; F = new fluorescence. All dyes were
−
6
−7
[
4 × 10 M] except SR-101 and SR-B which were [1 × 10 M]. Quencher : dye ratios and apparent binding constants are given in Table 2. The
number of anionic groups (acids and phenols) is given in parentheses next to the dye name. The physiological glucose range is boxed. The data were
distributed for improved clarity. Note: 10 mM [glucose] = 180 mg dL−
Table 2 Glucose sensing data
1
.
Optimal quencher : dye
(Q : D) ratio
Apparent glucose binding constants at
Signal increase from 0
to 30 mM (%)
optimal Q : D ratio (Kapp)/M−
1
Dye
TCPP
MPTS
Fluorescein-SA
HPTS
HPTS(Lys)
TSPP
188 : 1
250 : 1
1300 : 1
31 : 1
625 : 1
188 : 1
0.030 ± 0.005
38 ± 5
59
59
53
43
42
33
22
16
9
22 ± 3
27 ± 6
3
28 ± 5
14 ± 4
PTCA
38 : 1
50 ± 10
13 ± 3
Lucifer yellow-I
Sulforhodamine-101
CTR
1250 : 1
125 000 : 1
12 500 : 1
125 000 : 1
32 ± 3
60 ± 10
7
6
Sulforhodamine-B
23 ± 1
Optimal quencher : dye ratios for linear signal response to glucose in 0 to 30 mM concentration range and apparent glucose binding constants at that
ratio.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 0 8 – 1 7 1 3
1 7 1 1

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yellow-I = lucifer yellow iodoacetamide (MP#L1338); SR-B =
sulforhodamine-B (Aldrich#23,016-2); SR-101 = sulforhoda-
mine-101 (Aldrich#28,491-2); MPTS = methoxypyrenetrisul-
fitting the data with eqn. (1). Apparent glucose binding constants
were calculated by fitting the data with eqn. (4):
F
calc = (Fmin + FmaxK[glucose])/(1 + K[glucose])
(4)
fonate (MP#335); CTR
=
carboxytetramethylrhodamine
tetrakis(4-sulfophenyl)porphine
(
(
(
MP#C300);
MP#T6932); TCPP
MP#T6931). The preparation of HPTS(Lys)
TSPP
=
where Fcalc is the calculated fluorescence intensity, Fmin is the
initial fluorescence intensity of the quenched dye, Fmax is the
calculated intensity at which the fluorescence increase reaches
=
tetrakis(4-carboxyphenyl)porphine
has already been
3
9
described. Perylenetetracarboxylic acid (PTCA) was prepared
by simple alkaline hydrolysis of perylene dianhydride with
potassium hydroxide and used as the tetrapotassium carboxylate
salt. Details for the preparation and characterization of m-
BBVBP are given in the supporting information.‡ All other
reagents including HPTS dye were purchased from Aldrich Co.
and used as received. All solvents were HPLC grade. DMF was
27
its maximum and K is the apparent binding constant.
Quencher : dye ratio optimization for glucose sensing
4
+
The fluorescence measurements were done in situ by taking the
emission spectra of a 1 : 1 quencher : dye solution then adding
an aliquot of buffered 1 M glucose solution and measuring the
new fluorescence emission after shaking for 60 s. Additional
aliquots were added and measurements taken until a glucose
concentration of 30 mM was obtained. The overall process
was then repeated at successively higher ratios until an optimal
quencher : dye ratio could be determined.
stirred over CaH for 2 days and filtered immediately before
2
use. All reactions were conducted under an inert atmosphere of
argon using air sensitive techniques. Measurements of pH were
1
1
carried out on a Mettler Toledo MP 220 pH meter. B NMR
spectra were measured on a Bruker 250 using BF :Et O as an
3
2
1
3
1
external standard. C and H NMR spectra were measured on
a Varian 500 referenced to TMS.
Glucose sensing
The fluorescence measurements for each dye were done in situ
by taking the fluorescence emission spectra of the quencher :
dye solution at its optimized ratio. An aliquot of buffered 1 M
glucose solution was then added and the new fluorescence
emission was measured after shaking for 60 s. Additional
aliquots were added and measurements taken until a glucose
concentration of 30 mM was obtained. Representative spectra
are provided in supporting information.‡
Fluorescence emission and UV-vis absorption studies
All studies were carried out in pH 7.4 buffer solution prepared
with water purified via a Nanopure Ultrafiltration system.
Buffer solution (pH 7.4, 0.1 ionic strength) was freshly prepared
using KH
2
PO
4
and Na
2
HPO . Fluorescence spectra were taken
4
on a Perkin-Elmer LS50-B luminescence spectrometer. The
absorption spectra were taken on a Hewlett Packard 8452A
Diode Array Spectrophotometer. All studies were carried out
◦
at 20 C without exclusion of air. Excitation and emission
Acknowledgements
wavelengths for each dye are given in Fig. 1. For fluorescence
titration experiments, the added volume did not exceed 3% of
the total volume and the sample absorbance for fluorescent
We thank Glumetrics, Inc., operating through the BioStar
Industry–University Cooperative Research program, for con-
tinuing financial support.
24
measurements was below 0.1. All experiments except the
quencher : dye optimization studies were carried out in triplicate
and the error is reported as the standard deviation. All data was
analyzed using the Solver (non-linear least-squares curve fitting)
25
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