Exploring the use of APTS as a fluorescent reporter dye for continuous glucose sensing

Article abstract of DOI:10.1039/b821934f

The anionic fluorescent dye, aminopyrene trisulfonic acid (APTS), was synthesized and used in a solution-based two-component glucose-sensing system comprising the dye and a boronic acid-appended viologen. The fluorescence of the dye was quenched in the pr

Full text of DOI:10.1039/b821934f

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Exploring the use of APTS as a fluorescent reporter dye for continuous
glucose sensing†‡
Zachary Sharrett, Soya Gamsey, Lacie Hirayama, Boaz Vilozny, Jeff T. Suri, Ritchie A. Wessling and
Bakthan Singaram*
Received 8th December 2008, Accepted 27th January 2009
First published as an Advance Article on the web 28th February 2009
DOI: 10.1039/b821934f
The anionic fluorescent dye, aminopyrene trisulfonic acid (APTS), was synthesized and used in a
solution-based two-component glucose-sensing system comprising the dye and a boronic
acid-appended viologen. The fluorescence of the dye was quenched in the presence of the viologen and
the fluorescence restored upon glucose addition. An important feature of this fluorophore is that it can
be covalently bonded to a polymer through the amine group without a significant effect on optical
properties. Two APTS derivatives, functionalized with polymerizable groups, were synthesized and
immobilized in hydroxyethyl methacrylate (HEMA)-based hydrogels. The latter were used to
continuously monitor glucose. The fluorescence signal modulation, signal stability, reversibility,
reproducibility, and pH sensitivity of the hydrogels were evaluated. The APTS dyes described herein are
insensitive to pH changes within the physiological range, both in solution and when immobilized in a
hydrogel. When APTS is used in conjunction with boronic acid-appended viologens to sense glucose,
the system displays some pH sensitivity because of the presence of the boronic acid.
Introduction
The need for improved methods of glucose measurement in the
treatment of diabetes has long been recognized by scientists and
1
healthcare professionals. In addition to diabetics, critically ill
patients in the intensive care unit (ICU) also require improved
methods of glucose detection for implementation of tight glycemic
2
control (TGC). A method that provides accurate, real-time,
continuous monitoring of blood glucose levels is essential to
facilitate TGC. Commercial sensors are enzyme-based. They
employ enzymes such as glucose oxidase (GOX) or glucose
dehydrogenase (GDH), immobilized in a polymer matrix. The
3
enzymes function both as catalysts and as glucose receptors.
The glucose binding protein Concavalin A has also been used
as a glucose receptor in optical sensing systems. The drawbacks
Scheme 1 Equilibria between phenyl boronic acid and a generic diol in
aqueous media.
4
10
inherent in these approaches stimulated extensive research on
alternative systems employing synthetic saccharide receptors,
5
especially those comprising boronic acids and fluorescent dyes
These systems, when immobilized, offer promise as continuous
comprises a fluorescent anionic dye and a boronic acid-appended
cationic benzyl viologen. In the absence of glucose, the viologen
moiety quenches the fluorescence of the dye; it becomes a
far less effective quencher when bound to glucose. Thus, the
measurable fluorescence of the system is modulated in response
to varying glucose concentration. Using this combination, we
have demonstrated continuous and reversible glucose sensing
in vitro by immobilizing the sensing elements in a thin-film poly(2-
6
glucose monitors (CGM) for in vivo applications.
7–9
Interest in the use of boronic acid receptors is based on the
well-documented ability of arylboronic acids to reversibly bind
10
diols, including glucose, in water (Scheme 1).
Research in our laboratory has focused on the development of
a two-component, fluorescence-based saccharide-sensing system
11
that utilizes boronic acids as glucose receptors. The system
12
hydroxyethyl methacrylate) (p(HEMA)) hydrogel. These studies
utilized polymerizable derivatives of hydroxypyrene trisulfonic
acid (HPTS) (Fig. 1). HPTS-based dyes can be excited at 460 nm
and emit around 510 nm, which is advantageous for use in sensors.
Even though they perform remarkably well for glucose sensing at
pH 7.4, the fact that they are pH sensitive in the physiologically
relevant range (in the ICU, the pH of a patient’s blood can vary
Department of Chemistry and Biochemistry, University of Califonia, Santa
Cruz, California 95064, U. S. A. E-mail: singaram@chemistry.ucsc.edu
th
†
Dedicated to Professor Seiji Shinkai on the occasion of his 65 birthday.
‡
Electronic supplementary information (ESI) available: Fluorescence
profiles with varying pH for MABP with APTS and HPTS; excitation
and emission spectra of APTS and polymerizable derivatives; stability of
hydrogels 4 and 5; NMR spectra of APTS, APTS-BuMA, and APTS-
DEGMA. See DOI: 10.1039/b821934f
13
between 6.8 and 7.8) warranted a search for dyes that are less pH
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phosphate buffer giving an excitation maximum at 425 nm and
an emission maximum at 503 nm (Fig. 2). That the dye can be
excited in the visible region and has a fairly large Stokes shift
(78 nm) is advantageous for sensor applications.
Fig. 1 Structures of water-soluble, visibly excited dyes, HPTS and APTS.
sensitive in this range. This search led us to aminopyrene trisulfonic
acid (APTS) (Fig. 1).
APTS has excitation and emission maxima similar to those
of HPTS. The hydroxyl group on the pyrene ring in HPTS is
responsible for pH sensitivity. Since the substituent on APTS is an
amino group, we anticipated that APTS would only be affected at
low pH, and remain pH insensitive in the physiological range.
APTS has been used to form saccharide-dye conjugates by
14
reductive amination. We reasoned that this chemistry could
be utilized to synthesize a polymerizable derivative of APTS.
There is an advantage in using APTS rather than HPTS for
making dyes that can be immobilized into hydrogels, in that
the former can be easily appended with only one polymerizable
group by coupling through the amino group. In contrast, HPTS
cannot be functionalized by attaching a polymerizable group
through the phenolic oxygen via ester or ether formation because
the photophysical properties of the dye are drastically affected.
The excitation maximum, for example, is shifted from 460 nm
Fig. 2 Excitation and emission spectra of APTS (lex = 425 nm, lem
=
-6
5
03 nm) 4 ¥ 10 M in pH 7.4 phosphate buffer.
Fluorescence quenching studies
Benzyl viologens are known to effectively quench the fluorescence
17
15
of anionic dyes, such as HPTS in solution. Modification of benzyl
viologen-type quenchers with polymerizable groups has permitted
their immobilization in a polymer matrix and subsequent use in
a glucose-sensing system. For example, MABP and 1MABP have
been found to be effective fluorescence quenchers (Fig. 3).
to 400 nm. Instead, it must be functionalized through the
three sulfonic acid groups making mono-substitution difficult.
As a consequence, most of the HPTS derivatives that have been
synthesized are substituted with three polymerizable groups. We
reasoned that a single point of attachment of the dye is preferable
to multiple bonding because it would allow greater mobility in
12
the hydrogel. An added advantage is that the mono-functional
dye does not participate in the cross-linking of the hydrogel.
In this paper we report the synthesis and characterization
of APTS and the use of this dye in a soluble glucose-sensing
ensemble. We also report the synthesis of two polymerizable APTS
derivatives, the incorporation of the latter in thin-film hydrogels
and the evaluation of the gels as glucose sensors.
Results and discussion
APTS was synthesized by sulfonation of aminopyrene followed by
neutralization with sodium hydroxide (Scheme 2).
Fig. 3 Polymerizable quenchers MABP and 1MABP.
16
Fluorescence intensity measurements show that these quenchers
12
respond to glucose when immobilized in a hydrogel. Both MABP
and 1MABP were used in this study to evaluate the performance
of the APTS derivatives in solution and in thin film hydrogels. The
ability of MABP to quench the fluorescence of APTS and HPTS
was evaluated in pH 7.4 phosphate buffer (Fig. 4).
The Stern–Volmer quenching constants were determined to be
-
1
-1
K
1
s
= 51,400 M and V = 2,140 M for APTS compared to K
65,300 M and V = 5,690 M for HPTS.
The fluorescence of APTS is clearly quenched, albeit with a
s
=
-
1
-1
Scheme 2 Synthesis of APTS from 1-aminopyrene.
smaller quenching constant relative to HPTS. In part, this may
be a consequence of differences in coulombic attraction. We have
shown previously that quencher–dye interaction correlates with
This allowed for the preparation of gram quantities of the dye
in analytically pure form. APTS was characterized in pH 7.4
1
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pH Sensitivity
A potential benefit of using APTS as a reporter dye is its lack
of pH sensitivity. We verified this by measuring the fluorescence
of APTS as a function of pH; the emission maximum remains
essentially constant over the broad pH range of 4–10 (Fig. 6).
-6
Fig. 4 Stern–Volmer plot of 4 ¥ 10 M APTS (ꢀ) and HPTS (᭹) with
increasing concentrations of MABP.
15
the negative charge on the dye. At pH 7.4, the hydroxy group
on HPTS is partially ionized, increasing the number of negative
charges on the dye to greater than three. In contrast, APTS has
only three negative charges at pH 7.4.
HPTS is known to form a ground state complex with viologen-
type quenchers, including MABP, and this is reflected in the static
quenching constant. In order to determine if APTS shows similar
behavior, the formation of a ground-state complex between MABP
and APTS was studied spectroscopically (Fig. 5).
Fig. 6 Normalized fluorescence intensity of APTS (ꢀ) and HPTS (᭹)
-6
(
4 ¥ 10 M in phosphate buffer) as a function of pH. The pH was adjusted
with either HCl or NaOH. The fluorescence is normalized relative to
fluorescence at pH 7.4.
At pH less than 4, the fluorescence intensity drops markedly
as the amine group becomes protonated. Fortunately this pH is
well below the physiological pH, suggesting that APTS derivatives
could be used as a component in a CGM and would not be affected
by variations in pH. In contrast, HPTS is very sensitive to pH in
the physiological range, and for this reason can be used as a pH
18
indicator.
Glucose-sensing studies in solution
The glucose response of quencher–dye systems in pH 7.4 PBS
was compared. APTS and HPTS were each used with the
quencher MABP to sense glucose in aqueous solution. One of
the advantages of a two-component system over a one-component
system is the ability to alter the quencher–dye ratio. The optimum
quencher–dye ratio [Q/D] has been determined for many different
17a
combinations of quenchers and dyes. We chose a Q/D of 10:1
to compare the dyes in solution so that it would match the
ratios used in our hydrogel studies. At this ratio, APTS shows
a fluorescence increase (F/F
0
) of 1.25 between 0–20 mM glucose
whereas for HPTS it is 1.60. The combination of MABP:APTS
-
1
has an apparent glucose binding constant of 701 M versus
-
1
2
89 M for MABP:HPTS. The increased binding constant for
MABP:APTS could explain the diminished response compared to
MABP:HPTS. Nevertheless, these results indicate that APTS is a
suitable fluorophore to use in the two-component glucose-sensing
system (Fig. 7).
The sensitivity of the quencher–dye systems (MABP/APTS and
MABP/HPTS) to pH changes within the range of 6.8–7.8 in the
presence of glucose (10 mM) was determined (see ESI‡).
In this pH range, the system utilizing APTS as the fluorescent
reporter is less sensitive to changes in pH than the HPTS-
containing system. The HPTS system shows a 130% increase in
fluorescence intensity when the pH increases from 6.8 to 7.8. In
Fig. 5 (a) Absorbance spectrum of APTS alone (lmax 424 nm) and with
increasing concentrations of MABP. (b) Difference spectra showing the
new peak appearing at 464 nm as the quencher–dye complex forms.
[
APTS] = 10 mM in phosphate buffer (pH 7.4, 39 mM). MABP was added
with increasing concentrations of 0, 5, 10, 20, 40, 100, 150, 200, and
3
00 mM.
The absorbance spectrum of the dye is red-shifted upon addition
of the quencher, indicating formation of a quencher–dye complex.
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polymerizable group would result in greater mobility for the
dye. This would allow better ionic interaction between dye and
quencher and possibly greater signal modulation upon glucose
binding. The amino functionality on APTS serves as a reactive
site for attaching a single polymerizable group to the dye.
Initial synthetic efforts were focused on the much simpler
method of connecting the polymerizable group through an amide
bond (Scheme 3). However, this resulted in a derivative with
a drastically reduced fluorescence intensity, indicating that the
interaction of the free amine group with the pyrene ring is essential
for the fluorescence properties of APTS.
-6
Fig. 7 Glucose-sensing of 4 ¥ 10 M APTS (ꢀ) and HPTS (᭹) with
MABP (10:1 quencher–dye ratio) with increasing amounts of glucose.
contrast, the APTS system displays only a 30% increase over
that same range. Since the change in fluorescence seen in the
MABP/APTS system is not caused by the dye, it must be a result
of the inherent pH sensitivity of the boronic acid substituents
on the quencher. Nevertheless, relative to HPTS, APTS greatly
decreases the pH sensitivity of the quencher–dye combination.
Scheme 3 General scheme for converting APTS to an amide derivative.
19
We then shifted our attention to attaching an aldehyde func-
tionalized monomer to APTS by reductive amination, assuming
that the alkylated amine functionality would not significantly
change the fluorescence properties. This approach was success-
fully used to prepare two APTS derivatives, one tethered with
butylmethacrylate (APTS-BuMA) and one with diethyleneglycol
methacrylate (APTS-DEGMA). APTS-BuMA was synthesized as
follows (Scheme 4).
Mono-esterification of 1,4-butanediol with methacryloyl chlo-
ride gave alcohol 1, which was oxidized with pyridinium
chlorochromate (PCC) to afford aldehyde 2. Reaction of 2 with
APTS gave the imine in situ that was reduced with sodium
cyanoborohydride to give the desired product, APTS-BuMA, a
mono-functionalized anionic fluorescent dye with a methacrylate
polymerizable group.
Synthesis of polymerizable APTS derivatives
Solution studies showed that APTS would be a suitable flu-
orescent reporter dye for our two-component glucose-sensing
system. Although APTS works well in solution, to be useful
in a continuous glucose sensing device, it must be immobilized.
Our approach is to covalently bond dye and quencher to a
hydrogel polymer. Consequently, we have developed synthetic
routes for the preparation of polymerizable APTS derivatives. We
had previously synthesized polymerizable HPTS derivatives with
three polymerizable groups attached to the pyrene ring through
12
the sulfonic acid moieties. Even though APTS could also be
functionalized in a similar manner, we envisioned that a single
Scheme 4 Synthesis of the polymerizable dye APTS-BuMA.
464 | Org. Biomol. Chem., 2009, 7, 1461–1470
1
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For comparison, another mono-functionalized APTS dye,
APTS-DEGMA, was synthesized with a linking group that is
slightly longer and more hydrophilic. It was made from commer-
cially available diethyleneglycol monomethacrylate (DEGMA).
The synthetic route was similar to that used for APTS-BuMA
of the dye monomers into the hydrogel, two dye-containing gels
were prepared, 4 with APTS-BuMA and 5 with APTS-DEGMA.
The APTS monomers were copolymerized with 2-hydroxyethyl
methacrylate (HEMA), polyethylene glycol (1000) dimethacrylate
(PEGDMA), 3-sulfopropyl methacrylate potassium salt (SPM),
and 1MABP to form a hydrogels. The thermally activated radical
initiator, 2,2¢-azobis[2-(2-imidazolin-2-yl)propane] dihydrochlo-
ride (VA-044), was used to induce polymerization (Scheme 6).
The hydrophilic anionic co-monomer SPM was used to enhance
(
Scheme 5).
Spectral properties of polymerizable APTS derivatives
21
The excitation and emission spectra of both APTS monomers in
pH 7.4 buffer were recorded and the optical properties of the new
dyes and the parent APTS dye compared (see ESI‡). Alkylation of
the amine on APTS resulted in a red-shift of both the excitation
and emission maxima for APTS-DEGMA and APTS-BuMA.
Both dyes had very similar bathochromic shifts with APTS-BuMA
being slightly more red-shifted.
the swelling capacity of the gels. To verify that the fluorescent dyes
remained immobilized within the HEMA hydrogel matrices, they
were monitored for up to 24 hours while immersed in circulating
◦
pH 7.4 phosphate buffer maintained at 60 C. They were subjected
to the high temperature to ensure there was no leaching of the dyes
from the hydrogels. The emission intensity remained essentially
constant over the duration of the experiment, leading us to
conclude that the two polymerizable dyes were successfully bonded
to the polymer matrix (see ESI‡).
Hydrogel studies
The APTS-DEGMA hydrogel 5, was then tested for its sen-
sitivity to a pH change from 6 to 8. The emission of this gel
remained nearly constant over this range. In comparison, hydrogel
The hydrogels used in this study are cross-linked HEMA copoly-
mers containing more than 50% water. To verify incorporation
20
Scheme 5 Synthesis of the polymerizable dye APTS-DEGMA.
Scheme 6 Polymerization of APTS-derived monomers to form glucose-sensing hydrogels. Hydrogels contain the following dye (D) and quencher
components: APTS-BuMA (4), APTS-DEGMA (5), HPTS-Lys-MA (6), APTS-BuMA and 1MABP (7), APTS-DEGMA and 1MABP (8 and 9).
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6
, prepared from polymerizable HPTS derivative HPTS-Lys-
glucose. For this gel, F/F
0
= 2.8 at 20 mM. The signal at 20 mM
12
MA, was highly sensitive to changes in pH within this range
Fig. 8).
remained stable for 15 hours. The increased glucose modulation
compared to that of 7 could be a result of the longer, more
hydrophilic tether connecting the dye moiety to the polymer
matrix. The plot also demonstrates the reversibility of the sensor.
Decreasing the glucose concentration incrementally to zero (buffer
only) brought the signal back to the starting value (Fig. 10).
Average response times for this gel were 1.2 h for increasing analyte
and 2.0 h for decreasing analyte.
(
Fig. 10 Glucose response of hydrogel 8 monitored in a flow cell (lex
65 nm, lem = 528 nm). a) buffer, b) 2.5 mM glu, c) 5 mM glu, d) 10 mM
glu, e) 20 mM glu.
=
Fig. 8 Comparison of APTS-DEGMA dye-only hydrogel 5 (ꢀ) with an
HPTS-Lys-MA dye-only hydrogel 6 (᭹) at varying pH. The fluorescence
is normalized relative to intensity at pH 7.4.
4
To evaluate their ability to act as glucose-sensing materials, each
dye monomer was combined with the quencher, 1MABP, and
immobilized in a hydrogel by copolymerization. Three hydrogels,
Another hydrogel (9) was prepared with 1MABP and APTS-
DEGMA (Q/D = 30:1). The reproducibility of the glucose
response was tested by repeatedly exposing this gel to 20 mM
of glucose and returning to buffer (Fig. 11).
7, 8, and 9 were synthesized (Scheme 6). Hydrogel 7, which
contained the dye APTS-BuMA, was made with a Q/D of 10:1
◦
It was polymerized at 45 C for 24 hours. After polymerization,
the gel was allowed to swell in phosphate buffer for 24 hours.
12
It was then mounted in a flow cell and monitored at 528 nm
ex = 465 nm). After allowing the gel to equilibrate in the buffer,
glucose was added to the circulating stream incrementally (2.5,
.0, 10, and 20 mM) and the change in intensity monitored. An
increase in fluorescence intensity was observed that was directly
related to the glucose concentration, with F/F reaching 1.7 at
0 mM glucose (Fig. 9). The average response time for this gel,
(
l
5
0
2
measured as the time required to reach 95% signal for each analyte
concentration, was 0.66 h.
Fig. 11 Reproducibility of hydrogel 9. a) buffer, b) 20 mM glucose.
The glucose modulation F/F averaged 1.9 for the three runs
0
over a total period of 55 h. This demonstrates that the signal is
sufficiently reproducible for this system to be used as a practical
glucose sensor. The average response time for this gel was 1.6 h. It
should be noted that these gels were prepared with a thickness of
0.25 cm to prevent tearing during transfer and handling. Thinner
gels have more rapid response times. The decreased glucose
response of hydrogel 9 versus hydrogel 8 is probably caused by the
change in Q/D. At 30:1 Q/D the fluorescence of APTS-DEGMA
is quenched much better than at 10:1. Nevertheless, the gel still
functions as a glucose sensor with excellent reproducibility. Of the
gels tested, hydrogel 8 gave the highest signal modulation.
Fig. 9 Glucose response of hydrogel 7 monitored in a flow cell (lex
65 nm, lem = 528 nm). a) buffer, b) 2.5 mM glu, c) 5 mM glu, d) 10 mM
glu, e) 20 mM glu.
=
4
Hydrogel 8 was made in a similar manner using APTS-DEGMA
Q/D = 10:1) and was subjected to different concentrations of
(
1
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Conclusion
workstation. Measurement of pH was carried out using a Bruker
989 pH meter. UV-VIS spectra were recorded on Shimadzu
The water-soluble, anionic fluorescent dye, aminopyrene trisul-
fonic acid (APTS), has some photophysical properties similar to
those of the commonly used fluorescent indicator, HPTS, but
its sensitivity to pH is dramatically different. In particular, the
fluorescence intensity of APTS remains nearly constant over a pH
range from 4 to 10. This dye was used to make a solution-based,
two-component fluorescent probe comprising the dye as reporter
and a boronic acid-appended viologen as glucose receptor. The
fluorescence of the dye was quenched in the presence of the
viologen and the fluorescence restored upon glucose addition.
The performance of this system nearly equals that of an HPTS-
based probe, while the emission intensity of the quencher–APTS
combination is much less pH sensitive. Consequently, APTS
should prove useful as a reporter in our saccharide recognition
UV-1800 spectrophotometer using standard quartz cuvettes.
Fluorescence spectra were recorded on a Perkin-Elmer LS50-B
luminescence spectrometer. Standard quartz fluorescence cuvettes
were used in all studies. APTS was excited at 424 nm. HPTS was
excited at 460 nm. The emission spectra were collected from 460–
6
50 nm. All studies were carried out under ambient conditions
◦
(
25 C, in air) unless otherwise noted. For titration experiments
the added volume did not exceed 2% of the total volume for all
fluorescence measurements. Solution and hydrogel measurements
were made after the signal had stabilized for at least 5 minutes.
Synthesis
Aminopyrene trisulfonic acid trisodium salt (APTS). In an
oven-dried 100 mL round bottom flask equipped with a drying
22
system.
Two APTS-based dye monomers were synthesized and in-
corporated into a glucose-sensing hydrogel. APTS is readily
functionalized with polymerizable groups by reductive amination
with only a minor effect on optical properties. The dyes retain their
fluorescence characteristics when immobilized in a hydrogel by
copolymerization. The present study shows further that hydrogels
made with polymerizable APTS derivatives and viologen boronic
acid quenchers have good modulation over the physiological range
of 2.5–20 mM glucose. The signal is reproducible, reversible, and
stable over the time period studied. Since the only pH responsive
component in the hydrogel is the boronic acid receptor, the glucose
response is likely to be less sensitive to pH changes than that of
hydrogels made with HPTS derivatives. We conclude that these
dyes form an important new family of fluorophores that can be
used both in solution-phase probes and in polymer-based sensors.
tube (charged with drierite and NaOH pellets) was added Na
2
SO
4
(8 mmol, 1.13 g) and conc. H SO (5 mL). Aminopyrene (2 mmol,
2
4
434 mg) was added followed by fuming H
2
SO (20%, 6 mL) and
4
◦
the slurry was stirred at 60 C for 30 h. The mixture was carefully
poured into 50 mL of water and neutralized with 50% NaOH
solution to pH 7 (required about 12 g NaOH). The solution was
concentrated in vacuo and extracted with MeOH (via sonication)
and the solids removed via filtration. Concentration of the MeOH
solution gave 1.04 g (99% yield) of a brown powder that gave
only one spot by TLC. Further purification through a plug of
silica gel (NH OH: IPA, 1:2) yielded an orange powder. Yield:
4
-
1
-1
1.04 g (99%). UV–VIS (water) lmax 424 nm (e = 20,600 M cm ),
383 nm. Fluorescence (pH 7.4 buffer) excitation maximum 428 nm,
1
emission maximum 500 nm. H NMR (CD
3
OD, 500 MHz) d 8.17
(s, 1H), 8.42 (d, J = 9.5 Hz, 1H), 8.90 (d, J = 9.5 Hz, 1H),
.03 (d, J = 9.5 Hz, 1H), 9.13 (d, J = 9.5 Hz, 1H), 9.28 (s, 1H);
C NMR (CD OD, 125 MHz) d 113.39, 116.20, 117.88, 120.56,
22.78, 123.42, 124.61, 126.31, 126.56, 127.43, 129.96, 130.48,
9
1
3
3
Experimental section
1
Materials and methods
134.62, 135.00, 140.90, 144.48.
Reagents were used without further purification unless noted. The
dyes, 8-hydroxypyrene trisulfonate trisodium salt (HPTS, pyra-
nine) and aminopyrene, and the intermediates, 1,4-butane diol,
methacryloyl chloride, diethyleneglycol monomethacrylate. and
the mono-saccharide, D-glucose, were purchased from Aldrich.
The preparation of the components, HPTS-LysMA, MABP, and
4-Hydroxybutyl methacrylate (1). Methacryloyl chloride
(5.86 mL, 60 mmol) was added dropwise to a cooled (0 C)
◦
solution of 1,4-butanediol (5.33 mL, 60 mmol) and pyridine
(30 mL) in dichloromethane (30 mL). The reaction was stirred
at room temperature for 2 h, quenched with 1M HCl (50 mL),
and extracted with dichloromethane (3 ¥ 100 mL). The combined
DCM layers were washed with 3M HCl (3 ¥ 100 mL), dried
with magnesium sulfate, and evaporated to an oil which was
chromatographed on silica gel using hexanes/ethyl acetate (6:4)
12
1
MABP, was reported previously. The synthetic intermediates
(
1, 2, and 3) used in the preparation of polymerizable APTS dyes
were characterized by NMR and used without further purification.
Spectra of purified polymerizable dyes can be found in the ESI.‡
All solvents were reagent grade. All aqueous solutions were
prepared with water that was purified via a Barnstead NANOpure
system (17.7 MX/cm). Phosphate buffers (0.1 ionic strength)
1
to give 3.7 g (40% yield) of clear oil. Yield: 3.7 g (40%) H NMR
(CDCl
4.17 (t, J = 6.5 Hz, 2H), 5.54 (s, 1H), 6.08 (s, 1H); C NMR
(CDCl , 62.5 MHz) d 18.31, 25.12, 29.15, 62.22, 64.54, 125.45,
136.37, 167.59.
3
, 250 MHz) d 1.70 (m, 4H), 1.92 (s, 3H), 3.67 (br s, 2H),
13
3
were freshly prepared before use (pH 7.4: KH
Glucose solutions were freshly prepared in 0.1 ionic strength pH
.4 phosphate buffer. The dye HPTS is sensitive to pH changes
2
PO
4
, NaHPO
4
).
3
-Formylpropyl methacrylate (2). A solution of 1 (3.7 g,
7
2
3 mmol) in dichloromethane (10mL) was added to a suspension
and thus pH was kept with in ± 0.02 of pH 7.40 unless otherwise
noted.
of pyridinium chlorochromate (7.4 g, 34.5 mmol) and celite (5 g)
in dichloromethane (30 mL). The reaction was stirred at room
temperature for 4 h. Diethylether (200 mL) was added and the
reaction was filtered through celite. The dark brown filtrate was
evaporated to a black oil which was then chromatographed on
silica gel using 100% dichloromethane to yield 2.0 g (56% yield)
Instrumentation
1
H-NMR spectra were recorded on a Varian 500 MHz instrument.
Mass spec data was collected on a Mariner Biospectrometry
This journal is © The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1461–1470 | 1467

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1
of clear oil. Yield 2.0 g (56%). H NMR (CDCl
(
3
, 500 MHz) d 1.88
9.5 Hz, 1H), 8.88 (d, J = 9.5 Hz, 1H), 9.05 (d, J = 10 Hz, 1H), 9.13,
1
3
m, 3H), 1.98 (p, J = 6.5 Hz, 2H), 2.53 (dt, J = 7.0, 1.5 Hz, 2H),
(d, J = 9.5 Hz, 1H), 9.28 (s, 1H); C NMR (CD
3
OD, 125 MHz)
4
1
1
.13 (t, J = 6.5 Hz, 2H), 5.52 (m, 1H), 6.03 (m, 1H), 9.75 (t, J =
d 16.98, 43.10, 65.46, 68.73, 68.92, 108.39, 116.84, 117.53, 120.71,
121.77, 123.87, 124.69, 125.17, 126.26, 126.8, 127.45, 130.09,
1
3
.5 Hz, 1H); C NMR (CDCl
25.7, 136.2, 167.3, 201.3.
3
, 125 MHz) d 18.3, 21.4, 40.6, 63.7,
130.24, 134.82, 135.16, 136.04, 141.11, 143.70, 167.44; MS (ESI)
+
m/z calcd for C24
H
24NO12
S
3
(M + H) : 614.04, found 614.0.
N-Butylmethacrylateaminopyrenetrisulfonic acid trisodium salt
APTS-BuMA). To a solution of APTS (0.6 g, 1.15 mmol) in
(
Preparation of hydrogel containing APTS-BuMA (4). A 1 mL
volumetric tube was charged with HEMA (353 mg, 2.7 mmol),
PEGDMA (111 mg, 0.96 mmol), SPM (28 mg, 0.11 mmol),
APTS-BuMA (0.2 mL of a 0.01 M solution in Nanopure
water, 0.002 mmol), 2,2¢-azobis[2-(2-imidazolin-2-yl)propane]
dihydrochloride (VA-044) (2.4 mg, 7.4 mmol), and filled to the
dry methanol (20 mL) was added 2 (0.18 g, 1.15 mmol) and glacial
acetic acid (1 mL, 17 mmol). A solution of sodium cyanoborohy-
dride (0.3 g, 4.7 mmol) in dry methanol (10 mL) was then added,
and the reaction was left stir at room temperature overnight.
Starting material and product (~50:50) were observed by TLC, so
◦
the reaction was heated at 55 C for 4 h. The reaction mixture was
1
mL mark with Nanopure water. The vial was sealed with a
evaporated, and the resulting residue was re-dissolved in a minimal
amount of water and purified by flash column chromatography
septum, agitated with a vortex mixer until a clear red solution
was obtained, then deoxygenated by passing a stream of argon
through the solution for about 10 minutes. The solution was then
injected, by syringe, into a polymerization mold. The mixture was
on silica gel (isopropanol:NH
g (20% yield) of orange powder. TLC: fluorescent green spot,
OH 2:1. Yield: 0.15 g (20%) UV–VIS (water)
4
OH, 9:1–3:1 gradient) to give 0.15
R
l
f
= 0.5 in IPA/NH
4
◦
polymerized at 45 C for 24 h.
-
1
-1
max 457 nm (e = 19,300 M cm ). Fluorescence (pH 7.4 buffer)
1
excitation maximum 463 nm, emission maximum 516 nm. H
Preparation of hydrogel containing APTS-DEGMA (5).
A
NMR (CD OD, 500 MHz) d 1.90 (m, 3H), 1.95 (m, 4H), 3.60
3
1
2
0
mL volumetric tube was charged with HEMA (353 mg,
.7 mmol), PEGDMA (111 mg, 0.96 mmol), SPM (28 mg,
.11 mmol), APTS-DEGMA (0.2 mL of a 0.01 M solution in
(
6
t, J = 6.5 Hz, 2H), 4.26 (t, J = 6.0 Hz, 2H), 5.57 (m, 1H),
.07 (m, 1H), 8.12 (s, 1H), 8.48 (d, J = 9.5 Hz, 1H), 8.88 (d,
J = 9.5 Hz, 1H), 9.05 (d, J = 10 Hz, 1H), 9.13, (d, J = 9.5 Hz,
Nanopure water, 0.002 mmol), 2,2¢-azobis[2-(2-imidazolin-2-
yl)propane] dihydrochloride (VA-044) (2.4 mg, 7.4 mmol), and
filled to the 1 mL mark with Nanopure water. The vial was sealed
with a septum, agitated by a vortex mixer until a clear red solution
was obtained, then deoxygenated by passing a stream of argon
through the solution for about 10 minutes. The solution was then
injected, by syringe, into a polymerization mold. The mixture was
1
3
1
2
1
1
H), 9.28 (s, 1H); C NMR (CD OD, 125 MHz) d 16.98, 25.21,
3
6.09, 42.97, 64.34, 108.28, 116.67, 117.15, 120.44, 122.02, 123.55,
24.65, 124.72, 126.31, 126.64, 127.49, 130.19, 130.32, 134.52,
34.89, 136.33, 141.13, 144.05, 167.52; MS (ESI) m/z calcd for
+
C
24
H
24NO11
S
3
(M + H) : 598.04, found 598.1.
2
-(Formylmethoxy)ethyl methacrylate (3).
A solution of
◦
polymerized at 45 C for 24 h.
diethylene glycol monomethacrylate (4 g, 23 mmol) in
dichloromethane (10 mL) was added to a suspension of pyri-
dinium chlorochromate (7.4 g, 34.5 mmol) and celite (8 g) in
dichloromethane (40 mL). The reaction was stirred at room
temperature for 1 h. Diethyl ether (200 mL) was added and the
reaction was filtered through celite. The dark brown filtrate was
evaporated to a black oil which was then chromatographed on
silica gel using 0% to 5% ethyl acetate in dichloromethane to yield
.4 g (36% yield) of light green oil. Yield: 1.40 g (36%). H NMR
CDCl , 250 MHz) d 1.91 (s, 3H), 3.75 (m, 2H), 4.13 (m, 2H), 4.28
m, 2H), 5.54 (s, 1H), 6.09 (s, 1H), 9.67 (s, 1H).
Preparation of hydrogel containing HPTS-Lys-MA (6).
A
1
2
0
mL volumetric tube was charged with HEMA (353 mg,
.7 mmol), PEGDMA (111 mg, 0.96 mmol), SPM (28 mg,
.114 mmol), HPTS-Lys-MA (0.2 mL of a 0.01 M solution
in Nanopure water, 0.002 mmol), 2,2¢-azobis[2-(2-imidazolin-2-
yl)propane] dihydrochloride (VA-044) (2.4 mg, 7.4 mmol), and
filled to the 1 mL mark with Nanopure water. The vial was sealed
with a septum, agitated by a vortex mixer until a clear red solution
was obtained, then deoxygenated by passing a stream of argon
through the solution for about 10 minutes. The solution was then
injected, by syringe, into a polymerization mold. The mixture was
1
1
(
3
(
N-Ethoxyethyl methacrylate aminopyrenetrisulfonic acid tri-
sodium salt (APTS-DEGMA). To a solution of APTS (0.23 g,
◦
polymerized at 45 C for 24 h.
0
1
.44 mmol) in dry methanol (10 mL) was added 3 (0.3 g,
.77 mmol) and glacial acetic acid (0.4 mL, 6.6 mmol). A solution
Preparation of hydrogel containing 1MABP and APTS-BuMA
(7). A 1 mL volumetric tube was charged with HEMA (353 mg,
2.7 mmol), PEGDMA (111 mg, 0.96 mmol), SPM (28 mg,
0.114 mmol), APTS-BuMA (0.2 mL of a 0.01 M solution in
Nanopure water, 0.002 mmol), 1MABP (23 mg, 0.02 mmol),
2,2¢-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-
044) (2.4 mg, 7.4 mmol), and filled to the 1 mL mark with Nanopure
water. The vial was sealed with a septum, agitated by a vortex
mixer until a clear red solution was obtained, then deoxygenated
by passing a stream of argon through the solution for about
10 minutes. The solution was then injected, by syringe, into a
of sodium cyanoborohydride (0.12 g, 1.77 mmol) in dry methanol
◦
(
10 mL) was then added, and the reaction was left stir at 50 C for
2
h. The reaction mixture was evaporated, and the resulting residue
was re-dissolved in a minimal amount of methanol and purified by
flash column chromatography on silica gel (isopropanol:NH OH,
:1–4:1 gradient) to give 0.145 g (48% yield) of orange powder.
TLC: fluorescent green spot, R OH 3:1.
= 0.3 in IPA/NH
Yield: 0.145 g (48%). UV–VIS (water) lmax 454 nm (e = 21,300
M cm ). Fluorescence (pH 7.4 buffer) excitation maximum 462
4
7
f
4
-
1
-1
1
nm, emission maximum 513 nm. H NMR (CD OD, 500 MHz) d
3
◦
1
.90 (m, 3H), 1.95 (m, 4H), 3.60 (t, J = 6.5 Hz, 2H), 4.26 (t, J =
polymerization mold. The mixture was polymerized at 45 C for
24 h.
6
.0 Hz, 2H), 5.57 (m, 1H), 6.07 (m, 1H), 8.12 (s, 1H), 8.48 (d, J =
468 | Org. Biomol. Chem., 2009, 7, 1461–1470
1
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Preparation of hydrogel containing 1MABP and APTS-DEGMA
8). A 1 mL volumetric tube was charged with HEMA (353 mg,
.7 mmol), PEGDMA (111 mg, 0.96 mmol), SPM (28 mg,
.114 mmol), APTS-DEGMA (0.2 mL of a 0.01 M solution in
Nanopure water, 0.002 mmol), 1MABP (20.8 mg, 0.02 mmol),
to a cuvette and the absorbance spectrum measured. To the cuvette
were added successive aliquots of 0.5, 0.5, 1, 2, 4, 5, 5, and 10 mL
MABP solution (20 mM in water). The total volume change was
1.5%. The solution was mixed by gentle shaking for 10 s after each
addition. The spectrum was recorded after each titration.
(
2
0
2
0
,2¢-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-
44) (2.4 mg, 7.4 mmol), and filled to the 1 mL mark with Nanopure
Glucose response studies. Fluorescence measurements were
done in situ by taking the emission spectrum of the quencher–
dye solution at a series of increasing glucose concentrations.
The emission spectrum of dye (APTS or HPTS, 2 mL of 4 ¥
water. The vial was sealed with a septum, agitated by a vortex
mixer until a clear red solution was obtained, then deoxygenated
by passing a stream of argon through the solution for about
-
6
10
M in buffer) was taken and quencher (MABP) was added
1
0 minutes. The solution was then injected, by syringe, into a
(
16 mL of 5 mM in buffer) to obtain a MABP/dye ratio of 10:1.
◦
polymerization mold. The mixture was polymerized at 45 C for
After quencher addition, emission intensity was measured again.
Glucose solution was then added (0.5–10 mL aliquots of 1 M in pH
7.4 buffer), the mixture was shaken for 30 sec, and the resultant
intensity was measured after each addition. Fluorescence intensity
was taken as the area under the curve between 480 nm and 650 nm
for all studies. Apparent glucose binding constants were calculated
2
4 h.
Preparation of hydrogel containing 1MABP and APTS-DEGMA
9). A 1 mL volumetric tube was charged with HEMA (353 mg,
(
2
0
.7 mmol), PEGDMA (111 mg, 0.96 mmol), SPM (28 mg,
.114 mmol), APTS-DEGMA (0.1 mL of a 0.01 M solution in
24
Nanopure water, 0.002 mmol), 1MABP (31.2 mg, 0.06 mmol),
by fitting the data with eqn 2.
F/F
is the fluorescence intensity of the quenched dye, F is
2
0
,2¢-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-
44) (2.4 mg, 7.4 mmol), and filled to the 1 mL mark with Nanopure
0
= (1 + (Fmax/F
0
)K
b
[S])/(1 + K
b
[S])
(2)
water. The vial was sealed with a septum, agitated by a vortex
mixer until a clear red solution was obtained, then deoxygenated
by passing a stream of argon through the solution for about
where F
0
the fluorescence intensity after the addition of glucose, Fmax is the
intensity at which the fluorescence increase reaches its maximum,
1
0 minutes. The solution was then injected, by syringe, into a
K is the apparent binding constant, and [S] is the concentration
of glucose. Data was analyzed using Microcal Origin 7.5 from
OriginLab, Northampton, MA (USA).
b
◦
polymerization mold. The mixture was polymerized at 45 C for
2
4 h.
Solution-based studies
Hydrogel studies
pH Sensitivity studies. Fluorescence measurements were done
in situ by taking the emission spectrum of APTS or HPTS over
a pH range from 2.0–11.0. The pH was adjusted with either HCl
or NaOH and checked with a Bruker 989 pH meter. The emission
spectra of the dyes (4 ¥ 10 M phosphate buffer) were initially
measured at pH 7.4. The pH was then changed by addition of
acid or base and the emission intensity was measured again at the
resulting pH.
Synthesis of hydrogels. All HEMA hydrogels were prepared
in a similar manner except that the type and amount of dye
and quencher were varied as noted in the text. The synthesis
of hydrogel 7 is a representative example: a 1 mL volumetric
tube was charged with HEMA (353 mg, 2.7 mmol), PEGDMA
(111 mg, 0.96 mmol), SPM (28 mg, 0.114 mmol), dye (0.2 mL
of a 0.01 M solution in Nanopure water, 0.002 mmol), 1MABP
(23 mg, 0.02 mmol), 2,2¢-azobis[2-(2-imidazolin-2-yl)propane]
dihydrochloride (VA-044) (2.4 mg, 7.4 mmol), and filled to the
-
6
Stern–Volmer quenching studies. Fluorescence measurements
were done in situ by taking the emission spectra of APTS or
HPTS in pH 7.4 PBS containing a series of increasing quencher
concentrations. The emission spectrum of the dye (APTS or HPTS,
1
mL mark with Nanopure water. The vial was sealed with a
septum, agitated on a vortex mixer until a clear red solution was
obtained, then deoxygenated by passing a stream of argon through
the solution for about 10 minutes. The solution was then injected,
by syringe, into a polymerization mold, which was constructed in
the following manner. Two 3 mm-thick glass plates (8 ¥ 8 cm),
one containing two small holes for solution injection, were treated
with dichlorodimethylsilane (2% solution in toluene) to facilitate
removal of the gel after polymerization. The treated plates were
stacked atop each other separated by a 25.4 mm-thick Teflon
spacer. The plates were clamped together with a metal frame that
contained syringe injection ports aligned with the holes in the
upper glass plate to form the mold. After the mold was filled with
monomer solution and the holes closed with rubber septa, it was
-
6
2
mL of 4 ¥ 10 M in buffer) was first obtained. The quencher
(
MABP) was then added (0.5–10 mL aliquots of a 5 mM solution
in buffer), the solution was shaken for 30 sec, and a new emission
spectrum was recorded after each quencher addition. Emission
intensity was taken as the area under the curve from 480–650 nm
for all studies. Stern–Volmer quenching constants were calculated
23
using the sphere of action model, eqn (1), where K
static quenching constant, V represents the dynamic quenching
constant, and Q is the concentration of MABP. F represents
s
represents the
0
the initial fluorescence intensity of the dye only. F represents the
fluorescence intensity at a given quencher concentration.
ꢀ
R
sealed in a Ziploc bag that was purged and filled with argon,
V[Q]
◦
F
0
/F = (1 + K
s
[Q])e
(1)
and heated in a 45 C oven for 24 hours. The glass plates were
then removed from the metal frame, placed in pH 7.4 phosphate
buffer for 2 h, and separated to expose the thin orange/pink film.
The film was cut into 1 ¥ 3 cm pieces and immersed in phosphate
Data was analyzed using Microcal Origin 7.5 from OriginLab,
Northampton, MA (USA).
◦
UV–VIS spectrophotometry of APTS and MABP. A 2.00 mL
aliquot of APTS in phosphate buffer (pH 7.4, 39 mM) was added
buffer at 40 C for 16 hours. They were then stored in fresh buffer
◦
solution at 5 C until tested.
This journal is © The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1461–1470 | 1469

View Article Online
Fluorescence monitoring of hydrogel by front-face illumination.
7 (a) J. Yoon and A. W. Czarnik, J. Am. Chem. Soc., 1992, 114, 5874–
5
875; (b) S. Arimori, C. J. Ward and T. D. James, J. Chem. Soc., Chem.
Measurements were performed using a Perkin-Elmer LS50-B
Commun., 2001, 2018–2019; (c) H. Eggert, J. Frederiksen, C. Morin
and J. C. Norrild, J. Org. Chem., 1999, 64, 3846–3852; (d) W. Yang,
H. He and D. G. Drueckhammer, Angew. Chem., Int. Ed., 2001, 40,
1714–1718; (e) A. J. Tong, A. Yamauchi, T. Hayashita, Z. Y. Zhang,
D. B. Smith and N. Teramae, Anal. Chem., 2001, 73, 1530–1536; (f) D.
P. Adhikiri and M. D. Heagy, Tetrahedron Lett., 1999, 40, 7893–7896;
TM
luminescence spectrometer controlled by FL WinLab (version
2
.0) software. The hydrogels were mounted in a flow-though
cuvette, the cuvette was held in the spectrometer using a front
◦
surface accessory, and studies were carried out at 37 C. A piece
of hydrogel was mounted inside of the flow-through fluorescence
cuvette in the following manner. While the cuvette (1 ¥ 1 ¥ 4 cm)
was horizontal with both ends open, a piece of hydrogel was laid
flat inside the cuvette. A stiff piece of 2 mm-thick black plastic
(
g) N. DiCesare and J. R. Lakowicz, Tetrahedron Lett., 2001, 42, 9105–
9108.
8
(a) S. Arimori, C. J. Ward and T. D. James, Tetrahedron Lett., 2002,
4
3, 303–305; (b) G. Springsteen and B. Wang, J. Chem. Soc., Chem.
Commun., 2001, 17, 1608–1609; (c) G. Springsteen and B. Wang,
Tetrahedron, 2002, 58, 5291–5300; (d) S. L. Wilkur, H. Ait-Haddou,
J. J. Lavigne and E. V. Anslyn, Acc. Chem. Res., 2001, 34, 963–972;
(
0.9 ¥ 3 cm) with a window (0.7 ¥ 2 cm) cut out of it was positioned
on top of the gel. Using small forceps, two compression septa
were placed on each end of the plastic frame, pressing it against
the hydrogel. The polyethylene caps (appended with solvent inlet
ports) were tightly sealed onto the cuvette using vacuum grease
and Teflon tape. The cuvette was held in the spectrometer with
the front surface accessory and oriented so that solutions entered
through the bottom and flowed upward. The side of the cuvette
against which the gel was pressed, was facing the excitation source
(
e) S. Arimori, H. Murakami, M. Takeuchi and S. Shinkai, J. Chem.
Soc., Chem. Commun., 1995, 9, 961–962; (f) N. DiCesare, M. R. Pinto,
K. S. Schanze and J. R. Lakowicz, Langmuir, 2002, 18, 7785–7787.
9 (a) T. D. James, K. Sandanayake and S. Shinkai, Angew. Chem., Int.
Ed. Eng., 1994, 33, 2207–2209; (b) T. D. James, K. Sandanayake and
S. Shinkai, J. Chem. Soc., Chem. Commun., 1994, 477–478; (c) T. D.
James, K. Sandanayake, R. Iguchi and S. Shinkai, J. Am. Chem. Soc.,
1
995, 117, 8982–8987; (d) K. Sandanayake, S. Imazu, T. D. James, M.
Mikami and S. Shinkai, Chem. Lett., 1995, 139–140; (e) T. D. James,
K. Sandanayake and S. Shinkai, Angew. Chem., Int. Ed. Eng., 1996,
35, 1911–1922; (f) T. D. James, H. Shinmori and S. Shinkai, Chem.
Commun., 1997, 71–72.
◦
at a 30 angle relative to the incident beam. Phosphate buffer
◦
(
37 C) was pumped into the cell at a flow rate of 12 mL/min.
The film was excited at 470 nm by front-face illumination, and
the emission was monitored over time. A 515 nm cutoff filter was
used, and the excitation and emission slit widths were adjusted
between 7 and 10 nm to obtain a baseline intensity of ~400 intensity
units (1000 unit limit). Using the time drive application, the
integration time was set for 10 sec, and the source was pulsed every
10 J. P. Lorand and J. O. Edwards, J. Org. Chem., 1959, 24, 769–774.
11 (a) J. N. Camara, J. T. Suri, F. E. Cappuccio, R. A. Wessling and B.
Singaram, Tetrahedron Lett., 2002, 43, 1139–1141; (b) J. T. Suri, D. B.
Cordes, F. E. Cappuccio, R. A. Wessling and B. Singaram, Langmuir,
2
003, 19, 5145–5152; (c) J. T. Suri, D. B. Cordes, F. E. Cappuccio, R.
A. Wessling and B. Singaram, Angew. Chem., Int. Ed. Engl., 2003, 42,
5
857–5859; (d) F. E. Cappuccio, J. T. Suri, D. B. Cordes, R. A. Wessling
and B. Singaram, J. Fluoresc., 2004, 14, 521–533.
2
sec. After a stable signal was obtained (~1 h), buffered glucose
1
2 S. Gamsey, J. T. Suri, R. A. Wessling and B. Singaram, Langmuir, 2006,
solutions of varying concentrations were pumped through the
cuvette.
2
2, 9067–9074.
rd
13 C. A. Burtis and Tietz, in Textbook of Clinical Chemistry, 3 edn, WB
Saunders Co., Pennsylvania, 1998.
1
1
1
4 R. A. Evangelista, A. Guttman and F.-T. A. Chen, Electrophoresis,
Acknowledgements
1996, 17, 347–351.
5 D. B. Cordes, A. Miller, S. Gamsey, Z. Sharrett, P. Thoniyot, R. A.
Wessling and B. Singaram, Org. Biomol. Chem., 2005, 3, 1708–1713.
6 APTS can be purchased from Fluka as a fluorescent labeling reagent
for glycoproteins and saccharides for $158/10 mg. 1-Aminopyrene can
be purchased for $18/250 mg.
The authors would like to thank GluMetrics, Inc., operating
through the BioStar Industry-University Cooperative Research
Program (BioStar Grant Isi-bio04-10458), for continuing financial
support.
17 (a) D. B. Cordes, S. Gamsey, Z. Sharrett, A. Miller, P. Thoniyot,
R. A. Wessling and B. Singaram, Langmuir, 2005, 21, 6540–6547;
(
b) S. Gamsey, A. Miller, M. M. Olmstead, C. M. Beavers, L. C.
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2
rd
23 (a) J. R. Lakowicz, in Principles of Fluorescence Spectroscopy, 3 edn.,
(
Springer, 2006, ch. 8, pp. 278–286; (b) D. L. Wang, J. Wang, D. Moses,
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24 K. A. Connors, in Binding Constants- The Measurement of Molecular
Complex StabilityWiley-Interscience, 1987.
See for example: US Pat., 6 002 954, 1999; US Pat., 6 304 766, 2001;
US Pat., 6 387 672, 2002; US Pat., 6 627 177, 2003; US Pat., 7 297 548,
2
007.
1
470 | Org. Biomol. Chem., 2009, 7, 1461–1470
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