A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and biothiols

Article abstract of DOI:10.1016/j.saa.2019.117770

A new long-lived Donor–Acceptor (D–A) fluorophore based on carbazolyl dicyanobenzene was developed as an ON–OFF–ON multifunctional fluorescent probe 1 for sequential detection of Cu²⁺ and biothiols (Cys, Hcy and GSH). The fluorescence of probe

Full text of DOI:10.1016/j.saa.2019.117770

SAA-117770; No of Pages 8
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
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Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A long-lived Donor–Acceptor fluorescent probe for sequential detection
of Cu2+ and biothiols
b
c
Duobin Chao a, , Yaping Pan , Xue-Wang Gao
⁎
a
School of Materials Science and Chemical Engineering, Ningbo University, Zhejiang 315211, China
School of Petroleum and Chemical Engineering, Dalian University of Technology, Panjin, Liaoning 124221, China
Key Laboratory of Photochemical Conversion and Optoelectronic Materials Technical Institute of Physics and Chemistry, University of Chinese Academy of Sciences Chinese Academy of Sciences,
b
c
Beijing 100190, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new long-lived Donor–Acceptor (D–A) fluorophore based on carbazolyl dicyanobenzene was developed as an
ON–OFF–ON multifunctional fluorescent probe 1 for sequential detection of Cu2+ and biothiols (Cys, Hcy and
GSH). The fluorescence of probe 1 can be significantly and selectively quenched by Cu2+. Meanwhile, the fluores-
cence lifetime decreased from 2.1 μs to 18.5 ns. The limit of detection was determined to be 33.6 nM. Upon ad-
dition of biothiols (Cys, Hcy and GSH), the generated ensemble 1-Cu2+ displayed a “turn-on” fluorescent
response at 555 nm and an obvious recovery in fluorescence lifetime and UV–vis absorption within 1 min. The
limit of detection for Cys, Hcy and GSH were calculated by fluorescence titration experiments to be 0.19, 0.21
and 0.29 μM, respectively. The ensemble 1-Cu2+ was further successfully applied in bioimaging.
© 2019 Elsevier B.V. All rights reserved.
Received 25 August 2019
Received in revised form 25 October 2019
Accepted 4 November 2019
Available online xxxx
Keywords:
TADF
Donor–Acceptor
Sensor
Copper
Biothiol
1. Introduction
liver damage, muscle and fat loss [14,15]. GSH is an important tripeptide
in many biochemical processes, and GSH level in the cellular is associ-
The identification of transition metal ions has attracted significant
interest in recent years due to its crucial roles in environmental and bi-
ological systems [1,2]. Cu2+ is one of the most abundant and indispens-
able metal elements, playing important roles in many biological
systems, and metabolic processes [3]. However, the disordered level of
Cu2+ may destroy the balance of reactive oxygen species, and in turn
exhibit serious toxicity, [4] resulting in many serious health problems,
such as Wilson's, Parkinson's, and Alzheimer's disease [5–10]. Besides,
a large amount of Cu2+ in life and industry could also cause serious en-
vironmental pollution. Therefore, monitoring Cu2+ in the human body
and the environment is an urgent task.
On the other hand, biothiols such as cysteine (Cys), homocysteine
(Hcy) and glutathione (GSH) play important roles in many biological
processes, like the cellular antioxidant defense and the growth of tissues
in living system [11–13]. It has been reported that irregular level of
biothiols is related to some diseases. Cys deficiency may cause many
syndromes, such as retarded growth, hair depigmentation, lethargy,
ated with several diseases, such as leucocyte loss and HIV infection
[16,17]. High levels of Hcy in human plasma are risk factors for disorders
including cardiovascular and Alzheimer's diseases, neural tube defects
and mental disorders [18,19]. Therefore, it is of great interest to develop
efficient methods to detect biothiols in biological systems.
Up to date, a variety of methods have been established to detect Cu2
+
and biothiols (Cys, Hcy and GSH), including inductively coupled
plasma mass spectrometry, high-performance liquid chromatography,
electrochemical assays and fluorescence spectrometry [20–23]. Among
these methods, fluorescence spectrometry has been recognized as one
of the most promising techniques, owing to its high sensitivity, selectiv-
ity, simplicity of operation and short response time [24–27]. The re-
ported fluorescent probes for the detection of biothiols were designed
with various mechanisms, such as Michael addition reaction, [28–30]
cyclization reaction, [31,32] cleavage reaction, [33–35] and others
[36,37]. Recently, much attention has been paid to indicator displace-
ment assays (IDA) between biothiols and Cu2+ complex [38,39]. The
probe designed by this method can simultaneously detect Cu2+ and
biothiols. The procedure relies on the fluorescence quenching property
of Cu2+ and high affinity between Cu2+ and biothiols. That is to say, the
⁎
Corresponding author.
E-mail address: chaoduobin@nbu.edu.cn (D. Chao).
https://doi.org/10.1016/j.saa.2019.117770
1386-1425/© 2019 Elsevier B.V. All rights reserved.
Please cite this article as: D. Chao, Y. Pan and X.-W. Gao, A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and
biothiols, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117770

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D. Chao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
addition of biothiols induced the Cu2+ to detach from the ensemble, re-
leasing the probe and leading to the change of fluorescence. Taking ad-
vantage of this strategy, various luminescent probes were obtained with
different kinds of fluorophores, such as coumarin, [40,41] imidazole,
[42] benzothiazole derivative, [43] fluorescein [44]. However, these
fluorophores are mostly limited by short fluorescence lifetimes (in the
nanosecond range) and may display poor performance in fluorescence
lifetime imaging. In contrast, long-lived organic fluorophores are helpful
for avoiding the interference of autofluorescence background and dam-
age of cells in biological systems, which could also be good candidates in
fluorescence lifetime imaging and detection [45]. Thus, to develop or-
ganic fluorophores with long-lived is a matter of interest.
In 2012, Adachi's group reported thermally activated delayed
fluorescence (TADF) organic compounds for realizing organic light-
emitting diodes [45]. Thermally activated delayed fluorescence (TADF)
molecules undergo efficient reverse intersystem crossing (RISC) from a
triplet excited state to a singlet state, so these molecules generally ex-
hibit long fluorescence lifetimes. At present, thermally activated delayed
fluorescence (TADF) molecules, such as carbazolyl dicyanobenzene
(CDCBs) derivatives, which comprises carbazole as the electron donor
and dicyanobenzene as the electron acceptor, are mainly used in OLED,
[46] biosensing [47] and photocatalytic [48,49]. The long-lived D–A
fluorophores based on carbazolyl dicyanobenzene (CDCB) is an inspira-
tion for us to design fluorescent probes.
Therefore, we designed and synthesized a long-lived probe 1 with
D–A fluorophores using the distinguished properties of carbazolyl
dicyanobenzene (CDCB) for the efficient recognition of Cu2+ and
biothiols, in which carbazole and dicyanobenzene were used as the
electron donor and the electron acceptor, respectively (Scheme 1).
Terpyridine groups were introduced as a recognition group due to
good coordination ability. The interaction between the metal and the li-
gand results in a unique structure that the final structure does not pos-
sess [50]. In this work, in the presence of Cu2+, the fluorescence of probe
1 was selectively quenched and the fluorescence lifetime also de-
creased. Due to the high affinity between Cu2+ and biothiols, the spec-
tral signals and fluorescence lifetime were restored after the addition of
biothiols. Ensemble 1-Cu2+ has also been successfully applied in
bioimaging. To the best of our knowledge, this is the first report
regarding the use of carbazolyl dicyanobenzene (CDCB) derivatives as
long-lived probe for efficiently detecting biothiols and Cu2+
.
2. Experimental section
2.1. Materials and instruments
All solvents and starting materials were purchased from commercial
suppliers and used with no further purification. 1H NMR and 13C NMR
spectra were recorded by a Bruker Avance 500 MHz spectrometer at
room temperature. High–resolution mass spectra (HRMS) were mea-
sured by a Thermofisher Q–Exactive instrument equipped with an ESI
ion source. UV–vis absorption spectra were recorded with AOE instru-
ment UV–1800PC spectrophotometer. Fluorescence spectra studies
were carried out with Lengguang F97PRO spectrophotometer. Transient
photoluminescence decay spectra were recorded on K287 Full-featured
steady-state/transient fluorescence spectrometer. Confocal Laser Scan-
ning Microscope (CLSM) experiments were carried out using ZEISS
LSM 510.
2.2. Synthesis
2.2.1. Synthesis of Cz–tpy
Tpy1 and Tpy2 were synthesized according to previous proce-
dure, [51] as shown in Scheme 1. Tpy2 (1 mmol), K2CO3 (2 mmol)
and 9H-carbazol-4-ol (1.5 mmol) were dissolved in 5 mL anhydrous
dimethylformamide (DMF). The mixture was stirred at 75 °C for 24 h
under nitrogen atmosphere. After cooling to room temperature, the
solution was poured into 300 mL cold water and filtered. The crude
product was isolated by silica gel column chromatography using
CH2Cl2/CH3OH, (100:1, v/v) as eluent to produce a white solid.
Yield: 50%. 1H NMR (500 MHz, DMSO–d6), δ (ppm): 11.31 (s, 1H),
8.78 (d, J = 4.6 Hz, 4H), 8.69 (d, J = 7.9 Hz, 2H), 8.21 (d, J =
7.8 Hz, 1H), 8.05 (dd, J = 8.8, 7.2 Hz, 4H), 7.83 (d, J = 7.9 Hz, 2H),
7.54 (ddd, J = 7.6, 4.7, 1.2 Hz, 2H), 7.48 (d, J = 8.1 Hz, 1H),
7.38–7.30 (m, 2H), 7.20–7.14 (m, 1H), 7.12 (d, J = 8.0 Hz, 1H), 6.85
(d, J = 7.9 Hz, 1H), 5.48 (s, 2H). 13C NMR (125 MHz, DMSO–d6), δ
(ppm): 156.20, 155.42, 154.98, 149.83, 149.64, 141.67, 139.46,
137.97, 137.36, 128.83, 127.65, 126.93, 125.11, 125.04, 122.67,
122.12, 121.44, 119.24, 118.41, 112.06, 110.96, 104.67, 101.52,
69.36. HRMS (m/z): found 527.1843 for [M + Na]+ (calcd. for
C
34H24N4NaO: 527.1842).
2.2.2. Synthesis of 1
To a stirred solution of Cz–tpy (1 mmol) in dehydrated THF at room
temperature under a nitrogen atmosphere, NaH (4 mmol) was added.
After stirring for 30 min, 2,4,5,6-tetrafluoroisophthalonitrile (0.2 mmol)
was added to the above solution. The reaction mixture stirred at room
temperature for 18 h. The reaction was quenched with water (2 mL).
The mixture was concentrated under reduced pressure, extracted with
CH2Cl2, dried over Na2SO4 and evaporated in vacuo to obtain the crude
product. The crude product was isolated by alumina column chromatog-
raphy using n-hexane/dichloromethane as eluent to produce orange
solid. Yield: 45%. m.p. 200.3 °C. 1H NMR (500 MHz, DMSO–d6), δ
(ppm): 8.77 (d, J = 9.4 Hz, 4H), 8.74–8.56 (m, 18H), 8.37 (d, J =
7.8 Hz, 1H), 8.21 (d, J = 8.1 Hz, 1H), 8.09–7.61 (m, 30H), 7.58–7.08 (m,
23H), 6.99–6.70 (m, 6H), 6.54 (q, J = 7.5, 6.3 Hz, 1H), 5.59 (s, 2H), 5.32
(s, 4H), 5.17 (t, J = 8.6 Hz, 2H). 13C NMR (125 MHz, DMSO–d6). δ
(ppm): 156.17, 156.04, 155.95, 155.40, 155.35, 155.33, 154.42, 149.79,
149.70, 149.34, 146.43, 140.69, 139.69, 138.97, 138.74, 138.73, 138.66,
137.91, 137.80, 137.78, 137.73, 137.53, 137.28, 129.02, 128.73, 128.65,
128.30, 127.70, 127.43, 127.42, 127.22, 124.99, 124.89, 123.01, 122.67,
122.06, 121.40, 121.33, 121.28, 118.40, 118.26, 118.18, 117.67, 113.10,
112.88, 112.59, 111.34, 104.92, 104.68, 63.04, 52.48, 45.87, 40.49, 40.42,
40.33, 40.25, 40.16, 40.08, 39.99, 39.83, 39.66, 39.49. HRMS (m/z):
found 2175.7148 for [M + K]+ (calcd. for C144H92N18KO+4 : 2175.7186).
Scheme 1. Synthesis of fluorescent probe 1.
Please cite this article as: D. Chao, Y. Pan and X.-W. Gao, A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and
biothiols, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117770

D. Chao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
3
Elemental Anal. Calcd. for C144H92N18O4: C, 80.88; H, 4.34; N, 11.79.
Found: C, 80.62; H, 4.53; N, 11.27.
2.2.3. Measurement procedures
A stock solution of 1 (0.5 mM) was prepared in DMSO and stored in
dark at 4 °C. The solution of 1 was appropriately diluted to be (1.5 μM) in
CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) for all spectral mea-
surements. The solutions of various metal ions and anions were pre-
pared in purified water. 1-Cu2+ solution for the detection of biothiols
and other amino acids was prepared by addition of 5.0 equiv. Cu2+ to
1 (1.5 μM) solution in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1 v/v).
3. Results and discussion
3.1. Spectroscopic studies of 1 in the presence of Cu2+
Firstly, to verify the detection behaviour of 1 toward Cu2+, the fluo-
rescence titration experiments were conducted. As shown in Fig. 1a, 1
displayed a remarkable emission band at 555 nm (λex = 400 nm) in
CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v). Due to the strong
bonding ability of the terpyridine units toward Cu2+, the fluorescence
intensity of probe 1 (1.5 μM) continuously decreased with increasing
concentrations of Cu2+ (0–7.5 μM). The quenching effect can be attrib-
uted to effective combination between 1 and paramagnetic Cu2+
[52,53]. Moreover, the fluorescence intensity of probe 1 was linearly re-
lated to Cu2+ in the concentration range from 0.3 to 1.8 μM (R2 = 0.99)
(Fig. 1b). The limit of detection of 1 toward Cu2+ reached 33.6 nM based
on a 3σ/k, which is much lower than the limit specified by the World
Health Organization (WHO) guideline for drinking water (30 μM)
[54,55]. Then, the interaction between 1 and Cu2+ was investigated by
the UV–vis absorption spectra in CH3CN/HEPES buffer (10 mM, pH =
7.4, 1:1, v/v). As shown in Fig. 2, 1 exhibited strong absorption bands
at 325 nm and 282 nm, while a weak band at around 379 nm was ob-
served (Inset of Fig. 2), which could be attributed to the π − π* transi-
tion and intramolecular charge transition (ICT), respectively. Upon
addition of Cu2+ (0–7.5 μM), the absorption at 282 nm gradually de-
creased, accompanied by the emergence of new absorption bands
around 349 nm. The new absorption peak at 349 nm could be ascribed
to ligand-metal charge transfer (LMCT), [56] which indicated the forma-
Fig. 2. Absorption spectra of 1 (1.5 μM) in the presence of different amounts of Cu2+ (0–7.5
μM) in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v). Inset: the absorption spectra of 1
(1.5 μM).
and the solution containing Cu2+ showed no emission under UV light
(Fig. 3b). In addition, as shown in Fig. S1, in the presence of various in-
terfering ions, it does not affect the response of probe 1 toward Cu2+
.
These results indicate that compound 1 has high selectivity toward
Cu2+. Additionally, the influence of pH on the detection of Cu2+ is
shown in Fig. 4. The emission intensity of 1 remained stable over a
broad pH range of 6–10. After coordination with Cu2+, weak fluores-
cence from pH 3 to 10 was noticed. This result indicates that probe 1
can successfully recognize Cu2+ over a wide pH range.
Furthermore, time-dependent fluorescence experiments of 1 toward
Cu2+ was also investigated. As depicted in Fig. S2, upon the addition of
Cu2+, the fluorescence intensity at 555 nm was quickly decreased
within a few seconds. This result indicates the probe 1 can rapidly rec-
ognize Cu2+ at room temperature.
3.2. Spectroscopic studies of ensemble 1-Cu2+ to biothiols
tion of ensemble of 1 and Cu2+, destinated as 1-Cu2+
.
To investigate the selectivity of probe 1, the fluorescence response of
1 in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) toward various
Considering that Cu2+ has a high affinity toward the amino acid con-
taining thiol group, [57] we tested the possibility of ensemble 1-Cu2+ as
a turn-on fluorescent probe for the detection of biothiols via indicator
displacement assays (IDA). Firstly, the fluorescence response of 1-Cu2
+ to a series of analytes, including biothiols (Cys, Hcy and GSH), S2−
and other amino acids (His, Thr, Ser, Pro, Glu, Tyr, Trp, Gly, Lys) were
metal cations including Cu2+, Zn2+, Cd2+, Mn2+, Al3+, Ag+, Hg2+
,
Mg2+, Co2+, Ni2+, Fe3+, Cr3+, Ca2+, Pb2+, Fe2+ was detected. As
shown in Fig. 3, compared with other cations, only Cu2+ displayed sig-
nificant fluorescence quenching effect toward 1 at 555 nm (Fig. 3a),
Fig. 1. (a) Fluorescence emission spectra of 1 (1.5 μM) in the presence of different amounts of Cu2+ (0–7.5 μM) in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) at 555 nm (λex
400 nm); (b) Linear relationship of fluorescence intensity with concentration of Cu2+
=
.
Please cite this article as: D. Chao, Y. Pan and X.-W. Gao, A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and
biothiols, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117770

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Fig. 3. (a) Fluorescence emission spectra of 1 (1.5 μM) and (b) the change of the color under 365 nm UV lamp in the presence of Cu2+ (7.5 μM) and other various metal ions (15 μM) in
CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) at 555 nm (λex = 400 nm).
investigated by fluorescence spectroscopy (Fig. 5 and Fig. S3a). Only Cys,
Hcy and GSH could significantly enhance the fluorescence intensity of 1-
Cu2+ at 555 nm. In the presence of other interfering ions, 1-Cu2+ still
has strong selectivity for biothiols. The corresponding photographs of
1-Cu2+ in the presence of various analytes under a 365 nm UV lamp
were also collected, respectively (Fig. S3b). The results demonstrate
that 1-Cu2+ can selectively discriminate biothiols among a variety of
analytes.
final absorption spectrum is the same as that before the addition of
Cu2+ (Inset of Fig. 7). This phenomenon demonstrated that the recovery
of UV–vis absorption spectra was ascribed to the demetallation of 1-Cu2
+, induced by biothiols, which released free probe 1. Meanwhile, the
fluorescence intensity of 1-Cu2+ reached maximum within 1 min in
the presence of Cys and remained stable for 10 min (Fig. S4).
The reversibility of 1-Cu2+ in the detection of biothiols was demon-
strated by an alternate cycle experiment with titration of 1-Cu2+ with
Cu2+ and Cys. As shown in Fig. 8, the fluorescence intensity of 1-Cu2+
was recovered following the addition of further Cys. This reversible
cycle could be repeated at least 4 times. This result indicates that en-
semble 1-Cu2+ can be developed as a turn-on and reversible
chemosensor for the detection of biothiols.
Next, the relationship between the fluorescence intensity of 1-Cu2+
and the concentration of biothiols (Cys, Hcy and GSH) was studied by
the titration experiment in Fig. 6. Upon the addition of Cys (0–7.5
μM), the fluorescence intensity at 555 nm was gradually increased.
Moreover, the fluorescence intensity of 1-Cu2+ was linear correlation
to Cys concentration from 1 to 7.5 μM, and gave the limit of detection
to be 0.19 μM based on a 3σ/k. In addition, similar results were obtained,
when the same amount of Hcy and GSH were added to the solution of 1-
Cu2+, respectively. The limits of detection of 1-Cu2+ for Hcy and GSH
were calculated to be 0.21 μM and 0.29 μM. These results indicate that
1-Cu2+ can quantitatively detect biothiols with high sensitivity.
The above displacement mechanism was further confirmed by UV–
vis absorption spectra. Upon addition of Cys (0–7.5 μM) to the solution
of 1-Cu2+ (1.5 μM), the absorption band at 349 nm gradually faded, ac-
companied by an enhancement of absorption at 282 nm (Fig. 7). The
3.3. Fluorescence lifetime studies
As an important parameter, fluorescence lifetime can directly reflect
the nature of the environment around the fluorophore and long-lived
organic molecular probes can avoid the use of precious transition-
metal complexes [58,59]. Transient photoluminescence decay spectra
at different stages were measured at 25 °C in air. As shown in Fig. 9,
the fluorescence lifetime of 1 is calculated to be 2.1 μs, which is much
longer than previous organic probes for Cu2+ [41,42]. We suppose
that the relatively long lifetime is ascribed to TADF effect [60]. After
adding Cu2+, the lifetime of 1 was significantly shortened (18.5 ns) in
Fig. 4. Fluorescence intensity of probe 1 (1.5 μM) at 555 nm with (red line) and without
(black line) Cu2+ (7.5 μM) under different pH. λex = 400 nm. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this
article.)
Fig. 5. Fluorescence spectra of 1-Cu2+ (1.5 μM) in the presence of various analytes (7.5 μM)
at 555 nm in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) at 555 nm (λex = 400 nm).
Please cite this article as: D. Chao, Y. Pan and X.-W. Gao, A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and
biothiols, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117770

D. Chao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
5
Fig. 6. Fluorescence spectra of 1-Cu2+ (1.5 μM) in the presence of 0–7.5 μM (a) Cys, (c) Hcy, (e) GSH in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) at 555 nm (λex = 400 nm);
(b) Linear relationship of fluorescence intensity with concentration of (b) Cys, (d) Hcy, (f) GSH.
Fig. 7. Absorption spectra of 1-Cu2+ (1.5 μM) in the presence of 0–7.5 μM Cysin CH3CN/HEPES
buffer (10 mM, pH = 7.4, 1:1, v/v). Inset: absorption spectra before the addition of Cu2+
Fig. 8. Fluorescent intensity of 1-Cu2+ (1.5 μM) at 555 nm (λex = 400 nm) in CH3CN/
HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) upon the alternate addition of Cys/Cu2+
.
.
Please cite this article as: D. Chao, Y. Pan and X.-W. Gao, A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and
biothiols, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117770

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D. Chao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
comparison with its lifetime prior to binding with Cu2+ (2.1 μs), sug-
gesting its potential application in lifetime-based sensing. Upon the ad-
dition of Cys, free probe 1 could be released, resulting in a recovery in
fluorescence lifetime which was calculated to be 0.742 μs. These results
indicate that 1 can be used as a long-lived molecular probe to detect Cu2
+ and biothiols.
3.4. Sensing mechanism
Based on spectroscopic results and previous studies [61,62], we pro-
posed a possible sensing mechanism of 1 toward Cu2+ and biothiols
(Scheme 2). Probe 1 exhibits strong emission with long fluorescence
lifetime, while the emission turned off in the presence of Cu2+, due to
the combination between terpyridine units and Cu2+, generating en-
semble 1-Cu2+. The fluorescence quenching of 1 is mainly attributed
to ligand-metal charge transfer (LMCT) [56]. The HRMS of 1 after the
addition of Cu2+ was further carried out to prove the sensing mecha-
nism (Fig. S5). The three new signal peaks of m/z = 632.0863,
Fig. 9. Transient photoluminescence decay spectra of 1.5 μM of 1 upon addition of Cu2+ and
Cys in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) in air. λex = 400 nm. λem = 555 nm.
810.1438 and 1166.2726 were derived from the [1 + 4Cu + 4Cl]4+
,
[1 + 3Cu + 3Cl]3+, [1 + 2Cu + 2Cl]2+ species formed by the combina-
tion of probe 1 and Cu2+ at different stoichiometric ratios (1:4, 1:3, 1:2),
respectively. The ensemble 1-Cu2+ show strong emission with long life-
time again in the presence of biothiols, since biothiols have a high affin-
ity with Cu2+. As a result, 1-Cu2+ can be demetallized by Cys, Hcy and
GSH, releasing probe 1.
3.5. Application in cell imaging
Fluorescence microscopy imaging experiments was performed on
the feasibility of 1-Cu2+ in detecting biothiols in HeLa cells. As shown
in Fig. 10, HeLa cells were incubated with 1.5 μM 1-Cu2+ solution for
30 min at 37 °C (a–c). Then the cells were washed three times with
PBS buffer, and exhibited green fluorescence. However, in the control
experiment, the cells were first treated with NEM (a thiol scavenger)
for 30 min, then being treated with 1-Cu2+ solution, and the cells
were observed to have weak emission (d–f). These results indicate
that 1-Cu2+ can be used for the detection of biothiols in cells. However,
the cytotoxicity needs to be further investigated in detail for more
applications.
Scheme 2. Possible mechanism for the detection of Cu2+ and biothiols.
Fig. 10. Confocal fluorescence images of HeLa cells at 37 °C. (a–c) HeLa cells incubated with 1.5 μM 1-Cu2+ for 30 min; (b) brightfield image of a; (c) merged image of a and b; (d–f) Hela
cells were pre-incubated with 1 mM NEM for 30 min and then treated with 1.5 μM 1-Cu2+ for another 30 min; (e) brightfield image of d; (f) merged image d and e.
Please cite this article as: D. Chao, Y. Pan and X.-W. Gao, A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and
biothiols, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117770

D. Chao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
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4. Conclusions
In conclusion, a new long-lived organic fluorescent probe 1, bearing
a TADF core and terpyridine fragments, has been synthesized for the de-
tection of Cu2+ and biothiols. The fluorescence intensity of probe 1 can
be selectively quenched by Cu2+ and subsequently recovered in the
presence of biothiols, displaying an ON–OFF–ON switching process.
Meanwhile, the fluorescence lifetime (2.1 μs) decreased significantly
upon addition of Cu2+ (18.5 ns) due to the formation of ensemble 1-
Cu2+. However, a longer lifetime of 0.742 μs is found in the presence
of biothiols for the solution containing 1-Cu2+. Our results show organic
TADF compounds have promising applications in lifetime-based sens-
ing, in spite of a common sensing process.
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Declaration of competing interest
[25] R. Balasaravanan, V. Sadhasivam, G. Sivaraman, A. Siva, Triphenylamino α-
cyanovinyl- and cyanoaryl-based fluorophores: solvatochromism, aggregation-
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The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
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This work has been supported by the National Natural Science Foun-
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MagnaFund in Ningbo University.
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