Rhodol-based far-red fluorescent probe for the detection of cysteine and homocysteine over glutathione

Article abstract of DOI:10.1002/bio.3152

The simultaneous discrimination of cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) is of great importance due to their roles in biology and close link to many diseases, especially via the development of a far-red fluorescent probe that could be used for rapid, selective, and sensitive detection of all three. Herein, we report the characterization of a far-red fluorescent probe with turn-on fluorescence properties and visible color changes that could be used for the detection of cysteine and homocysteine over glutathione. In this study we found that the sensor could discriminate cysteine and homocysteine over glutathione within 20?min. Function of this probe was based on the conjugate addition–cyclization reaction and showed a low detection limit to cysteine and homocysteine. Upon the addition of cysteine and homocysteine, the absorption band at 592?nm rose gradually and fluorescence was detected at 645?nm. The color changed from colorless to blue and fluorescence changed from absent to strong red fluorescence, which could be differentiated by the naked eye. All these unique features make this probe particularly potentially favorable for use in cysteine/homocysteine sensing and bioimaging applications. Copyright

Full text of DOI:10.1002/bio.3152

Research article
Received: 13 January 2016,
Revised: 01 April 2016,
Accepted: 01 April 2016
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.3152
Rhodol-based far-red fluorescent probe for the
detection of cysteine and homocysteine over
glutathione
Yunchang Liu, Kaiqiang Xiang, Baozhu Tian and Jinlong Zhang*
ABSTRACT: The simultaneous discrimination of cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) is of great importance due
to their roles in biology and close link to many diseases, especially via the development of a far-red fluorescent probe that could be
used for rapid, selective, and sensitive detection of all three. Herein, we report the characterization of a far-red fluorescent probe
with turn-on fluorescence properties and visible color changes that could be used for the detection of cysteine and homocysteine
over glutathione. In this study we found that the sensor could discriminate cysteine and homocysteine over glutathione within
20 min. Function of this probe was based on the conjugate addition–cyclization reaction and showed a low detection limit to
cysteine and homocysteine. Upon the addition of cysteine and homocysteine, the absorption band at 592 nm rose gradually and
fluorescence was detected at 645 nm. The color changed from colorless to blue and fluorescence changed from absent to strong
red fluorescence, which could be differentiated by the naked eye. All these unique features make this probe particularly potentially
favorable for use in cysteine/homocysteine sensing and bioimaging applications. Copyright © 2016 John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: cysteine; homocysteine; far-red emission; fluorescent probe; rhodol
cysteine and homocysteine using spirocyclization of xanthene
dyes. The ratiometric sensing of this probe toward Cys/Hcy was
Introduction
Biological thiols, including cysteine (Cys), homocysteine (Hcy) and
glutathione (GSH), play crucially important roles in many physiolog-
ical processes such as maintaining the appropriate redox status of
biological systems (1–3) and are closely related to many diseases
realized
by
utilizing
a
tandem
native
chemical
ligation/spirocyclization reaction to interrupt the large
π-conjugated system of the CB fluorophore, which caused a re-
markable blue shift in the spectra of the sensing system. Liu et al.
(4–6). Cys has been associated with neurotoxicity (7) and the lack
(31) applied a visible-light excitable excited-state intramolecular
of Cys could induce many diseases such as neurotoxicity, liver dam-
age, leucocyte loss and other syndromes (8). Conversely, elevated
Hcy levels are well known to be associated with vascular diseases
such as cardiovascular disease and Alzheimer’s disease (9). Due to
their important roles in biology and their close link to so many dis-
eases, it is of significant importance to develop a rapid, selective
and sensitive method for the detection of these two amino acids.
Fluorescent probes have always played important roles in the
detection of different biomolecules because of their significant
features such as spatiotemporal resolution in living systems (10),
high sensitivity, relatively simple analysis protocols (11–13) and
low cost. As a result, fluorescent sensors have become a hot topic
in the detection of different metal ions (14–16), cations (17), anions
proton transfer (ESIPT) dye (4′-dimethylamino-3-hydroxyflavone)
as the fluorophore and an acrylate group as the ESIPT blocking
agent to synthesize a ratiometric fluorescent sensor for the detec-
tion of Cys. Zhang et al. (32) presented a new selective fluorescent
and colorimetric chemosensor for the detection of GSH. The probe
could discriminate GSH from Cys and Hcy through two emission
channel detection systems and the detection limit of probe for
GSH reached 10 nM (3 ppb). Niu et al. (33) synthesized a ratiometric
fluorescent sensor based on monochlorinated BODIPY for highly
selective detection of glutathione (GSH) over cysteine (Cys)/homo-
cysteine (Hcy) and applied the probe for the detection of GSH in
cells. Finally, Han et al. (34) developed a NIR fluorescent probe with
high sensitivity and selectivity for the rapid detection of Cys over
homocysteine (Hcy) and glutathione (GSH) with an ultralow detec-
tion limit of 14.5 nM.
(18–20), and biomolecules (21–23). To date, the use of fluorescent
sensors for the detection of biomolecules (24) has received much
attention and the development of sensitive and specific probes
for thiols is of critical importance for understanding their roles in
disease. Several reviews on fluorescent probes for the detection
of thiols have been published previously (25–29). They have
discussed proposed mechanisms and strategies for the design of
fluorescent probes. However, it is still a challenge to discriminate
among thiol-containing molecules with their similar structures
and reactivities, especially between the Cys/Hcy and GSH. Re-
cently, several papers have reported the use of sensors for the dis-
crimination of Cys, Hcy and GSH. Lv et al. (30) designed a
ratiometric and selective fluorescence detection system for
*
Correspondence to: Jinlong Zhang, Key Lab for Advanced Materials and Insti-
tute of Fine Chemicals, East China University of Science and Technology,
Shanghai, 200237, China. Tel: Fax: +86 21 64252062. E-mail: jlzhang@ecust.
edu.cn
Key Lab for Advanced Materials and Institute of Fine Chemicals, East China
University of Science and Technology, Shanghai 200237, China
Abbreviations: ESIPT, excited-state intramolecular proton transfer; PBS, phos-
phate-buffered saline
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Copyright © 2016 John Wiley & Sons, Ltd.

Y. Liu et al.
Rhodol dyes have several advantages that make them good
candidates as fluorescent sensors in biological systems (35,36).
Their relatively high molar absorption coefficients and quantum
yields, high-intensity emission peaks, relative inertness under
physiologically relevant conditions and relatively low toxicity make
them excellent dyes for the design of sensors. The emission of
most rhodol dyes, however, is below 600 nm. The design and syn-
thesis of a far-red rhodol sensor for the detection of Cys/Hcy over
GSH is of great interest because it is very difficult to distinguish
GSH/Cys/Hcy due to the similar structures and reactivities of these
biothiols (37). Conversely, longer wavelength fluorescence would
not be disturbed by auto-fluorescence from endogenous biomole-
cules in living systems (38–40).
In this study, we designed and synthesized a far-red rhodol
probe for the discrimination of Cys/Hcy over GSH. Cys and Hcy in-
duced a substitution–rearrangement–cyclization reaction, but GSH
could not, which enabled Cys/Hcy be selectively distinguished
from GSH. The spirocyclic form of rhodol was colorless and nonflu-
orescent due to separation of the π-conjugation, whereas the ring
opening form showed strong spectroscopic signals in the absorp-
tion and fluorescence spectra by reacting with Cys/Hcy, as shown
in Scheme 1. The reaction increased sharply the absorption at
other chemicals were of the highest grade available and were used
without further purification. All employed solvents were analytical
pure and were employed without any further drying or purification.
Reactions were monitored by thin layer chromatography (TLC)
1
using Merck Millipore DC Kieselgel 60 F-254 aluminum sheets. H
13
nuclear magnetic resonance (NMR) and C NMR spectra were
recorded on Brucker AM-400 MHz instruments (Karlsruhe,
Germany) with tetramethylsilane as the internal standard. Low-
resolution electrospray ionization (ESI) mass analyses were
performed on a Waters LCT Premier XE spectrometer. UV–vis
absorption spectra were recorded on a Shimadzu UV–vis
spectrophotometer (Kyoto, Japan). Fluorescence spectra were
measured on a Shimadzu-5301PC (Kyoto, Japan) fluorescence
spectrophotometer.
Preparation and characterization of RH1
2-(4-Diethylamino-2-hydroxybenzoyl)benzoic acid 1 was prepared
according to the reported literature procedures (41,42). Next, a sus-
pension of 1 (469 mg, 1.5 mmol) and 1,6-dihydroxynaphthalene
(240 mg, 1.5 mmol) in methanesulfonic acid (10 mL) was stirred
at 90 °C for 12 h. The reaction mixture was cooled to room temper-
ature and then poured into ice-cold water (45 mL). The precipitate
was filtered, washed with brine (3 × 15 mL) and dried under
vacuum. The target compound was isolated by flash column
chromatography on silica gel using dichloromethane:methanol
5
92 nm and fluorescence at 645 nm, which could be differentiated
by the naked eye. The detection limit calculated for Cys and Hcy
reached 0.6 μM and 0.69 μM, respectively.
Experiments
(
20:3 v/v) for elution. The product produced was dark red in color,
1
which is the solid state. Yield: 300 mg. H NMR (400 MHz, DMSO-
d6) δ 10.13 (s, 1H), 8.45 (d, J = 8, 1H), 8.03 (d, J = 8, 1H), 7.75
(m, 2H), 7.36 (d, J = 8, 1H), 7.26 (m, 2H), 7.15 (d, J = 4, 1H), 6.73
(s, 1H), 6.59 (d, J = 8, 1H), 6.53 (m, 2H), 3.41 (q, 4H), 1.13 (t, J = 8,
Chemicals and instrumentals
3-(Diethylamino)phenol was purchased from Energy Chemical
(Shanghai, China) and used directly without any purification. The
Scheme 1. Equilibrium between the open form and spirocyclic form of rhodol.
Scheme 2. Synthesis procedure for the PRH probe.
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Rhodol-based far-red fluorescent probe
1
3
6
1
1
3
H) (Fig. S1). C NMR (100 MHz, DMDO-d6) δ 168.83, 157.27,
35.88, 135.47, 130.02, 128.65, 126.34, 124.67, 124.01, 123.74,
21.83, 118.90, 117.11, 109.29, 97.12, 56.02, 43.80, 40.08, 39.87,
9.67, 39.46, 39.25, 39.04, 38.84, 18.51, 12.34 (Fig. S2).
solvent, the residue was dissolved in dichloromethane
(50 mL). The solution was washed with brine (3 × 20 mL), dried
over anhydrous Na SO and evaporated. The crude product
2 4
was purified by chromatography on silica gel with
dichloromethane/alcohol (100:1 v/v) as the eluent. The probe
HRMS:438.1705, calcd for: 438.1705 (Fig. S5). Yield: 45.7%.
1
was in the solid state and white in color. Yield: 140 mg. H
NMR (400 MHz, CDCl ) δ 8.65 (d, J = 8, 1H), 8.05 (dd, J = 4,
3
Preparation and characterization of probe PRH
1
H), 7.61 (m, 2H), 7.41 (m, 2H), 7.16 (m, 1H), 6.78 (d, J = 8,
RH1 (218 mg, 0.5 mmol) and triethylamine 208 μL (1.5 mmol)
was dissolved in 15 mL dichloromethane in a 50 mL flask.
The mixture was stirred in an ice bath. Next, 128 μL (1.5 mmol)
acryloyl chloride in 5 mL dichloromethane was added to the
above solution under stirring. The whole mixture was stirred
in an ice bath for another 30 min and then continued to be
stirred overnight (43,44) (Scheme 2). After evaporation of the
1H), 6.66 (m, 3H), 6.4 (m, 2H), 6.07 (dd, J = 12, 1H), 3.40 (q,
J = 8, 4H), 1.21 (t, J = 4, 6H) (Fig. S3). C NMR (100 MHz, CDCl )
3
1
3
δ 135.00, 134.95, 133.00, 129.56, 128.95, 127.83, 127.00, 125.17,
124.91, 124.19, 124.09, 122.61, 122.01, 121.37, 118.55, 112.79,
108.94, 104.96, 97.68, 84.36, 77.38, 77.06, 76.74, 44.51, 12.57
(Fig. S4). HRMS: 492.1804, calcd for: 492.1811 (Fig. S6). Yield:
57%.
Figure 1. (a) pH effect on the fluorescence behavior of probe PRH (black line) and probe PRH with Cys (red line). (b) pH effect on the fluorescence behavior of probe PRH (black
line) and probe PC with Hcy (red line). Fluorescence was measured with excitation at 592 nm taken after 30 min. The spectroscopic properties of probe PRH were tested in aqueous
À5
PBS solutions (10 mM, pH = 7.4) containing 50% DMSO at a concentration of 10 M.
Figure 2. (a) Time-dependent absorption changes of probe PRH after addition of 5 equiv. Cys. (b) Change in absorption intensity at 592 nm over the time range 0–25 min. (c)
Time-dependent absorption changes of probe PRH following addition of 5 equiv. Hcy. (d) Changes of absorption intensity at 592 nm over the time range 0–30 min. The spectro-
À5
scopic properties of probe PRH were tested in aqueous PBS solutions (10 mM, pH = 7.4) containing 50% DMSO at a concentration of 10 M.
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Y. Liu et al.
produce a product capable of detecting Cys and Hcy. PRH ex-
hibited no fluorescence because of the spirocyclization of rho-
damine dyes, while strong fluorescence appeared when it
reacted with Cys or Hcy. The spectroscopic properties of probe
PRH were all tested in aqueous phosphate-buffered saline (PBS)
solutions (10 mM, pH = 7.4) containing 50% DMSO at a concen-
Results and discussion
Synthesis
The synthesis of probe PRH is outlined in Scheme 2. Relatively
high yields of PRH were achieved by a three-step process. 2-(4-
Diethylamino-2-hydroxybenzoyl) benzoic acid 1 was prepared
according to the literature procedures. RH was synthesized to
good yields and displayed excellent fluorescence. The PRH
probe was synthesized conveniently via condensation of RH
À5
tration of 10 M.
Effect of pH on the probe PRH
2 2
with acryloyl chloride in CH Cl , and its structure was con-
1
13
firmed by H NMR, C NMR, and HRMS spectra. Regulation
of the spirocyclization of rhodamine dyes could therefore
It is very important for a fluorescent probe to be stable over a wide
range of pH. At least, it must stable in a physiological environment
Figure 3. (a) Time-dependent fluorescence intensity changes of probe PRH after addition of 5 equiv. Cys. (b) Fluorescence intensity changes at 645 nm excited by 592 nm in the
time range 0–22 min. (c) Time-dependent fluorescence intensity changes of probe PRH after addition of 5 equiv. Hcy. (d) The fluorescence intensity changes at 645 nm excited by
5
92 nm in the time range 0–30 min. The spectroscopic properties of probe PRH were tested in aqueous PBS solutions (10 mM, pH = 7.4) containing 50% DMSO at a concentration
À5
of 10 M.
Figure 4. (a) Absorption of probe PRH in the presence of different amino acids including Ala, Alg, Asp, Gln, Glu, Gly, His, Ile, Met, Phe, Pro, Ser, Thr, Trp, 3-nitrobenzenthiol, 3-
aminobenzenthiol and 2-mercaptoethanol. (b) Fluorescence intensity of probe PRH in the presence of different amino acids excited at 592 nm. The tested amino acids are the
same as used in the absorption experiment. The spectroscopic properties of probe PRH were tested in aqueous PBS solutions (10 mM, pH = 7.4) containing 50% DMSO at a con-
À5
centration of 10 M.
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Rhodol-based far-red fluorescent probe
because pH varies over a wide range in nature including a slight
variation in the human body. Ester groups are susceptible to pH
change, as both acidic and basic media can promote the hydrolysis
of ester groups. Therefore, the effect of pH on a probe is the first
variable tested. The pH effect on the fluorescence behavior of
probe (PRH) and probe PRH with Cys or Hcy is shown in Fig. 1.
PBS solutions were adjusted to a wide pH range from 2 to 12
The response time towards Cys and Hcy
To evaluate when the probe would become saturated after Cys or
Hcy addition, time-dependent absorption and fluorescence inten-
sity changes of probe PRH after the addition of 5 equiv. Cys or Hcy
À5
were tested. The probe at a concentration of 10 M was dissolved
in aqueous PBS solutions (10 mM, pH = 7.4) containing 50% DMSO.
Next, 5 equiv. Cys or Hcy was added to the solution, the absorption
changes are shown in Fig. 2. It can be seen in Fig. 2(a) that probe
PRH exhibits nearly no absorption at 592 nm before Cys addition.
However, absorption at 592 nm increases sharply after 1 min fol-
lowing the addition of 5 equiv. Cys, after which time absorption
at 592 nm increased gradually. The absorption changes at
592 nm versus time were measured and are shown in Fig. 2(b). It
can be seen that the probe became saturated after 10 min. Simi-
larly, 5 equiv. Hcy was also added to another solution and the ab-
sorption changes recorded. Hcy addition had similar effects on
probe PRH, shown in Fig. 2(c), however in this case PRH became
saturated after 25 min. The relationship between absorption
changes and time was measured and is shown in Fig. 2(d).
(
10 mM, pH = 7.4). Then, probe PRH was dissolved in the prepared
PBS solutions (10 mM, pH = 7.4) using 50% DMSO as good solvent;
equiv. of Cys (Fig. 1a) or Hcy (Fig. 1b) were added to the above
5
solutions, respectively. The spectra of probe PRH and probe PRH
with Cys or Hcy were recorded after 20 min and the fluorescence
spectra were collected as shown in Fig. 1. It can be seen that probe
PRH is stable over a wide pH range from 2 to 10. When the pH was
higher than 10, hydrolysis occurred, which resulted in an increase
in fluorescence. This result proves that probe PRH can be used in a
physiological environment. Probe PRH with Cys or Hcy over a wide
pH range was also tested. Over a pH range of 6 to 12, the probe
could detect Cys or Hcy, and therefore it could be used in physio-
logical environments.
Figure 5. (a) Color changes of solutions following the addition of different amino acids. These are, from left to right, Ala, Arg, Asp, Gln, Glu, Gly, His, Cys, Hcy, GSH, Ile, Met, Phe, Pro,
Ser, Thr and Trp. Other amino acids were added at 20 equiv. to the probe and Cys, Hcy or GSH were added at 5 equiv. (b) Fluorescence changes of the solutions following the
addition of different amino acids. The tested amino acids are the same as in the absorption experiment.
Figure 6. Effect of different amino acids. The fluorescence intensity changes of probe PRH in the presence of 20 equiv. of various of amino acids. The short bar on the left is the
fluorescence intensity of probe PRH with other amino acids at a concentration of 10 μM. The ions include Ala, Alg, Asp, Gln, Glu, Gly, His, Ile, Met, Phe, Pro, Ser, Thr and Trp. The high
bar on the right is the fluorescence intensity of probe PRH following the addition of 5 equiv. Cys and Hcy to the above solutions respectively. (a) Effect of different amino acids on
Cys. (b) Effect of different amino acids on Hcy. The spectroscopic properties of probe PRH were measured in aqueous PBS solutions (10 mM, pH = 7.4) containing 50% DMSO at a
À5
concentration of 10 M.
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Y. Liu et al.
À5
Likewise, fluorescence was also tested. Here, 5 equiv. Cys or Hcy
was added to the solution and the fluorescence spectra recorded.
Fluorescence followed the same trend subsequent to the addition
of the tested species. It can be seen in Fig. 3(a) that probe PRH had
nearly no fluorescence prior to Cys addition. However, fluores-
cence at 645 nm increased sharply at 1 min after Cys addition
and continued to increase with time. The relationship between
fluorescence intensity and time is shown in Fig. 3(b). It can be seen
that the probe became saturated after 10 min. Also, Hcy had a sim-
ilar effect on probe PRH, as can be seen in Fig. 3(c). The plot of fluo-
rescence intensity versus time demonstrated that the probe
became saturated after 25 min as shown in Fig. 3(d). All of these
results demonstrated the rapid detection of Cys/Hcy and therefore
this probe could be used for practical application. The color
changed from colorless to blue, as seen by the naked eye and
fluorescence changed from absent to strong red fluorescence.
10 M concentration in aqueous PBS solutions (10 mM, pH = 7.4)
containing 50% DMSO. Next, 20 equiv. of other amino acids and 5
equiv. of Cys, Hcy or GSH were added respectively. Each spectrum
was recorded 20 min following addition of the amino acid and the
fluorescence spectra were collected with the excitation at 592 nm.
It can be seen from the absorption shown in Fig. 4(a) that the ab-
sorption spectra showed little change following the addition of 20
equiv. of other amino acids; addition of GSH had a slight effect on
the absorption. However, absorption at 592 nm clearly increased
following the addition of Cys/Hcy and the color changed from col-
orless to blue which could be differentiated by the naked eye, as
shown in Fig. 5(a). The fluorescence spectra followed the same
trend as the absorption spectra. It can be seen in Fig. 4(b) that
nearly no fluorescence could be seen after the addition of other
amino acids except for GSH; when excited at 592 nm, probe PRH
showed strong red fluorescence (Fig. 5b) after the addition Cys
or Hcy. There was an emission band at 645 nm. All these data dem-
onstrated that probe PRH has good selectivity towards Cys/Hcy
over other amino acids. Thus, the probe has Cys/Hcy specificity.
Specificity for one good probe is not sufficient. Many amino
acids are mixed together in the human body, therefore it is very
important to be able to detect specific species without interfer-
ence from other amino acids, especially when other amino acids
are present at higher concentrations. The effect of the presence
of other amino acids was therefore tested. First, probe PRH was dis-
Selectivity towards different amino acids
It is very important for a probe to selectively detect certain amino
acids. The selectivity towards different amino acids and sulfhydryl
is tested. The selectivity towards different amino acids is shown in
Fig. 4. Ala, Arg, Asp, Gln, Glu, Gly, His, Phe, Met, Ile, Pro, Ser, Thr or
GSH was used as the source of other tested species,
3
-nitrobenzenthiol, 3-aminobenzenthiol and 2-mercaptoethanol
À5
wer used as sulfhydryl and Cys and Hcy were used as the tested
species. All the amino acids were dissolved in distilled water and
sulfhydryl was dissolved in ethanol. The probe was dissolved at a
solved in PBS containing 50% DMSO at a concentration of 10 M;
20 equiv. of different amino acids were added to the above solu-
tion respectively. The fluorescence was tested and recorded after
Figure 7. (a) Fluorescence intensity changes with addition of different equivalents of Cys with excitation at 592 nm taken after 30 min. (b) Fluorescence intensity changes versus
concentration of Cys at 645 nm with excitation at 592 nm taken after 30 min. (c) Fluorescence intensity changes with addition of different equivalents of Hcy with excitation at
5
92 nm taken after 30 min. (d) Fluorescence intensity changes versus concentration of Hcy at 645 nm with excitation at 592 nm taken after 30 min. The spectroscopic properties
À5
of probe PRH were tested in aqueous PBS solutions (10 mM, pH = 7.4) containing 50% DMSO at a concentration of 10 M.
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Rhodol-based far-red fluorescent probe
Figure 8. Proposed response mechanism of PRH to Cys and Hcy.
2
2
0 min, shown in Fig. 6. The fluorescence intensity after addition of
0 equiv. of other amino acids is shown on the left bar of the
et al. (3) amd Han et al. (45). Rhodol exhibited no fluorescence
when connected to an acrylate group, because rhodamine is in
the spirocyclic form which breaks the π-conjugation, whereas the
ring opening form showed strong spectroscopic signals in the fluo-
rescence spectra by reacting with Cys/Hcy, as shown in Fig. 8. The
mechanism of PRH responding to Cys and Hcy is based on conju-
gated addition of Cys/Hcy to an α,β-unsaturated carbonyl moiety,
which generated intermediate and intramolecular cyclization giv-
ing the desired fluorescence dye of far-red rhodol. To further prove
the mechanism of the reaction, the reaction of probe PRH with Cys
was conducted under the same conditions and the products were
subjected to mass spectral analysis. The peak at m/z 438.1710
corresponding to compound 1 was observed, which proved the
proposed mechanism (Fig. S7).
figure. All solutions showed close to no fluorescence; 5 equiv. of
Cys (Fig. 6a) and Hcy (Fig. 6b) were added to the above solutions
respectively. Fluorescence intensity investigated and collected
after 20 min is shown in Fig. 6. The fluorescence intensity after
adding 5 equiv. of Cys and Hcy corresponds to the right bar in
the figure. It can be seen that probe PRH can still detect Cys and
Hcy even with addition of quantities of amino acids. These results
proved that sensing for Cys/Hcy was hardly affected by the pres-
ence other amino acids, as can be seen in Fig. 6. There was little
influence on the detection of Cys/Hcy even with quantities of other
amino acids.
Determination of the detection limit
To calculate the detection limit, titration of Cys (0.2–1 μmol) and
Hcy (0.2–1 μmol) with the probe PRH solution was tested. Probe
Conclusions
PRH is dissolved in PBS solutions (10 mM, pH = 7.4) containing
In summary, we report a far-red fluorescent probe with a turn-on
fluorescence property and visible color changes, which can be dif-
ferentiated by the naked eye for the detection of Cys and Hcy over
GSH. The probe was based on the conjugate addition–cyclization
reaction and showed a low detection limit for Cys and Hcy. Upon
the addition of Cys or Hcy to the solution, the absorption band
at 592 nm increased gradually and fluorescence was seen at
645 nm. The color changed from colorless to blue and the fluores-
cence from no fluorescence to strong red fluorescence, which
could be differentiated by the naked eye. All these unique features
make it a particularly potential favorite for use in Cys/Hcy sensing
and bioimaging applications.
À5
5
0
0% DMSO at a concentration of 10 M. 0.2 μmol, 0.4 μmol,
.6 μmol, 0.8 μmol or 1 μmol Cys or Hcy was added to the solution
respectively. Upon the titration of Cys and Hcy, the fluorescence at
45 nm increased gradually as shown in Fig. 7(a) and Fig. 7(c), re-
6
spectively. The detection limit was calculated based on the
method reported in the literature (40). The detection limit was cal-
culated using the equation: detection limit =3σ/k. Where σ is the
standard deviation of blank measurement, k is the slope between
the fluorescence intensity versus amino acids concentration. The
fluorescence emission spectrum of PRH was measured 10 times
to obtain the standard deviation σ of the blank measurement. To
get the plot, the fluorescence intensity at 645 nm was plotted as
a concentration of Cys (0.2–1 μM) and Hcy (0.2–1 μM) as shown
in Fig. 7(b) and Fig. 7(d), respectively. The detection limit was calcu-
lated to be 0.6 μM for Cys and 0.69 μM for Hcy, respectively.
Acknowledgements
This work has been supported by the National Natural Science
Foundation of China (21277046, 21173077, 21377038), the
Shanghai Committee of Science and Technology (13NM1401000),
the Research Fund of Education of the People’s Republic of China
( JPPT-125-4-076), and the National Basic Research Program of
China (973 Program, 2013CB632403).
The proposed mechanism for the detection of Cys/Hcy
The acrylate group in probe PRH has been known as an efficient
reaction site for Cys and Hcy since it was reported by Zhang
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Y. Liu et al.
2
5. Niu LY, Chen YZ, Zheng HR, Wu LZ, Tung CH, Yang QZ. Design strate-
gies of fluorescent probes for selective detection among biothiols.
Chem Soc Rev 2015;44:6143–60.
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Additional supporting information may be found in the online ver-
sion of this article at the publisher’s web site.
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