Highly specific-glucose fluorescence sensing based on boronic anthraquinone derivatives via the GOx enzymatic reaction

Article abstract of DOI:10.1016/j.tet.2012.08.037

The boronic acid anthraquinones, oHAQB and pHAQB, have been designed, and demonstrated to serve as fluorogenic biosensors for glucose. The sensory molecule, oHAQB, has exhibited the specific-glucose sensing via the GOx enzymatic reaction. In this contribu

Full text of DOI:10.1016/j.tet.2012.08.037

Tetrahedron 68 (2012) 8899e8904
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journal homepage: www.elsevier.com/locate/tet
Highly specific-glucose fluorescence sensing based on boronic anthraquinone
derivatives via the GOx enzymatic reaction
,
,
*
Krittithi Wannajuk a b, Matinee Jamkatoke c, Thawatchai Tuntulani a, Boosayarat Tomapatanaget a
a Department of Chemistry, Faculty of Science, Chulalongkorn University, Phyathai Road, Bangkok 10330, Thailand
b Center for Petroleum, Petrochemicals and Advanced Materials, Chulalongkorn University, Bangkok 10330, Thailand
c Faculty of Science and Arts, Burapha University, Chanthaburi Campus, Chanthaburi 22170, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 May 2012
Received in revised form 27 July 2012
Accepted 13 August 2012
Available online 21 August 2012
The boronic acid anthraquinones, oHAQB and pHAQB, have been designed, and demonstrated to serve as
fluorogenic biosensors for glucose. The sensory molecule, oHAQB, has exhibited the specific-glucose
sensing via the GOx enzymatic reaction. In this contribution, the fluorescence changes of oHAQB rea-
sonably correspond to the concentration of glucose upon the conversion of boronic acid to hydroxy based
sensor by H2O2 generated from glucose along with GOx enzymatic reaction. Our sensing ensemble was
then successfully applied to determine the glucose concentration in the range of 0.08e0.42 mM. The
limit of detection (LOD) of oHAQB for glucose detection using the GOx enzymatic probe is approximately
0.011 mM.
Keywords:
Boronic anthraquinone
GOx enzymatic reaction
Fluorescence sensing
Glucose sensing
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
commercial glucose test is electrochemical, including a monitor
based on the electrical wiring of glucose oxidase, which underlies
Diabetes is a disease, which involves abnormal blood sugar
metabolism due to inadequate insulin production. Blood sugar,
especially glucose, has regularly been used to diagnose diabetes for
over a century. Nowadays, the capability to accurately monitor
blood glucose concentrations has proven essentially for diagnosing
and curing many metabolic disorders, mainly diabetes.1 Therefore,
reliable and accurate saccharide sensors are continually developed
in order to search for better ones for quantitative analysis and de-
tection of saccharides and their derivatives in physiological media.
Moreover, the indispensability of glucose monitoring in the incur-
able disease is a critical requirement for determination of the glu-
cose levels in nondiabetic acute care patients because high
variations in blood glucose level have also been observed in non-
diabetic patients as a common sign of acute illness. Patients can
modify their lifestyle to better control their diabetes and can use
insulin in achieving effective dosing and timing.2 The important
demands for continuous, accurate, and relatively noninvasive glu-
cose sensing methods have motivated methodologies as well as the
design of various sensing materials.3 For specific-glucose sensing,
various analytical methods such as amperometry, potentiometry,
spectrophotometry, or fluorometry are utilized for the enzymatic
measurements of glucose concentration.4 The traditional
the direct conversion of the concentration-dependent glucose flux
to an electrical current. In this method, the generated hydrogen
peroxide is oxidized under a constant working potential, and the
extent of oxidation corresponds to the glucose concentration.5
However, it needs careful calibration because other oxidized
products may be easily adsorbed onto the metal electrode surfaces,
causing a decrease in activity of the electrode surface and the
sensitivity will dramatically decrease.6
The development of specific sensors for biochemically relevant
analytes via chemical reaction has high impact and challenging.
Fluorescence sensors with preferential binding to glucose have
been developed in the past decade. The obvious advantages of
a fluorescence sensor are the extreme sensitivity and lack of
damage to the host system. The methodology involves the in-
corporation of synthetic based receptors, such as boronic acid.
Boronic acid based sensors are the best candidate for saccharide
probes due to the covalent binding properties of boronic acid with
the diol group of sugars in water as widely reported for the last
decade.7 Moreover, most boronic acid derivatives based fluo-
rophores showed the fluorescence emission band at a relatively
long wavelength, which is an important role in detecting glucose in
blood or serum samples. However, the disadvantages of some bo-
ronic acid based fluorescence sensor are the selectivity and a high
pKa values, which is not appropriate for physiological pH.8 More-
over, a number of publications have reported the glucose-specific
* Corresponding author. E-mail address: tboosayarat@gmail.com (B. Tomapatanaget).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.08.037

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K. Wannajuk et al. / Tetrahedron 68 (2012) 8899e8904
fluorescence changes with the interference of other saccharides
especially fructose and ribose.9 A few reports exist regarding the
selective fluorogenic boronic acid for glucose sensing.10 As the basic
knowledge of boronic acid, aryl boronic acid and boronic ester react
selectively with H2O2 to produce a phenolic compound.11 To over-
come the disadvantage of using a boronic-based fluorescence
sensor for selective detection of glucose, we took an effort to
combine the basic knowledge of the specific-glucose sensing by
GOx enzymatic reaction with the new boronic-based anthraqui-
none for fluorescence changes. Our proposition for the specific-
glucose fluorescence sensor via the GOx enzymatic reaction is
schematically illustrated in Fig. 1.
directly. Herein, new anthraquinone based fluorosensors, oHAQB
and pHAQB containing boronic acid as the enzymatic probe for glu-
cose has been designed and synthesized. The sensing affinity for
glucose was investigated by fluorescence spectrophotometry.
Among both sensors, oHAQB permits the selective detection of glu-
cose upon the GOx enzymatic reaction with a detection limit of
11.4 mMandaverage%recoveryof104, whichisindicativeofpowerful
glucose sensing.
2. Results and discussion
2.1. Synthesis and physical properties of sensors pHAQB and
oHAQB
Synthetic methodologies for the new probes, pHAQB and
oHAQB are shown in Scheme 1. The heterocyclic precursors, Boc-
pHAQB and Boc-oHAQB were achieved from oxidative condensa-
tion between diamino anthraquinone and formyl phenylboronated
ester with 70% and 60% yields, respectively. The characterization of
all sensors was carried out using 1H as well as 13C NMR and HRMS.
For 1H NMR spectra, the appearance of a broad singlet signal at
around 13.76 and 12.97 ppm for pHAQB and oHAQB, respectively,
supports the formation of intramolecular hydrogen bonding be-
tween the NH of the imidazole ring and the neighboring quinone
carbonyl group.12
Fig. 1. Schematic illustration of the GOx enzymatic mechanism for glucose detection
using boronic-based fluorescence sensors.
Scheme 1. Synthesis of sensory molecules, pHAQB, oHAQB, pHAQ and oHAQ.
For our hypothesis, the H2O2 generated by the reaction between
glucose and glucose oxidase would convert the boronic acid to the
phenol based fluorescence sensor and the fluorescence change
would be related to the concentration of glucose. Although, most
organic fluorophores can exhibit fluorescence quenching by H2O2,
we designed the organic fluorophore containing boronic acid for
specific detection of glucose upon the GOx enzymatic reaction
without the interference of glucose binding to the boronic acid
Sensors oHAQB and pHAQB in 10% DMSO with 0.1 M HEPES
buffer at pH 7.4 showed a strong fluorescence intensity at 609
and 589 nm, respectively, with quantum yields of 0.322 and
0.231 with respect to a typical quinone compound as shown in
Fig. 2. As expected, fluorescence responses of pHAQ and oHAQ
consisting of hydroxy groups were relatively low in aqueous
media and consequently their quantum yields could not be
determined.

K. Wannajuk et al. / Tetrahedron 68 (2012) 8899e8904
8901
the high efficiency of enzymatic probe was performed by six units
of GOx at a temperature of 37 ꢀC and reaction time of 60 min. These
appropriate conditions were utilized for all manipulations related
to the enzymatic reaction of GOx.
From the literature review13 reporting the binding ability be-
tween GOx and boronic acid, we thus tested the binding ability of
oHAQB and GOx by fluorescence spectrophotometry. We found
unchanged emission spectra of oHAQB in the presence of GOx as
shown in Fig. S5. This indicated unbound oHAQB with GOx sug-
gesting no influence of the complexation of oHAQB and GOx to-
ward emission spectral changes. Hence, we have focused on
studying the use of oHAQB for detection of glucose. Hypothetically,
H2O2 generated from the reaction of glucose and glucose oxidase
would convert the boronic acid based sensor to the hydroxy group
and subsequently induced the fluorescent quenching of anthra-
quinone derivatives. To confirm this hypothesis, the product of the
boronate-based fluorescent probe (oHAQB) hydrolyzed by H2O2
generated from the reaction of the glucose and GOx was in-
vestigated by ESI-MS (shown in ESI), which showed the peak of m/z
340.08 corresponding to the structure of oHAQ (the hydrolyzed
boronate-based oHAQB).
Fig. 2. Emission spectra of pHAQB, oHAQB, pHAQ and oHAQ in 10% DMSO with 0.1 M
HEPES buffer at pH 7.4.
These positive preliminary results encouraged us to investigate
the boronic acid based sensors as a glucose sensing under the en-
zymatic mechanism mentioned above.
We first examined the comparison of fluorescence changes of
oHAQB upon direct addition of H2O2 and the addition of glucose
along with GOx as shown in Fig. 4.
To study the binding properties between sensors pHAQB and
oHAQB toward glucose without glucose oxidase, the fluorescence
titration curves exhibited large changes of fluorescence quenching
for pHAQB but the fluorescence quenching of oHAQB in the pres-
ence of glucose displayed insignificant change as shown in Fig. 3. As
a result of steric hindrance of boronic binding site of oHAQB toward
glucose, pHAQB preferred to bind glucose over oHAQB. Moreover,
the binding constants of oHAQB and glucose cannot be determined.
We, therefore, reasoned that oHAQB was a better candidate for
enzymatic activation as it would be no competition from direct
binding.
Fig. 4. Fluorescence titration curves (at 609 nm) of 20 mM oHAQB upon addition of
direct H2O2 and addition of glucose and GOx in 100 mM HEPES buffer (pH 7.4).
As anticipated, the fluorescence responses of oHAQB in the
presence of H2O2 by direct addition, and H2O2 generated by the
reaction of glucose and glucose oxidase were consistent and almost
overlapped. The small difference of fluorescence responses from
both systems is acceptable. In this contribution, monitoring based
on the fluorescent changes of oHAQB stemmed from the direct
conversion of the glucose by glucose oxidase corresponding to the
schematic illustration in Fig. 1. Therefore, we envisaged the use of
oHAQB as the enzymatic GOx probe for glucose. To prove the se-
lectivity of oHAQB for specific-glucose sensing, the effects of in-
terfering saccharides (2.0 mM) including galactose, fructose, sucrose
and maltose were evaluated in the determination of 0.2 mM of
glucose with and without GOx in 100 mM HEPES buffer (pH 7.4).
Regarding the fluorescence responses of oHAQB shown in Fig. 5,
poor responses were readily observed for galactose and fructose
with fluorescent enhancement as well as sucrose and maltose with
fluorescence quenching. On the other hand, the observation of
a large fluorescent enhancement of oHAQB in the presence of
Fig. 3. Fluorescence titration curves of 20 mM oHAQB and pHAQB at 590 and 560 nm,
respectively, with glucose in 100 mM HEPES buffer (pH 7.4).
2.2. Enzymatic detection of glucose using oHAQB
Prior to the use of enzymatic mechanism of GOx and glucose,
the optimized conditions of the system were investigated in terms
of amount of GOx unit, temperature and reaction time. From the
fluorescence results as shown in ESI, the appropriate condition for

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K. Wannajuk et al. / Tetrahedron 68 (2012) 8899e8904
In analytical applications using this spectrofluorometric
method, oHAQB was applied to detect glucose in drinking water.
The % recoveries (shown in Table 1) of the spike samples of oHAQB
were in the range of 91e116 and the average % recovery of 104,
which is acceptable for analytical application.
Table 1
Analysis of glucose using oHAQB with the enzymatic reaction of GOx in drinking
water
Added glucose (mM)
Found (mM)
% Recovery
0.05
0.10
0.15
0.20
0.30
0.51
102
91
116
108
104
0.091
0.175
0.216
0.313
Fig. 5. Fluorescent responses of oHAQB (20 mM) in the presence of 0.2 mM glucose [1]
or 2 mM of the competitive saccharides: galactose [2], galactoseþGOx [3], fructose [4],
fructoseþGOx [5], sucrose [6], sucroseþGOx [7], maltose [8], maltoseþGOx [9], glu-
coseþGOx [10] and the mixture of gluþGOx with galactose [11], fructose [12], sucrose
[13], and maltose [14].
3. Conclusions
A fluorescence sensory molecule, oHAQB, has been successfully
synthesized, and its glucose sensing capability has been developed
under enzymatic mechanism of GOx. All corresponding results are
consistent with the hypothesis that H2O2 generated from the en-
zymatic reaction of glucose and GOx showed high affinity to hy-
drolyze the boronic acid group to the hydroxy moiety with the
proportional fluorescence changes of the sensor, oHAQB. To the
best of our knowledge, this is the first report of the use of boronic-
based fluorescence biosensor to detect glucose under the GOx en-
zymatic reaction. Consequently, oHAQB serves as an excellent
biosensor for glucose sensing with high selectivity. The proposed
mechanism highlighted the benefits of further use of the simple
fluorescence sensor as the GOx enzymatic probe for detection of
glucose in real-time analysis in the field of medical diagnosis and
food safety.
glucose along with GOx as shown in Fig. 5 could indicate a highly
specific recognition for glucose. Interestingly, other saccharides did
not affect the glucose detection under the enzymatic GOx
mechanism.
To verify the sensing application of the boronic anthraquinone
based sensors (oHAQB), the fluorescence titration between oHAQB
and glucose in the presence of glucose oxidase was investigated in
the optimum conditions. As depicted in Fig. 6, the emission band at
609 nm of oHAQB was gradually decreased upon the increment of
glucose. To normalize the fluorescence titration of enzymatic probe
system of oHAQB, the calibration curve was plotted between I0eI
and the glucose concentration (shown in inset of Fig. 6). The fluo-
rescence response was linear in the range of 0.08e0.42 mM with an
acceptable correlation coefficient (R2) of 0.99082. Therefore, the
range of concentration for glucose sensing by the GOx enzymatic
probe system is 0.08e0.42 mM. The limit of detection (LOD) of
oHAQB for glucose detection using GOx enzymatic probe is ap-
4. Experimental section
4.1. Materials and general methods
proximately 0.0114 mM or 11.4 mM.
Nuclear Magnetic Resonance (NMR) spectra were recorded on
a Varian Mercury Plus 400 NMR spectrometer. All chemical shifts
were reported in parts per million (ppm) using the residual proton
or carbon signal in deuterated solvents as internal references.
MALDI-TOF mass spectra were carried out on Bruker Daltonics
MALDI-TOF using 2-cyano-4-hydroxy cinnamic acid (CCA) as
matrix.
All materials and solvents chemicals were purchased from
Aldrich, Fluka and Merck as standard analytical grade and were
used without further purification. Commercial grade solvents such
as dichloromethane, ethyl acetate, hexane, and methanol were
purified by distillation. Anhydrous solvents such as dichloro-
methane were dried over CaH2 and freshly distillation under ni-
trogen atmosphere. Column chromatography was carried out on
silica gel (Kieselgel 60, 0.063e0.200 mm, Merck). Thin layer chro-
matography (TLC) was performed on silica gel plates (Kieselgel 60,
F254, 1 mm). Compounds on TLC plates were detected by the UV-
light.
For UV/vis and Fluorescence analysis, compounds were dis-
solved in dimethyl sulfoxide (DMSO, fluorometric grade, Dojindo)
to obtain 1 mM stock solutions. These stock solutions were diluted
with buffer as specified in the figure legends to the desired con-
centration. For determination of the quantum efficiency of fluo-
rescence (Ff), were calculated using the integrated emission
intensity of quinine sulfate as standard. All absorption spectra were
obtained with Hewlett Packard 8452A Diode Array Spectrometer.
Fig. 6. Fluorescence titration curve of 20 mM oHAQB and six units of glucose oxidase
titrated with glucose in 10% DMSO with 0.1 M HEPES buffer at pH 7.4. The excitation
wavelength was 415 nm. The reaction was performed at 37 ꢀC and 1 h and inset: liner
calibration curve of oHAQB in the same condition.

K. Wannajuk et al. / Tetrahedron 68 (2012) 8899e8904
8903
All fluorescence spectra were obtained with Varian Cary Eclipse
Fluorescence Spectrophotometer. The boronate esters were syn-
thesized according to the previously published procedure.14
in 10% DMSO aqueous solution, the mixtures was stirred at room
temperature for 10 min and placed in a 100.0 mm width quartz cell
and then fluorescence spectra were recorded at 25 ꢀC.
4.2. Synthesis
4.3.2. Fluorescence measurement of boronic anthraquinone based
sensors (oHAQB) in optimum condition of the enzymatic probe sys-
tem for glucose detection by glucose oxidase. Typically, the sensors
were prepared in spectroscopic grade DMSO at concentrations of
2.00ꢂ10ꢁ4 mol/L as a stock solution. The stock solutions of glucose
were prepared at concentrations of 6.00ꢂ10ꢁ3 mol/L in 0.1 M HEPES
buffer pH 7.4. In a 10.0 mL vial, 0.30 mL of the stock solution of sensor
was mixed with the portions of stock solution of glucose to give final
concentration of glucose. After volumes were adjusted to 3.00 mL
with 0.1 M HEPES buffer pH 7.4 to give a final concentration of
sensors at 2.00ꢂ10ꢁ5 mol/L in 10% DMSO aqueous solution, then six
units of glucose oxidase were added to the solution mixture of the
sensor and the glucose. The mixtures was stirred at 37 ꢀC for 60 min
and placed in a 100.0 mm width quartz cell and then, the fluores-
cence spectra were recorded at 25 ꢀC. For kinetic studies, the fluo-
rescence spectra were recorded at 25 ꢀC every 5 min.
Preparation of 2-(R-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)phe-
nyl)-1H-anthra[1,2-d]imidazole-6,11-dione (R¼2, 4). Into a two-neck
round bottom flask equipped with a magnetic bar, the corre-
sponding formyl phenylboronate ester (1 mmol) in nitrobenzene
(25 mL) was added dropwise to a solution of 1,2-diamino-1,4-
anthraquinone (1 mmol) in nitrobenzene (75 mL).
The reaction mixture was slowly heated to 150 ꢀC for 1 day
under nitrogen atmosphere. The solution was cooled to room
temperature and then the precipitate slowly formed. The pre-
cipitate was filtered and washed with diethylether to give a brown
solid of the protecting products (Boc-oHAQB¼45%, Boc-
pHAQB¼60%). 1H NMR (DMSO, 400 MHz) Boc-oHAQB,
d (ppm):
13.56 (s, 1H), 8.41 (d, 1H, J¼6.4 Hz), 8.25e8.13 (m, 4H), 7.94e7.92
(m, 2H), 7.57e7.50 (m, 3H), 3.70 (s, 4H), 1.07 (s, 6H). Boc-pHAQB,
d
(ppm): 10.18 (s, 1H), 8.40 (t, 1H, J¼8.2 Hz), 8.29e8.21 (m, 4H),
8.13e8.05 (m, 2H), 7.97e7.93 (m, 3H), 3.78 (s, 2H), 3.11 (s, 2H), 0.97
(s, 3H) 0.72 (s, 3H).
4.3.3. Competitive studies of other saccharides. In a 10.0 mL vial,
0.30 mL of 2.00ꢂ10ꢁ4 mol/L of a sensor in spectroscopic DMSO was
Preparation of R-(6,11-dioxo-6,11-dihydro-1H-anthra[1,2-d]imi-
dazol-2-yl)phenylboronic acid (R¼2: oHAQB, 4: pHAQB). Protecting
groups of 2-(R-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)phenyl)-1H-
anthra[1,2-d]imidazole-6,11-dione (R¼2:2o, 4: 2p) (1 mmol) were
removed by refluxing in 30 mL 0f 30% H2O:CH3CN. The yellow solid
of product oHAQB and brown solid of product pHAQB was filtered
off and washed with diethylether to provide the desired product in
a quantitative yield.
mixed with 100
m
L of 6.00ꢂ10ꢁ2 mol/L of stock solutions including
D-glucose, D-galactose, D-fructose, D-sucrose and D-maltose in 0.1 M
HEPES buffer pH 7.4 and all solutions was adjusted to 3.00 mL to
give final concentration of sensors at 2.00ꢂ10ꢁ5 mol/L in 10% DMSO
aqueous solution. Then six units of glucose oxidase were added to
the solution mixture of the sensor and the saccharides. The mixture
solution was stirred at 37 ꢀC for 60 min and placed in a 100.0 mm
width quartz cell and then fluorescence spectra were recorded at
room temperature.
Characterization data for oHAQB: Melting point 350e354 ꢀC; 1H
NMR (DMSO-d6, 400 MHz)
d (ppm): 13.76 (s, 1H), 8.26e8.21 (m,
2H), 8.14 (d, 2H, J¼6.8 Hz), 7.99 (d, 2H, J¼6.0 Hz), 7.94e7.92 (m, 1H),
4.3.4. Glucose detection of drinking water sample. The commer-
cially drinking water sample was used for volume adjustment with
0.1 M HEPES buffer pH 7.4 to give a final concentration of 10% DMSO
aqueous solution of sample. The glucose stock solution was spiked
to the mixture solution of oHAQB to give the final concentration of
0.05e0.3 mM of glucose and 2.00ꢂ10ꢁ4 M of oHAQB and then 6
units of glucose oxidase was added to the solution mixture of the
sensor and the glucose. After stirring the mixture solution at tem-
perature of 37 ꢀC for 60 min, the sample mixture was then recorded
at the room temperature.
7.75 (d, 1H, J¼8.4 Hz), 7.68 (d, 2H, J¼6.8 Hz). 13C NMR (100.6 MHz,
DMSO-d6)
d
¼182.36, 182.00, 181.50, 159.60, 148.58, 133.60, 132.59,
132.33,131.91,129.68,129.23,128.49,128.04,126.84,126.66,126.23,
126.12, 124.74, 121.60, 120.93, 118.58. IR spectrum (KBr, (cmꢁ1)):
3421 (NeH (2ꢀ-amines) stretching, wk), (2935 (C]C, stretching)
1657 (C]O stretching, str), 1584, 1490 (C]C, stretching, str), 1300
(CeN) stretching). HRESI-MS calculated for C21H13N2O4: 369.12
[M], observed: 387.09 [MþH2O]¼419.123.
Characterization data for pHAQB: Melting point 366e367 ꢀC;
1H NMR (400 MHz, DMSO-d6)
d
¼12.97 (s, 1H), 8.34 (d, 1H,
J¼7.6 Hz), 8.26 (s, 2H), 8.06e8.25 (m, 2H), 7.96 (m, 4H), 7.80 (m,
2H); 13C NMR (100.6 MHz, DMSO-d6)
¼183.37, 182.56, 158.60,
Acknowledgements
d
137.53, 134.76, 134.54, 133.36, 133.23, 133.160, 128.33, 127.47,
127.09, 126.50, 125.27, 121.33, 118.91; IR spectrum (KBr, (cmꢁ1)):
3415 (NeH (2ꢀ-amines) stretching, wk), (1658 (C]O stretching,
str), 1523, 1470 (C]C, stretching, str), 1298 (CeN) stretching, med),
723 (CeH bending, str); Elemental analysis: Anal. Calcd for:
C21H13N2O4: C, 68.51; H, 3.56; N, 7.61; found: C, 68.57; H, 3.56; N,
7.62. MALDI-TOF: m/z for (Mþ3H)þ¼382.11.
K.W. is a M.Sc. Student supported by Research Fund from Faculty
of Science, Chulalongkorn University (RES-A1B1-24). We also
appreciately acknowledge the TRF and CHE (RMU5380003 and
RTA5380003), the 90th Anniversary of Chulalongkorn University
fund (Ratchadaphisek Somphot Endowment Fund) and Higher
Education Research Promotion and National Research University
Project of Thailand, Office of the Higher Education Commission and
the Ratchadaphisaksomphot Endowment Fund (AM1009I-55) for
financial supports.
4.3. Glucose sensing studies
4.3.1. The complexation studies of the boronic anthraquinone based
sensors with glucose using fluorescence titration experiments. Typi-
cally, the sensors were prepared in spectroscopic grade DMSO at
concentrations of 2.00ꢂ10ꢁ4 mol/L as a stock solution. The stock
solutions of glucose were prepared at concentrations of
6.00ꢂ10ꢁ3 mol/L in 0.1 M HEPES buffer pH 7.4. In a 5.0 mL beaker,
0.30 mL of the stock solution of sensor was mixed with the portions
of stock solution of glucose to give final concentration of glucose.
After volumes were adjusted to 3.00 mL with 0.1 M HEPES buffer
(pH 7.4) to give a final concentration of sensors at 2.00ꢂ10ꢁ5 mol/L
Supplementary data
1H NMR spectrum of oHAQB and pHAQB. Fluorescence spectra
and Mass spectroscopy. Supplementary data related to this article
can be found online at http://dx.doi.org/10.1016/j.tet.2012.08.037.
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