A reaction-based turn-on fluorescent sensor for the detection of Cu (II) with excellent sensitivity and selectivity: Synthesis, DFT calculations, kinetics and application in real water samples

Article abstract of DOI:10.1016/j.dyepig.2019.02.043

A reaction-based turn-on fluorescent chemosensor RhB-Cu, starting from rhodamine B (RhB), for Cu²⁺ was easily synthesized in two steps. The sensor could selectively detect Cu²⁺ with a 100-fold fluorescence enhancement among the common metal ions, exhibiting an extremely low detection limit of 4.7 nM. To the best of our knowledge, this was the best record for the detection of Cu²⁺ with organic fluorescent sensors. There was a 1:1 binding stoichiometry between RhB-Cu and Cu²⁺ with an association constant of 6.42 × 10⁴ M^?1. Noteworthy, it could distinguish Cu²⁺ from Cu⁺, which was hard to realize in the previous studies. In addition, the detection mechanism was proposed based on mass spectrometric analysis and density functional theory (DFT) calculations. Kinetic studies were conducted to obtain the activation energy, enthalpy and entropy, so as to elucidate the solvent effect. Interestingly, the kinetic compensation effect (KCE) was uncovered in this work. Finally, RhB-Cu was proved to have the capability to work in real water samples. It would highly contribute to the even better design of fluorescent sensor for Cu²⁺ in future.

Full text of DOI:10.1016/j.dyepig.2019.02.043

Accepted Manuscript
A reaction-based turn-on fluorescent sensor for the detection of Cu (II) with excellent
sensitivity and selectivity: Synthesis, DFT calculations, kinetics and application in real
water samples
Ming Tian, Huan He, Bei-Bei Wang, Xi Wang, Yi Liu, Feng-Lei Jiang
PII:
S0143-7208(18)32723-2
https://doi.org/10.1016/j.dyepig.2019.02.043
DYPI 7377
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 14 December 2018
Revised Date: 21 February 2019
Accepted Date: 24 February 2019
Please cite this article as: Tian M, He H, Wang B-B, Wang X, Liu Y, Jiang F-L, A reaction-based turn-
on fluorescent sensor for the detection of Cu (II) with excellent sensitivity and selectivity: Synthesis,
DFT calculations, kinetics and application in real water samples, Dyes and Pigments (2019), doi: https://
doi.org/10.1016/j.dyepig.2019.02.043.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
1
2
3
A Reaction-based Turn-on Fluorescent Sensor for the Detection of Cu
(II) with Excellent Sensitivity and Selectivity: Synthesis, DFT
Calculations, Kinetics and Application in Real Water Samples
4
5
6
Ming Tian a, Huan He a, Bei-Bei Wang a, Xi Wang, Yi Liu a,b,c and Feng-Lei Jiang a,*
7
8
a State Key Laboratory of Virology & Key Laboratory of Analytical Chemistry for
Biology and Medicine (Ministry of Education), College of Chemistry and Molecular
Sciences, Wuhan University, Wuhan 430072, P. R. China
9
b
10
11
12
13
14
School of Chemistry and Chemical Engineering, Wuhan University of Science and
Technology, Wuhan 430081, P. R. China
c
College of Chemistry and Materials Science, Nanning Normal University, Nanning
530001, P. R. China
*Corresponding Author. E-mail: fljiang@whu.edu.cn. Tel: +86-27-68756667.
15
16
1

ACCEPTED MANUSCRIPT
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Abstract:
A reaction-based turn-on fluorescent chemosensor RhB-Cu, starting from
rhodamine B (RhB), for Cu2+ was easily synthesized in two steps. The sensor could
selectively detect Cu2+ with a 100-fold fluorescence enhancement among the common
metal ions, exhibiting an extremely low detection limit of 4.7 nM. To the best of our
knowledge, this was the best record for the detection of Cu2+ with organic fluorescent
sensors. There was a 1:1 binding stoichiometry between RhB-Cu and Cu2+ with an
association constant of 6.42 × 104 M-1. Noteworthy, it could distinguish Cu2+ from
Cu+, which was hard to realize in the previous studies. In addition, the detection
mechanism was proposed based on mass spectrometric analysis and density functional
theory (DFT) calculations. Kinetic studies were conducted to obtain the activation
energy, enthalpy and entropy, so as to elucidate the solvent effect. Interestingly, the
kinetic compensation effect (KCE) was uncovered in this work. Finally, RhB-Cu was
proved to have the capability to work in real water samples. It would highly contribute
to the even better design of fluorescent sensor for Cu2+ in future.
Keywords: fluorescent sensor, copper, PET mechanism, density functional theory
34
35
(DFT), solvent effect, kinetic compensation effect (KCE)
2

ACCEPTED MANUSCRIPT
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
1. Introduction
Copper, the third most abundant transition metal in the body [1], not only is the
static metabolic cofactor at the center of enzymatic activity, but also can serve as
dynamic signal that binds and regulates protein function at external allosteric sites
[2,3]. Interestingly, copper is recently found to play a crucial role in neural activity
toward a chemistry of consciousness [4]. Moreover, diseases such as Alzheimer’s
disease [5-7], Parkinson’s disease [8,9], Menkes [10-12], Wilson disease [13,14],
Huntington’s [15], prion [17], obesity and diabetes [18,19] are suggested to be related
to abnormal levels of copper ions. The limit of copper in drinking water, 1.3 ppm
(∼20 µM), is set by the US Environmental Protection Agency (EPA), and the average
concentration of blood copper in the normal group is 15.7-23.6 µM [20] and of brain
copper is ~ 0.1 mM [21]. Therefore, an efficient method for the detection of copper
ions is full of significance. Traditional methods, including atomic absorption
spectrometry (AAS) [22,23], surface plasmon resonance spectroscopy (SPR) [24],
inductively coupled plasma atomic emission spectrometry (ICP-AES) [25],
chromatography [26], voltammetry [27] and so on, have advantage of considerable
sensitivity but shortcoming of time-consuming and complicated protocols. Thus,
fluorescent chemosensors have been emerging for detection and are widely studied
due to their simple instrumentation, good reproducibility, easy operation as well as
better sensitivity[28-30].
In recent years, rhodamine, a well-known fluorophore, exhibits excellent
photophysical properties as an ideal candidate for the design of fluorescent sensors
[31-33], which include long-wavelength emission, large molar extinction-coefficient
and high quantum yield. Most sensors based on rhodamine framework have
successfully detected copper ion by utilizing the property of spirocyclic closed
(non-fluorescent) and ring-opened (fluorescent) forms, which exhibits a “turn-on”
signal response [34-37]. Nevertheless, there are few studies involved in chemical
information about the mechanism, reaction kinetics and solvent effect.
Therefore, bearing these facts in mind, we designed and synthesized a
3

ACCEPTED MANUSCRIPT
65
66
67
68
69
70
71
72
73
74
75
chemodosimeter RhB-Cu, in which Rhodamine B (RhB) as the fluorophore
responded to Cu2+ based on the “turn-on” mechanism. The target product was easily
prepared via a two-step procedure (Scheme 1). First, RhB-1a was prepared by the
reaction of Rhodamine B with hydrazine hydrate. It had no fluorescence indicating its
spirocycle form due to the quenching mechanism of photo-induced electron transfer
(PET). Second, RhB-Cu was subsequently synthesized by reacting RhB-1a with
2,4-dinitrobenzenesulfonyl chloride (DNBS), a well-known fluorescence quencher.
Consequently, the introduction of DNBS would further quench the fluorescence of
RhB, which led to extremely low background and thereafter very high fluorescence
turn-on ratio. Both RhB-1a and RhB-Cu were fully characterized by NMR and mass
spectrometry (see the Supporting Information, Fig. S1-S6).
76
77
Scheme 1. Synthetic route of RhB-Cu.
78
79
80
81
82
83
84
85
86
87
2. Materials and methods
2.1. Chemicals and instrumentation
All regents and chemicals, unless otherwise stated, were used without further
purification from the commercial resources. All glassware was oven-dried before used.
NMR spectra were recorded on a Bruker AVANCE III HD 400 MHz spectrometer,
using tetramethylsilane (TMS) as the internal standard. Mass spectra were measured
with the Finnigan LCQ advantage mass spectrometer and the Agilent Technologies
6530 Q-TOF LC/MS. Fluorescent spectra were measured using a Perkin-Elmer LS-55
fluorescence spectrometer. A Cary 100 spectrophotometer was used to measure
4

ACCEPTED MANUSCRIPT
88
89
absorption spectra. Fluorescent lifetimes were measured with a FLS1000 fluorescence
spectrometer. Melt points were measured using a SGW X-4 Micromelting Point
Apparatus.
90
91
2.2. Computational methods
92
The ground-state structures of the RhB-Cu and complexes based on RhB-Cu were
optimized using the TD-DFT and DFT methods with the hybrid-generalized gradient
approximation (HGGA) functional B3LYP. The 6-31g(d) basis set and the effective
core potential LanL2DZ basis sets were respectively assigned to nonmetal elements
(C, H, O, N and S) and metal elements [38]. A polarized continumm model (PCM)
method was employed to treat the solvent effects of water and acetonitrile. All the
calculations were performed with the Gaussian09 program package.
2.3. Preparation for solutions
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
The stock solution of RhB-Cu was prepared at 2 mM in dimethyl sulfoxide
(DMSO). Testing solutions were prepared by diluting 4 µL of stock solution into 2 mL
CH3CN/H2O (1:1, v/v) solution. Stock solutions (0.05 M, 4 µL) of metal ions
including Ni2+, Co2+, Ca2+, Li+, Mg2+, Zn2+, Pb2+, Cd2+, Ag+, Hg2+, Fe3+, Al3+, Fe2+,
Mn2+, Cr3+ and Cu+ were respectively added into the testing solutions. According to
the previous report [39], stock solution for Cu+ was prepared by dissolving
Cu(MeCN)4PF6 in CH3CN under nitrogen conditions, while other metal ion stock
solutions were derived from chloride salts. The fluorescence spectra were measured
after the addition of each analytes.
3. Results and discussion
3.1. Computational study
The DFT calculations were performed for predicting the selectivity of RhB-Cu by
calculating the energy of RhB-Cu and several representative complexes RhB-Cu-M
(M: Cu2+, Cu+, Pb2+, Al3+ and Hg2+). It is found, except for Cu2+ and Cu+, no
converged complex between RhB-Cu and Pb2+, Al3+ or Hg2+ could be obtained,
indicating that potential complexes of RhB-Cu-Pb2+, RhB-Cu-Al3+ and
5

ACCEPTED MANUSCRIPT
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
RhB-Cu-Hg2+ may not exist. However, RhB-Cu and RhB-Cu-Cu+ are speculated
non-fluorescent due to the PET mechanism, which is explained by the molecular
orbitals calculated from TD-DFT. Four molecular orbitals (from HOMO to HOMO-2,
LUMO+2) of RhB-Cu are mainly located at the RhB moiety, while another two
molecular orbitals (LUMO and LUMO+1) are mainly located at the DNBS group.
Thus, after the electron is excited from HOMO, HOMO-1 or HOMO-2 to the
LUMO+2 orbital of RhB group, the electron may be transferred to LUMO+1 or
LUMO orbital due to the fact that the energy of LUMO (LUMO+2 orbital of the
whole molecule) of RhB moiety is higher than the LUMO (LUMO orbital of the
whole molecule) of the DNBS moiety (Fig. 1a). As a result, the electron of higher
electron state could be transferred to the lowest vibrational state which provides the
non-emissive path for electron transition [38]. However, when RhB-Cu is complexed
with Cu2+, on the contrary, the HOMO and LUMO orbitals of RhB-Cu-Cu2+ are
mainly distributed on RhB moiety, while the LUMO+1 and LUMO+2 orbitals are
distributed on the DNBS moiety (Fig. 1b). When the electron is excited from HOMO
to the LUMO orbital of RhB moiety, it goes back directly accompanied by
fluorescence, owing to the fact that the unoccupied molecular orbitals with DNBS
character are higher in energy than the LUMO with RhB character. In another word,
the PET process is blocked and the fluorescence is recovered. Moreover, when
RhB-Cu is complexed with Cu+, the mechanism of the fluorescence quenching by
Cu+ is similar to the RhB-Cu due to a similar distribution of molecular orbitals to
RhB-Cu (Fig. 1c). It is worth noting that the transitions of electrons in molecular
orbitals under excitation mentioned here all have a large oscillator strength (Table S1).
6

ACCEPTED MANUSCRIPT
140
141
Fig. 1. Molecular orbitals calculated at B3LYP/6-31g(d)/LANL2DZ level for (a)
RhB-Cu, (b) RhB-Cu-Cu2+ and (c) RhB-Cu-Cu+.
142
143
144
145
3.2. Selective reaction of RhB-Cu with Cu2+ and possible sensing mechanism
The fluorescence spectra further suggest that RhB-Cu can distinguish Cu2+ from
7

ACCEPTED MANUSCRIPT
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
other metal ions, including Ni2+, Co2+, Ca2+, Li+, Mg2+, Zn2+, Pb2+, Cd2+, Ag+, Hg2+,
Fe3+, Al3+, Fe2+, Mn2+, Cr3+ and Cu+. Obviously, only Cu2+ leads to a 100-fold
fluorescence enhancement due to the turn-on mechanism with ring-opening of
RhB-Cu induced by Cu2+ (Fig. 2a-c), which is in perfect agreement with the above
theoretical calculations. Visually, only Cu2+ can trigger the occurrence of distinct pink
color and orange fluorescence (Fig. 2e). Moreover, different types of anions including
2-
-
Cl-, SO4 , CH3COO-, NO3 and Br- don’t affect the fluorescence intensity of the
RhB-Cu in response to Cu2+ (Fig. S7). A coordination reaction with the 1:1 binding
stoichiometry between RhB-Cu and Cu2+ is confirmed by the Job’s plot (Fig. 2d).
Besides, a peak at m/z 443.3 in the mass spectrum corresponds to RhB and a peak at
m/z 749.9 corresponds to the complex of RhB-Cu with Cu2+ (Fig. S8). Moreover, the
results of HPLC-MS showed that the retention times of RhB-Cu and RhB were 0.696
min and 0.513 min, and their corresponding molecular weights were 687.2231 and
443.2232, respectively, while the retention time and corresponding molecular weight
were 0.520 min and 443.2333 after the reaction of RhB-Cu with 25 equiv. of Cu2+,
which also indicated the formation of RhB (Fig. S9-S11). Furthermore, DFT was
performed for calculating the bonding length of RhB-Cu-Cu2+, which implied that N1
is too far away from the copper atom to form a coordination bond (Table 1). Therefore,
a possible sensing mechanism can be proposed as follows. The coordination reaction
with the 1:1 binding stoichiometry may proceed firstly by means of a copper atom
coordinating with N2, O1 and O2 atoms of RhB-Cu, which promotes the PET and
subsequently causes the ring opening of the RhB-Cu accompanied by fluorescence
enhancement (Fig. 3).
8

ACCEPTED MANUSCRIPT
170
171
172
173
174
175
176
177
178
179
180
181
Fig. 2. (a) UV-vis and fluorescence spectra of RhB-Cu (4 µM) in the absence and
presence of 25 equiv. of Cu2+ in CH3CN/H2O (1:1, v/v) solution. The insets show the
photo of RhB-Cu (left) and RhB-Cu in the presence of Cu2+ (right) under ambient (i)
and UV light (ii), respectively. (b) Fluorescence spectra of RhB-Cu (4 µM, λex = 500
nm) exposed to 25 equiv. of Cu2+ and other metal ions in CH3CN/H2O (1:1, v/v)
solution at room temperature. Other metal ions: blank, Ni2+, Co2+, Ca2+, Li+, Mg2+,
Zn2+, Pb2+, Cd2+, Ag+, Hg2+, Fe3+, Al3+, Fe2+, Mn2+, Cr3+ and Cu+ ions. (c) The black
bar is the histogram of (b), the red bar is the fluorescence response of the sensor with
Cu2+ (25 equiv.) and different analytes. (d) Job’s plot according to the method for
continuous variations. The total concentration of RhB-Cu and Cu2+ is 10 µM in
9

ACCEPTED MANUSCRIPT
182
183
184
185
186
187
188
CH3CN/H2O (1:1, v/v) solution. (e) Photos of RhB-Cu with different metal ions under
ambient light (top) and UV irradiation at 365 nm (bottom). From left to right: Cu2+,
Fe3+, Ni2+, Co2+, Ca2+, Li+, Mg2+, Al3+, Fe2+, Zn2+, Mn2+, Cr3+, Pb2+, Cd2+, Hg2+, Ag+,
Cu+.
Table 1 The distances between Cu atom and coordination atoms of RhB-Cu-Cu2+
performed by DFT.
Atom-atom
Cu-O1
Bond distance (Å)
1.84424
Cu-N1
2.95103
Cu-N2
2.49361
Cu-O2
1.92089
189
190
191
192
193
194
195
196
197
198
199
200
Fig. 3. (a) Proposed mechanism for the reaction of RhB-Cu with Cu2+. (b) The
optimized structure of RhB-Cu-Cu2+ by DFT.
3.3. Fluorescence titration with Cu2+and pH effects
More attractively, after titration of various amounts of Cu2+, the detection limit of
RhB-Cu for sensing Cu2+ was found to be as low as 4.7 nM with a signal-to-noise
ratio of 3 (Fig. 4a and b). Compared with the performances of the recently reported
sensors for Cu2+ (Table 2), the RhB-Cu herein not only has an extremely low
10

ACCEPTED MANUSCRIPT
201
202
203
204
205
206
207
detection limit for Cu2+ but also can effectively distinguish Cu2+ from Cu+, indicating
the excellent sensitivity and selectivity of the RhB-Cu. Moreover, after addition of
Cu2+, the solution of RhB-Cu changed from colorless to pink immediately and the
complete enhancement of the fluorescence intensity occurred within 5 min at 25 ºC.
This response time is quite competitive.
According to previous studies [40,41], modified Benesi-Hildebrand expression has
been given as:
ꢀ
ꢁꢂꢃ − ꢀꢄ
1
= 1 +
(1)
ꢀ − ꢀꢄ
ꢅ[ꢆ]ꢇ
208
209
210
211
212
213
214
215
216
where F0, F and Fmax are the fluorescence intensities of RhB-Cu in the absence of
Cu2+, in the presence of different concentrations of Cu2+ and for complete reaction,
respectively, K is the binding constant, [M] is the concentration of Cu2+ and n is the
number of Cu2+ bound per RhB-Cu.
The linear relationship between 1 / (F – F0) and 1 / [M] confirms the 1:1
stoichiometry for binding of Cu2+ by RhB-Cu with an association constant of 6.42 ×
104 M-1 (Fig. 4c). Otherwise, the pH effect on the detection capability suggests that
the RhB-Cu is stable in the pH range from 4 to 8 (Fig. 4d).
11

ACCEPTED MANUSCRIPT
217
218
219
220
221
222
223
Fig. 4. (a) Fluorescence spectra of RhB-Cu (4 µM) with increasing concentration of
Cu2+ (0 - 20 equiv.) in CH3CN/H2O (1:1, v/v) solution (λex = 500 nm, λem = 580 nm).
(b) The plot of the fluorescence intensity of RhB-Cu titrated with increasing
concentrations of Cu2+ at 580 nm. The inset shows the linear range of the curve. (c)
Benesi-Hildebrand plot for the reaction of RhB-Cu with Cu2+. (d) Fluorescence
intensity of RhB-Cu (4 µM) and RhB-Cu with 25 equiv. of Cu2+ at 580 nm under
different pH conditions (λex = 500 nm).
224
225
12

ACCEPTED MANUSCRIPT
226
Table 2 The performances of the recently reported sensors for Cu2+.
Sensor
Target
Cu2+
λex/λem (nm)
745/770
425/522
520/555
515/585
520/617
380/570
420/570
370/508
254/342
470/530
LOD (nM)
50
Ka (M-1)
2.70 × 106
NA
Response to Cu+ mechanism
Ref.
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
1
2
NA
No response
NA
PET
ICT
Cu2+
40
3
Cu2+, Al3+
Cu2+
9.9
1.10 × 106
9.32 × 104
NA
PET
PET
NA
4
110
NA
5
Cu2+
310
42
NA
6
Cu2+
NA
NA
FRET
FRET
NA
7
Cu2+
18.6
140
615
677
NA
NA
8
Cu2+, Fe3+
Cu2+, Hg2+
Cu2+, Al3+
8.60 × 104
5.65 × 104
3.94 × 107
NA
9
NA
PET
NA
10
NA
This
Cu2+
500/580
4.7
6.42 × 104
No response
PET
RhB-Cu
work
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
LOD: Limit of detection. NA: Not available. Ka is association constant.
3.4. The reaction kinetics in solution
The physicochemical properties of solvent molecules should be considered to study
the reaction kinetics in solutions, which could be affected by many factors including
dielectric constant, polarity, ionic strength and solvation. On the other hand, the
change of kinetic parameters in different solvents to some extent also reflects changes
of the reaction itself. In this regard, DFT was performed to calculate the total energy
of the reaction between RhB-Cu and Cu2+ in H2O and CH3CN, whose values were
-2784341.40 and -2784354.16 kcal mol-1, respectively, indicating that the energy
barrier required for this reaction with CH3CN as solvent was lower than that with H2O
as solvent. It should be noting that, the DFT calculation without considering entropy
change would have deviation from the experimental results.
So, further investigation of RhB-Cu exposed to Cu2+ in a mixture solution with
different ratios of CH3CN and H2O (v/v) was conducted (Fig. 5a-e). The rate constants
could be obtained by fitting the fluorescence intensity (F) as a function of reaction
13

ACCEPTED MANUSCRIPT
243
times (t) with a first order kinetic equation (2):
ꢈ
ꢈ
(
)
ꢀ = A + B exp −ꢉꢊ
(2)
244
245
246
where F is the fluorescence intensity, k is the pseudo-first-order kinetic rate constant, t
is the reaction time, and A′ and B′ are constants. Then, the relationship between rate
constant and temperature can be described by Arrhenius equation (3):
ꢌꢂ
lnꢉ = lnꢋ −
(3)
Rꢍ
247
248
249
250
251
where A is the pre-exponential factor, Ea is the apparent activation energy, R is the gas
constant (8.314 J mol-1 K-1), and T is the absolute temperature. According to the
Transition State Theory (TST), the enthalpy and entropy of activation are calculated to
help elucidate the solvent effect. For the reaction in the condensed phase, standard
molar enthalpy of activation (Δꢏꢎ ꢐꢁѲ ) could be obtained by the equation (4):
Δꢏꢎ ꢐꢁѲ = ꢌꢑ − ꢒꢍ
(4)
252
253
Furthermore, according to the thermodynamic functions at the given temperature,
standard molar entropy of activation (Δꢏꢎ ꢓꢁѲ ) could be obtained by the equation (5):
Ѳ
Ѳ
Ѳ ( )
Δꢏꢎ ꢓꢁ
Ѳ ( )
Δꢏꢎ ꢐꢁ
ꢉꢔꢍ
ℎ
ꢕ
ꢕ
ꢉ =
(ꢕѲ)ꢖꢗꢘ exp ꢙ
ꢚ exp ꢙ−
ꢚ
(5)
ꢒ
ꢒꢍ
254
255
256
257
258
259
260
261
262
263
264
265
266
where kB is the Boltzmann constant (1.38 × 10-23 J K-1), h is the Planck constant
(6.626 × 10-34 J S-1) and ꢕѲ= 1mol dm-3.
Then, a series of kinetic parameters were obtained with different percentage of
CH3CN (20% ~ 50%) in H2O (v/v) (Table 3). The activation entropy (Δꢏꢎ ꢓꢁѲ ) and
activation energy (Ea) increase with the increasing percentage of CH3CN in the mixed
solvent indicating that the solvent effect really exists. Actually, in the mixed solvent
system of CH3CN and H2O, the RhB-Cu, lower solubility in water, was surrounded
by acetonitrile molecules, that is a form of solvation compound. When Cu2+ is added,
solvent effect could be destroyed due to complexation. From this perspective, the
higher percentage of CH3CN in the mixed solvent, the more obvious the solvent effect
and the more difficult the reaction between RhB-Cu and Cu2+, which could lead to
increased disorder, entropy, and energy barrier. The experimental results also showed
Δꢏꢎ ꢐꢁѲ > 0, demonstrating that the formation of transition state is endothermic. Taking
14

ACCEPTED MANUSCRIPT
267
268
269
270
271
272
into account the Δꢏꢎ ꢓꢁѲ and Δꢏꢎ ꢐꢁѲ , a conclusion could be drawn that as the CH3CN
content in the mixture solution increases, both the Δꢏꢎ ꢓꢁѲ and Δꢏꢎ ꢐꢁѲ will increase
and the Δꢏꢎ ꢓꢁѲ substantially increase, which suggests that the formation of transition
state gradually changes from enthalpy driven to entropy driven due to the solvent
effect.
273
274
Fig. 5. First order kinetic fitting curves for the reaction of the RhB-Cu with Cu2+ at
different ratio (v/v) of CH3CN and H2O mixture. (a) CH3CN: H2O, 2:8 (v/v), (b)
CH3CN: H2O, 3:7 (v/v), (c) CH3CN: H2O, 4:6 (v/v), and (c) CH3CN: H2O, 5:5 (v/v). (e)
275
276
15

ACCEPTED MANUSCRIPT
277
278
279
280
281
The effect of temperature on the rate constant according to Arrhenius equation, where
k is rate constant and T is temperature. (f) The relationship between ln A and Ea.
Table 3 Kinetic parameters obtained from the pseudo-first-order at different ratio of
CH3CN and H2O (v/v) mixture.
k (min-1)
∆ꢏꢛ ꢐꢁѲ
∆ꢏꢛ ꢓꢁѲ
CH3CN :
Ea
lnA
R2
(kJ·mol-1)
(kJ·mol-1·K-1)
H2O (v/v)
(kJ·mol-1)
(min-1)
310 K
304 K
298 K
2:8
3:7
4:6
5:5
0.5766
1.0606
1.5954
3.2853
0.3405
0.5506
0.7776
1.3945
0.1881
0.2794
0.3645
0.6022
0.9989
0.9999
0.9999
0.9994
71.7
85.4
27.3
33.2
37.1
43.3
69.2
82.9
-60.5
-11.4
21.5
72.8
94.5
92.0
108.6
106.1
282
283
284
285
286
287
288
3.5. The kinetic compensation effect (KCE)
Interestingly, the logarithm of preexponential factor A has a positive linear
correlation with the Ea indicating that the kinetic compensation effect (KCE) possibly
exist in the reaction system (Fig. 5f). According to previous reports [52,53], the
pre-exponential factor A and the activation energy Ea often obey the following
relationship:
ln ꢋ = ꢜ × ꢌꢂ + ꢝ
(6)
289
290
291
292
293
294
295
Where a and b are constants for a series of related process. This relationship is
referred to as the kinetic compensation effect (KCE), which means that according to
Arrhenius eq. (3), an increase of activation energy will not result in a decrease of the
reaction rate due to a compensatory increase of A. Basically, KCE is accompanied by
the appearance of isokinetic point (Tiso, ln kiso), which in turn verifies the validity of
the kinetic compensation effect. According to eq. (6), ln kiso could be calculated by eq.
(7):
ln ꢉꢞꢟꢠ = ꢝ
(7)
296
Further, Tiso can be calculated by eq. (8):
16

ACCEPTED MANUSCRIPT
1
ꢍ
ꢞꢟꢠ
=
(8)
ꢒ × ꢜ
297
298
Besides, ln kiso could also be calculated by eq. (3) at Tiso. In this work, the mean value
of (lnꢉ)|ꢡꢢꢡꢣꢤꢥ is -3.49, which is very close to -3.80 as indicated in the compensation
299
300
301
302
303
plot (Fig. 5f). This result confirms the KCE obtained in the reactions of RhB-Cu with
Cu2+ at different percentage of CH3CN (20% ~ 50%) in H2O.
3.6. Calculation of PL decay parameters
Moreover, the radiative and nonradiative rates of RhB-Cu in the presence of Cu2+
were obtained according to the following equations [54]:
ꢦ
ꢉꢎ =
(9)
(10)
ꢧ
1 − ꢦ
ꢉꢘꢎ
=
ꢧ
304
305
306
307
308
309
310
311
where kr and knr are the radiative and nonradiative rates, respectively, and Φ is the
relative quantum yield and τ is the average lifetime. Φ, τ, kr and knr for the reaction of
the RhB-Cu with Cu2+ have little change at different ratio (v/v) of CH3CN and H2O
(Table 4), which suggests that the change of solvent has little effect on the ring
opening of RhB-Cu and fluorescence emission.
Table 4 Relative quantum yields, lifetimes, radiative rates and nonradiative rates for
the reaction of the RhB-Cu and Cu2+ at different ratio (v/v) of CH3CN and H2O.
CH3CN: H2O (v/v)
Φ
R2
τ (ns)
χ2
kr (108 s-1) knr (108 s-1)
1:9
2:8
3:7
--
--
1.87 ± 0.00289
1.97 ± 0.00297
1.97 ± 0.00295
1.98 ± 0.00297
0.951
0.935
0.898
--
--
0.504
0.498
0.9988
0.9989
0.9993
2.56
2.53
2.52
2.55
4:6
5:5
0.503
0.493
0.912
0.952
2.54
2.43
2.51
2.50
0.9974 2.03 ± 0.00299
312
313
314
315
3.7. Application of RhB-Cu for the detection of Cu2+ in water samples
With the above results in hand, we applied RhB-Cu for Cu2+ detection in real
water samples. Under the optimized conditions, East Lake (in Wuhan city, Hubei
17

ACCEPTED MANUSCRIPT
316
317
318
319
320
321
province, P. R. China) water samples were analyzed by the proposed fluorescence
spectroscopic method. The recovery rates of Cu2+ in the lake water are in the range of
108.4% - 113.5% (Table 5), which indicates that the RhB-Cu could be used for the
highly sensitive detection of Cu2+ in real water samples.
Table 5 Determination of Cu2+ in real water samples by RhB-Cu.
Samples
Added (µM) Detected (mean ± SD, n = 4) Recovery (%)
1.500
2.000
2.500
3.000
1.626 ± 0.067
2.250 ± 0.078
2.837 ± 0.045
3.315 ± 0.081
108.4
112.5
113.5
110.5
Lake
Water
3.500
3.822 ± 0.078
109.2
322
323
4. Conclusion
324
325
326
327
328
329
330
331
332
333
334
335
336
337
In conclusion, a Rhodamine B-based turn-on fluorescent chemodosimeter (RhB-Cu)
for Cu2+ was designed and synthesized with a facile method. This fluorescent sensor
had an extremely low detection limit of 4.7 nM with a fast response time of 5 min. It
could distinguish Cu2+ from other metal ions, including Ni2+, Co2+, Ca2+, Li+, Mg2+,
Zn2+, Pb2+, Cd2+, Ag+, Hg2+, Fe3+, Al3+, Fe2+, Mn2+, Cr3+ and Cu+, which was proved
by fluorescence spectrometry and TD-DFT calculation. Moreover, a possible
mechanism was proposed based on fluorescence spectroscopy, HPLC-MS and DFT
calculations. The coordination reaction with the 1:1 binding stoichiometry may
proceed firstly by means of a copper atom coordinating with N2, O1 and O2 atoms of
RhB-Cu, which promotes the PET and subsequently causes the ring opening of the
RhB-Cu accompanied by fluorescence enhancement. In addition, a series of kinetic
parameters for the reaction of the RhB-Cu with Cu2+ were obtained in the mixed
solvent indicating that the solvent effect really exists. Furthermore, kinetic studies
uncovered the kinetic compensation effect. Finally, RhB-Cu had good performances
18

ACCEPTED MANUSCRIPT
338
339
340
341
in the detection of Cu2+ in real water samples. Future efforts should be devoted to
further improvement of the specific response to copper ions by increasing the water
solubility and reversibility of the sensor as well as the detection of endogenous copper
ions in the organism.
342
Acknowledgments
343
344
345
346
347
348
The authors gratefully acknowledge the financial support from the National Natural
Science Foundation of China (21573168, 21773178, 21873075), the Natural Science
Foundation of Hubei Province (2018CFA090), Guangxi Science and Technology
Project (GuiKeAD17195081), Bagui Scholar Program of Guangxi Province, the
Fundamental Research Funds for the Central Universities (2042017kf0170) and
Large-scale Instrument and Equipment Sharing Foundation of Wuhan University.
349
350
351
352
References
353
354
355
356
357
358
359
360
361
362
363
364
365
366
[1] G.T. Sfrazzetto, C. Satriano, G.A. Tomaselli, E. Rizzarelli, Synthetic fluorescent
probes to map metallostasis and intracellular fate of zinc and copper, Coord.
Chem. Rev. 311 (2016) 125-167.
[2] C.J. Chang, Searching for harmony in transition-metal signaling, Nat. Chem. Biol.
11 (2015) 744.
[3] A.T. Aron, K.M. Ramos-Torres, J.A. Cotruvo Jr, C.J. Chang, Recognition-and
reactivity-based fluorescent probes for studying transition metal signaling in
living systems, Acc. Chem. Res. 48 (2015) 2434-2442.
[4] C.J. Chang, Bioinorganic life and neural activity: toward a chemistry of
consciousness? Acc. Chem. Res. 50 (2017) 535-538.
[5] P.S. Donnelly, Z. Xiao, A.G. Wedd, Copper and Alzheimer's disease, Curr. Opin.
Chem. Biol. 11 (2007) 128-133.
[6] M.G. Savelieff, S. Lee, Y. Liu, M.H. Lim, Untangling amyloid-β, tau, and metals
in Alzheimer’s disease, ACS Chem. Biol. 8 (2013) 856-865.
19

ACCEPTED MANUSCRIPT
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
[7] A. Pal, M. Siotto, R. Prasad, R. Squitti, Towards a unified vision of copper
involvement in Alzheimer's disease: a review connecting basic, experimental,
and clinical research, J. Alzheimer’s Dis. 44 (2015) 343-354.
[8] C.G. Dudzik, E.D. Walter, G.L. Millhauser, Coordination features and affinity of
the Cu2+ site in the α-synuclein protein of Parkinson’s disease, Biochemistry 50
(2011) 1771-1777.
[9] E. Carboni, P. Lingor, Insights on the interaction of alpha-synuclein and metals in
the pathophysiology of Parkinson's disease, Metallomics 7 (2015) 395-404.
[10] M. DiDonato, B. Sarkar, Copper transport and its alterations in Menkes and
Wilson diseases, Biochim. Biophys. Acta, Mol. Basis Dis. 1360 (1997) 3-16.
[11] I.D. Goodyer, E.E. Jones, A.P. Monaco, M.J. Francis, Characterization of the
Menkes protein copper-binding domains and their role in copper-induced protein
relocalization, Hum. Mol. Genet. 8 (1999) 1473-1478.
[12] S.G. Kaler, ATP7A-related copper transport diseases-emerging concepts and
future trends, Nat. Rev. Neurol. 7 (2011) 15.
[13] S. Lutsenko, Atp7b-/- mice as a model for studies of Wilson's disease, Biochem.
Soc. Trans. 36 (2008) 1233–1238.
[14] M.L. Graper, D. Huster, S.G. Kaler, Lutsenko, S.; Schilsky, M. L.; Thiele, D. J.
Introduction to human disorders of copper metabolism, Ann. N. Y. Acad. Sci.
1314 (2014) 37-44.
[15] G. Xiao, Q. Fan, Wang, X.; Zhou, B. Huntington disease arises from a
combinatory toxicity of polyglutamine and copper binding, Proc. Natl. Acad.
Sci. U. S. A. 110 (2013) 14995-15000.
[16] P. Davies, P.C. McHugh, V.J. Hammond, F. Marken, D.R. Brown, Contribution of
individual histidines to prion protein copper binding. Biochemistry 50 (2011)
10781-10791.
[17] A.J. McDonald, J.P. Dibble, E.G. Evans, G.L. Millhauser, A new paradigm for
enzymatic control of α-cleavage and β-cleavage of the prion protein, J. Biol.
Chem. 289 (2014) 803–813.
20

ACCEPTED MANUSCRIPT
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
[18] D. Huster, S. Lutsenko, Wilson disease: not just a copper disorder. Analysis of a
Wilson disease model demonstrates the link between copper and lipid
metabolism, Mol. BioSyst. 3 (2007) 816-824.
[19] T.E. Engle, Copper and lipid metabolism in beef cattle: A review, J. Anim. Sci.
89 (2011) 591-596.
[20] H.S. Jung, P.S. Kwon, J.W. Lee, J.I. Kim, C.S. Hong, J.W. Kim, S. Yan, J.Y. Lee,
J.H. Lee, T. Joo, J.S. Kim, Coumarin-derived Cu2+-selective fluorescence sensor:
synthesis, mechanisms, and applications in living cells, J. Am. Chem. Soc. 131
(2009) 2008-2012.
[21] J.R. Prohaska, Functions of trace elements in brain metabolism, Physiol. Rev. 67
(1987) 858-901.
[22] M. Soylak, I. Narin, M. Dogan, Trace enrichment and atomic absorption
spectrometric determination of lead, copper, cadmium and nickel in drinking
water samples by use of an activated carbon column, Anal. Lett. 30 (1997)
2801-2810.
[23] M. Ghaedi, F. Ahmadi, A. Shokrollahi, Simultaneous preconcentration and
determination of copper, nickel, cobalt and lead ions content by flame atomic
absorption spectrometry, J. Hazard. Mater. 142 (2007) 272-278.
[24] E.S. Forzani, H. Zhang, W. Chen, N. Tao, Detection of heavy metal ions in
drinking water using a high-resolution differential surface plasmon resonance
sensor, Environ. Sci. Technol. 39 (2005) 1257-1262.
[25] Y. Liu, P. Liang, L. Guo, Nanometer titanium dioxide immobilized on silica gel
as sorbent for preconcentration of metal ions prior to their determination by
inductively coupled plasma atomic emission spectrometry, Talanta. 68 (2005)
25-30.
[26] A. Ali, H. Shen, X. Yin, Simultaneous determination of trace amounts of nickel,
copper and mercury by liquid chromatography coupled with flow-injection
on-line derivatization and preconcentration, Anal. Chim. Acta. 369 (1998)
215-223.
21

ACCEPTED MANUSCRIPT
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
[27] A. Bobrowski, K. Nowak, J. Zarębski, Application of a bismuth film electrode to
the voltammetric determination of trace iron using a Fe (III)-TEA-BrO3-catalytic
system, Anal. Bioanal. Chem. 382 (2005) 1691-1697.
[28] Y. Zhao, Y. Ren, H. Li, T. Han, H. Chen, W. Guo, A new far-red
naphthorhodamine dye: Synthesis, fluorescent probe and bioimaging applications,
Dyes Pigments 132 (2016) 255-261.
[29] X. Gu, Q. Chen, Z. Fang, A novel fluorescent probe based on β-C-glycoside for
quantification of bovine serum albumin, Dyes Pigments 139 (2017) 334-343.
[30] F. Xu, W. Tang, S. Kang, J. Song, X. Duan, A highly sensitive and photo-stable
fluorescent probe for endogenous intracellular H2O2 imaging in live cancer cells,
Dyes Pigments 153 (2018) 61-66.
[31] J. Liu, Y. Qian, A novel naphthalimide-rhodamine dye: intramolecular
fluorescence resonance energy transfer and ratiometric chemodosimeter for Hg2+
and Fe3+, Dyes Pigments 136 (2017) 782-790.
[32] M. Hong, S. Lu, F. Lv, D. Xu, A novel facilely prepared rhodamine-based Hg2+
fluorescent probe with three thiourea receptors, Dyes Pigments 127 (2016)
94-99.
[33] A.A. Soares-Paulino, L. Giroldo, G. Celante, C. Lodeiro, A.A. Dos Santos,
Formation of an emissive telluroxide promoted by Hg2+ in aqueous environment:
A new naked-eye and ratiometric rhodamine dimer fluorescent Mercury (II)
probe, Dyes Pigments 159 (2018) 121- 127.
[34] V. Dujols, F. Ford, A.W. Czarnik, A long-wavelength fluorescent chemodosimeter
selective for Cu (II) ion in water. J. Am. Chem. Soc. 119 (1997) 7386-7387.
[35] X. Chen, T. Pradhan, F. Wang, J.S. Kim, J. Yoon, Fluorescent chemosensors
based on spiroring-opening of xanthenes and related derivatives. Chem. Rev. 112
(2011) 1910-1956.
[36] S. Pu, L. Ma, G. Liu, H. Ding, B. Chen, A multiple switching diarylethene with a
phenyl-linked rhodamine B unit and its application as chemosensor for Cu2+.
Dyes Pigments 113 (2015) 70-77.
22

ACCEPTED MANUSCRIPT
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
[37] H. Kang, C. Fan, H. Xu, G. Liu, S. Pu, A highly selective fluorescence switch for
Cu2+ and Fe3+ based on a new diarylethene with a triazole-linked rhodamine 6G
unit. Tetrahedron 74 (2018) 4390-4399.
[38] T. Rasheed, C. Li, Y. Zhang, F. Nabeel, J. Peng, J. Qi, L. Gong, C. Yu,
Rhodamine-based multianalyte colorimetric probe with potentialities as on-site
assay kit and in biological systems. Sens. Actuators, B 258 (2018) 115-124.
[39] S.C. Dodani, A. Firl, J. Chan, C.I. Nam, A.T. Aron, C.S. Onak, K.M.
Ramos-Torres, J. Paek, C.M. Webster, M.B. Feller, C.J. Chang, Copper is an
endogenous modulator of neural circuit spontaneous activity. Proc. Natl. Acad.
Sci. 111 (2014) 16280-16285.
[40] M. Barra, C. Bohne, J.C. Scaiano, Effect of cyclodextrin complexation on the
photochemistry of xanthone. Absolute measurement of the kinetics for
triplet-state exit, J. Am. Chem. Soc. 112 (1990) 8075-8079.
[41] M. Sohrabi, M. Amirnasr, H. Farrokhpour, S. Meghdadi, A single chemosensor
with combined ionophore/fluorophore moieties acting as a fluorescent “Off-On”
Zn2+ sensor and a colorimetric sensor for Cu2+: Experimental, logic gate behavior
and TD-DFT calculations, Sens. Actuators, B 250 (2017) 647-658.
[42] P. Li, X. Duan, Z. Chen, Y. Liu, T. Xie, L. Fang, X. Li, M. Yin, B.A Tang,
near-infrared fluorescent probe for detecting Copper (II) with high selectivity
and sensitivity and its biological imaging applications, Chem. Commun. 47 (2011)
7755-7757.
[43] M. Xu, C. Yin, F. Huo, Y. Zhang, J. Chao, A highly sensitive “ON-OFF-ON”
fluorescent probe with three binding sites to sense copper ion and its application
for cell imaging, Sens. Actuators, B 204 (2014) 18-23.
[44] V.K. Gupta, N. Mergu, L.K. Kumawat, A new multifunctional rhodamine-derived
probe for colorimetric sensing of Cu (II) and Al (III) and fluorometric sensing of
Fe (III) in aqueous media, Sens. Actuators, B 223 (2016) 101-113.
[45] B. Zhang, Q. Diao, P. Ma, X. Liu, D. Song, X. Wang, A sensitive fluorescent
probe for Cu2+ based on rhodamine B derivatives and its application to drinking
23

ACCEPTED MANUSCRIPT
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
water examination and living cells imaging, Sens. Actuators, B 225 (2016)
579-585.
[46] H. Ding, B. Li, S. Pu, G. Liu, D. Jia, Y. Zhou, A fluorescent sensor based on a
diarylethene-rhodamine derivative for sequentially detecting Cu2+ and arginine
and its application in keypad lock, Sens. Actuators, B 247 (2017) 26-35.
[47] Y. Ge, X. Zheng, R. Ji, S. Shen, X. Cao, A new pyrido [1,2-a]
benzimidazole-rhodamine FRET system as an efficient ratiometric fluorescent
probe for Cu2+ in living cells, Anal. Chim. Acta. 965 (2017) 103-110.
[48] J. Tang, S. Ma, D. Zhang, Y. Liu, Y. Zhao, Y. Ye, Highly sensitive and fast
responsive ratiometric fluorescent probe for Cu2+ based on a naphthalimide-
rhodamine dyad and its application in living cell imaging, Sens. Actuators, B
236 (2016) 109-115.
[49] B. Zhang, H. Liu, F. Wu, G. Hao, Y. Chen, C. Tan, Y. Tan, Y. Jiang, A
dual-response quinoline-based fluorescent sensor for the detection of Copper (II)
and Iron (III) ions in aqueous medium, Sens. Actuators, B 243 (2017) 765-774.
[50] J. Dong, Y. Liu, J. Hu, H. Baigude, H. Zhang, A novel ferrocenyl-based
multichannel probe for colorimetric detection of Cu (II) and reversible
fluorescent “turn-on” recognition of Hg (II) in aqueous environment and living
cells, Sens. Actuators, B 255 (2018) 952-962.
[51] X. Zhang, P. Sun, F. Li, H. Li, H. Zhou, H. Wang, B. Zhang, Z. Pan, Y. Tian, X.
Zhang, A tissue-permeable fluorescent probe for Al (III), Cu (II) imaging in vivo,
Sens. Actuators, B 255 (2018) 366-373.
[52] N. Liu, R. Zong, L. Shu, J. Zhou, W. Fan, Kinetic compensation effect in thermal
decomposition of cellulosic materials in air atmosphere. J. Appl. Polym. Sci. 89
(2003) 135-141.
[53] E.L. Mui, W.H. Cheung, V.K. Lee, G. McKay, Compensation effect during the
pyrolysis of tyres and bamboo. Waste Manage. 30 (2010) 821-830.
24

ACCEPTED MANUSCRIPT
510
511
512
[54] R. Prajapati, S. Chatterjee, A. Bhattacharya, T.K. Mukherjee, Surfactant-induced
modulation of nanometal surface energy transfer from silicon quantum dots to
silver nanoparticles, J. Phys. Chem. C. 119 (2015) 13325-13334.
25

ACCEPTED MANUSCRIPT
Highlights:
ò The probe RhB-Cu for Cu (II) had an extremely low detection limit of 4.7 nM.
ò The probe RhB-Cu can distinguish Cu (II) from Cu (I).
ò The kinetic compensation effect (KCE) was proved in this work.