A selective and sensitive fluorescent sensor for cysteine detection based on bi-8-carboxamidoquinoline derivative and Cu2+ complex

Article abstract of DOI:10.1002/bio.3385

In this paper, a novel fluorescent sensor 1 for selective and sensitive detection of cysteine was developed based on a complex between bi-8-carboxamidoquinoline derivative ligand (L) and Cu²⁺. The interaction of Cu²⁺ with the ligand causes a dramatic fluorescence quenching most likely due to its high affinity towards Cu²⁺ and a ligand–metal charge transfer (LMCT) process. The in situ generated L–Cu² complex was utilized as a chemosensing ensemble for cysteine. In the presence of cysteine, the fluorophore, L, was released from L–Cu² complex because of the strong affinity of cysteine to Cu²⁺ via the Cu–S bond, leading to the fluorescence recovery of the ligand. The proposed displacement mechanism was confirmed by the results of mass spectrometry (MS) study. Under optimized conditions, the recovered fluorescence intensity is linear with cysteine concentrations in the range 1?×?10^?6?mol/l to 8?×?10^?6?mol/l. The detection limit for cysteine is 1.92?×?10^?7?mol/l. Furthermore, the established method showed a highly sensitive and selective response to cysteine among the 20 fundamental α-amino acids used as the building blocks of proteins, after Ni²⁺ was used as a masking agent to eliminate the interference of His. The proposed sensor is applicable in monitoring cysteine in practical samples with good recovery rate.

Full text of DOI:10.1002/bio.3385

Received: 28 April 2017
DOI: 10.1002/bio.3385
Revised: 2 July 2017
Accepted: 18 July 2017
R E S E A R C H A R T I C L E
A selective and sensitive fluorescent sensor for cysteine
detection based on bi‐8‐carboxamidoquinoline derivative and
2
+
Cu complex
Guoping Chai | Qingwen Liu | Qiang Fei | Jinling Zhang | Xiaoxiao Sun | Hongyan Shan |
Guodong Feng | Yanfu Huan
College of Chemistry, Jilin University,
Abstract
Changchun, People's Republic of China
In this paper, a novel fluorescent sensor 1 for selective and sensitive detection of cysteine was
Correspondence
Yanfu Huan, College of Chemistry, Jilin
University, Changchun 130023, People's
Republic of China.
developed based on a complex between bi‐8‐carboxamidoquinoline derivative ligand (L) and
2+
2+
Cu . The interaction of Cu with the ligand causes a dramatic fluorescence quenching most
2+
likely due to its high affinity towards Cu and a ligand–metal charge transfer (LMCT) process.
Email: yfhuan@jlu.edu.cn
2
The in situ generated L–Cu complex was utilized as a chemosensing ensemble for cysteine. In
Funding information
2
the presence of cysteine, the fluorophore, L, was released from L–Cu complex because of the
The State Major Project for Science and
Technology Development, China, Grant/
Award Number: No. 2013YQ47078102‐3;
The Program for the State Key Laboratory of
Inorganic Synthesis and Preparative Chemistry
Open Project, Jilin University, China, Grant/
Award Number: No. 2014‐07; The Natural
Science Foundation of Jilin Province, Grant/
Award Number: 20160101314JC; The
National Natural Science Foundation of China,
Grant/Award Number: No. 21207047; The
Science‐Technology Development Project of
Jilin Province of China, Grant/Award Number:
No. 20130206014GX, 20140623007TC and
2
+
strong affinity of cysteine to Cu via the Cu–S bond, leading to the fluorescence recovery of
the ligand. The proposed displacement mechanism was confirmed by the results of mass
spectrometry (MS) study. Under optimized conditions, the recovered fluorescence intensity is
−6
−6
linear with cysteine concentrations in the range 1 × 10 mol/l to 8 × 10 mol/l. The detection
−
7
limit for cysteine is 1.92 × 10 mol/l. Furthermore, the established method showed a highly
sensitive and selective response to cysteine among the 20 fundamental α‐amino acids used as
2+
the building blocks of proteins, after Ni was used as a masking agent to eliminate the
interference of His. The proposed sensor is applicable in monitoring cysteine in practical samples
with good recovery rate.
KEYWORDS
2
0150204017YY
bi‐8‐carboxamidoquinoline, cysteine, fluorescent sensor
[
7]
1
|
INTRODUCTION
and weakness. Elevated levels of Cys have been linked to neurotox-
[
8]
icity. It is also widely used in the food industry as an antioxidant and
[
9]
Among the 20 α‐amino acids used as the building blocks of proteins,
cysteine (Cys), a thiol‐containing amino acid, is nucleophilic and
easily oxidized, and plays numerous roles in biological processes. For
example, Cys plays a crucial role in maintaining biological redox
in drug formulations in the pharmaceutical industry. Therefore, the
detection of Cys with high specificity and sensitivity is significant in
both academic research and clinical applications.
Thus far, various methods based on analytical techniques have
been reported for the determination of Cys, including high perfor-
[
1,2]
[3,4]
homeostasis,
protein synthesis,
detoxification and metabo-
[5]
[10,11]
[12,13]
lism.
post‐translational modifications.
Cys is also related to biocatalysis, metal binding, and
mance liquid chromatography (HPLC),
[14]
colorimetric method,
[3,6]
[15,16]
More specifically, the abnormal
mass spectrometry,
electrochemical,
capillary electrophoresis
[18]
[
17]
levels of Cys are closely related to certain diseases. For instance, Cys
deficiency is implicated in slow growth in children, depigmentation of
hair, edema, lethargy, liver damage, loss of muscle and fat, skin lesions,
(CE)
and Fourier transform infra‐red (FT‐IR) spectroscopy.
These
approaches usually require sophisticated instrumentation and involve
cumbersome laboratory procedures, thereby limiting the scope of their
practical applications. Recently, great attention has focused on
fluorescent probes for Cys detection owing to their apparent advan-
tages of high sensitivity and specificity, low detection limit and cost,
Abbreviations used: DMF, dimethyl formamide; DMSO, dimethyl sulphoxide;
ESI, electrospray ionization; FTIR, Fourier transform infra‐red; NMR, nuclear
magnetic resonance; PBS, phosphate‐buffered saline; RSD, relative standard
deviation.
[
19–21]
and easy operation.
Therefore, several fluorescent probes for
Luminescence. 2017;1–8.
wileyonlinelibrary.com/journal/bio
Copyright © 2017 John Wiley & Sons, Ltd.
1

2
CHAI ET AL.
[
22]
the detection of Cys have been extensively investigated.
Various
Trap2000 (Applied Biosystems Corporation, American) without using
the liquid chromatography (LC) part.
fluorescent probes for Cys, utilizing the strong nucleophilicity of Cys
and their high binding affinity towards metal ions, have been
[
23–28]
reported.
Due to the strong affinity of Cys for metal ions, the
addition of Cys into a fluorophore–metal ion complex system may
release free fluorophores, and induce changes in the fluorescence
intensity and/or colour of the system.
2
.3
|
Synthesis of compound 2
Compound 2 was synthesized according to the published methods in
[
32]
Sensors are always designed containing an organic fluorophore as
a signaling unit linked with a binding unit. The N, O donors are among
the literature with slight modification.
(4.46 mmol) and potassium carbonate 1.849
were mixed in 10 ml anhydrous CH Cl , then the solution of the
2‐chloroacetyl chloride 0.504 ml (6.69 mmol) in 5 ml anhydrous
CH Cl was added dropwise under cooling by ice. The resulting
8‐Aminoquinoline 0.6430 g
g
(13.38 mmol)
[
29,30]
the most widely used binding units.
derivatives containing O, N, coordinating sites exhibit high
Furthermore, they exhibit good photostability
Bi‐8‐carboxamidoquinoline
2
2
N
[
31]
affinity to metal ions.
and can be synthesized easily.
2
2
[
32,33]
Consequently, the bi‐8‐
mixture was allowed to stir 2 h at room temperature. After filtration,
the solvent was concentrated under vacuum. The crude products were
carboxamidoquinoline derivatives seem to be ideal ligands in company
with metal ions to form the high selectivity sensing ensemble system
for Cys.
2 2
purified by column chromatography using CH Cl as eluant to afford a
white solid as compound 2 (Scheme 1). Yield: 0.7566 g (76.9%).
1
Herein, we report a new fluorescent sensor for the detection of
Cys based on a complex between bi‐8‐carboxamidoquinoline deriva-
3
H NMR (400 MHz, CDCl ) δ (ppm): 10.91 (s, 1H), 8.87 (dd, J = 4.1,
1.5 Hz, 1H), 8.76 (dd, J = 6.2, 2.6 Hz, 1H), 8.18 (dd, J = 8.3, 1.5 Hz,
2
+
tive ligand (L) and Cu
.
The ligand (L) was synthesized by the combina-
1H), 7.55 (dd, J = 9.3, 5.4 Hz, 2H), 7.48 (dd, J = 8.2, 4.2 Hz, 1H), 4.32
+
tion of 2,2′‐dihydroxybiphenyl and compound 2. Upon the addition of
(s, 2H). ESI‐MS: m/z [M + H] calc. For C11
H
9
2
N OCl, 221.7; found,
2+
2+
Cu , the complexation of Cu with the ligand (L) lead to fluorescence
221.1 (Figures and S3).
quenching most likely due to a ligand–metal charge transfer (LMCT)
2+
process. In the presence of Cys, the decomplexation of Cu from
2
2+
the L–Cu complex by forming a Cu –S bond lead to significant fluo-
2
.4
|
Synthesis of the ligand L
rescence enhancement. The proposed displacement mechanism was
2
2
,2′‐Dihydroxybiphenyl 0.0763 g (0.41 mmol), potassium carbonate
.2267 g (1.64 mmol) and compound 2 0.1990 g (0.902 mmol) were
confirmed by the results of MS study. Therefore, the L–Cu complex
0
can be utilized as a chemosensing ensemble for Cys, and shows a
highly sensitive and selective response to Cys among the 20 funda-
mental α‐amino acids.
mixed in 5 ml anhydrous dimethyl formamide (DMF) the resulting
mixture was kept stirring under a N atmosphere at 100°C for 1 h.
The mixture was then diluted by water and extracted with
Cl . The organic layer was dried over magnesium sulfate and
concentrated under reduced pressure. The crude products were
2
CH
2
2
2
2
|
EXPERIMENTAL
purified by column chromatography (CH
Cl
2 2
3
/CH OH = 20:1) to
1
.1
|
Reagents and chemicals
afford the ligand L (Scheme 1). Yield: 0.1610 g (70.8%). H NMR
(
400 MHz, CDCl
.57 (d, J = 2.6 Hz, 2H), 8.06 (d, J = 7.9 Hz, 2H), 7.55 (d,
J = 6.6 Hz, 2H), 7.46 (d, J = 4.2 Hz, 4H), 7.41–7.29 (m, 4H), 7.15
3
) δ (ppm): 10.48 (s, 2H), 8.68 (t, J = 4.3 Hz, 2H),
All chemicals were of analytical reagent grade and used without fur-
ther purification. 2,2′‐Dihydroxybiphenyl and 8‐aminoquinoline were
purchased from J&K Technology Co., Ltd (Beijing, China). Twenty α‐
amino acids including cysteine (Cys), glutamic acid (Glu), aspartic acid
8
13
(
t, J = 7.4 Hz, 2H), 7.06 (t, J = 10.4 Hz, 2H), 4.71 (s, 4H).
C
3
NMR (101 MHz, CDCl ) δ (ppm): 166.92, 154.89, 148.37, 138.61,
(
(
Asp), asparagine (Asn), phenylalanine (Phe), alanine (Ala), glutamine
Gln), threonine (Thr), serine (Ser), tyrosine (Tyr), lysine (Lys), trypto-
1
1
35.93, 133.54, 132.31, 129.08, 128.05, 127.80, 126.98, 122.22,
+
21.41, 117.07, 113.30, 68.75. ESI‐MS: m/z [M + H] calc. For
phan (Trp), proline (Pro), glycine (Gly), valine (Val), methionine (Met),
leucine (Leu), isoleucine (Ile), arginine (Arg), histidine (His) were also
purchased from J&K Technology Co., Ltd (Beijing, China). All the other
chemicals and reagents were purchased from commercial suppliers and
used as received without further purification. Double‐distilled water
was used throughout all experiments.
+
C
34
H
26
N
4
O
O
4
,
555.6, found, 555.2; [M
+
Na]
calc. For
C
H
34 26
N
4
4
Na, 577.6, found, 577.2 (Figures S2, S4 and S5).
2
.2
|
Apparatus
Fluorescence measurements were performed on
a RF‐5301PC
spectrophotofluorometer (Shimadzu Corporation, Japan). All pH
measurements were made with a Sartorius PB‐10 digital pH meter.
Nuclear magnetic resonance (NMR) spectra were performed on a
Varian Mercury YH‐300 spectrometer operated at 400 MHz.
Electrospray ionization (ESI) mass spectra were obtained using a Q‐
SCHEME 1 Synthesis of the ligand L

CHAI ET AL.
3
2
.5
|
Preparation of fluorescence spectroscopy
ligand L displayed a strong fluorescence maximum at 400 nm in PBS
2+
buffer solution. When 10 μM Cu was added into a 10 μM L solution,
measurements
2+
the complexation of L with Cu led to an obvious fluorescence
quenching, which could be ascribed to a ligand–metal charge transfer
The ligand L was dissolved in dimethyl sulphoxide (DMSO) to get a
1
mmol/l stock solution. The amino acids including cysteine, glutamic
(
LMCT) process. Upon the following addition of 8 μM Cys into the
acid, aspartic acid, asparagine, phenylalanine, alanine, glutamine, threo-
nine, serine, tyrosine, lysine, tryptophan, proline, glycine, valine, methi-
onine, leucine, isoleucine, arginine, histidine were prepared by
dissolving them in deionized water to get the corresponding concen-
tration of the solution. The solutions of copper ion and nickel ion were
prepared from the corresponding sulfate and nitrate salts. For real
samples detection, the miscellaneous flower honey and linden honey
produced by ChangchunTianyi Apiculture Co., Ltd were obtained from
supermarket. The sample solutions of honey were prepared by dissolv-
ing 5.0 g corresponding honey in deionized water and diluting it to the
mark in a 100 ml volumetric flask respectively. The sample solutions
were filtered through 0.45‐μm membrane prior to measurement. The
solutions were added in the following sequence with a total volume
2
L–Cu system, the fluorescence intensity increased significantly, indi-
2
+
cating the release of the fluorophore L by forming a Cu –S bond.
Thus, a turn‐on fluorescence sensor for Cys detection could be
established.
2
The sensing mechanism of L–Cu towards Cys was further con-
firmed by the ESI mass spectra. The ESI mass spectra of L (Figure S3)
showed two molecular ion peaks at m/z 555.2 and 577.2, which
+
+
corresponded to [L + H] and [L + Na] . As displayed in Figure S6, upon
2+
the addition of Cu into solution of L, the molecular ion peaks
2
+
corresponding to L–Cu
were observed in 616.0–620.0 and
6
38.0–641.9 Da mass range with a 1 Da mass difference due to the
[
34,35]
isotopic distributions of carbon and copper,
they could be
+
+
assigned to [L–H + Cu] and [L–H + Cu + Na] , respectively. This result
2+
of 3 ml: 30 μl ligand L (1 mM), acetonitrile, 30 μl Cu (1 mM), 30 μl
Cys or 500 μl honey sample solution, phosphate‐buffered saline
2
+
revealed a 1:1 stoichiometry complex between L and Cu . Further-
more, the Job's plot also demonstrated a 1:1 coordination ratio of L
(
PBS) buffer solution (water/acetonitrile, 9:1 v/v; PBS 0.02 mol/l;
2+
2+
and Cu (Figure S7). Upon the addition of Cys to the L–Cu system,
pH = 8.0), and then the resulting solutions were sonicated to make it
mixed evenly and incubated for 30 min at room temperature before
fluorescence spectroscopy measurement. The excitation wavelength
was 247 nm, and the emission spectra were scanned over the spectra
range of 340–473 nm. A quartz cell of 1 cm path length was used. The
slit widths of excitation and emission were both 5 nm.
ESI mass spectra analysis (Figure S8) displayed the peaks at 185.2,
+
+
5
55.2, and 577.3, which were assigned to [Cys + Cu + H] , [L + H]
+
and [L + Na] ; and the peaks in the 616.1–621.2 Da mass range with
+
a 1 Da mass difference were assigned to [L–H + Cu] . All this evidence
2+
firmly supported the complexation of
L
with Cu
and the
2
decomplexation of L–Cu by Cys, which led to the liberation of
fluorophore L.
3
3
|
RESULTS AND DISCUSSION
Fluorescence properties and sensing
2
+
3
.2
|
Fluorescence quenching of L by Cu
.1
|
2+
In order to investigate the quenching effect of Cu on the fluores-
mechanism
cence intensity of L, the fluorescence spectra of L in the presence of
2+
2
+
2
The fluorescence emission spectra of L, L–Cu and the L–Cu ‐Cys
system in a water/acetonitrile mixture (9:1 v/v; PBS 0.02 mol/l;
pH = 8.0) were recorded and the results are shown in Figure 1. The
serial concentrations of Cu in PBS buffer solution (water/acetoni-
trile, 9:1 v/v; PBS 0.02 mol/l; pH = 8.0) were recorded, and the results
are shown in Figure 2. The results demonstrate that the fluorescence
2+
FIGURE 1 Fluorescence emission spectra of 10 μM L (black line),
FIGURE 2 The quenching effect of a series concentrations of Cu on
the fluorescence intensity of 10 μM L in PBS buffer solution (water/
acetonitrile, 9:1 v/v; PBS 0.02 mol/l; pH = 8.0). The concentrations of
2+
2
1
0 μM L and 10 μM Cu (red line), and the fluorescent probe L–Cu
in the presence of 8 μM Cys (blue line) in PBS buffer solution (water/
acetonitrile, 9:1 v/v; PBS 0.02 mol/l; pH = 8.0)
2+
Cu are 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 μM, respectively

4
CHAI ET AL.
intensity of L decreases gradually with the increasing concentration of
2+
Cu and then remains approximately constant after addition of 1
2+
2+
equiv. of Cu (10 μM). Therefore, the 10 μM Cu concentration
was selected for future experiments.
3
.3
|
Optimization of experimental conditions for
Cys detection
For the sake of a steadier as well as a better fluorescence response,
several experimental conditions such as solvents, solvent composition,
pH values, buffer solutions, incubation time and incubation tempera-
ture were optimized.
3.3.1
|
Effect of solvents on the fluorescence response
FIGURE 4 Effect of different ratios of CH
3
CN to H
2
O solvents (PBS
To investigate the effect of solvents on the fluorescence response of
2
0.02 mol/l, pH = 8.0) on the fluorescence intensity of L–Cu in the
2
2
L–Cu to Cys, the fluorescence intensity changes of L–Cu in the
absence or presence of Cys were explored in five different common
laboratory organic solvents, including methanol, ethanol, acetonitrile,
DMSO, and DMF. The results are shown in Figure 3. From Figure 3,
absence (black line) and presence (red line) of Cys (8 μM)
3
.3.3
|
Effect of different pH on the fluorescence response
2
The fluorescence intensities of L–Cu in the absence or presence of
2
we can see that the fluorescence intensity of L–Cu in the presence
Cys within the pH range 4.0 to 12.0 (mediated by NaOH and a mixture
of Cys was most obviously enhanced in the acetonitrile system. Thus
we chose acetonitrile as the system solvent for subsequent
experiments.
3 4 3 3 3
of acid comprising H PO , CH COOH, H BO ) were explored. As
depicted in Figure 5, in the presence of Cys, there was an obvious
2
fluorescence off–on change of L–Cu with different fluorescence
enhancement in the pH range from 7.0 to 10.0. And the fluorescence
enhancement reached the maximum at pH 8.0. At lower pH, the
fluorescence intensity of L displayed an inconspicuous quenching
3
.3.2
|
Effect of solvent composition on the fluorescence
response
It was necessary to investigate the effect of different ratios between
2+
effect by Cu . At higher pH, an inconspicuous fluorescence enhance-
2
acetonitrile and water on the fluorescence response of L–Cu in the
2
ment of L–Cu system was displayed by Cys. Therefore, pH 8.0 was
absence or presence of Cys. In this part, the contents of acetonitrile
ranged from 0 to 70% were investigated and experimental results are
shown in Figure 4. Under the acetonitrile contents range from 0 to
chosen for the fluorescence intensity determination for further
experiments.
4
0%, it can be seen that the most fluorescence enhancement was
obtained when the acetonitrile content was 10% (acetonitrile:
water = 1:9). At acetonitrile contents ranging from 50% to 70%, the
fluorescence intensity displayed no obvious enhancement. Thus, the
acetonitrile content of 10% was chosen as an optimum experimental
condition.
3.3.4
response
|
Effect of buffer solutions on the fluorescence
In this section, the effect of the following buffer solutions on the fluo-
2
rescence response of L–Cu in the absence or presence of Cys was
2
2
FIGURE 3 The fluorescence intensity of L–Cu before (black bars) and
FIGURE 5 Effect of different pH on fluorescence intensities of L–Cu
after (red bars) the addition of Cys (8 μM) in different solvent/PBS
buffer solution (1:9, v/v), PBS 0.02 mol/l, pH = 8.0
in the absence (black line) or presence (red line) of Cys (8 μM) in
CH CN‐buffer solution (1:9, v/v)
3

CHAI ET AL.
5
determined and compared, including HEPES, PBS and Tris–HCl. The
results are shown in Figure 6. The PBS buffer solution could foster
the highest fluorescence enhancement in the presence of Cys at
pH 8.0. Therefore, pH 8.0 PBS buffer solution was selected as the
working buffer for the following experiments.
increasing. Therefore, an incubation time of 30 min was selected in
subsequent experiments.
3
.3.6
|
Effect of incubation temperature on the fluores-
cence response
In order to study the effect of incubation temperature on the fluores-
3.3.5
|
Effect of incubation time on the fluorescence
2
cence response of L–Cu in the absence or presence of Cys, experi-
response
ments were carried out in the temperature range 20–70°C. From
2
The effect of incubation time on the fluorescence response of L–Cu in
Figure 8, we found that temperature had no obvious interference on
2
the absence or presence of Cys was studied in PBS buffer solution
the fluorescence response of L–Cu system. In the presence of Cys,
(
water/acetonitrile, 9:1 v/v; PBS 0.02 mol/L; pH = 8.0) at room tem-
the fluorescence intensity remained nearly constant from 20 to 30°C.
However, with further increasing in temperature, the fluorescence
intensity began to decrease. Thus, 25°C was selected as the optimal
reaction temperature for subsequent experiments.
perature. As shown in Figure 7, the fluorescence intensity of L was
2+
sharply quenched by Cu within 1 min and remained nearly constant
2+
thereafter. Upon the addition of Cys after Cu accomplished the task
of quenching, it can be seen that the fluorescence intensity of the sys-
tem increased immediately and reached the maximum value within
In summary, taking every factor into consideration, PBS buffer
solution (water/acetonitrile, 9:1 v/v; PBS 0.02 mol/l; pH = 8.0) was uti-
lized for the following determinations after incubation for 30 min at
room temperature.
3
0 min, and then remained nearly constant with the incubation time
3
.4
|
Cys titration experiments
Under the optimum conditions, we studied the relationship between
2
the fluorescence recovery of the L–Cu system and the concentration
of Cys and the results are shown in Figure 9. Figure 9a shows that the
2
fluorescence intensity of the L–Cu system recovered gradually with
−
6
−6
increasing Cys concentration (from 1 × 10 mol/l to 8 × 10 mol/l)
and there was no obvious maximum fluorescence emission wavelength
shift. Figure 9b shows the linear relationship between the fluorescence
2
recovery of the L–Cu system and the Cys concentration in the range
−
6
−6
1
× 10 mol/l to 8 × 10 mol/l. The linear regression equation is as
−6
follows: F = 189.01 + 53.04[Cys] (10 mol/l), with the correlation
2
FIGURE 6 Effect of different buffer solutions on the fluorescence
coefficient (R) of 0.9988 (R = 0.9972). The detection limit for Cys is
−7
2
intensities of L–Cu in the absence (black bars) or presence (red bars)
1
.92 × 10 mol/l defined by the equation LOD = (3σ/s), where σ is
of Cys (8 μM); pH value of buffer solutions, 8.0; CH
solution (1:9, v/v)
3
CN:Buffer
the standard deviation of the blank signals (n = 20) and s is the slope
of the calibration curve.
FIGURE 7 Effect of incubation time on the fluorescence response of
FIGURE 8 Effect of incubation temperature on the fluorescence
2
2
L–Cu in the absence (black line) or presence (red line) of Cys (8 μM)
response of L–Cu in the absence (black line) or presence (red line) of
in PBS buffer solution (water/acetonitrile, 9:1 v/v; PBS 0.02 mol/l;
Cys (8 μM) in PBS buffer solution (water/acetonitrile, 9:1 v/v; PBS
pH = 8.0) at room temperature
0.02 mol/l; pH = 8.0)

6
CHAI ET AL.
2
FIGURE 10 Fluorescence intensity of L–Cu and its fluorescence
responses to Cys (8 μM) and other α‐amino acids (8 μM) in PBS
buffer solution (water/acetonitrile, 9:1 v/v; PBS 0.02 mol/l pH = 8.0)
2+
further addition of 8 μM Cys to the solution containing Ni and His
led to significant fluorescence enhancement. The fluorescence inten-
2
sity was comparable with the solution of L–Cu with equal amounts
2+
of Cys only. Thus Ni was chosen as a masking agent to eliminate
the interference of His.
To further check the practical applicability of this fluorescence
2
chemosensor L–Cu for Cys detection, competition experiments were
performed with above‐mentioned 20 α‐amino acids (Figure 11). When
2
L–Cu was treated with 8 μM Cys in the presence of the same concen-
tration of other α‐amino acids, the fluorescence intensity of the system
remained almost unchanged before and after the addition of other
interfering α‐amino acids, indicating that there was no interference
2
2
FIGURE 9 (a) the fluorescence spectra of L–Cu with the addition of
for the detection of Cys by L–Cu in PBS buffer solution (water/aceto-
−
6
−6
increasing concentrations of Cys (1 × 10 mol/l to 8 × 10 mol/l) in
PBS buffer solution (water/acetonitrile, 9:1 v/v; PBS 0.02 mol/l;
pH = 8.0). (b) the linear relationship between the fluorescence recovery
nitrile, 9:1 v/v; PBS 0.02 mol/l; pH = 8.0). These experimental results
2
also demonstrated that L–Cu could be an excellent fluorescence sen-
sor with a high selectivity for Cys over other competing α‐amino acids.
2
of L–Cu system and the concentrations of Cys
3
.5
|
Selectivity and competition studies
A significant feature of the sensor was its high selectivity towards Cys
over other competitive species. To evaluate the selectivity of the pro-
posed sensor, 20 naturally occurring α‐amino acids were determined.
2
The fluorescence intensities study of the L–Cu complex with 20 nat-
urally occurring α‐amino acids such as Cys, Glu, Asp, Asn, Phe, Ala, Gln,
Thr, Ser, Tyr, Lys, Trp, Pro, Gly, Val, Met, Leu, Ile, Arg, and His was car-
ried out in PBS buffer solution (water/acetonitrile, 9:1 v/v; PBS
0
.02 mol/l; pH = 8.0). As shown in Figure 10, significant fluorescence
enhancement was observed with the addition of 8 μM Cys. Mean-
while, no obvious changes in fluorescence intensities were observed
in the presence of equal amounts of other α‐amino acids except for
His. It is worthwhile to mention that sulfur‐containing methionine
had no apparent interference on the detection. As for the interference
2+
caused by His, we selected Ni as a masking agent due to the strong
2
FIGURE 11 Fluorescence intensity of L–Cu upon the addition of
8
2+
affinity between Ni and the imidazole group of His. In Figure S9, it
μM different α‐amino acids (black bars) and the subsequent
addition of 8 μM Cys to the mixture (red lines) in PBS buffer solution
(water/acetonitrile, 9:1 v/v; PBS 0.02 mol/; pH = 8.0)
2
can be seen that the fluorescence intensity of L and L–Cu showed
2+
negligible change following the addition of 10 μM Ni . Moreover,

CHAI ET AL.
7
TABLE 1 Results of the sample analysis and recoveries of spiked Cys
Technology Development, China (No. 2013YQ47078102‐3), the
Science‐Technology Development Project of Jilin Province of China
(Nos. 20130206014GX, 20140623007TC, and 20150204017YY), the
Natural Science Foundation of Jilin Province (20160101314JC)
and the Program for the State Key Laboratory of Inorganic Synthesis
and Preparative Chemistry Open Project, Jilin University, China
(No. 2014‐07).
in real samples
Cys added/
mol l
Recovery RSD(%)
(%)
−
1
−1
Sample
Cys found/mol l
Miscellaneous
flower
0
Not detected
—
—
honey
−
−
−
6
6
6
−6
3.00 × 10
5.00 × 10
7.00 × 10
2.99 × 10
5.08 × 10
7.06 × 10
99.7
101.6
100.9
1.27
1.49
1.52
−6
−6
ORCID
Linden honey
0
Not detected
—
−
6
6
6
−6
Yanfu Huan http://orcid.org/0000-0002-5111-3561
3.00 × 10
5.00 × 10
7.00 × 10
3.01 × 10
100.3
99.2
102.1
1.21
0.94
1.49
−
−
−6
−6
4.96 × 10
7.15 × 10
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