A fluorescent probe for biothiols based on the conjugate addition of thiols to α,β-unsaturated ester

Article abstract of DOI:10.1002/bio.1257

A sensitive fluorogenic probe 1 for biothiols was developed based on the Michael addition reaction. The probe 1 was readily synthesized via the reaction of 2-(4′-hydroxyphenyl) benzimidazole (HPBI) with acryloyl chloride and shows weak fluorescence emission. Upon mixing with biothiols, the fluorescence of 1 is significantly enhanced due to the conjugate addition of thiols to the α,β-unsaturated carbonyl moiety, thus eliminating the photoinduced electron transfer (PET) quenching of the fluorophore by the intramolecular carbon-carbon double bond. Cysteine (Cys) was selected as the representative thiol in the spectral experiment. A good linear relationship was obtained from 1.0 to 30.0 μmol L^-1 for Cys and the detection limit was 0.17 μmol L^-1. Furthermore, probe 1 was highly selective for biothiols without the interference of some biologically relevant analytes and has been applied to detecting biothiols in human urine samples. Copyright

Full text of DOI:10.1002/bio.1257

Research article
Received: 08 May 2010,
Revised: 28 July 2010,
Accepted: 08 September 2010,
Published online in Wiley Online Library: 20 October 2010
(wileyonlinelibrary.com) DOI 10.1002/bio.1257
A fluorescent probe for biothiols based on the
conjugate addition of thiols to
a,b-unsaturated ester
Jun Du, Zhe Yang, Haiping Qi and Xiao-Feng Yang*
ABSTRACT: A sensitive fluorogenic probe 1 for biothiols was developed based on the Michael addition reaction. The probe 1
was readily synthesized via the reaction of 2-(4Ј-hydroxyphenyl) benzimidazole (HPBI) with acryloyl chloride and shows weak
fluorescence emission. Upon mixing with biothiols, the fluorescence of 1 is significantly enhanced due to the conjugate
addition of thiols to the a,b-unsaturated carbonyl moiety, thus eliminating the photoinduced electron transfer (PET) quench-
ing of the fluorophore by the intramolecular carbon–carbon double bond. Cysteine (Cys) was selected as the representative
thiol in the spectral experiment. A good linear relationship was obtained from 1.0 to 30.0 mmol L-1 for Cys and the detection
limit was 0.17 mmol L-1. Furthermore, probe 1 was highly selective for biothiols without the interference of some biologically
relevant analytes and has been applied to detecting biothiols in human urine samples. Copyright © 2010 John Wiley & Sons,
Ltd.
Keywords: biothiols; benzimidazole; fluorescent probe; Michael addition
much current interest, especially systems that provide an optical
Introduction
signal upon conjugation.
Low-molecular-weight aminothiols such as cysteine (Cys),
On the other hand, it was reported that the conjugate 1,4-
homocysteine (Hcy) and glutathione (GSH) continue to be of
interest because the low/high level of aminothiols in the biologi-
cal fluid is involved in various clinical disorders such as AIDS,
heart disease, Alzheimer’s and cardiovascular disease (1–3). Thus,
addition of thiols to a,b-unsaturated carbonyl compounds to
form the b-sulfido carbonyls can performed smoothly in aqueous
media (35). These results stimulate us to explore whether a,b-
unsaturated esters could be employed as a platform for the con-
the rapid, sensitive and selective detection of thiols is of consid-
struction of fluorescent thiol probes that may be potentially
erable importance and significant interest. Among the various
useful for thiol detection at physiological pH conditions.
reported methods, fluorescent chemosensors and probes are
Herein, we developed a new fluorescent thiol probe based on
more desirable since they are simple, sensitive and efficient. In
the past few years, various fluorescent probes for thiols based on
different mechanisms have been exploited, including Michael
addition (4–9), cyclization reaction with aldehyde (10–18), cleav-
age reaction by thiols (19–27), and others (28–32). However, most
of the fluorescent thiol probes developed so far are associated
with some limitations, such as limited pH range (9), high fluores-
cence background (11), long response time (13), etc. Therefore,
the conjugate addition of thiols to a,b-unsaturated esters. The
probe 1 was synthesized via the one-step reaction of HPBI with
acryloyl chloride and it displayed high selectivity toward thiols in
neutral buffer solutions. In probe 1, the a,b-unsaturated carbonyl
moiety acted as a quencher of fluorophores’ fluorescence. The
chemosensing of fluorogenic probe 1 involved the conjugate
addition of thiols to a,b-unsaturated carbonyl moiety to generate
the corresponding b-sulfido carbonyl adduct, thus eliminating
the development of fluorescent probes specifically recognizing
the photoinduced electron transfer (PET) quenching of the
biothiols under physiological conditions is of current interest.
fluorophore by the intramolecular carbon–carbon double bond
More recently, the conjugate addition of thiols to a,b-
unsaturated carbonyl compounds has been utilized for the
developed fluorescent thiol probes (4–7). Among them, the
maleimide-based probes have been widely reported (4,6,7),
based on the attachment of a maleimide group to dye frag-
ments, resulting in quenching of the fluorophore due to the
and leading to a dramatic increase in fluorescence intensity
simultaneously (Scheme 1). The proposed method proves to be
intramolecular PET process, and the fluorescence quenching is
* Correspondence to: Xiao-Feng Yang, Key Laboratory of Synthetic and
relieved when the p-system of the maleimide is disrupted.
Natural Functional Molecule Chemistry of Ministry of Education, Institute of
Analytical Science, Department of Chemistry, Northwest University, Xi’an
However, maleimide synthesis can be challenging in some
710069, China. E-mail: xfyang@nwu.edu.cn
cases, and the deactivation maleimides by water (giving ring-
opened amic acids) can compete significantly with thiol modi-
fication, particularly above pH 8.0 (33,34). For these reasons, the
development of selective thiol-reactive acceptors is a subject of
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of
Ministry of Education, Institute of Analytical Science, Department of Chem-
istry, Northwest University, Xi’an 710069, China
Copyright © 2010 John Wiley & Sons, Ltd.
Luminescence 2011; 26: 486–493

A fluorescent probe for biothiols
O
O
C
SH
R
N
N
R
C
S
O
O
N
H
N
H
1
1-thiol adduct
Weakly fluorescent
Strongly fluorescent
Scheme 1. Reaction of probe 1 with thiol-containing compound.
O
O
R
R
N
Et3N
N
C
C
OH
+
O
Cl
N
H
CH2Cl2
N
H
1: R= -H
2: R=
Scheme 2. Synthesis of 1 and 2 from HPBI
sensitive and selective and has been successfully used to detect
biothiols in human urine samples.
added and the above mixture was stirred at room temperature
for 4 h. After that, 1.08 g (10 mmol) of o-phenylenediamine dis-
solved in 5 mL DMF was added and the mixtures were refluxed
for 5 h. After cooling, the mixtures were poured into ice–water.
The precipitate was filtered off, washed with water several times
and dried to give the desired product as white solids (1.6 g, 70%
Experimental
1
Apparatus
yield). H NMR (400 MHz, DMSO), d 9.97 (s, 1H), 7.99 (d, 2H, J =
8.4 Hz), 7.54 (d, 2H, J = 2.4 Hz), 7.16 (m, 2H) 6.91 (d, 2H, J = 3.2 Hz).
The fluorescence spectra and relative fluorescence intensity were
performed on a Sanco CRT-970 spectrofluorimeter (Shanghai,
China) with a 10 mm quartz cuvette. Absorption spectra were
determined with a Shimadzu UV-1700 spectrophometer (Tokyo,
Japan). Infrared spectra (IR) were taken in KBr pellets on a Bruker
Tensor 27 IR spectrometer. Mass spectra were obtained with a
Compound 1. Acryloyl chloride was prepared following the
method described previously (37). Then, compound 1 was pre-
pared by a simple one-step reaction of HPBI with acryloyl chlo-
ride. To a stirred solution of HPBI (1.4 mmol, dissolved in 7 mL of
dichloromethane), acryloyl chloride (0.1 mL, 1.5 mmol) and tri-
ethylamine (0.5 mL) were added dropwise. The mixture was
stirred at room temperature for 4 h. After that, the organic layer
was dried in vacuo and the crude product was then purified by
silica gel (200–300 mesh) column chromatography eluted with
ethyl acetate–petroleum ether (1:3, v/v) to give the desired
product as a white solid (0.15 g, 31.5% yield). MS: m/z = 264.0
(M+); M+ calculated 264.26; 1H NMR (400 MHz, CDCl3), d 8.04 (d, 2H,
J = 8.8 Hz), 7.58 (q, 2H, J = 3.2 Hz), 7.22–7.13 (m, 4H), 6.63 (d,1H, J
= 16 Hz), 6.33 (m,1H), 6.05(d,1H, J = 10.4 Hz); IR (KBr, cm-1): 3442,
3056, 2365, 1738, 1608, 1498, 1404, 1295, 1205, 1168, 1020, 981,
903, 850, 748.
1
Shimadzu GCMS-QP2010 system without separation. H NMR
spectra were recorded on aVarian Unity INOVA-400 spectrometer
with tetramethylsilane (TMS) as the internal standard. All of the
measurements were operated at room temperature at about
298 K.
Reagents
Unless otherwise stated, all reagents were purchased from com-
mercial suppliers and used without further purification. Solvents
were purified and dried by standard methods prior to use.
Twice-distilled water was used throughout the experiments.
L-Cys, GSH (reduced form), 4-hydroxybenzaldehyde and cin-
namoyl chloride were bought from Shanghai Chemical Reagent
Company (Shanghai, China); DL-Hcy was purchased from Fluka;
o-phenylenediamine was obtained from Xi’an Chemical Reagent
Co.(Xi’an, China).
Compound 2. Compound 2 was prepared by HPBI and cin-
namoyl chloride by the same method as that used to obtain
compound 1. MS: m/z = 339.95 (M +); M+ calculated 340.16; IR (KBr,
cm-1): IR (KBr, cm-1), 3480, 3058, 1768, 1712, 1632, 1496, 1477,
1449, 1394, 1274, 1248, 1167, 1134, 1078, 968, 929, 860, 764, 749,
703, 678; 1H NMR (400 MHz, CDCl3), d 8.08 (d,1H, J = 8.8 Hz), 7.89
(dd,1H, J = 3.6 Hz), 7.74 (d,1H, J = 8.4Hz), 7.62 (m,3H), 7.44 (m, 4H),
7.27–7.31(m, 3H,), 7.17(s,1H) d 6.64 (m,1H).
Synthesis of compound 1
Compound 1 was synthesized in two steps as shown in Scheme 2.
Procedure
Synthesis of HPBI. HPBI was synthesized following the method
described in the literature (36). Briefly, 1.2 g (10 mmol) of
4-hydroxybenzaldehyde was dissolved in 10 mL of ethanol. To
this, 2.0 g of sodium bisulfite dissolved in 7 mL of water was
Typically, to a 10 mL test tube containing 1.0 mL of NaH2PO4–
Na2HPO4 buffer (0.2 mol L-1, pH = 7.4), 2.0 mL of ethanol and
1.0 mL of compound 1 (0.2 mmol L-1), an appropriate aliquot of
Luminescence 2011; 26: 486–493
Copyright © 2010 John Wiley & Sons, Ltd.
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J. Du et al.
240
210
180
150
120
90
0.75
0.60
0.45
0.30
0.15
0.00
1
2
a
b
c
d
HPBI
60
30
0
5
10 15 20 25 30 35 40 45 50 55
260
280
300
320
340
Time / min
Wavelength / nm
Figure 1. Time dependent fluorescence intensity changes observed during reac-
tion of 1 (or 2) (20.0 mmol L-1) with Cys (6.0 mmol L-1) in ethanol–water (20:80,v/v) at
pH 7.4 phosphate buffer solution. (a) 1 only; (b), 1 + Cys; (c), 2 only; (d), 2 + Cys.
Figure 2. Absorption spectra of compounds 1 and 2 and HBPI in ethanol–water
(20:80, v/v) at pH 7.4 phosphate buffer solution. The concentrations of 1, 2 and HBPI
are all 20.0 mmol L-1
.
Cys was added and the reaction mixture was diluted to 10.0 mL
with water. The resulting solution was allowed to stand at room
temperature (298 K) for 30 min, and then the absorption or emis-
sion spectra were recorded. For fluorescence intensity measure-
ments, the excitation and emission wavelengths were at 306 and
348 nm, respectively.
700
600
500
400
300
200
100
0
1
2
HPBI
Results and discussion
Effect of substituent on the fluorescence response of
the probe
In this study, compounds 1 and 2 were synthesized and the reac-
tivity of the probe toward biothiols were investigated by mixing
them with Cys at 20% (v/v) buffered (phosphate buffer, pH 7.4)
water–ethanol solution, respectively. As can be seen from Fig. 1,
the fluorescence intensity of solution 1 increased dramatically
upon mixing with Cys (the observed rate constant kobsd at 20°C
was found to be 58.4 M-1 s-1) (38), whereas the fluorescence inten-
sity of compound 2 remained almost unchanged under identical
conditions. The significant difference between them lies in the
substituent on the C=C bond of the probe. As for compound 2,
the addition of thiol to a,b-unsaturated carbonyl moiety of the
probe is relatively difficult because of the steric hindrance effect,
which provides us with some empirical rules on developing thiol-
selective probes. Moreover, it was observed that the rate of con-
jugate addition to the a,b-unsaturated ester was relatively slower
compared with that of a,b-unsaturated ketones. This can be
explained by the b-carbon atom of a,b-unsaturated ketone being
more electrophilic than the corresponding ester derivatives (35).
340
360
380
400
420
440
Wavelength / nm
Figure 3. Fluorescence spectra (excitation at 304 nm) of compounds 1 and 2 and
HPBI in ethanol–water (20:80, v/v) at pH 7.4 phosphate buffer. The concentrations of
1, 2 and HPBI are all 8.0 mmol L-1
.
Sensing response of probe 1 toward biothiols
The sensing response of probe 1 toward the representative bio-
logical thiol (Cys) was examined with absorption and fluores-
cence spectroscopy. Addition of 30 mmol L-1 of Cys to a solution
of probe 1 (20 mmol L-1) in ethanol–water (20:80, v/v) at pH 7.4
phosphate buffer exhibited a slight increase in the absorbance at
304 nm (Fig. 4). Furthermore, the changes in the fluorescence
spectra of probe 1 in the presence of increasing concentrations of
Cys are shown in Fig. 5. The probe 1 itself was weakly fluorescent
in 20% ethanol–phosphate buffer solution with a quantum yield
(F) of 0.020 [using 2-(2′-hydroxyphenyl) benzimidazole as a stan-
dard] (39); however, the introduction of Cys caused a dramatic
change in the fluorescence emission. The fluorescence emission
at 348 nm increased linearly with increasing Cys concentration.
The similar experimental phenomena were also observed when
Hcy or GSH was added to the solution of 1 (see Supporting Infor-
mation, Figs S1 and S2). Based on the previous studies (4,5,35),
the fluorescence increase is most likely due to the formation of
strongly fluorescent 1-thiol adducts (F1-Cys = 0.49). Here, we
provide some further evidence to confirm it. Firstly, upon addi-
tion of Cys to the solution of 1, TLC data of the reaction mixture
indicated that a new low Rf substance was formed [Rf(1) = 0.52,
Rf(1-Cys) ª 0, ethyl acetate–petroleum ether (v/v) = 1:1], indicating
the interaction of an a,b-unsaturated carbonyl group with Cys.
Secondly, the reaction product was separated byTLC and its mass
Spectra characteristics of compounds 1 and 2
The absorption spectra of compounds 1 and 2 and HBPI in a
mixture of ethanol and phosphate buffer solution at pH 7.4
(20 mmol L-1) were recorded and are shown in Fig. 2. It was
observed that maximum absorption of compounds 1 and 2 and
HPBI were both centered at about 304 nm. However, the absor-
bance followed the order 2 > HPBI > 1. Furthermore, the fluores-
cence emission spectra of 1, 2 and HPBI were also compared
(Fig. 3). As expected, the introduction of a,b-unsaturated carbo-
nyl group into HPBI led to almost nonfluorescent compounds 1
and 2. The weakly fluorescence emission of 1 and 2 can be
explained by PET quenching of fluorophore by the intramolecu-
lar C=C bond.
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Luminescence 2011; 26: 486–493

A fluorescent probe for biothiols
0.36
a
0.30
Blank
0.24
0.18
0.12
0.06
0.00
b
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
270
300
330
360
1 H (ppm)
Wavelength / nm
Figure 6. 1H NMR spectra of probe 1 (7.6 mmol L-1) in the absence (a) and pres-
ence of 4 equiv. of Cys (b) in D2O–CD3CN (1:1) solution. The spectrum b was mea-
sured after the addition of Cys for 40 min.
Figure 4. The absorption changes of 1 (20.0 mmol L-1) in ethanol–water (20/80,
v/v) at pH 7.4 phosphate buffer solution after addition of Cys (30 mmol L-1) for
30 min.
toward Cys was also pH-dependent and the fluorescence signal
of probe 1 in the absence or presence of Cys both increased with
increasing pH. The F/F0 obtained the maximum value at pH 7.4
(Fig. 7, insert curve). Therefore, pH 7.4 phosphate buffer was
selected for the proposed fluorogenic reaction in the subsequent
experiments.
1000
800
Cys
600
400
200
0
Optimization of the conditions
The effect of reaction time on the fluorescence signal of the
system was studied and the results are shown in Fig. 1. It can be
seen that, upon mixing with Cys, the fluorescence signal of the
system increased gradually with increasing the reaction time.
Although the sensitivity of the present method could be
increased with increasing the incubation time, a 30 min reaction
time was selected as a compromise of sensitivity and analytical
frequency.
330 345 360 375 390 405 420 435
Wavelength / nm
Figure 5. Fluorescence emission spectra (excitation at 306 nm) of
1
(20.0 mmol L-1) upon addition of increasing concentration of Cys (0–20 equiv) in
ethanol–water (20:80,v/v) buffered at pH 7.4. Each spectrum was obtained after
addition of Cys for 30 min.
In the present study, the organic solvent–water solution was
selected as the fluorogenic reaction medium. The effect of
ethanol concentration on the fluorescence emission of the
system was further studied, and the results are shown in Fig. 8.
It can be seen that fluorescence increased with increasing the
concentration of ethanol from 0 to 20%, and remained stable
when ethanol concentration was above 20%. Therefore,
20% ethanol–water solution was selected in the subsequent
experiment.
spectrum displayed peaks at m/z 386.1 [M+ + H], indicating that
1-Cys adduct is generated. Finally, the 1H NMR spectra of probe 1
in the absence or presence of Cys were recorded under the iden-
tical conditions. As observed from Fig. 6, the resonance signal at
d 6.0–6.7 ppm, corresponding to the C=C bond proton disap-
peared, which is consistent with the formation of 1-Cys adduct.
Based on the aforementioned experimental results, we conclude
that the fluorescence increase of the present system is due to the
formation of 1-Cys adduct.
Selectivity studies
For an excellent fluorescent probe, high selectivity is a matter of
necessity. The selectivity of probe 1 for thiols was investigated
by adding some biologically relevant analytes under the same
conditions. As shown in Fig. 9, probe 1 shows a ‘turn-on’ fluo-
rescence response to biothiols (Cys, Hcy and GSH). In contrast,
no significant changes in fluorescent emission were observed
upon addition of other amino acids (Glu, Asp, Met, Leu, Ala, Tyr,
Gly, His, Val, Thr, Arg, Iso, Phe, Lys), metal ions (K+, Ca2+, Mg2+,
Fe2+, Zn2+), hydrogen peroxide or ascorbic acid, indicating that
the selectivity of 1 toward biothiols is remarkably high. To
Effect of pH
The effect of pH on the fluorescence spectrum of probe 1 was
studied and the results are shown in Figure 3S (Supporting Infor-
mation). It can be observed that both fluorescence spectra of 1
and 1-Cys adduct were red-shifted with increasing pH, indicating
that fluorescence spectra of 1 and 1-Cys adduct are pH depen-
dent. Furthermore, the influence of pH on the fluorogenic reac-
tion was studied in the range 4.9–9.2, and the results are shown in
Fig. 7. It can be seen that the fluorescence response of probe 1
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J. Du et al.
900
750
600
450
300
150
0
10
8
b
6
4
2
0
5
6
7
8
9
10
pH
a
5
6
7
8
9
10
pH
Figure 7. Effect of pH on fluorescence intensity of the Cys-induced conjugated addition of
1
(20.0 mmol L-1). (a) 1 only; (b) 1 + Cys (10.0 mmol L-1). Insert curve, the F/F0 at different pH values, where F0 and
F are fluorescence intensity of probe 1 in the absence and presence of Cys, respectively.
Cys in the range 1.0–30.0 mmol L-1 (r = 0.9925, Figure S4). The
detection limit was 0.17 mmol L-1 (3d). The relative standard
deviation was 3.1% (n = 7) for 10.0 mmol L-1 of Cys.
400
320
240
160
80
a
b
Sample analysis
In order to evaluate the applicability of the proposed probe, a
standard addition method with Cys as the standard (9) was
employed to estimate the unknown concentration of thiols in a
human urine sample from a healthy volunteer. The raw urine was
collected from a healthy male volunteer. A 1.0 mL aliquotof this
solution was transferred into a 10 mL volumetric flask and diluted
to volume with water. Meanwhile, different concentrations of
standard Cys solution were added to the aliquot of diluted urine
samples. Then, the above samples were determined according to
the procedure described above. The total content of biothiols in
the urine sample was analyzed to be 25.0 Ϯ 0.48 mmol L-1
(Table 1), which was within the concentration range correspond-
ing to normal levels of thiols in urine samples (40).
0
0
10
20
30
40
50
Ethanol content/%, V/V
Figure 8. Effect of ethanol concentration on fluorescence intensity of the Cys-
induced conjugated addition of
(10.0 mmol L-1). Other reaction and detection conditions are the same as those
1 1 only; (b) 1 + Cys
(20.0 mmol L-1). (a)
described in the procedure.
further confirm the high selectivity of the probe to biothiols,
competition experiments were also carried out to test biothiols
selectivity response in the presence of other competitors such
as amino acids or metal ions. As displayed in Fig. 10, all of the
biologically relevant analytes tested have virtually no obvious
interference on the fluorescence detection of Cys. These facts
proved that probe 1 merits a high selectivity of for sensing bio-
thiols at physiological pH conditions, even with the involvement
of some biologically relevant analytes.
Conclusion
In conclusion, we have developed a highly sensitive and selective
fluorescent probe 1 for biothiols under physiological pH condi-
tions. The fluorescence enhancement was attributed to the
Michael addition of thiols to a,b-unsaturated carbonyl moiety in
probe 1 to form the corresponding b-sulfido carbonyl adduct.
Owing to the simplicity and sensitivity of the analysis, we believe
that this probe will find many applications in a variety of settings.
Analytical characteristics of 1 for biothiols
Supporting information
Under the selected conditions given above, the fluorescence
increment showed a linear relationship with the concentration of
Supporting information can be found in the online version of this
article.
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Copyright © 2010 John Wiley & Sons, Ltd.
Luminescence 2011; 26: 486–493

A fluorescent probe for biothiols
Table 1. Determination of thiols concentrations in urine samples
Sample
Urine
Cys added
Total thiols
Total thiols in
Recovery (%)
RSD (%) (n = 3)
(mmol L-1)
found (mmol L-1)
urine (mmol L-1)
0.0
2.0
4.0
2.5
4.6
6.5
25.0Ϯ0.48
—
103.5
97.6
1.9
1.0
5.5
—
—
420
350
280
210
140
70
a
Cys
Hcy
GSH
Other amino acids, Vc, H2O2 and metal ions
0
330 345 360 375 390 405 420 435
Wavelength / nm
400
350
300
250
200
150
100
50
b
0
Figure 9. (a) Fluorescence spectra (excitation at 304 nm) of 1 (20.0 mmol L-1) with various bio-
logically relevant analytes (10 mmol L-1) in ethanol–water (20:80, v/v) buffered at pH 7.4. (b) Fluo-
rescence intensity changes of 1 (20.0 mmol L-1) after addition of (10 mmol L-1) of various biologically
relevant analytes in ethanol–water (20:80, v/v) at pH 7.4 phosphate buffer solution. From left to
right: 1 only (blank), Cys, Hcy, GSH, Glu, Asp, Met, Leu, Ala, Tyr, Gly, His, Val, Thr, Arg, Iso, Phe, Lys,
ascorbic acid, H2O2, K+, Ca2+, Mg2+, Fe2+, Zn2+
.
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J. Du et al.
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350
300
250
200
150
100
50
0
1 2 3 4 5 6 7 8 9 101112131415161718
Figure 10. Fluorescence intensity changes of 1 (20.0 mmol L-1) after addition of
different analytes (100 mmol L-1) in the presence of Cys (10 mmol L-1) in ethanol–
water (20:80, v/v) at pH 7.4 phosphate buffer solution. Grey bars represent the
addition of the appropriate amino acids, metal ions or H2O2 to the solution of 1.
White bars represent the subsequent addition of Cys to the solution: (1) 1 only; (2)
Cys; (3) Arg; (4) Glu; (5) His; (6) Val; (7) Asp; (8) Gly; (9) Met; (10) Tyr; (11) Thr; (12) Leu;
(13) Lys; (14)K+; (15) Ca2+; (16) Mg2+; (17) Zn2+; (18) H2O2.
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We are grateful to the Science and Technology Department (no.
SJ08B05), the Education Department of Shaanxi Province of
China (no. 09JK787) and NWU Graduate Experimental Research
Funds (09YSY19). Z. Yang was supported by the National Science
Foundation for FosteringTalents in Basic Research of the National
Natural Science Foundation of China (no. J0830417).
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