A simple and effective ratiometric fluorescent probe for the selective detection of cysteine and homocysteine in aqueous media

Article abstract of DOI:10.3390/molecules21081023

Biothiols such as cysteine (Cys) and homocysteine (Hcy) are essential biomolecules participating in molecular and physiological processes in an organism. However, their selective detection remains challenging. In this study, ethyl 2-(3-formyl-4-hydroxyphenyl)-4-methylthiazole-5-carboxylate (NL) was synthesized as a ratiometric fluorescent probe for the rapid and selective detection of Cys and Hcy over glutathione (GSH) and other amino acids. The fluorescence intensity of the probe in the presence of Cys/Hcy increased about 3-fold at a concentration of 20 equiv. of the probe, compared with that in the absence of these chemicals in aqueous media. The limits of detection of the fluorescent assay were 0.911 μM and 0.828 μM of Cys and Hcy, respectively. ¹H-NMR and MS analyses indicated that an excited-state intramolecular proton transfer is the mechanism of fluorescence sensing. This ratiometric probe is structurally simple and highly selective. The results suggest that it has useful applications in analytical chemistry and diagnostics.

Full text of DOI:10.3390/molecules21081023

molecules
Article
A Simple and Effective Ratiometric Fluorescent Probe
for the Selective Detection of Cysteine and
Homocysteine in Aqueous Media
Risong Na , Meiqing Zhu , Shisuo Fan , Zhen Wang , Xiangwei Wu , Jun Tang , Jia Liu 1,2,
1
,2
1
3
3
1
1
1
,
1,
Yi Wang * and Rimao Hua *
1
Department of Science of Pesticides, School of Resources and Environment, Anhui Agricultural University,
No. 130 Changjiang West Road, Hefei 230036, China; risongna@163.com (R.N.);
zhumeiqing@ahau.edu.cn (M.Z.); wxw@ahau.edu.cn (X.W.); tangjun@ahau.edu.cn (J.T.);
ljia198346@163.com (J.L.)
2
Collaborative Innovation Center of Henan Grain Crops, National Key Laboratory of Wheat and Maize Crop
Science, College of Plant Protection, Henan Agricultural University, Wenhua Road No. 95,
Zhengzhou 450002, China
Department of Environmental Engineering, School of Resources and Environment,
3
Anhui Agricultural University, No. 130 Changjiang West Road, Hefei 230036, China;
fanshisuo@ahau.edu.cn (S.F.); zwang@ahau.edu.cn (Z.W.)
*
Correspondence: wangyi@ahau.edu.cn (Y.W.); rimaohua@ahau.edu.cn (R.H.);
Tel./Fax: +86-551-6578-6320 (Y.W. & R.H.)
Academic Editor: Zhen Cheng
Received: 18 July 2016; Accepted: 2 August 2016; Published: 5 August 2016
Abstract: Biothiols such as cysteine (Cys) and homocysteine (Hcy) are essential biomolecules
participating in molecular and physiological processes in an organism. However, their selective
detection remains challenging. In this study, ethyl 2-(3-formyl-4-hydroxyphenyl)-4-methylthiazole-
5-carboxylate (NL) was synthesized as a ratiometric fluorescent probe for the rapid and selective
detection of Cys and Hcy over glutathione (GSH) and other amino acids. The fluorescence intensity
of the probe in the presence of Cys/Hcy increased about 3-fold at a concentration of 20 equiv. of
the probe, compared with that in the absence of these chemicals in aqueous media. The limits of
1
detection of the fluorescent assay were 0.911
µM and 0.828
µ
M of Cys and Hcy, respectively. H-NMR
and MS analyses indicated that an excited-state intramolecular proton transfer is the mechanism of
fluorescence sensing. This ratiometric probe is structurally simple and highly selective. The results
suggest that it has useful applications in analytical chemistry and diagnostics.
Keywords: fluorescent probe; cysteine and homocysteine; detection
1
. Introduction
Biothiols such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are essential
biomolecules and participate in molecular and physiological processes in living systems. Recently, they
have received much research attention [ ]. Cys is an important amino acid that not only plays a pivotal
1,2
role in many physiological processes including protein synthesis, detoxification, and metabolism but
is also very closely related to many serious diseases such as slow growth in children, liver damage,
muscle and fat loss, skin lesions, and weakness [
with the risk of Alzheimer’s disease [ ], inflammatory bowel disease, and osteoporosis [
the detection of Cys and Hcy is very important for potential disease diagnosis.
Various detection techniques such as fluorescence assays, high-performance liquid chromatography
3
–
5
]. Hcy has been proposed to be closely associated
6
7
]. Therefore,
,
capillary electrophoresis, immunoassays, and colorimetry [
8,
9] have been developed. Fluorescence
Molecules 2016, 21, 1023; doi:10.3390/molecules21081023
www.mdpi.com/journal/molecules

Molecules 2016, 21, 1023
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detection is particularly attractive because of its high selectivity, high sensitivity, low cost, and
its great potential for bioimaging with fluorescent probes [ 10 20]. Distinguishing Cys and Hcy
from other biothiols, particularly GSH, remains a challenge because of their similar structures and
reactivity [21 22]. Furthermore, the intracellular concentration of Cys (30–200 M) is more than
0 times lower than that of GSH (1–10 mM), which makes the detection of Cys more difficult [23 25].
Many fluorescent probes have been developed for the detection of Cys/Hcy [ 26 30], however,
4, –
,
µ
1
–
4, –
most of these Cys/Hcy probes were based on fluorescence measurements at a single wavelength,
which may be limited by many factors such as the instrument detection efficiency and environmental
conditions. In contrast, ratiometric fluorescent probes allow the measurement of emission intensities at
two different wavelengths, which provides a built-in correction of environmental effects and can also
increase the dynamic range of the fluorescence measurement. Therefore, there is a need for ratiometric
fluorescent probes for selective detection of Cys/Hcy to be developed.
In the present study, we synthesized ethyl 2-(3-formyl-4-hydroxyphenyl)-4-methylthiazole-5
-
carboxylate (NL) (Figure 1) that can readily discriminate Cys/Hcy from other biothiols in approximately
2
.5 h of detection. This probe undergoes an excited-state intramolecular proton transfer (ESIPT) process
,
1
which is confirmed by H-NMR and MS spectra. The NL probe is easy to synthesize (Supplementary
Materials), versatile, and further structural modifications can improve its properties for biothiol detection.
Figure 1. Structure of the fluorescent probe NL.
2
. Results and Discussion
NL was characterized and optimized as a fluorescent probe via studies of its UV-Vis absorption,
fluorescence selectivity, reaction time of biothiols, quantification of Cys/Hcy, effect of pH on detection
of Cys, as well as its detection mechanisms.
2
.1. UV-Vis Absorption and Fluorescence Selectivity Studies of the NL Probe
First, the effect of dimethylsulfoxide (DMSO) content on the fluorescence intensity of the
free NL probe and the behavior of the probe towards Cys were investigated. The fluorescence
intensity of NL increased with increasing content of DMSO (Figure S3). In the presence of a polar
aprotic solvent, the proton of the hydroxyl group is most likely strongly bonded with the nearest
carbonyl group (Scheme 1). Buffer solutions were optimized based on the fluorescence signal-to-
background ratio of NL with or without Cys in aqueous DMSO. The optimized solution was DMSO in
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (8:2, v/v, pH 7.4) and was used in
the subsequent experiments.
Scheme 1. Probable isomers of NL.

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The selectivity of NL for Cys/Hcy measurements was evaluated with the DMSO/HEPES solution
containing each of the 20 interference chemicals, in addition to the analytes of Cys and Hcy. The
interference chemicals were GSH, arginine, aspartic acid, glutamic acid, glycine, histidine, lysine,
+
2+
+
2+
2+
proline, threonine, tryptophan, tyrosine, K , Ca , Na , Mg , Zn , H O , H S, HSCH CH OH
2
2
2
2
2
(MCH), and glucose, each at a concentration of 20-fold compared to the NL probe. Cys/Hcy increased
the absorbance of the solutions at 327 nm, while the other chemicals did not increase the absorbance of
the solutions (Figure 2a). In addition, fluorescence intensity at λem 501 nm was markedly enhanced in
the presence of Cys/Hcy (Figure 2b). The other analytes caused negligible effects on the fluorescence
intensity of NL. In particular, GSH did not affect the UV-Vis absorbance and fluorescence intensity
of the probe (Figure 2a,b, Figures S4 and S5). The presence of a wide range of interferences had little
effect on the detection of Cys/Hcy. Therefore, NL possesses good selectivity toward Cys/Hcy in the
presence of other amino acids and metal ions as well as GSH.
(
a)
(b)
Figure 2.
(a) UV-vis absorption spectra of the NL probe (10
µM) with various analytes (200
µM) in
HEPES buffer solution (DMSO/HEPES = 8:2, pH 7.4); (
various analytes (200 M) in HEPES buffer solution (DMSO/HEPES = 8:2, pH 7.4, λex = 327 nm, slit:
.0 nm/2.0 nm). Inset is a photograph of NL without and with Hcy upon excitation at 365 nm.
b) Fluorescence spectra of NL (10 µM) with
µ
2
2
.2. Reaction Time of Biothiols
To investigate the reaction time of NL with biothiols, the fluorescence intensity at λem 501 nm was
recorded at varying periods of time after additon of Cys, Hcy, and GSH to the NL solutions (Figure S6).
The reaction of NL with Cys or Hcy was nearly completed within 2.5 h at ambient temperature.
These reactions are relatively fast compared with those reported in the literature [31,32]. In contrast,
there was no obvious change in the fluorescence intensity of the solution containing GSH during
the measurement time. Some reported probes can detect biothiols rapidly but with large amounts of
biothiols, such as 30 equiv. or even more than 1000 equiv. of the probe [33–36]. In this study, we used
2
0 equiv. to achieve an accurate measurement of the biothiols.
2
.3. Quantification of Cys/Hcy
Figure 3a,b show the change in the UV-Vis absorbance of the solutions containing the NL probe
with increasing content of Cys/Hcy. The absorbance intensity at 327 nm increased, but the intensity at
26 nm decreased with increasing Cys/Hcy concentration. Figure 3c,d reveal that the fluorescence
4
intensity of the probe solution increased at λem 501 nm with increasing content of Cys/Hcy. When
Cys/Hcy was at 20 equiv. of NL, the fluorescence intensity and the ratio of the absorption intensities
at 501 and 426 nm (I501/I426) were almost at their maximum values, which is a superior performance
2
in comparison with some other probes [33
between the fluorescence intensity and Cys concentration from 1–7
of Cys was 0.911 M (Figure 3e), which was calculated by S/N = 3. A similar response of the NL probe
,37]. In addition, we found a linear correlation (R = 0.9888)
µM. The limit of detection (LOD)
µ

Molecules 2016, 21, 1023
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to Hcy was also observed, as shown in Figure 3f. In this case, the LOD was 0.828
linear relationship (R = 0.9971) between the fluorescence intensity and Hcy concentration in the range
µ
M based on the
2
of 1–8 µM. In comparison with the previously reported probes [20,38–40], NL provides a much lower
LOD of Cys/Hcy for qualitative analysis of Cys/Hcy despite the presence of GSH (Figure S7), making
it attractive for use in human plasma samples [28].
(
a)
(b)
(
c)
(d)
(
e)
(f)
Figure 3.
(
a,
b) UV-vis absorption spectra of NL (10
µM) with Cys/Hcy (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
1
2, 13, 14, 15, 16, 17, 18, 19 and 20 equiv.) in HEPES buffer solution (DMSO/HEPES = 8:2, pH 7.4);
) Fluorescence spectra of NL (10 M) with Cys/Hcy (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
6, 17, 18, 19 and 20 equiv.) in HEPES buffer solution (DMSO/HEPES = 8:2, pH 7.4, λex= 327 nm, slit:
.0 nm/2.0 nm). Inset is the corresponding ratiometric response (I501/I426) of NL to the addition of
Cys/Hcy; ( ) Plot of the ratiometric response (I501/I426) of NL (10 M) against Cys/Hcy equiv.; data
are mean ˘ standard error (bars) (n = 3).
(
c
,d
µ
1
2
e,
f
µ

Molecules 2016, 21, 1023
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2
.4. Effect of pH on Detection of Cys
The effect of pH on the response of NL to Cys is illustrated in Figure S8. In the presence or absence
of Cys, NL was stable under acidic conditions. With increasing pH from 2.0–8.0, the fluorescence
intensity of the solution increased until it reached its maximum value at pH 8.0. Weak alkaline
conditions possibly promoted the reaction between Cys and NL. With a further increase of the pH, the
fluorescence intensity of the solution decreased until it was quenched, which might be caused by the
hydrolysis of ester groups or the opening of the thiazolidine ring under strong alkaline conditions.
Therefore, we chose pH 7.4 as a suitable pH for the detection experiments. Unlike the early and even
recently reported probes in the literature that require use at pH
physiological pH range.
ě
9.0 [28,41,42], NL can function in the
2
.5. Mechanism of NL in Detection of Cys/Hcy
To verify the detection mechanism between NL and Cys/Hcy as shown in Scheme 2, we
1
investigated the H-NMR spectrum of NL upon the addition of Cys and compared it with that
of the probe itself. Under the same conditions, Hcy also reacted similarly with NL and formed the
six-membered thiazinane ring. After the reaction of NL with Cys/Hcy in DMSO-d for 4 h, the reaction
6
mixture was analyzed with NMR, which indicated that the aldehyde proton at around
had disappeared, and that the thiazolidine methane proton at around 5.5 ppm had newly appeared
Figure S2). Furthermore, UPLC-MS/MS were carried out to confirm that the thiazolidine ring was
δ 10.3 ppm
δ
(
formed by the reaction of Cys/Hcy with the aldehyde group of NL. The mass spectrum (ESI MS) of
+
the NL probe shows a peak at m/z 292.09 (NL + H) . The peak of the product of NL + Cys is 394.93,
which also proves a single mononuclear addition of Cys to NL. A peak at m/z 408.92 corresponding to
the product of NL + Hcy was also observed (Figure S2).
Scheme 2. Proposed reaction mechanism of NL and Cys/Hcy.
NL at λex 327 nm produced two emission peaks at ~426 nm and ~501 nm. Adding Cys/Hcy
to NL enhanced the ESIPT process to shift the emission signal to a longer wavelength. As a result,
the fluorescence intensity at λem 501 nm, which was attributed to the tautomer (T* emission) of the

Molecules 2016, 21, 1023
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Cys/Hcy-products, was enhanced strongly, and the normal isomer (N* emission) of the products
caused the fluorescence intensity at 426 nm to increase slowly. The two emission peaks can be used for
the ratiometric fluorescent measurement to determine the presence of the analytes more accurately
with the minimization of the background signal [43].
3
. Experimental Section
3
.1. General Methods and Materials
Commercial reagents were used as obtained from Sigma-Aldrich (St. Louis, MO, USA), Alfa
Aesar (Ward Hill, MA, USA), and J&K (Beijing, China). Twice-distilled water was used throughout
all the experiments. Nuclear magnetic resonance (NMR) spectra were recorded with a 600-MHz
spectrometer (Agilent Technologies, Inc., Santa Clara, CA, USA) and mass spectrometry (MS) was
performed with a UPLC/XevoTQ MS/MS spectrometer (Waters, Milford, MA, USA). All fluorescence
measurements were carried out on a 970CRT fluorescence spectrophotometer (Inesa, Shanghai, China)
equipped with a xenon lamp source. UV-Vis spectra were measured on a UV-1800 spectrophotometer
(Shimadzu, Kyoto, Japan). Quartz cuvettes with a 1-cm path length and 3-mL volume were used for
all measurements. All pH measurements were completed with a PHS-25 digital pH meter (Shanghai
REX Instrument Factory, Shanghai, China).
3
.2. Optical Studies of the NL Probe upon Addition of Various Analytes
A stock solution of the NL probe with a concentration of 12.5
acids (Cys, Hcy, GSH, arginine, aspartic acid, glutamic acid, glycine, histidine, lysine, proline, threonine,
µ
M was prepared in DMSO. Amino
+
2+
+
2+
2+
tryptophan, and tyrosine), cations (K , Ca , Na , Mg and Zn ), H S, and HSCH CH OH (MCH)
2
2
2
were all dissolved in distilled water at a concentration of 5 mM for the absorption and fluorescence
spectra analyses. The stock solutions of the analytes were diluted to various concentrations with
HEPES buffer. Test solutions were prepared by adding 2400
concentration of NL was 10 M) and an appropriate aliquot of test analyte into a 3-mL volumetric
cuvette. Each solution was diluted to 3 mL using a mixture of DMSO and HEPES buffer (8:2, v/v) at pH
µL of the NL stock solution (the final
µ
7
.4. The resulting solution was shaken well and incubated for 2.5 h at room temperature. Fluorescent
´
1
spectra were then recorded using an excitation wavelength of 327 nm and scan speed of 450 nm
¨min
.
4
. Conclusions
In order to find a simple method to distinguish Cys/Hcy from other amino acids at physiological
pH, an effective ratiometric fluorescent NL probe was developed. The fluorescence intensity of the
probe was enhanced 3-fold upon addition of Cys/Hcy at a concentration of 20 equiv. of the probe, and
its absorbance in buffer solution was also increased. NL can detect Cys at concentrations of 0.911 to
2
0 µM and Hcy at concentrations of 0.828 to 20 µM.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
/1023/s1.
8
Acknowledgments: This work was supported by the National High Technology Research and Development
Program of China (2013AA102804B), the National Natural Science Foundation of China (NSFC) (No. 31272062),
the Anhui Agricultural University Youth Fund Project (2014zr003, yj2015-25), the Natural Science Foundation of
Anhui Province of China (1408085MKL36), and the Foundation for the Excellent Youth Scholars of Anhui Province
(2013SQRL016ZD).
Author Contributions: This research was carried on by all the authors. Risong Na, Yi Wang, and Rimao Hua
designed the theme of the study and carried out the probe synthesis as well as the manuscript preparation.
Meiqing Zhu, Shisuo Fan, Zhen Wang, Xiangwei Wu, and Jun Tang participated in UV-Vis and fluorescence
experiments. Jia Liu contributed greatly to the manuscript revision.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2016, 21, 1023
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Sample Availability: Samples of the ethyl 2-(3-formyl-4-hydroxyphenyl)-4-methylthiazole-5-carboxylate (NL
)
are available from the authors.
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