A label-free turn-on fluorescence probe for rapidly distinguishing cysteine over glutathione in water solution

Article abstract of DOI:10.1016/j.ab.2016.01.027

A novel label-free fluorescent chemodosimeter (C1) was synthesized, based on coumarin and N-(4-aminobenzoyl)-β-alanine, for the selective detection of cysteine (Cys) over glutathione (GSH), which involved a click reaction of Cys to C=N of a Schiff base. T

Full text of DOI:10.1016/j.ab.2016.01.027

Accepted Manuscript
A label-free turn on fluorescence probe for rapidly distinguishing cysteine over
glutathione in water solution
Liqiang Yan, Zhineng Kong, Wei Shen, Wenqi Du, Yan Zhou, Zhengjian Qi
PII:
S0003-2697(16)00052-X
10.1016/j.ab.2016.01.027
YABIO 12306
DOI:
Reference:
To appear in: Analytical Biochemistry
Received Date: 4 November 2015
Revised Date: 25 December 2015
Accepted Date: 25 January 2016
Please cite this article as: L. Yan, Z. Kong, W. Shen, W. Du, Y. Zhou, Z. Qi, A label-free turn on
fluorescence probe for rapidly distinguishing cysteine over glutathione in water solution, Analytical
Biochemistry (2016), doi: 10.1016/j.ab.2016.01.027.
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ACCEPTED MANUSCRIPT
A label-free turn on fluorescence probe for rapidly distinguishing
cysteine over glutathione in water solution
Liqiang Yan, Zhineng Kong, Wei Shen, Wenqi Du, Yan Zhou and Zhengjian Qi *
College of Chemistry and Chemical engineering, Southeast University, Nanjing, Jiangsu, 211189, P.R. China.
E-mail: qizhengjian@seu.edu.cn (Z-J Qi), liqiangyan2014@163.com (L-Q Yan);
A novel label-free fluorescent chemodosimeter (C1) based on coumarin and N-(4-Aminobenzoyl)-β-alanine for the
selective detection of cysteine (Cys) over glutathione (GSH) was synthesized, which was involved by click
reaction of Cys to C=N of schiff base. The probe C1 featured a fast response (about 3 min), emission in the visible
region and high selectivity. Addition of Cys in HEPES-NaOH solution (pH = 7.4) to C1 in water resulted in
appearance of a new emission peak at 445 nm in company with remarkable enhancement of fluorescence intensity,
while other amino acids did not induce any significant fluorescence change. Meanwhile, the addition reaction of
Cys to C1 elicited 90.8-fold fluorescence intensity enhancement, which resulted in emission color change from
orange to blue.
Keywords: fluorescence probe, turn on, coumarin, N-(4-Aminobenzoyl)-β-alanine, cysteine, Schiff base
Introduction
As one of the most important components among the bio-relevant proteins, cysteine has attracted
much attention in recent years due to its crucial role in biological body, such as catalysis, heavy
metal binding, protein turnover, and signal transduction. [1-4] Therefore, a number of diseases can
result from abnormalities in cysteine levels. Cysteine deficiency is known to be involved in many
syndromes, such as edema, lethargy, hair depigmentation, liver damage, muscle and fat loss,
slowed growth, and skin lesions. [5-6] Additionally, the increased cysteine levels can lead to a
higher risk of cancer, Alzheimer’s, and cardiovascular diseases. [7-10] Therefore, it is of great
importance to develop rapid, sensitive, selective, and quantitative analysis of the cysteine level.
To date, many strategies have been developed for the measurement of cysteine have been
studied, including mass spectrometry, [11-12] high performance liquid chromatography (HPLC),
[
13-14] capillary electrophoresis, [15-16] optical assays, [17-18] and the electrochemical analysis.
19-20] Nevertheless, most of above-mentioned methods require professional experts, or
[
expensive instruments, or cumbersome separation and purification procedures of samples. As a
highly sensitive, non-destructive technique, fluorescence analysis has also been applied
extensively for the measurement of cysteine. [21-25] However, some of them reveal some
limitations in the process of detections, including the introduction of heavy metal ions, [26-28] or
a long response time. More importantly, only a few sensors able to detect Cys over GSH have
been reported to date. [29-31] Therefore, it is still high interest to explore low-cost and
time-saving methods for measuring cysteine.
Herein we report a new turn on fluorescence probe C1 (Scheme1) based on coumarin,
which can effectively discriminate Cys from GSH in aqueous solution.

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Results and discussion
Selective response of probe C1 to Cys
In order to investigate the effect of various amino acids on the fluorescence spectra of C1, the
amino acid Cys, His, Lys, Arg, Cys-Cys, Thr, Gly, Ala, Asp, Val, Met, Glu, Ser, GSH, Tyr and
Na S were used to evaluate the response properties of C1 in aqueous solution (HEPES-NaOH,
2
1
0.0 mM, pH=7.4). Figure.1 shows the changes in the fluorescence spectra of C1 upon addition of
various analytes. Compound C1 had a large effect only with Cys among the analytes examined. In
the presence of Cys, C1 showed fluorescence enhancement (about 90.8-fold enhancement) so
strong and a dramatic fluorescence color change from orange to light blue which could easily be
identified by the naked eye under UV lamp during the detection process. Other analytes gave no
distinct response to C1 in the fluorescence spectra. This obvious feature reveals that compound C1
has high selectivity for Cys.
Sensitivity of C1 for Cys detection
The fluorescence spectra of C1 upon titration with Cys were then performed (Fig.2). The
fluorescence intensity of C1 (5.0 ꢀM) at 445 nm was gradually increased with the addition of an
increasing amount of Cys (0-11.0 ꢀM). The enhanced intensity of C1 displayed a good linear
2
regression relationship (Stern–Volmer equation) [32]: y = 1.84E4[Cys]+3.50E3 (R = 0.996) with
the concentration of Cys in the range of 0-7.0 ꢀM based on the titration experiment (Fig.3). The
limit of detection (LOD) of C1 for Cys was estimated to be as low as 0.15 ꢀM based on 3σ/S,
where σ is the standard deviation of blank measurements, and S is the slope between fluorescence
intensity versus sample concentration. The results indicate that the probe C1 can be used as a
sensitive chemosensor for the detection of Cys in aqueous solution.
Competition experiments
To further investigate the interference of other analytes on the detection of Cys, competition
experiments were performed in which various analyte (10.0 µM) were added to a solution of
C1(5.0 ꢀM) in the presence of Cys. As shown in Fig.4, all of the competing analytes tested have
virtually no influence on the fluorescence detection of Cys. The result suggested that C1 could be
an effective sensor for the detection of Cys in aqueous solution.
The effect of pH
The fluorescent response of probe C1 toward Cys in HEPES-NaOH buffer (10.0 mM) at
different pH conditions were further investigated to apply probe C1 in complicated systems, such
as environmental or biological systems. The results indicate that the response of probe C1 towards
Cys was pH dependent. The fluorescence intensity of probe C1 had a relatively weak emission
band at 445 nm and remained unaffected between pH levels 5.0 and 10.0, as shown in Fig.5. Upon
addition of Cys, the maximal fluorescence signals were observed in the pH range of 6.6-8.0
without the interference by protons, and the stable fluorescence of the probe at a pH level of
approximately 7.4 was favorable for the sensing assays of Cys in environmental and biological
samples.
Response time studies

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Response time is also an important property of fluorescence probe. The time course for
fluorescence intensity changes of probe C1 (5.0 ꢀM) in the presence of Cys (10.0 µM) at 445 nm
(excited at 380 nm) was investigated (Fig.6). The result showed that the fluorescence response
occurs immediately after addition of Cys, and the recognition interaction completed within 3 min,
indicating that probe C1 might be used for the real-time monitoring of Cys conveniently.
Reaction mechanism
Coumarin was selected as the fluorophore due to its desirable photophysical properties, such
as a large Stokes’ shift, and visible emission wavelengths. The N atom of C=N acts as an electron
donor for the photo-induced electron transfer (PET) process from C=N to coumarin ring, leading
to a low fluorescence of probe C1. After adding Cys, the double bond C=N is broken, and
blocking of the PET process can cause fluorescence recovery. In order to investigate the
1
mechanism we contrastively analysed the H NMR spectra (Fig.7). With addition of Cys to probe
C1 in D O, the CH=N protons at 9.14 ppm (H1) of probe C1 dramatically disappear while new
2
peaks at 3.80 ppm (H1’) and 3.90 ppm (H2) were observed. The changes of these peaks should be
attributed to the mercapto group of Cys added to the carbon atom of C=N group with shift from
CH=N to CH-NH. Therefore, the CH=N proton (H1) shifted to high-field and a peak appeared at
3
.90 ppm because of the existence of the NH proton of CH-NH.
Experimental
Apparatus and Chemicals
Melting point was determined using an SGW X-4 digital melting point apparatus (Shanghai
Instrument Physical Optics Instrument Co., Ltd). 1H NMR spectrum was run on a Varian
Mercury-Plus 400 NMR spectrometer using TMS as the internal standard (Varian, San Diego).
Elemental analysis was taken with a Vario EL CHNS elemental analyzer (Elementar, Germany).
Mass spectrum was recorded with a VG ZAB-HS double focusing mass spectrometer (Thermo
Onix Ltd, UK). Fluorescence spectra, fluorescence lifetime and quantum efficiency were
measured with a Fluorolog 3-TSCPC (Horiba Jobin Yvon Inc. France). All chemicals were
purchased from Aladdin Industrial Corporation (China) and used without further purification. The
solvents were purified using standard methods.
All solutions of C1 (5.0 ꢀΜ ) and various amino acid and Na S (10.0 ꢀΜ) were prepared in
2
HEPES-NaOH buffer solution (10.0 mM, pH 7.4) and allowed to equilibrate at room temperature
for 3 minutes before spectral measurements. Every measurement was performed three times at
same testing conditions in a quartz cell with a 1 cm path length with 380 nm excitation
wavelength.
Synthesis of 8-formyl-7-hydroxy-4-methylcoumarin
A solution of 8-Formyl-7-hydroxy-4- methylcoumarin was prepared by the known method.
[33] 7-Hydroxy-4-methylcoumarin (5.0 g, 0.0284 mol) and hexamine (10.0 g, 0.071 mol) in acetic
acid (37 mL) were stirred for 5.5 h at 95 ºC. Then, hydrochloric acid (75 mL, HCl: H O = 84:100,
2
v/v) was added and further heated for 45 min. After cooling, the mixture was poured into
ice-water (375 mL) and extracted with ethyl acetate (150 mL×3). The organic layer was dried over

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sodium sulfate and the solvent removed. The residue was purified by column chromatography on
silica gel using dichloromethane as eluent to provide the product as a light yellow solid. Yield 0.9
1
g (15.5 %). H NMR (CDCl , δ ppm): 2.45 (s, 3H); 6.22 (s, 1H); 6.92 (d, J = 9.2 Hz, 1H); 7.74 (d,
3
+
J = 8.8 Hz, 1H); 10.63 (s, 1H); 12.23(s, 1H). MS m/z: 205[M+H] .
Synthesis of 7-hydroxy-4-methyl-8-(4ˊ-(N-carboxylethylformamide)phenylimino)methyl-2H-1-
benzopyran-2-one (C1)
8
-formyl-7-hydroxy-4-methylcoumarin (0.21 g, 0.1 mmol) and N-(4-Aminobenzoyl)-β-
alanine (0.20 g, 0.11 mmol) were dissolved in anhydrous ethanol (20 mL). The reaction mixture
was refluxed for 2 h then the mixture was cooled to room temperature. The precipitate was filtered
off, washed with cold ethanol two times and dried in vacuum to give the desired product as a
1
yellow solid. Yield 0.34 g (85.1 %). m. p 265.4-267.2℃. H NMR (d -DMSO, 400MHz, δ ppm):
6
2
8
1
1
1
.33 (s, 3H); 2.51-2.55 (t, J = 8.0, 2H) ; 3.44-3.48 (t, J = 8.0, 2H); 6.17 (s, 1H); 6.84-6.86 (d, J =
.0, 1H), 7.50-7.52 (d, J = 8.0, 2H); 7.70-7.72 (d, J = 8.0, 1H); 7.92-7.94 (d, J = 8.0, 2H); 8.60 (s,
1
3
H); 9.14 (s, 1H); 12.20 (s, 1H), 14.65 (s, 1H). C NMR (d -DMSO, δppm): 18.3, 33.7, 35.6,
6
06.0, 110.4, 110.9, 114.0, 121.2, 128.6, 130.6, 133.0, 148.5, 153.8, 154.0, 157.6, 159.0, 165.2,
65.2, 172.9.
Conclusions
In conclusion, a new label-free and Cys-specific probe was successfully developed. The
probe showed a rapid response and selectivity for detection of Cys over GSH. Addition of Cys in
HEPES-NaOH (pH = 7.4) to C1 in water resulted in a fluorescence intensity enhancement about
9
0.8-fold, while other amino acids did not induce significant emission change. In addition, it is
worth noting that these fluorescence experiments were performed in a pure aqueous solution,
which is different from other probes determined in organic solvents or mixed solutions, and has
potential application in the detection and analysis of various Cys-related issues in biological and
medical areas.
Acknowledgements
This work was supported by the Fundamental Research Funds for the Central Universities
(
KYLX15_0125) and National Major Scientific Instruments and Equipment Development Projects
2014YQ060773).
(
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Scheme and Figures
Scheme 1 Synthetic route of compound C1
Fig.1. Fluorescence emission and fluorescence color changes (Insert) of C1 (5.0 µM) upon
addition of various analytes (10.0 µM) in aqueous solution. (HEPES-NaOH, 10.0 mM, pH = 7.4,
λex = 380 nm). Analytes:1: C1, 2: His, 3: Lys, 4: Arg, 5: Cys-Cys, 6: Thr, 7: Gly, 8: Ala, 9: Asp,
1
0: Na S, 11: Met, 12: Glu, 13: Ser, 14: GSH 15: Val, 16: Tyr, 17: Cys.
2

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Fig.2. The change in fluorescence intensity of probe C1 (5.0 ꢀM) at 445nm against varied
concentrations of Cys from 0 to 11.0 ꢀM in aqueous solution. Insert: The fluorescence colors of
C1 (5.0 ꢀM) and C1 (5.0 ꢀM)+Cys (11.0 ꢀM) in aqueous solution under 365 nm UV irradiation.
(
HEPES-NaOH, 10.0 mM, pH = 7.4, λ = 380 nm).
ex
Fig.3. Calibration curve based on fluorescence intensities (445 nm) as a function of Cys
concentrations in aqueous solution (HEPES-NaOH, 10.0 mM, pH = 7.4, λ = 380 nm).
ex
Fig.4. Changes in fluorescence intensity of C1 (5.0 ꢀM) toward Cys (2.0 equiv.) in the presence of
the competing analytes (amino acids and Na S, 2.0 equiv., respectively), λ = 380 nm, λ = 445
2
ex
em
nm, HEPES-NaOH, 10.0 mM, pH = 7.4.

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Fig.5. The fluorescence intensity (445 nm) of probe C1 (5.0 ꢀM) with 10.0 µM Cys was obtained
at different pH values ( HEPES-NaOH, 10.0 mM, λ = 380 nm).
ex
Fig.6. Time dependent fluorescence emission (445 nm) change of C1 (5.0 ꢀM) upon addition of
Cys (10.0 ꢀM) in HEPES-NaOH buffer solution, λ = 380 nm.
ex
Fig.7. The comparative trial of H NMR spectra of C1 (d -DMSO, 400 MHz) and C1+1.0 equiv.
6
Cys (D O, 400 MHz).
2