A coumarin derivative-Cu2+complex-based fluorescent chemosensor for detection of biothiols

Article abstract of DOI:10.1039/d0ra05651k

Herein, a novel fluorescent sensor has been developed for the detection of biothiols based on theoretical calculations of the stability constant of the complex between a Cu2+ ion and (E)-3-((2-(benzo[d]thiazol-2-yl)hydrazono)methyl)-7-(diethylamino) coumarin (BDC) as a fluorescent ligand. In this study, on the basis of density functional theory method, the Gibbs free energy of ligand-exchange reaction and the solvation model were carried out using thermodynamic cycles. The obtained results are in good agreement with the experimental data. The BDC-Cu2+ complex can be used as a fluorescent sensor for the detection of biothiols in the presence of non-thiol containing amino acids, with a detection limit for cysteine at 0.3 μM. Moreover, theoretical calculations of excited states were used to elucidate variations in the fluorescence properties. The computed results show that the excited doublet states D2 and D1 are dark doublet states, which quench the fluorescence of the complex.

Full text of DOI:10.1039/d0ra05651k

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PAPER
A coumarin derivative-Cu2+ complex-based
fluorescent chemosensor for detection of
biothiols†
Cite this: RSC Adv., 2020, 10, 36265
Nguyen Khoa Hien, ‡a Mai Van Bay,‡bc Phan Diem Tran,a Nguyen Tan Khanh,d
e
g
Nguyen Dinh Luyen,b Quan V. Vo,
Dang Ung Van,f Pham Cam Nam
*
b
*
and Duong Tuan Quang
Herein, a novel fluorescent sensor has been developed for the detection of biothiols based on theoretical
calculations of the stability constant of the complex between a Cu2+ ion and (E)-3-((2-(benzo[d]thiazol-2-
yl)hydrazono)methyl)-7-(diethylamino) coumarin (BDC) as a fluorescent ligand. In this study, on the basis of
density functional theory method, the Gibbs free energy of ligand-exchange reaction and the solvation
model were carried out using thermodynamic cycles. The obtained results are in good agreement with
the experimental data. The BDC–Cu2+ complex can be used as a fluorescent sensor for the detection of
biothiols in the presence of non-thiol containing amino acids, with a detection limit for cysteine at 0.3
mM. Moreover, theoretical calculations of excited states were used to elucidate variations in the
fluorescence properties. The computed results show that the excited doublet states D2 and D1 are dark
doublet states, which quench the fluorescence of the complex.
Received 29th June 2020
Accepted 14th September 2020
DOI: 10.1039/d0ra05651k
rsc.li/rsc-advances
associated with some diseases including immune diseases,
inammatory, cancer, metabolic diseases, diseases of aging,
1. Introduction
cystic brosis, cardiovascular, and neurodegenerative
diseases.1,7 As a result, the development of selective and sensi-
tive detection methods for biothiols has been attracting the
attention of scientists. In particular, analytical methods based
on uorescence technique have been widely developed due to
their outstanding advantages.8,9 This technique, in particular,
can be used to detect biothiols in living cells.10,11 A number of
uorescent sensors have been reported based on various
interactions with biothiols, such as Michael addition,12,13
addition-cyclization with acrylates or aldehydes,14,15 cleavage
reactions of sulfonamide, sulfonate ester, disuldes,16–18
substitution reactions,19 and disulde exchange reactions.20
Recently, sensors for biothiols have been widely synthesized
and reported; their working mechanism is based on the reac-
tions between biothiols and complexes of uorescent ligands
with metal ions.21,22 This approach has opened a new research
direction taking advantage of complexes between uorescent
sensors and metal ions towards the detection of biothiols.
These works become more convenient if the stability constants
of complexes can be determined. Recently, some theoretical
computational models for determining the stability constants
of complexes have been proposed with the aim of replacing
traditional experimental methods.23–25 However, the accuracy of
these models needs to be veried before their application. This
problem can be effectively solved by combining theoretical
calculations and empirical investigations to determine the
stability constant of complexes.
Cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are
thiol biomolecules that play an important role in human bio-
logical processes. Some diseases are believed to be related to
abnormalities in the concentrations of these compounds in
biological systems. A low level of Cys may cause a decline in the
immune system, resulting in diseases such as infections,
cancer, Parkinson's, Alzheimer's, retarded growth, liver
damage, and skin lesions.1–4 A high level of Hcy is believed to be
associated with heart disease, thromboembolic disease, stroke,
atherosclerosis, renal and thyroid dysfunction, psoriasis, dia-
betes, and cancer.5,6 Disturbances in GSH homeostasis are
aMientrung Institute for Scientic Research, Vietnam Academy of Science and
Technology, Hue 530000, Vietnam
bUniversity of Education, Hue University, Hue 530000, Vietnam. E-mail: dtquang@
hueuni.edu.vn
cThe University of Danang ꢀUniversity of Science and Education, Danang 550000,
Vietnam
dFaculty of Pharmacy, Hue University of Medicine and Pharmacy, Hue 530000,
Vietnam
eFaculty of Chemical Technology-Environment, The University of DanangꢀUniversity
of Technology and Education, 48 Cao Thang, Danang 550000, Vietnam
fHoa Binh University, Hanoi 100000, Vietnam
gThe University of Danang ꢀUniversity of Science and Technology, Danang 550000,
Vietnam. E-mail: pcnam@dut.udn.vn
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra05651k
‡ These authors contributed equally to the work.
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In the present study, a complex between a Cu2+ ion and
a uorescent ligand is reported as a novel chemosensor for the
detection of biothiols based on complex exchange reactions.
The stability constant of the complex, also known as the
complexation equilibrium constant in aqueous solutions, was
calculated using the Gibbs free energy of the ligand-exchange
reaction determined by a solvation model based on density
(SMD), a density functional theory (DFT) method, and ther-
modynamic cycles.26,27 The calculated results are in good
agreement with the experimental data obtained by a nonlinear
curve-tting method.28,29 From the value of the stability
constant, the applicability of the complex as a uorescent
sensor for the detection of biothiols based on the complex
exchange reaction was predicted. This was further conrmed by
the experimental data obtained by applying this complex as
a uorescent sensor. In addition, herein, theoretical calcula-
tions of the excited states were used to explain the uorescence
properties of the substances.
2.3 Computational methodology
2.3.1 Optimization of the geometries, energies, and
absorption and uorescence properties. Quantum chemical
calculations were performed using the Gaussian 09 program
package.30 The optimization of the geometries of related moie-
ties was carried out using density functional theory (DFT) at the
PBE0/6-31+G(d) level of theory.31–34 Single-point energies of the
optimized geometries in the gas phase were calculated at the
higher 6-311++G(d,p) basis set.35–39 The variation in the Gibbs
free energy (DG) of reactions was determined by the difference
between the sums of the electronic and thermal free energies of
products and reactants. The absorption and uorescence
properties were investigated based on the electron excited states
of the optimized geometries using the time-dependent density
functional theory (TD-DFT) at the PBE0/6-311++G(d,p) level.40–43
2.3.2 Theoretical method for determining the stability
constants of the complex. The stability constant of the complex,
also known as the complexation equilibrium constant in
aqueous solutions (log b), is calculated by the Gibbs free ener-
gies of ligand-exchange reactions determined using thermody-
namic cycles, the DFT theory method, and the SMD solvent
model (Scheme 1).23–27 Accordingly, the complexation equilib-
rium constant in aqueous solutions (log b) is calculated by the
following equation:
2. Materials and methods
2.1 Instruments
The Shimadzu RF-5301 spectrouorophotometer, Shimadzu
UV-1800 UV-Vis spectrophotometer, Varian Unity NMR instru-
ments (200 MHz), and electrospray ionization mass spectrom-
eter (ESI MS) were used to obtain the uorescence spectra, UV-
Vis absorption spectra, NMR spectra, and MS spectra,
respectively.
DGaq
log b ¼
þ log bref ꢀ ðx ꢀ yÞlog½H2Oꢁ
(1)
RT lnð10Þ
where [H2O] is the concentration of water in the standard state,
which can be approximated to be 55.56 M.44,45 log bref is the
experimental equilibrium constant in the aqueous solution of
[CuLref(H2O)2]2+, which was used as a reference complex for
calculation. DGaq is the Gibbs free energy of the ligand-
exchange reaction in the aqueous solution, which was deter-
mined by Scheme 1 and calculated using eqn (2):
2.2 Reagents
All chemicals were purchased from Aldrich and used directly
without further purication. In particular, 4-(diethylamino)sal-
icylaldehyde, 2-hydrazinobenzothiazole, diethyl malonate, and
triethylamine were synthesis grade reagents. Cysteine, gluta-
thione, homocysteine, alanine, aspartic acid, arginine, glycine,
glutamic acid, isoleucine, leucine, lysine, methionine, threo-
nine, serine, tyrosine, tryptophan, valine, histidine, HEPES,
acetic acid, HCl, NaOH, POCl3, and nitrate salts of K+, Ca2+, Na+,
Mg2+, Zn2+, Fe3+, Ni2+, Pb2+, Hg2+, Co2+, Cd2+, and Cu2+ were
analytical-grade reagents.
DGaq ¼ DGg + DDGsolv
(2)
DGg and DDGsolv were calculated by eqn (3) and (4), respectively:
P
P
DGg ¼ (D0 + Gcorr products
)
ꢀ
(D0 + Gcorr)reactants
(3)
DDGsolv ¼ (x ꢀ y)DGsolv(H2O2)g + DGsolv([CuL(H2O)y]g2+) +
2+
DGsolv(Lref)g ꢀ DGsolv(L)g ꢀDGsolv([CuLref(H2O)x]g
)
(4)
All solvents were purchased from Merck. Ethanol was HPLC-
grade without uorescent impurities and used without further
purication. DMF was also HPLC-grade; however, it was re-
distilled before use. H2O was two times distilled water.
where DGg(the variation in the Gibbs free energy of the reaction)
is the difference between the sums of the electronic (30) and
Scheme 1 Thermodynamic cycle for the calculation of the Gibbs free energy of a ligand-exchange reaction in an aqueous solution, DGaq
.
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Fig. 1 Schematic of the synthesis route of BDC.
thermal free energies (Gcorr) of products and reactants in the gas where
phase at the PBE0/6-311++G(d,p) level of theory and DGsolv is the
free energy of solvation of each compound, which was calcu-
lated using the solvent model density (SMD) model at the
M052x/6-31+G(d) level of theory.46–49
1
P1 ¼
(8)
bex
CM is the total concentration of the Cu2+ ions added to the
2.3.3 Experimental method for determining the stability
solution, F0 is the uorescence intensity of the free L solution
(concentration of L is CL) at the time when the concentration of
Cu2+ ions was zero, and F is the uorescence intensity of the L
solution at the time when the concentration of Cu2+ ions was
CM.
constants of the complex. The experimental complexation
equilibrium constant can also be determined by a nonlinear
curve-tting method.28,29 In this case, the experimental
complexation equilibrium constant (bex) of reaction (5) is
calculated by a nonlinear curve-tting method based on the
uorescence titration spectra of a uorescent ligand solution (L)
with the gradual addition of Cu2+ ions.
3. Results and discussion
3.1 Characterization of the uorescent ligand and its
complex with a metal ion
Cu2+ + L ¼ [CuL]2+
(5)
(6)
2þ
½CuLꢁ
(E)-3-((2-(Benzo[d]thiazol-2-yl)hydrazono)methyl)-7-
bex
¼
½Cu2þꢁ½Lꢁ
(diethylamino) coumarin (BDC) was synthesized in about 30%
overall yield using 4-(diethylamino)salicylaldehyde via the four
steps shown in Fig. 1. The structural characteristics of BDC were
According to this method, the experimental complexation
equilibrium constant (bex) is detected by the nonlinear curve-
tting method based on the relationship between the two
quantities y ¼ CM and x ¼ F/F0, as shown below (see details in
the ESI†):
1
conrmed by the H NMR, 13C NMR, and mass spectra (ESI†).
The experimental results show that the free ligand BDC is
a uorescent compound. It exhibits a characteristic emission
band peaked at 536 nm, with a uorescence quantum yield (F)
of 0.11 calculated using uorescein in 0.1 N NaOH (F ¼ 0.85) as
a ref. 50. When 1 equivalent of Cu2+ was added to the BDC
solution, the uorescence intensity of the solution was almost
ꢀ
ꢁ
1 ꢀ x
y ¼ CLð1 ꢀ xÞ þ P1
(7)
x
Fig. 2 (a) Fluorescence spectra of BDC after the addition of 1 equiv of various metal ions, i.e. Na+, K+, Ca2+, Mg2+, Fe3+, Co2+, Ni2+, Zn2+, Pb2+
,
Cd2+, Hg2+, and Cu2+ ions, and (b) fluorescence titration spectra of BDC with the gradual addition of Cu2+ ions (BDC: 5 mM in ethanol/HEPES, pH:
7.4; 1/1, v/v; and excitation wavelength: 460 nm).
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Fig. 3 Optimized geometries of BDC at the PBE0/6-31+G(d) level of theory.
completely quenched, over 95%. However, the uorescence results indicated that the reaction between Cu2+ ions and BDC
intensity of the BDC solution was quenched only about 40% was a reversible reaction and led to the formation of a complex
when 1 equivalent of Hg2+ was added. On the other hand, the with a 1 : 1 stoichiometry.
addition of 1 equivalent of other metal ions, i.e. K+, Ca2+, Na+,
The optimization of the geometries of BDC and its 1 : 1
Mg2+, Zn2+, Fe3+, Ni2+, Pb2+, Co2+, and Cd2+ ions, hardly changed complex with Cu2+ was performed using the PBE0 functional
the uorescence spectra of the BDC solution (Fig. 2a). The with the 6-31+G(d) basis set. The calculated results are pre-
uorescence titration spectra of BDC with the gradual addition sented in Fig. 3 and 4 and Tables S1–S7.†
of Cu2+ ions, as shown in Fig. 2b, indicated that the reaction
Herein, three optimized geometries of BDC were dened and
between BDC and Cu2+ occurred in a 1 : 1 stoichiometry. In denoted as L, L-1, and L-2. In each of these geometries, most
another experiment, when 1 equivalent of EDTA was added to atoms are in the same plane except for the atoms of the ethyl-
the solution obtained aer adding 1 equivalent of Cu2+ to the amino groups. In the L geometry, the conguration of the two
BDC solution, the uorescence intensity was almost restored to chain bonds C16–C15–C17–N36 and N36–N35–C41–N34 is
the original uorescence intensity of the free ligand BDC. These similar to trans–trans conguration. Moreover, in the L-1
Fig. 4 Optimized geometries of the BDC–Cu2+ complex at the PBE0/6-31+G(d) level of theory.
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geometry, the conguration of these two chain bonds is similar
to cis–trans conguration. In the L-2 geometry, the congura-
tion of these two chain bonds is similar to cis–cis conguration.
The calculated results show that L is the most energetically
favorable geometry. Consequently, the L geometry was used to
dene the conguration of the complex as well as calculate the
Gibbs free energy of the ligand-exchange reaction in the
aqueous solution.
Moreover, four optimized geometries of the 1 : 1 complexes
between L and Cu2+ ion were dened and denoted as S-1, S-2, S-
3, and S-4. The molecular formulas of the S-1, S-2, S-3, and S-4
geometries are [Cu(L)]2+
,
[Cu(L)]2+
,
[Cu(L)(H2O)]2+, and
[Cu(L)(H2O)2]2+, respectively. The calculated results show that
the reactions for the formation of S-1, S-2, S-3, and S-4 geome-
tries are energetically favorable (Table S8†).
Fig. 5 Nonlinear curve-fitting method for the determination of the
complexation equilibrium constants in the aqueous solution of the
[CuL]2+ complex. CM is the total concentration of the Cu2+ ion added
to the solution: 0–10 mM; F0 is the fluorescence intensity of the free
BDC solution (CL ¼ 5 mM, in ethanol/HEPES, pH: 7.4, 1/1, v/v); F is the
fluorescence intensity of the BDC solution at the time when the
concentration of Cu2+ ions was CM; excitation wavelength: 460 nm;
and emission wavelength: 536 nm.
The molecular structures of the S-1, S-2, S-3, and S-4 geom-
etries are presented in Table S9,† including bond lengths, bond
angles, and dihedral bond angles, and the coordination
numbers of the S-1, S-2, S-3, and S-4 geometries are 3, 3, 4, and
5, respectively. These values are consistent with those reported
in previous studies.41,51–54 All contact distances of the coordi-
nation bonds are signicantly shorter than the sum of the van
˚
˚
der Waals radii of relevant atoms (Cu: 1.40 A, N: 1.55 A, O: 1.52
complexation equilibrium constant of 107.16. Consequently, the
S-1 conguration was used for all subsequent studies of the
BDC–Cu2+ complex.
In order to verify the calculated complexation equilibrium
constant before studying the applications of the complex, the
experimental complexation equilibrium constant was also
determined by a nonlinear curve-tting method. The uores-
cent titration and nonlinear curve-tting results are shown in
Fig. 5. Accordingly, the experimental complexation equilibrium
constant was determined to be 107.15 (Mꢀ1).
This result is in good agreement with the above calculated
complexation equilibrium constant. These results provide
further important evidence conrming the correctness of the
proposed method for the determination of the complexation
equilibrium constants in aqueous solutions using DFT theory
and the SMD solvent model.
˚
˚
A, and S: 1.80 A). All the four complexes have three coordination
bonds between a Cu atom and three atoms of the L ligand.
These four atoms are roughly on the same plane, forming the
bond angles of approximately 180ꢂ and 90ꢂ. The formation of
complexes mainly leads to changes in the congurations of the
two chain bonds C16–C15–C17–N36 and N36–N35–C41–N34 in
the BDC moieties. The congurations of the S-2 and S-1, S-3,
and S-4 geometries are similar to “cis–trans” and “cis–cis”
congurations, respectively.
The complexation equilibrium constants in the aqueous
solutions of the S-1, S-2, S-3, and S-4 complex congurations
were calculated via the Gibbs free energies of ligand-exchange
reactions determined using the thermodynamic cycles, DFT
theory method, and SMD solvent model (Scheme 1). Herein, the
reference complex [CuLref(H2O)2]2+ was used for calculation,
which is a complex between the Cu2+ ions and histamine (Lref
)
In terms of application, this value of the complexation
equilibrium constant of Cu2+ ions with BDC is signicantly
smaller than that of the Cu2+ ions with biothiols. For example,
the Cu2+ ions react with Cys to form a [CuX2]2+ complex with an
Moreover, the Cu ions react
with Hcy to form a [CuHY]2+ complex with an equilibrium
as a ligand, and the experimental equilibrium constant in the
aqueous solution (log bref) of this complex was 9.55 (ref. 55 and
56) (the optimized geometry of histamine (Lref) and cartesian
coordinate are presented in Fig. S5, S6 and Tables S10 and S11
in the ESI†). The calculation results presented in Table 1 indi-
cate that S-1 is the most stable conguration with a calculated
2+
equilibrium constant of 1016.
57,58
Table 1 The calculated complexation equilibrium constants in the aqueous solutions of various complex configurations (energies in kcal)a
DGsolv
A
B
C
D
E
DDGsolv
DGg
DGaq
log bref
log b
S-1
S-2
S-3
S-4
ꢀ201.61
ꢀ201.61
ꢀ201.61
ꢀ201.61
ꢀ15.35
ꢀ15.35
ꢀ15.35
ꢀ15.35
ꢀ172.79
ꢀ152.85
ꢀ157.3
ꢀ8.84
ꢀ8.84
ꢀ8.84
ꢀ8.84
ꢀ13.50
ꢀ13.50
ꢀ13.50
ꢀ13.50
12.99
32.93
37.32
54.71
ꢀ14.47
ꢀ2.08
ꢀ1.48
30.85
3.29
9.55
9.55
9.55
9.55
7.16
ꢀ16.56
5.40
ꢀ34.03
ꢀ42.46
ꢀ148.75
12.25
0.57
a
A: [CuLref(H2O)x](g)2+, B: L(g), C: [CuL(H2O)y](g)2+, D: H2O(g), E: Lref(g)
.
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Fig. 6 (a) Fluorescence titration spectra of BDC–Cu2+ with the gradual addition of Cys and (b) variation in the fluorescence intensity of BDC–
Cu2+ with the gradual addition of Cys at the emission wavelength of 536 nm. BDC–Cu2+: 5 mM, in ethanol/HEPES, pH: 7.4, 1/1, v/v; Cys: 0, 1.0, 2.0,
3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 13.0, 17.0, and 20.0 mM; and excitation: 460 nm.
Fig. 7 Schematic of the reaction between the BDC–Cu2+ complex and Cys.
constant of 1013.5
.
Compared to the abovementioned reac- also consistent with those reported in previous studies on the
59
tions, the reactions between the Cu2+ ions and GSH are more composition of the complex of Cys and Cu2+ ion.57,58
complicated. In particular, the main reaction occurs between
To use the BDC–Cu2+ complex as a uorescent sensor for the
the Cu2+ ions and GSH to generate a complex of Cu+ ions with determination of Cys, the interactions of the BDC–Cu2+ complex
GSH in the form of [CuZ2]+ and introduce the GSSG (oxidized with H2S and other amino acids were also investigated. The
glutathione). Herein, the stability constant of the [CuZ2]+ experimental results in Fig. 8 show that thiol-containing amino
complex is substantially large, equal to 1038.8
.
Based on acids, such as GSH and Hcy, also cause uorescence recovery.
60–62
these results, it can be anticipated that the complex of BDC with Moreover, H2S and non-thiol containing amino acids, such as
the Cu2+ ions can be used as a uorescent sensor for the
detection of biothiols based on the complex exchange reactions.
3.2 Application of the BDC–Cu2+ complex in the detection of
biothiols
The application of the BDC–Cu2+ complex as a uorescent
sensor for the detection of cysteine was investigated. As shown
in Fig. 6, when Cys was gradually added to the solution of the
BDC–Cu2+ complex, the uorescence intensity at 536 nm
accordingly changed and increased. It almost restored to the
original uorescence intensity of free BDC when Cys was added
to the solution at a concentration greater than or equal to twice
that of the BDC–Cu2+ complex. These results show that the
stoichiometry of the reaction between Cys and the BDC–Cu2+
complex is 2 : 1, and this reaction leads to the formation of the
Fig. 8 Fluorescence spectra of BDC (5 mM); BDC (5 mM)+Cu2+ (5 mM);
BDC (5 mM)+ Cu2+ (5 mM) + Cys/Hcy/GSH/Ala, Asp, Arg, Gly, Glu, Ile,
[CuX2]2+ complex (X: Cys) and the release of free BDC, resulting
in the restoration of uorescence intensity (Fig. 7). This result is Leu, Lys, Met, Thr, Ser, Tyr, Trp, Val, His, and H2S (10 mM).
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Fig. 9 The characteristics of the main transition between the electron excited states and ground states of BDC and BDC–Cu2+ at the PBE0/6-
31+G(d) level.
alanine (Ala), arginine (Arg), aspartic acid (Asp), glutamic acid using a calibration curve with a range of low concentrations of
(Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine Cys (0–5 mM).68 The values of these parameters were 0.3 and 1.1
(Leu), lysine (Lys), methionine (Met), serine (Ser), threonine mM, (Fig. S8†). Moreover, the intracellular concentration of Cys
(Thr), tyrosine (Tyr), tryptophan (Trp) and valine (Val), did not was 30–200 mM.63 These results indicate that the BDC–Cu2+
cause any changes in the uorescence spectrum of the BDC– complex can be used as a uorescent sensor for the quanti-
Cu2+ solution. These results indicate that the BDC–Cu2+ cation of intracellular Cys and has greater sensitivity than that
complex can be used as a uorescent sensor for the detection of of the similar previously reported sensors.69,70
thiol-containing amino acids in the presence of H2S and non-
thiol containing amino acids in the concentration range from
0 to 10 mM (from 0 to 2 equivalent). In addition, the experi-
3.3 Theoretical investigation of the changes in the
uorescence properties of the complexes and ligands
mental results show that the presence of H2S at concentrations
The uorescence properties of the ligand and complex were
greater than 10 mM affects the performance of BDC–Cu2+ in the
investigated based on the electronic excited states using time-
dependent density-functional theory (TD-DFT) at the PBE0/6-
detection of biothiols (Fig. S7†).
Similar to the cases of previous studies reported on uores-
31+G(d) level. The obtained results are presented in Table 2 and
cent sensors based on complex exchange reactions,63,64 the
Fig. 9.
BDC–Cu2+ complex also reacted with thiols, including Cys, Hcy,
The results provided in Table 2 indicate that in BDC, the S0
/ S1 transition plays a key role in all the singlet electronic
and GSH, and provided similar results. However, in many actual
samples, these thiols do not occur simultaneously. For example,
transitions from ground state (S0) to excited states (Sn). This is
in human whole blood, the concentration of reduced gluta-
due to the fact that the oscillator strength (f) of this transition is
thione is signicantly higher (up to 1 mM) than that of the other
1.2659, substantially larger than those of the other transitions.
thiols. Furthermore, in human plasma, the total concentration
The S0 / S1 transition energy is 2.74 eV (452.9 nm). The
of Cys species is up to 250 mM, whereas the concentrations of
calculated result is completely consistent with the empirical
GSH and Hcy are almost negligible.65–67 Therefore, the BDC–
investigations, indicating that BDC exhibits a maximum
Cu2+ complex can be used as a uorescent sensor for the
absorption peak at 460 nm. The HOMO / LUMO transition
plays a key role in all the orbital transitions from S0 to S1, with
a percentage contribution of 98.58%. This transition occurs
between two adjacent MOs. Therefore, it can be rmly affirmed
detection of Cys, HCy, or GSH depending on the actual cases.
The applicability of the BDC–Cu2+ complex as a uorescent
sensor for the quantication of Cys was also investigated. As
shown in Fig. 6b, there is a good linear relationship between the
that photoinduced electron transfer (PET) does not occur in
uorescence intensity of the BDC–Cu2+ solution and the
BDC, and the abovementioned transition leads to a green
concentration of Cys when Cys is added to the BDC–Cu2+
emission at 536 nm, as observed via empirical
solution (5 mM). In the Cys concentration range from 0 to 10 mM,
investigations.71,72
the calibration curve equation was determined as follows: FI536
The formation of the BDC–Cu2+ complex leads to a signi-
¼ (3.8 ꢃ 10.4) + (87.1 ꢃ 1.8) ꢄ [Cys], R ¼ 0.996. The limit of
cant decrease in the energy gap between the ground state and
detection (LOD) and the limit of quantitation (LOQ) of the
the excited states of BDC. As a result, the energy gap between
the excited state and the D0 state reaches 2.88 eV (430.7 nm)
method were also evaluated by the linear regression method
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Table 2 The calculated excitation energy (E), wavelength (l), and oscillator strength (f) for BDC and BDC–Cu2+ at the PBE0/6-31+G(d) level of
theory
Contribution of orbital transition
Percentage contribution
Compound
State
E (eV)
L (nm)
f
Composition
(%)
BDC
S0 / S1
S0 / S2
S0 / S3
S0 / S4
S0 / S5
S0 / S6
2.74
3.60
3.98
4.12
4.14
4.40
452.9
346.4
311.6
301.1
299.2
281.8
1.2659
0.0585
0.3340
0.1155
0.0325
0.0293
HOMO / LUMO
98.58
97.59
80.64
69.20
90.33
15.72
18.96
49.74
87.88
15.15
42.81
30.25
27.33
47.21
21.27
34.64
32.03
15.67
26.27
40.68
24.45
23.65
39.49
41.68
63.47
66.61
22.56
17.29
21.26
16.81
HOMOꢀ1 / LUMO
HOMO / LUMO+1
HOMOꢀ3 / LUMO
HOMOꢀ2/LUMO
HOMOꢀ3 / LUMO
HOMO / LUMO+2
HOMO / LUMO+3
HOMOb / SOMOb
HOMOꢀ2b / SOMOb
HOMOꢀ1b / SOMOb
SOMOa / LUMOa
HOMOb / LUMOb
HOMOꢀ3b / SOMOb
HOMOꢀ2b / SOMOb
HOMOꢀ1b / SOMOb
HOMOꢀ21b / SOMOb
HOMOꢀ28b / SOMOb
HOMOꢀ21b / SOMOb
HOMOꢀ2b / SOMOb
SOMOa / LUMO+1a
HOMOb / LUMO+1b
SOMOa / LUMOa
BDC–Cu2+
D0 / D1
D0 / D2
0.92
1.44
1350.8
861.9
0.0054
0.0099
D0 / D3
1.83
676.7
0.0011
D0 / D4
D0 / D5
1.86
1.94
666.2
637.3
0.0025
0.0124
D0 / D6
D0 / D7
2.14
2.16
579.3
574.2
0.0035
0.0019
D0 / D8
D0 / D9
2.27
2.68
545.6
462.4
0.0021
0.0014
D0 / D10
2.88
430.7
1.2645
HOMOb / LUMOb
HOMOꢀ3b / SOMOb
HOMOꢀ4b / SOMOb
HOMOa / LUMOa
SOMOa / LUMO+1a
HOMOꢀ1b / LUMOb
HOMOb / LUMO+1b
D0 / D11
D0 / D12
D0 / D13
2.89
2.97
3.13
428.7
417.9
396.5
0.1655
0.0949
0.0001
only when the D10 excited state is attained. The D0 / D10 uorescent radiation process, which quenches the Dn / D0
transition has an oscillator strength of 1.2645, signicantly uorescence (n > 2).75,76 Only the energy gap between the D2 and
bigger than that of the other transitions; therefore, this transi- D1 states is quite large, 0.52 eV, which should be considered.
tion plays a key role in all the electronic transitions. The D0 / The D2 / D0 transition is mainly contributed by the SOMO /
D
10 transition is mainly contributed by the SOMO / LUMO and HOMOꢀ2 and SOMO / HOMOꢀ1 transitions. However, the
HOMO / LUMO transitions, with the percentage contributions lack of overlapping between the SOMO and HOMOꢀ2 or
of 39.49% and 41.68%, respectively. These transitions are HOMOꢀ1 makes the radiation transition D2 / D0 strongly
believed to lead to a strong absorption spectrum, as observed in forbidden. As a result, the IC process is also preferred over the
the experiments. Regarding the uorescence properties of the uorescent radiation process, which quenches the D2 / D0
BDC–Cu2+ complex, on the basis of Kasha's rule, the uores- uorescence. In summary, in the BDC–Cu2+ complex, since the
cence occurs from the lowest-lying electron excited state D1,73,74 energy gap between adjacent excited states is small, the tran-
in which the D1 / D0 transition is mainly contributed by the sitions from the excited states Dn (n > 2) to the excited state D2
LUMO / SOMO. However, the lack of overlapping between the are dominated by the internal conversion processes. Moreover,
LUMO and SOMO makes the radiation transition D1 / D0 due to the lack of overlapping between MOs during each tran-
strongly forbidden. Instead, the internal conversion process sition, the D1 / D0 and D2 / D0 transitions are dominated by
occurs from D1 to D0 as a radiationless transition. Considering the internal conversion processes. The D2 and D1 states are dark
the exceptions of the Kasha rule, from the D2 state to the D10 doublet states that quench the uorescence.77
state, the energy gap between adjacent excited states is quite
small, less than 0.42. Thus, according to the energy gap law for
4. Conclusions
radiationless transitions, the internal conversion processes
from the excited states at higher energy level to the excited state
at adjacent low energy levels quickly occur, competing with the
In summary, herein, a complex exchange reaction-based uo-
rescent chemosensor was investigated for the detection of
36272 | RSC Adv., 2020, 10, 36265–36274
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RSC Advances
biothiols. This sensor was designed based on the theoretical 14 F. Kong, R. Liu, R. Chu, X. Wang, K. Xu and B. Tang, Chem.
calculations of the stability constants of the complex between Commun., 2013, 49, 9176–9178.
the Cu2+ ions and uorescent ligands. The obtained results are 15 J. Shi, Y. Wang, X. Tang, W. Liu, H. Jiang, W. Dou and W. Liu,
completely consistent with the experimental results. The BDC– Dyes Pigm., 2014, 100, 255–260.
Cu2+ complex can be used for the detection of biothiols in the 16 S. Ji, H. Guo, X. Yuan, X. Li, H. Ding, P. Gao, C. Zhao, W. Wu,
presence of non-thiol containing amino acids. The limit of W. Wu and J. Zhao, Org. Lett., 2010, 12, 2876–2879.
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mosensor for Cys are 0.3 and 1.1 mM, respectively. This result Chem., 2012, 10, 1966–1968.
opens a new research direction toward the utilization of the 18 M. Wei, P. Yin, Y. Shen, L. Zhang, J. Deng, S. Xue, H. Li,
complexes between metal ions and uorescent ligands for the
detection of biothiols based on the theoretical calculations of
the stability constants.
B. Guo, Y. Zhang and S. Yao, Chem. Commun., 2013, 49,
4640–4642.
19 Y.-S. Guan, L.-Y. Niu, Y.-Z. Chen, L.-Z. Wu, C.-H. Tung and
Q.-Z. Yang, RSC Adv., 2014, 4, 8360–8364.
In this study, the theoretical calculations of the excited states
were also used to elucidate the changes in the uorescence 20 M. H. Lee, J. H. Han, P.-S. Kwon, S. Bhuniya, J. Y. Kim,
properties of compounds. The quenching of the uorescence of
the BDC–Cu2+ complex occurred because the internal conver-
J. L. Sessler, C. Kang and J. S. Kim, J. Am. Chem. Soc., 2012,
134, 1316–1322.
sion processes dominated the uorescence process due to the 21 Y. Bao, Q. Li, B. Liu, F. Du, J. Tian, H. Wang, Y. Wang and
small energy gap between adjacent excited states, from the R. Bai, Chem. Commun., 2012, 48, 118–120.
excited states Dn (n > 2) to the excited state D2. Moreover, the D2 22 D. T. Nhan, N. K. Hien, H. Van Duc, N. T. A. Nhung,
and D1 states are dark doublet states because of the lack of
overlapping between MOs during each transition.
N. T. Trung, D. U. Van, W. S. Shin, J. S. Kim and
D. T. Quang, Dyes Pigm., 2016, 131, 301–306.
23 D. Devarajan, P. Lian, S. C. Brooks, J. M. Parks and
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24 O. Gutten and L. Rulisek, Inorg. Chem., 2013, 52, 10347–
10355.
Conflicts of interest
There are no conicts to declare.
25 S. Vukovic, B. P. Hay and V. S. Bryantsev, Inorg. Chem., 2015,
54, 3995–4001.
Acknowledgements
´
´
26 R. Flores, L. I. Reyes-Garcıa, N. Rodrıguez-Laguna and
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R. Gomez-Balderas, Theor. Chem. Acc., 2018, 137, 125.
This research was funded by the Vietnam National Foundation
for Science and Technology Development (NAFOSTED) under 27 A. T. Afaneh, G. Schreckenbach and F. Wang, J. Phys. Chem.
grant number 104.06-2016.32 (Nguyen Khoa Hien).
B, 2014, 118, 11271–11283.
28 J.-S. Wu, F. Wang, W.-M. Liu, P.-F. Wang, S.-K. Wu, X. Wu
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