Ultrasensitive turn-on fluorescence detection of Cu2?+ based on p-dimethylaminobenzamide derivative and the application to cell imaging

Article abstract of DOI:10.1016/j.saa.2016.09.011

A new p-dimethylaminobenzamide derivative based compound BDIH has been synthesized. Cu^2?+ turned on the fluorescence of compound BDIH with a 1:2 binding stoichiometry. The fluorescent color of compound BDIH shows an evident change from colorless to bright blue upon the addition of Cu^2?+, which could be visibly detected by the naked eye under UV light at 365?nm. More importantly, the detection limit was found to be 0.64?nM which is far lower than the maximal allowed concentration of the WHO limit (31.5?μM) for drinking water. This selective “turn-on” fluorescence sensor was used to identify Cu^2?+ in living cells using confocal fluorescence microscopy, indicating that compound BDIH has a potential application for selective detection of Cu^2?+ in organism.

Full text of DOI:10.1016/j.saa.2016.09.011

Ultrasensitive turn-on fluorescence detection of Cu2 + based on p-
dimethylaminobenzamide derivative and the application to cell imaging
Peng-Cheng Huang, Hao Fang, Jing-Jing Xiong, Fang-Ying Wu
PII:
DOI:
Reference:
S1386-1425(16)30529-7
doi:10.1016/j.saa.2016.09.011
SAA 14657
To appear in:
Received date:
Revised date:
Accepted date:
20 April 2016
7 September 2016
11 September 2016
Please cite this article as: Peng-Cheng Huang, Hao Fang, Jing-Jing Xiong, Fang-
Ying Wu, Ultrasensitive turn-on fluorescence detection of Cu2 + based on p-
dimethylaminobenzamide derivative and the application to cell imaging, (2016),
doi:10.1016/j.saa.2016.09.011
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
Ultrasensitive turn-on fluorescence detection of Cu2+ based on
p-dimethylaminobenzamide derivative and the application to cell imaging
Peng-Cheng Huang, Hao Fang, Jing-Jing Xiong, Fang-Ying Wu
College of Chemistry, Nanchang University, Nanchang 330031, China
Abstract A new p-dimethylaminobenzamide derivative based compound BDIH has been
synthesized. Cu2+ turned on the fluorescence of compound BDIH with a 1: 2 binding
stoichiometry. The fluorescent color of compound BDIH shows an evident change from colorless
to bright blue upon the addition of Cu2+, which could be visibly detected by the naked eye under
UV light at 365 nm. More importantly, the detection limit was found to be 0.64 nM which is far
lower than the maximal allowed concentration of the WHO limit (31.5 μM) for drinking water.
This selective “turn-on” fluorescence sensor was used to identify Cu2+ in living cells using
confocal fluorescence microscopy, indicating that compound BDIH has a potential application for
selective detection of Cu2+ in organism.
Keywords p-dimethylaminobenzamide derivative, fluorescence, Cu2+, living cells
Corresponding author: Fang-Ying Wu, Tel: + 86 7913969882(O). E-mail address: fywu@ncu.edu.cn
1

ACCEPTED MANUSCRIPT
1. Introduction
Acting as one of the most abundant transition metal ions in the biological environment and
essential trace elements in the human body, Cu2+ has a significant influence on Cu2+ binding
enzyme activities, such as oxidase, salivary amylase, tyrosinase, which related enzymes can play a
critical role as an isomerous component and as a catalytic co-factor 1-3. Deficiency intake of Cu2+
4
5
6
leads to Parkinson’s disease , Alzheimer’s disease and Wilson’s disease . However, under
overloading conditions, Cu2+ exhibits toxicity to the human body and leads to neurodegenerative
diseases and oxidative stress probably by its involvement in the procedure of reactive oxygen
species 7. Furthermore, Cu2+ is a crucial environmental contaminant owing to its widespread use in
8
agriculture and industries. It can be incorporated in the environment, water and biologic chain .
The World Health Organization (WHO) has set the maximum allowable level of copper in
drinking water at 2 ppm (31.5 μM) 9. Hence detection and recognition of Cu2+ has garnered much
attention owing to its importance in biological and environmental systems in recent years.
Compared with some analytical methods including atomic absorption spectrometry 10, atomic
emission spectrometry 11, and inductively coupled plasma-mass spectrometry 12, fluorescent
techniques are powerful tools to monitor ions due to simplicity, convenience, low cost, high
sensitivity, specificity, low background, fast response time, and rapid monitoring of target ions in
practical biological and environmental applications 13-15, 16. Metal-selective fluorescent sensors act
as useful tools for exploitation of metal ions and thus have been extensively studied to explore
biologically and environmentally relevant metal ions 17-22. On surveying the literature, most of
reported and investigated Cu2+ selective fluorescence probes have some drawbacks, for example,
long response times 23, tedious synthetic procedures 24, large molecular structure 25, interference
from other transition metal ions and high background with weak enhancement 26. Particularly,
there are still few examples of “turn-on” type sensors practicable 1, 27-30. These would badly
restrict their further applications in biological or environmental systems. Thus, to explore simple,
easy to-make and highly sensitive and selective fluorescent probes for Cu2+ in biological and
environmental system is still in an intense demand.
To fulfill the above-mentioned requirements, we herein report a compound of
p-dimethylaminobenzamide
derivative,
benzoic
acid-4-(dimethylamino)-
2

ACCEPTED MANUSCRIPT
2-(1H-indol-3-ylmethylene)hydrazide (BDIH) (Scheme 1), to construct a fluorescent assay for
Cu2+ with a very low detection limit, one-step synthetic process, and high chemical stability
during at least 3 months. Compound BDIH displayed a selective response toward Cu2+ via
significant turn-on fluorescence in aqueous media. Moreover, compound BDIH can also be used
to quantify Cu2+ on the paper test strips and as a practical, visible fluorescent detection kit for Cu2+.
Finally, compound BDIH was further applied to cell imaging of Cu2+ using hepatoma carcinoma
cells.
Scheme 1
2. Experimental Section
2.1 Reagents
Indole-3-carboxaldehyde is bought from Sinopharm Chemical Reagent Co. Ltd. (Shanghai,
China). Cu(NO3)2, FeCl3, NaCl, Ca(NO3)2, MgCl2, NiCl2, CdCl2, AlCl3, CrCl3, HgCl2, AgNO3,
Mn(NO3)2, CoCl2, Pb(NO3)2 and ZnCl2 were purchased from Shanghai Qingxi Technology Co.
Ltd. (Shanghai, China). All other reagents were of analytical grade and were used as supplied
without further purification. All aqueous solutions were prepared using ultrapure water from a
Milli-Q system.
2.2 Apparatus
All fluorescence measurements were carried out on an F-4500 spectrofluorimeter (Hitachi,
Japan) equipped with a xenon lamp source and a 1.0 cm quartz cell, the scan speed was 2400 nm
min-1 and the band pass of excitation and emission was set as 10 nm, respectively. Voltage was
400 V. Excitation wavelength was set at 352 nm. Absorption spectra were performed on a
Shimadzu-2550 UV-Vis spectrophotometer (Shimadzu, Japan) using a 1.0 cm quartz cell. ESI-MS
data were recorded on a Waters ZQ4000/2695 mass spectrometer (Waters, America). NMR
spectra were recorded on an Agilent Technologies Plus-400 MR spectrometer (Bruker BioSpin,
America) with DMSO-d6 as the solvent and tetramethylsilane (TMS) as internal standard.
Elemental analyses (C, H and N) were performed on an Elementary Vario EL analyzer (Elementar,
Germany). Infrared spectra were taken in KBr disks on a Nicolet 5700 FTIR spectrometer (Nicolet,
3

ACCEPTED MANUSCRIPT
America). Fluorescence imaging was performed using a Zeiss LSM 710 fluorescence microscope
(Carl Zeiss, Germany).
2.3 Methods
General UV–vis and fluorescence spectra measurements: All measurements were carried out
at room temperature. Stock solution of compound BDIH was prepared by dissolving 0.1 mM
compound BDIH in DMSO. Further dilution was made to prepare 1.0 μM solution for the
fluorescence experiments with MeCN-H2O (v/v=4:1, pH=5) HAc-NaAc buffer solution. The
UV–vis spectra were conducted with 10 μM of BDIH in MeCN-H2O (v/v=4:1, pH=5) HAc-NaAc
buffer solution.
General procedure for 1H NMR experiments: For 1H NMR titration, both compound BDIH and
Cu2+ were prepared in DMSO-d6. First of all, only compound BDIH in DMSO-d6 was added into
the NMR tube, and then Cu2+ was added. All solutions were mixed directly in the NMR tube.
2.4 Cell culture
The hepatoma carcinoma cells were seeded in the petri dish. The cells were incubated with
BDIH overnight at 37 °C to determine the cell permeability of BDIH. After being washed with
phosphate buffer solution (PBS) three times to remove the remaining BDIH, Cu2+ was added into
the cells to incubate for 30 min at 37 °C as the case of BDIH (without PBS). Confocal
fluorescence imaging was performed with a Zeiss LSM 710 microscope.
2.5 Synthesis of BDIH
p-dimethylaminobenzamide BDIH (CAS number: 540789-01-3) was prepared following a
literature method 31,32. The structure and synthesis procedure of compound BDIH is shown in
Scheme 1. Indole-3-carboxaldehyde (0.1596 g, 1.1 mmol) and p-dimethylaminobenzamide
(0.1791 g, 1.0 mmol) were dissolved in 25 mL of dry ethanol solution under reflux conditions at
77 °C for 3 h. A white precipitate was found. The reaction mixture was allowed to attain room
temperature, and then the precipitate was collected through filtration. The residue was washed
thoroughly with ethanol to isolate BDIH in pure form with 86% yield (the yield was calculated
4

ACCEPTED MANUSCRIPT
based on the starting reagents). Compound BDIH was characterized by IR, NMR, ESI-mass
spectrometry and element analysis data, which were consistent with the proposed formula. IR
1
(KBr, cm-1): ν=1608 cm-1 (C=O), 1577 cm-1 (C=N); H NMR (400 MHz, DMSO-d6) δ(ppm):
11.50 (s, 1H, N-H), 11.19 (s, 1H, NH), 8.57 (s, 1H, Ar-H), 8.28 (d, J = 6.6 Hz, 1H, Ar-H), 7.78
(dd, J = 22.5, 3H, Ar-H), 7.41 (d, J = 7.9 Hz, 1H, Ar-H), 7.27-7.09 (m, 2H,Ar-H), 6.74 (d, J = 8.7
Hz, 2H, Ar-H), 3.03-2.92 (s, 6H, N-CH3); 13C NMR (400 MHz, DMSO-d6) δ(ppm): 162.75,
152.64, 143.93 137.43, 130.07, 129.26, 124.81, 122.94, 122.49, 120.64, 112.42, 112.17, 111.30.
ESI-MS: calcd for [C18H18N4O + H]+ 307.2, found 307.2. Anal. calcd for C18H18N4O: C 70.53, H
5.87, N 18.28; Found: C 70.75, H 5.79, N 18.14.
3. Results and discussion
3.1 Fluorescence spectral change of compound BDIH in the presence of various ions
To investigate binding properties of compound BDIH (1.0 μM) with the cations and anions,
various ions including Na+, Ni2+, Pb2+, Cr3+, Zn2+, Cu2+, Ag+, Al3+, Mg2+, Ca2+, Cd2+, Mn2+, Co2+,
-
-
2-
-
2-
2-
-
Fe3+, Hg2+, F-, Cl-, Br-, I-, HCO3 , H2PO4 , HPO4 , HSO4 , SO4 , SO3 , NO3 , SCN-, Ac-, CN-
were examined in the MeCN-H2O (pH=5, v/v=4:1) HAc-NaAc buffer solution (Fig. 1).
Fluorescence spectra were recorded after 8 min upon the addition of each of these ions.
Compound BDIH itself exhibited no fluorescence. It was found that while the addition of 11 μM
other ions did not show no fluorescence enhancement, Cu2+ could cause an evident fluorescence
enhancement at 419 nm (excitation wavelength is set at 352 nm, Fig. S1). Meanwhile, upon the
addition of Cu2+, compound BDIH solution changed from colorless to blue under a UV lamp at
365 nm, and such a color change can be visibly observed by the naked eyes (Fig. 1 inset). The
above observations support that compound BDIH is highly efficient in detecting Cu2+ over other
foreign cations and anions existing in physiological and environmental samples. Therefore,
compound BDIH can be potentially used to design a Cu2+-selective turn-on fluorescent probe
worked in aqueous solution.
Fig. 1
3.2 Optimization of the assay for Cu2+
5

ACCEPTED MANUSCRIPT
To estimate the high selectivity in the organic-aqueous mixture, we added various amounts of
H2O to the MeCN solution, achieving the percentage of 0 to 90 as indicated. As shown in Fig. S2,
we explored the influence of Cu2+ on the fluorescence of BDIH with varying of H2O: MeCN ratios
(0:100-90:10). It can be seen that the most sensitive fluorescence response was observed at a
MeCN: H2O ratio of 4:1.
To study the practical applicability of the Cu2+-probe in environmental or biological samples,
the effect of various pH values on the fluorescence spectral change of compound BDIH towards
Cu2+ was investigated. As revealed in Fig. S3, free BDIH showed no fluorescence at the pH range
3.5-8.1. The increase in the fluorescence intensity at low pH values (3.5-5) is owing to the
formation of BDIH-Cu2+ complex. Under high pH conditions (pH> 5), the emission intensity at
419 nm gradually decreased, presumably due to the dissociation of complex BDIH-Cu2+ by the
formation of the precipitation of Cu(OH)2, reducing the concentration of Cu2+ in the sample
solution. It should be noted that the probe could also give a similar response to Cu2+ in
MeCN-H2O (v/v=4:1) solution even if the buffer solution is not utilized. However, in order to
maintain the solution stability, the pH of 5 was chosen to evaluate the response of compound
BDIH to Cu2+. We also studied the time course of the response of compound BDIH in the
presence of 11 μM Cu2+, and found that the fluorescence intensity remarkably increased to a
maximum within 8 min (Fig. S4).
3.3 Fluorescence titration of compound BDIH with Cu2+
Fluorescence spectra changes of BDIH upon titration with Cu2+ is shown in Fig. 2. Upon
excitation at 352 nm, compound BDIH alone showed very weak fluorescence. The fluorescence
titration experiment of BDIH for Cu2+ led to a remarkable enhancement at 419 nm with the
solution emission color change from colorless to blue, implying a Cu2+-selective turn-on
fluorescence signal behavior. A good linear correlation was obtained for the fluorescence intensity
of compound BDIH in the concentration range of Cu2+ between 0 and 15 μM with the detection
limit as low as 0.64 nM, indicating this probe could be employed for quantitative detection of Cu2+
with ultrahigh sensitivity.
Fig. 2
6

ACCEPTED MANUSCRIPT
3.4 Effect of coexisting components
To further study the selectivity of BDIH to Cu2+ in complicated systems, competitive
experiments were performed with other ions in the case of 11 μM Cu2+. As shown in Fig. 3, no
obvious fluorescence change happened in the presence of 1000 equiv. different competitive
-
cations (Na+, Mg2+, Al3+, Cd2+, Co2+, Ca2+, Zn2+, Ni2+, Mn2+) and anions (F-, Cl-, Br-, I-, HCO3 ,
-
-
2-
-
2-
2-
NO3 , H2PO4 , HPO4 , HSO4 , SO4 , Ac-, SO3 , SCN-, CN-), and the data also displayed that Cr3+
(100 equiv.) Ag+, Hg2+, Pb2+ (10 equiv.), and Fe3+ (4 equiv.) caused negligible response to the
fluorescence of BDIH-Cu2+. The competitive experiments indicated that compound BDIH was
capable of detecting Cu2+ in environmental and biological applications without the interference
from most cations and anions.
Fig. 3
3.5 Absorption spectra of compound BDIH to Cu2+
The absorption spectrum of BDIH showed the maximum peak at 336 nm in MeCN-H2O
(v/v=4:1, pH=5) HAc-NaAc buffer solution. Upon gradual addition of Cu2+ ions to the solution of
BDIH, the absorption peak gradually decreased and reached a minimum at 24 equiv. of Cu2+ at
336 nm. At the same time a new absorption band at 265 nm slowly increased. Moreover, a clear
isosbestic point at 293 nm was observed, implying the conversion of the free receptor BDIH to a
copper complex (Fig. S5). To further study the sensing selectivity of compound BDIH toward
Cu2+, we carried out the selectivity experiments to various ions (Fig. S6). The absorption spectrum
varied little by the presence of other cations and anions, except that Cu2+ induced an obvious
decrease of the absorption peak at 336 nm.
3.6. “Naked-eye” and test strips measurement
As depicted in Fig. 4, color change was observed by separate addition of various metal ions
(11 μM) into compound BDIH (1.0 μM) in MeCN-H2O (v/v=4:1, pH=5) HAc-NaAc buffer
solution. Under a UV lamp at 365 nm, compound BDIH showed an obvious color change from
colorless to blue only upon binding with Cu2+, whilst other metal ions did not induce any
7

ACCEPTED MANUSCRIPT
remarkable color change under identical conditions. So the selective visual color change can be
used for the “naked eye” detection of Cu2+ in aqueous media.
Fig. 4
To study the practical application of proposed compound BDIH, paper test strips were
prepared for on-site detection of Cu2+. In our experiment, Cu2+ solutions of five different
concentrations, 0, 10-6, 5×10-5, 10-5, 10-4 M, were prepared. For this purpose, filter papers were
dipped in a 1.0 μM BDIH in MeCN-H2O (v/v=4:1, pH=5) HAc-NaAc buffer solution for about 10
min and then dried in air. Under UV light at 365 nm, an obvious emission color change was
observed clearly by naked eyes when the test strips were applied for sensing different Cu2+
concentrations. As revealed in Fig. 5, the color of paper test strips was converted from colorless to
intense blue gradually as the concentration of Cu2+ increased, which was the same as that for the
corresponding test vial. This enabled the sensor quite useful for immediate on-site detection of
Cu2+.
Fig. 5
3.7 The proposed reaction mechanism
The binding stoichiometry of BDIH with Cu2+ was investigated by a Job’s plot (Fig. S7) in
which the total concentration of BDIH and Cu2+ kept at 10 μM. The fluorescence [1/(F - F0)] at
419 nm varied as a function of 1/[Cu2+]2 in a linear relationship (R = 0.992), and the analytical
result confirmed a 2 : 1 stoichiometry of complex BDIH-Cu2+ with a binding constant of
1.33×1010 M-2 (Fig. S8). Moreover, the ligand BDIH was inherently poorly fluorescent owing to
rapid isomerization of the O=C-N (-O-C=N) bond in the excited state. In the presence of Cu2+,
compound BDIH could coordinate to Cu2+ with a strong fluorescence enhancement phenomenon.
As shown in Scheme 2, these properties indicated that nitrogen atoms of the benzazole and Schiff
base moiety and oxygen atoms of the carbanyl group bearing the lone pair of electrons in BDIH
can efficiently chelate to Cu2+. The binding of BDIH with Cu2+ obviously increased the
-
fluorescence at 419 nm in the excited state due to the combined effect of suppression of O-C=N
isomerization. The fluorescence quantum yield of complex BDIH-Cu2+ was as high as 0.89, while
8

ACCEPTED MANUSCRIPT
that of compound BDIH was only 0.15, both obtained with quinine sulfate solution in 0.05 M of
H2SO4 (φ=0.55 at 363 nm excitation) as the reference 33, which also supporting significant
fluorescence enhancement.
Scheme 2
To demonstrate the proposed binding of compound BDIH with Cu2+, more direct evidence
1
was provided by IR spectroscopy, H NMR titrations and ESI-MS titration experiments. The IR
spectrum of compound BDIH-Cu2+ showed a change in the bending vibration band of the
secondary N-H corresponding to indole fragment relative to compound BDIH, that is, the peaks at
1577 and 766 cm-1 disappeared completely. Meanwhile the stretching vibration band of C=O
corresponding to amide fragment shifted to a higher wavenumber from 1608 cm-1 to 1615 cm-1
(Fig. 6). The stretching vibration band of C=N group at 1528 cm-1 shifted to 1509 cm-1. These
changes suggested that Cu2+ coordinated to oxygen atoms of carbanyl group, the secondary amine
of indole fragment and nitrogen atoms of Schiff base fragment, respectively.
Fig. 6
1H NMR titration analysis was carried out to further illustrate the characteristic structural
changes occurring upon interaction between BDIH and Cu2+ in DMSO-d6. As shown in Fig. 7, for
BDIH the imine proton in 11.21 ppm and methylene proton in 8.57 ppm consistent to the amide
and Schiff base moiety gradually decreased and disappeared completely upon the addition of Cu2+
while the imine proton in 11.50 ppm corresponding to the indole moiety shifted to upfield at 11.49
ppm along with a slight decline of the proton signal. This result indicates that the binding with
Cu2+ takes place with the carbanyl oxygen atom and nitrogen atom of Schiff base and amide
fragment. Moreover, the ESI mass spectrum of BDIH (Fig. S9) displayed a peak at m/z = 307.2
(calcd=307.26) corresponding to [BDIH+H]+. The complex BDIH-Cu2+ with the 2 : 1 complex
formation between BDIH and Cu2+ was supported by showing a peak at m/z 675.2 (calcd.=675.02)
corresponding to [2BDIH+Cu-H]+ (Fig. S10).
Fig. 7
9

ACCEPTED MANUSCRIPT
3.8 Fluorescence imaging of Cu2+ in living cells
The properties of BDIH for high selectivity and sensitivity towards Cu2+ have further
prompted us to extend our research to detect Cu2+ in biological systems by live cell imaging
experiments. The fluorescence imaging was carried out in hepatoma carcinoma living cells as
showed in Fig. 8. When incubated with 10 μM of BDIH for 12 h at 37 °C, the cells showed no
intracellular fluorescence. The cells were washed with PBS three times to remove free BDIH.
Upon treating above cells with Cu2+ (20 μM) for 30 min at 37 °C (here PBS is discarded as its
presence would influence fluorescence intensity), the intracellular blue fluorescence was
significantly observed in hepatoma carcinoma cells. This result indicated that compound BDIH
can be used as a fluorescent probe for the recognition of intracellular Cu2+ in living cells.
Fig. 8
4. Conclusion
In summary, we have successfully synthesized compound BDIH with a high yield and easy
to-make which exhibited an obvious fluorescence enhancement response to Cu2+ at 419 nm
enabling highly selective and sensitive detection of Cu2+ at a very low detection limit. Its
application in quantitative detection of Cu2+ using simple paper-made test kits was performed.
Accordingly, the probe was successfully used for fluorescence imaging of Cu2+ in living cells. It
can be envisaged that BDIH could be one of the most promising alternatives for the detection of
Cu2+ in biological and environmental systems.
Acknowledgement
This work is financially supported by Natural Science Foundation of China (No. 21365014
and 21505067). We greatly thank Dr. Xiaohong Zhao, College of Chemistry, Nanchang
University, China, for helpful discussion.
References
10

ACCEPTED MANUSCRIPT
[1] K.M.K. Swamy, S.-K. Ko, S.K. Kwon, H.N. Lee, C. Mao, J.-M. Kim, I. Shin, J. Yoon, Chem.
Commun. 44 (2008) 5915-5917.
[2] E. Gaggelli, H. Kozlowski, D. Valensin, G. Valensin, Chem. Rev. 106 (2006) 1995-2044.
[3] B.P. Zietz, J. Dassel de Vergara, H. Dunkelberg, Germany, Environ. Res. 92 (2003) 129-138.
[4] J.C. Lee, H.B. Gray, J.R. Winkler, J. Am. Chem. Soc. 130 (2008) 6898-6899.
[5] Y.H. Hung, A.I. Bush, R.A. Cherny, Inorg. Chem. 15 (2010) 61-76.
[6] E. Madsen, J.D. Gitlin, Annu. Rev. Neurosci. 30 (2007) 317-337.
[7] K.J. Barnham, C.L. Masters, A.I. Bush, Nat. Rev. Drug Discovery 3 (2004) 205-214.
[8] C. Zong, K. Ai, G. Zhang, H. Li, L. Lu, Anal. Chem. 83 (2011) 3126-3132.
[9] G.R. You, G.J. Park, J.J. Lee, C. Kim, Dalton Trans. 44 (2015) 9120-9129.
[10] Y. Yamini, N. Alizadeh, M. Shamsipur, Anal. Chim. Acta 355 (1997) 69-73.
[11] S.L.C. Ferreira, A.S. Queiroz, M.S. Fernandes, H.C. dos Santos, Spectrochim. Acta, Part B,
57B (2002) 1939-50.
[12] C.F. Harrington, S.A. Merson, T.M. D' Silva, Anal. Chim. Acta 505 (2004) 247-254.
[13] W. Zhu, X. Huang, Z. Guo, X. Wu, H. Yu, H. Tian, Chem. Commun. 48 (2012) 1784-1786.
[14] Y. Kubo, T. Ishida, A. Kobayashi, T.D. James, J. Mater. Chem. 15 (2005) 2889-2895.
[15] Y.H. Lee, N. Park, Y.B. Park, Y.J. Hwang, C. Kang, J.S. Kim, Chem. Commun. 50 (2014)
3197-3200.
[16] H.N. Kim, W.X. Ren, J.S. Kim, J. Yoon, Chem. Soc. Rev. 41 (2012) 3210-3244.
[17] A. Coskun, E.U. Akkaya, J. Am. Chem. Soc. 128 (2006) 14474-14475.
[18] X. Peng, J. Du, J. Fan, J. Wang, Y. Wu, J. Zhao, S. Sun, T. Xu, J. Am. Chem. Soc. 129 (2007)
1500-1501.
[19] S.-K. Ko, Y.-K. Yang, J. Tae, I. Shin, J. Am. Chem. Soc. 128 (2006) 14150-14155.
[20] Y. Xiang, A. Tong, Org. Lett. 8 (2006) 1549-1552.
[21] J.-S. Wu, I.-C. Hwang, K.S. Kim, J.S. Kim, Org. Lett. 9 (2007) 907-910.
[22] X. Chen, S.-W. Nam, M.J. Jou, Y. Kim, S.-J. Kim, S. Park, J. Yoon, Org. Lett. 10 (2008)
5235-5238.
[23] J.-H. Ye, J. Xu, H. Chen, Y. Bai, W. Zhang, W. He, Tetrahedron Lett. 55 (2014) 6269-6273.
11

ACCEPTED MANUSCRIPT
[24] J. Huang, M. Liu, X. Ma, Q. Dong, B. Ye, W. Wang, W. Zeng, RSC Adv. 4 (2014)
22964-22970.
[25] L. Liu, A. Wang, G. Wang, J. Li, Y. Zhou, Sens. Actuators, B 215 (2015) 388-395.
[26] Q. Liu, Q. Fei, Y. Fei, Q. Fan, H. Shan, G. Feng, Y. Huan, Spectrochim. Acta, Part A, 151
(2015) 785-789.
[27] Z. Xu, Y. Xiao, X. Qian, J. Cui, D. Cui, Org. Lett. 7 (2005) 889-892.
[28] S.H. Kim, J.S. Kim, S.M. Park, S.-K. Chang, Org. Lett. 8 (2006) 371-374.
[29] L. Zeng, E.W. Miller, A. Pralle, E.Y. Isacoff, C.J. Chang, J. Am. Chem. Soc. 128 (2006)
10-11.
[30] M. Royzen, Z. Dai, J.W. Canary, J. Am. Chem. Soc. 127 (2005) 1612-1613.
[31] F. Palace-Berl, K.F.M. Pasqualoto, S.D. Jorge, B. Zingales, R.R. Zorzi, M.N. Silva, A. K.
Ferreira, R. A. d. Azevedo, S. F. Teixeira and L. C. Tavares, Eur. J. Med. Chem. 96 (2015)
330-339.
[32] A.R.B. Rao, S. Pal, J. Organomet. Chem. 696 (2011) 2660-2664.
[33] P. Huang, Q. Jiang, P. Yu, L. Yang, L. Mao, ACS Appl. Mater. Interfaces 5 (2013) 5239-5246.
12

ACCEPTED MANUSCRIPT
Captions
Scheme 1 The structure and synthesis process of compound BDIH.
Fig. 1 Fluorescence spectra of compound BDIH (1.0 μM) upon the addition of 11 μM of Cu2+ and
other cations and anions in the MeCN-H2O (v/v=4:1, pH=5) HAc-NaAc buffer solution. The inset
shows the photographs of the solution of BDIH before and after the addition of Cu2+ (11 μΜ)
under a UV lamp at 365 nm.
Fig. 2 Fluorescence titration spectra of compound BDIH (1.0 μM) upon the addition of Cu2+ (0, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 equiv.) in MeCN-H2O (v/v=4:1,
pH=5) HAc-NaAc buffer solution. Inset: Linear plot of fluorescent intensity for BDIH upon the
addition of Cu2+ concentration (0-15 equiv.) at 419 nm.
Fig. 3 Fluorescent selectivity of BDIH toward Cu2+ over various competitive cations and anions in
MeCN-H2O (v/v=4:1, pH=5) HAc-NaAc buffer solution.
Fig. 4 Photographs of the color of BDIH (1.0 μM) in MeCN-H2O (v/v=4:1, pH=5) HAc-NaAc
buffer solution in the presence of different metal ions under a UV lamp at 365 nm.
Fig. 5 Fluorescent photographs of the test strips according to the solution color of BDIH (1.0 μM)
for the detection of various concentrations of Cu2+ from left to right: 0, 10-6, 5×10-5, 10-5, 10-4 M.
Scheme 2 The proposed binding mode of compound BDIH with Cu2+.
Fig. 6 IR spectra of BDIH and BDIH-Cu2+.
Fig. 7 1H NMR spectra of BDIH and BDIH + Cu2+ measured in DMSO-d6.
13

ACCEPTED MANUSCRIPT
Fig. 8 Fluorescence microscopic images of hepatoma carcinoma cells incubated with 10 μM
BDIH, adding 20 μM of Cu2+ to the BDIH-treated cells.
14

ACCEPTED MANUSCRIPT
Scheme 1
Fig. 1
Fig. 2
15

ACCEPTED MANUSCRIPT
Fig. 3
Fig. 4
Fig. 5
Scheme 2
16

ACCEPTED MANUSCRIPT
Fig. 6
Fig. 7
17

ACCEPTED MANUSCRIPT
Fig. 8
Graphical abstract
18

ACCEPTED MANUSCRIPT
19

ACCEPTED MANUSCRIPT
Highlights
1. An
ultrasensitive
fluorescence
“turn-on”
assay
for
Cu2+
based
on
p-dimethylaminobenzamide-based derivative was presented.
2. The probe exhibits excellent selectivity and a very high tolerance to other common ions by the
naked eye and fluorescence spectra.
3. The assay was applied to detect Cu2+ on the test kits and in living cells.
4. Compound BDIH showed a very low background.
20