Chromogenic and fluorogenic detection of copper ions in the solution and intracellular media

Article abstract of DOI:10.1002/jccs.201800319

The development of a fluorescent sensor for the trace detection of the metallic contamination in the ecosystem and biological media have been a demand to reduce the health risk and environmental protection. Herein, we report the synthesis of 2-formyl-5-pi

Full text of DOI:10.1002/jccs.201800319

Received: 15 August 2018
Revised: 13 November 2018
Accepted: 15 November 2018
DOI: 10.1002/jccs.201800319
A R T I C L E
Chromogenic and fluorogenic detection of copper ions
in the solution and intracellular media
Muhammad Hanif1 | Muhammad Rafiq2 | Muhammad Saleem3 | Muhammad Mustaqeem3 |
Saba Jamil4 | Muhammad Ramzan Saeed Ashraf Janjua5,6
1Department of Chemistry, GC University
The development of a fluorescent sensor for the trace detection of the metallic con-
Faisalabad, Sub campus Layyah, Pakistan
2Department of Biochemistry and Physiology,
Cholistan University of Vaterniray and Animal
Sciences, Bahawalpur, Pakistan
3Department of Chemistry, University of
Sargodha, Sub campus Bhakkar, Bhakkar,
Pakistan
4Laboratory of Superlight Materials and Nano
Chemistry, Department of Chemistry, University
of Agriculture, Faisalabad, Pakistan
5Department of Chemistry, King Fahd University
of Petroleum and Minerals (KFUPM), Dhahran,
Kingdom of Saudi Arabia
tamination in the ecosystem and biological media have been a demand to reduce
the health risk and environmental protection. Herein, we report the synthesis of
2-formyl-5-picolinyl-substituted rhodamine B derivative through Schiff base for-
mation for the trace detection of the copper ions in the aqueous organic media as
well as inside the live cells via bioimaging experiments. The ligand was success-
fully employed to detect the minute copper level ratiometrically through the chro-
mogenic and fluorogenic response. The detection limit of the sensor was found to
be 0.54 × 10−9 mol/L using 3 σ/slope relations. The reversible behavior of the
ligand copper complex was determined using sulfide ions that regenerate the entire
native properties of the ligands by snatching copper from the complex. The bioima-
ging experimental results represent the efficient membrane permeability of ligands
with the minimum level of toxicity in the measured range found by the MTT assay.
The appearance of bright red fluorescence from the BHK-21 (hamster kidney fibro-
blast) cells upon treatment of the ligand incubated cells with the copper ions repre-
sents the utility of the proposed sensor for the copper detection probe in the
intracellular media.
6Department of Chemistry, University of
Sargodha, Sargodha, Pakistan
Correspondence
Muhammad Saleem, Department of Chemistry,
University of Sargodha, Sub-campus Bhakkar,
Bhakkar, Pakistan.
Email: saleemqau@gmail.com
Muhammad Ramzan Saeed Ashraf Janjua,
Department of Chemistry, King Fahd University
of Petroleum and Minerals (KFUPM), Dhahran
31261, Kingdom of Saudi Arabia.
Email: janjua@kfupm.edu.sa
KEYWORDS
bioimaging, chromogenic, copper sensor, cytotoxicity, fluorogenic, imine
processes in organisms ranging from bacteria to mammals.[2]
Copper ions also catalyze the formation of reactive oxygen
1
| INTRODUCTION
The design and synthesis of artificial sensors for biologically
important metal ions attracts the attention from the analytical
chemists for the fast, accurate, reproducible, and selective
determination of various species due to their fundamental
roles in a wide range of chemical, environmental, and bio-
logical processes involved in multidisciplinary fields includ-
ing life science, environmental, agricultural, industrial,
medicinal analysis, and biotechnology.[1]
species (ROS) that can damage lipids, nucleic acids, and
proteins.[3] It is well known that it serves as a catalytic cofac-
tor for a variety of metalloenzymes including superoxide dis-
mutase, cytochrome c oxidase, and tyrosinase, and its
homeostasis is critical for the metabolism and development
of living organisms.[4] Copper-catalyzed enzymes regulate
several body functions including skin pigmentation via mel-
anin transformation, facilitate the formation of crosslinks in
collagens, elastin, and involved in repair of connective tis-
sues.[5] Unregulated overloading of copper neuronal cyto-
plasm can be toxic and can cause oxidative stress and
Copper (II) ion, being the third adequate element in the
human body among the essential metals following iron and
zinc, plays crucial roles in many fundamental physiological
© 2018 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
J Chin Chem Soc. 2018;1–6.
http://www.jccs.wiley-vch.de
1

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HANIF ET AL.
disorders associated with neurodegenerative diseases such as
Alzheimer's, Parkinson's, Menkes, Wilson, familial amyo-
trophic lateral sclerosis, and prion diseases.[6,7] Therefore,
the limit of copper in drinking water as set by the US Envi-
ronmental Protection Agency (EPA) is 1.3 ppm (~20 μM).[8]
Designing heterocycle-based fluorescent sensors that is,
rhodamine based,[9,10] benzimidazole based,[2,7] anthracene
based,[11] biphenyl based,[12] dipyrromethene (BODIPY),[8]
calix[4]arene based,[13,14] chromenone based,[5] cyclodextrin
based,[15] indene-1,3-dione based,[16] phenylenediamine
based,[17] thiazole based,[18] carbazole based,[19] ferrocene
based,[20] and pyrrole based,[21] is an interesting strategy for
probing a minute copper level in the sample. Fluorescent
probes made up of organic materials for the heavy metal ion
detection attracted widespread interest for environmental
detection of contaminants. It has the advantageous features
including high sensitivity, quick response, and nondestruc-
tive nature. There are several reports on the organic
material-based fluorescent sensors for trace copper detection
but only a few exhibited the applications of the proposed
sensor toward imaging of live cells.[11,22–28]
Schiff bases are the important scaffold employed broadly
in multiple dimensions for chemical production and scien-
tific research as chelating agents, stabilizers, biologically
active agents, analytic, and catalytic reagents.[29] In the pre-
sent investigation, we have synthesized a 2-formyl-5-picoli-
nyl-substituted rhodamine B derivative by treatment of
rhodamine B hydrazide with 2-formyl-5-picoline and evalu-
ated their complexation tendency toward several metal ions.
Rhodamine B substituted with the picoline acid via amide
linkage was reported by Liu et al.[30] for the copper ion
detection by the reaction of rhodamine B acid chloride and
pyridine-2-carboxylic acid hydrazide through the amide
bond formation. The present Schiff base derivative reported
possesses longer electronic dense cloud over the lower loop
of the molecular skeleton, which is of benefit to bind effi-
ciently with the copper ions. Furthermore, it is more facile to
generate the Schiff base ligand via imine formation reaction
under gentle experimental conditions. We have further
employed our reported ligands for copper signaling inside
the live cells and determined the toxicity profile of the
ligand. We believe that the present findings will be helpful
for the future designing of a metal ion detector via imine
linkage, which is compatible for live cell imaging due to its
membrane permeable efficacy. High product yield, ease of
synthesis, and low toxicity of the probe are the features that
inspire us to select the target compound.
SCHEME 1 Synthesis of ligand 3; reagents and conditions. (i) Hydrazine,
methylene chloride, reflux; (ii) 2-formyl-5-picoline, methylene chloride, and
reflux
oxychloride, which was then treated with the hydrazine
hydrate to afford the corresponding hydrazide. The treatment
of 2-formyl-5-picoline with the freshly prepared rhodamine
hydrazide affords the targeted Schiff base derivative. The
disappearance of the broad acidic hydroxyl peak in the range
of 3,400–2,500 cm−1 in the FT-IR spectrum indicates the
conversion of rhodamine acid into its chloride. The rhoda-
mine hydrazide formation was visually detected by the
change of color of the material into off while solids, which
affords a broad signal in the range of 3,389–3,340 cm−1 for
primary amide stretching vibration. The targeted Schiff base
formation was indicated by the FT-IR spectral investigation
by the emergence of a signal at 1,586 cm−1, which was
assigned to the C=N stretching vibration. The synthetic
pathway adopted to get the target molecule is shown in
Scheme 1.
2.2 | Ligand–metal chelation
The ligand–metal complexation mechanism was assessed
using optical measurements as well as FT-IR spectroscopic
analysis. The colorimetric change in the ligand solution after
copper ion addition as well as the emergence of new absorp-
tion and emission signals represent the conformational
changes in the ligand skeleton triggered by the copper ions.
There was slight shifting of the amide carbonyl as well as
C=N signal position after complexation with the copper ions
indicating the attachment of the copper to the receptor liga-
tion site. In the case of nuclear magnetic resonance (NMR)
spectral analysis, there was no noticeable disappearance or
shifting in the signals after copper complexation representing
that the molecular skeleton remains the same involving only
the electronic rearrangement. The reversibility of the recep-
tor with that of the sulfide ions confirms the complexation
process via colorimetric as well as florigenic variation. Col-
orimetric as well as chromogenic features of the ligand-
copper complex were recovered upon induction of the sul-
fide ion, which was due to the formation of copper sulfide,
as copper was displaced from its position, it will regenerate
the entire ligand features (Scheme 2).
2
| RESULTS AND DISCUSSION
2.3 | Spectroscopic properties
The spectroscopic properties of the ligand were investigated
in aqueous/acetonitrile (40:60, v/v) and the corresponding
spectra are shown in Figure 1. The free ligand solution was
colorless and nonemissive due to its existence in the
2.1 | Synthesis of ligand 3
The rhodamine B acidic functionality was transformed into
acid chloride by the treatment with phosphorous

HANIF ET AL.
3
The addition of the copper ion into the ligand solution trig-
gered an intense emission signal at 591 nm whose intensity
continues to increase upon successive copper ion addition
from 0.2–1.4 equivalent. The fluorescent titration graph was
also utilized to find out the limit of detection of
0.54 × 10−9 mol/L for the copper ions via 3 σ/slope. The
slope was obtained utilizing the inset graph (Figure 2). From
the titration graph, the association constant value was deter-
SCHEME 2 Proposed spirolactam ring-opening mechanism of ligand
3 upon copper chelation in acetonitrile/water (6:4, v/v) at neutral pH and its
reversibility with sulfide ions
mined to be 1.19 × 105 M−1
.
spirolactam conformation while upon copper ion addition
there was a chromogenic response in the reaction solution
from the colorless to the light pink. This chromogenic
change was due to the conformation changes in the ligand
molecule upon chelation with the metal ions. The UV–
visible properties of the ligand were investigated upon treat-
ment of the ligand solution with 0.5 μM copper ion and the
other metallic species including Sc3+, Yb3+, In3+, Ce3+, Sm3
+, Cr3+, Sn2+, Pb2+, Ni2+, Co2+, Ba2+, Ca2+, Mg2+, Ag+,
Cs+, Cu+, and K+ in aqueous/acetonitrile (40:60, v/v,
pH 7.0.) at ambient temperature. The ligand shows no
The quick response by the sensor toward the metal ion
recognition is a prerequisite to obtain better results. To
understand this, the fluorescent emission signal intensity was
observed with different time intervals from 1 to 10 min. The
results showed that the maximum emission signal intensity
was observed around 1–2 min of the sample preparation and
these results were quite satisfactory for a good sensing mate-
rial. Above 2 min of incubation, the emission spectral results
become stable as shown in Figure S1, Supporting
Information.
The practical applicability of the sensor toward the cop-
per ions was determined by utilizing the distilled water as
well as tape water and the same emission response was
observed by either of the solvent representing that proposed
material can be utilized directly for the metallic contaminant
assessment in the tape water, lake, or river water
(Figure S2). Moreover, this experiment represents that there
was no metallic contamination in our tap water used.
response upon induction of Sc3+, Yb3+, In3+, Ce3+, Sm3+
,
Cr3+, Sn2+, Pb2+, Ba2+, Ca2+, Mg2+, Ag+, Cs+, Cu+, and
K+ while less intense absorption spectrum was appeared in
the case of Ni2+ and Co2+. Interestingly, the high-intensity
absorption band was observed with the maximum absorption
at 558 nm upon copper ion induction, which was utilized for
the sensing of the copper ions. The ratiometric response of
the sensor toward the copper ions was determined by the
successive induction of the copper ions from 0.2 to 1.4
equivalent into the 0.5 μM ligand solution. The results
showed that there was constant increment in the absorption
signal intensity at 558 nm upon successive copper ion induc-
tion and these results might be utilized for the ratiometric
copper ion detection (Figure 1).
The effect of the pH was studied in the buffer solution
(PBS and Tris HCL buffer) ranging from pH 2–8 as shown
in Figure S3. The ligand exhibited the absorption and emis-
sion signals in the acidic pH range, which represent that the
spirolactam ring of the synthesized probe was fragile and
underwent ring opening involving the protonation pathway.
In the neutral pH range, the ligand was supposed to exist in
The similar response of the ligand toward the copper
ions was observed upon fluorescent spectral measurement.
FIGURE 2 The fluorescence titration of ligand 3 (0.5 μM) at emission
maxima of 591 nm as a function of copper concentration (0.2–1 eq), the
inset describes the fluorescence enhancement at emission maxima of
591 nm
FIGURE 1 UV–visible absorption spectral titration of ligand 3 (0.5 μM) in
the presence and absence of Cu2+ (0.2–1 eq) in acetonitrile/water (6:4, v/v)
at pH = 7.0

4
HANIF ET AL.
the spirolactam ring closure conformation. The copper ion +
ligand solution was still emissive in the neutral pH range
representing the conformational change in the ligand trig-
gered by the copper ions. In the basic environment, there
was again downfall in the emission signal intensity of the
ligand copper complex because of decreasing the copper ion
concentration due to involvement of the copper in the gener-
ation of Cu(OH)2.
FIGURE 3 Confocal fluorescence microscopic images of BHK-21 cells;
(a) BHK-21 cells without incubation with the probe (control); (b) bright
filed images of BHK-21 cells incubated with probe (0.5 μM);
(c) fluorescence images of BHK-21 cells incubated with probe (0.5 μM) in
the presence of (1 eq) copper ion; and (d) merged images of BHK-21 cells
incubated with probe (0.5 μM) in the presence of (1 eq) copper ion
Fluorescence quantum yield of ligand-Cu2+ complex
solution was calculated to be ФFL = 0.53 (relative to the
standard rhodamine B in acetonitrile, Фstd = 0.59), using
Equation 2. The ligand showed 0.0071 values for the fluo-
rescence quantum yield.[31–33]
level of probe toxicity toward the tested cell lines. Moreover,
there was bright red fluorescence coming from the probe-
incubated cells upon treatment with the copper ions. The
increasing concentration of the copper ions brings more red-
dishness from the cells. This increment in the fluorescence
brightness upon successive copper induction can be utilized
to find out the metal contamination level inside the cells in
the ratiometric manner (Figure 3).
Фunk ¼ Фstd ðIunk=AunkÞðAstd=IstdÞðnunk=nstdÞ,
ð1Þ
where Фunk is the fluorescence quantum yield of the sample,
Фstd is the quantum yield of the standard, and Iunk and Istd
are the integrated fluorescence intensities of the sample and
the standard, respectively, Aunk and Astd are the absorbances
of the sample and the standard at the absorption wavelength,
respectively, nunk and nstd are the refractive indices of the
corresponding solutions.
The effect of the solvent on the ligand-copper complexa-
tion process was established by utilizing different percentage
of the organic and mixed aqueous organic solvents. The
ligand becomes precipitated in the pure water while becomes
soluble upon increasing the percentage of the organic sol-
vent. The suitable ratio of the aqueous-organic solvent was
determined by the series of experiments and the results are
shown in Figure S4.
3.1 | Cytotoxicity analysis
The toxicity analysis of the receptor was carried out by the
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] analysis. The assay was performed after 4 and
24 hr of ligand treatment with BHK-21 (hamster kidney
fibroblast). The result represents 100% cell viability toward
the tested cell lines (Figure 4).
4
| EXPERIMENTAL
The experimental results including absorption signal
intensity at the absorption maxima of 558 nm and molar
absorptivity, obtained by varying solvents and their ratios,
are tabulated in Table 1. The minimum absorption signal
intensity was observed in the pure aqueous media due to the
ligand precipitation while there was the same results for the
mixed organic-aqueous solvents including acetonitrile:
water, methanol: water, and ethanol: water.
4.1 | Substrate and reagents
Rhodamine B, 2-formyl-5-picolyl, hydrazine hydrate and
hydrazine in THF, glacial acetic acid, phosphorous oxy-
chloride, and NaHCO3 were purchased from Aldrich. Etha-
nol, methanol, 1,2-dichloroethane, methylene chloride,
acetonitrile, acetone, water, dimethyl sulfoxide, hexane, and
3
| BIOIMAGING ANALYSIS
To trace the copper contamination in the intracellular media,
the confocal fluorescent microscopic analysis was conducted
utilizing BHK-21 (hamster kidney fibroblast) cells. The
experimental results exhibited that there wasno deformation
in the cells during the course of study showing the minimum
TABLE 1 The effect of solvents on the copper ligand complexation
Abs. Intensity
S. No.
Solvents
at 558 nm
0.019
1.24
1
2
3
4
H2O
MeCN:H2O
MeOH:H2O
EtOH:H2O
FIGURE 4 Cell viability of BHK-21 cells cultured in complete media with
ligand 3 (0.5 μM) while the control cells were cultured in the medium
without ligand
1.24
1.24

HANIF ET AL.
5
ethyl acetate (Samchun Chemicals, Korea), and Sc(OTF)3,
YbCl3ꢀ6H2O, InCl3, CeCl3, SmCl3ꢀ6H2O, CrCl3ꢀ6H2O,
SnCl2, PbCl2, FeCl3ꢀnH2O, NiCl2ꢀ6H2O, CoCl2ꢀ6H2O,
CuCl2ꢀ2H2O, BaCl2ꢀ2H2O, CaCl2ꢀ2H2O, CsCl, CuCl, and
KCl (Aldrich and Alfa Aesar) were used during experiment.
The major chemicals utilized for biological studies include
minimum essential media (MEM, Wel Gene, Korea), fetal
bovine serum (FBS, Bio West USA), Tripsin (Thermo Sci-
entific, South Loga, UT), PBS (Wel Gene), and MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide, Sigma-Aldrich, USA].
1H, s), 6.43–6.31 (aromatic, 5H, m), 3.35–3.29 (aliphatic,
8H, q, J = 10 Hz), 2.84–2.80 (aliphatic, 1H, q, J = 8.5 Hz),
1.28 (NH, 2H, bs), 1.09–1.07 (aliphatic, 12H, t, J = 9 Hz),
and 2.28 (aliphatic, 3H, s); 13C NMR (125 MHz, DMSO-d6)
δ175.9, 152.3, 152.2, 151.8, 149.7, 149.3, 148.3, 135.6,
136.1, 129.9, 128.8, 128.4, 127.8, 126.9, 117.3, 116.1, 48.1,
17.7, and 12.8; MS for C35H37N5O2(ESI, m/z), 560.6
[M + H]+. Anal. Calcd. For C35H37N5O2: C, 75.07; H, 6.54;
and N, 12.31; Found: C, 75.10; H, 6.62; and N, 12.49%.
4.4 | General procedure for spectroscopic assay
Stock solution of the ligand (300 μM) was prepared by dis-
solving 1.679 mg ligand in methanol (total volume 10 mL).
Similarly, to prepare Cu2+ stock solution (300 μM),
1.344 mg of copper (II) chloride was dissolved in distilled
water (total volume 10 mL) to get 1 mmol solution, then
3 mL of this solution was diluted to 10 mL with distilled
water to get 300 μM Cu2+ stock solution. All the metal ion
solution was prepared using the procedure similar to that used
for preparation of Cu2+ stock solution. For spectroscopic
measurements, test solution of 1 mL was prepared with
780 μL of 40% aqueous methanol, 10 μL of ligand stock
solution, 0.1 mL of buffer solution (10 mM), and 10 μL of
Cu2+ stock solution. The resulting solution was mixed before
measurement and the final volume was fixed as 1 mL for
UV–visible and Fluorescent measurement using a [SCINCO]
UV–Vis Spectrophotometer “S-3100” and a FS-2 fluores-
cence spectrometer (SCINCO, Korea), respectively.
4.2 | Instrumentations
The reaction progress was monitored by thin layer chromato-
graphic (TLC) analysis, and the Rf values were determined
by employing precoated silica gel aluminum plates, Kiesel-
gel 60F254, from Merck (Germany), using dichloromethane:
methanol, 9:1, as an eluent. TLC was visualized under a UV
lamp (VL-4. LC, France). The FT-IR spectra were recorded
in KBr pellets on a SHIMADZU FTIR-8400S spectrometer
(Kyoto, Japan). Proton and carbon nuclear magnetic reso-
nance (1H NMR &13C NMR) spectra were recorded on a
Bruker Avance 500 MHz spectrometer with TMS as an
internal standard. The chemical shifts are reported as δ
values (ppm) downfield from the internal tetramethylsilane
of the indicated organic solution. Peak multiplicities are
expressed as follows: s, singlet, bs, broad signal, d, doublet,
t, triplet, q, quartet, and m, multiplet. The coupling constants
(J values) are given in hertz (Hz). Mass spectra were
recorded on the AB SCIEX Co. 4000 QTRAP LC/MS/MS
System. Abbreviations are used as follows: DMSO-d6,
Dimethyl sulfoxide-d6; FT–IR spectroscopy, Fourier trans-
form infrared spectroscopy, MC, Methylene chloride, and
DMAP, 4-Dimethylaminopyridine.
4.5 | General procedure for the MTT assay
The MTT assay was carried out following the reported pro-
cedure.[34] Briefly, the cells were incubated with Cu2
+(50 μM) and ligand 3 (60 μM) for 24 hr. Then, cells were
washed with phosphate-buffered saline (PBS), and incu-
bated with Dulbecco's Modified Eagle's medium (DMEM
medium, 200 μL/well) containing 50 μL of MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide, 5 mg/mL] solution. Following 2 hr incubation at
37ꢁC, growth medium was removed gently from the plate
and 200 μL/well dimethyl sulfoxide was added to solubi-
lize the produced purple formazan crystals. Later, the
absorbance for each well was measured at 560 nm using
microplate spectrophotometer systems (BioTek, synergy
HT) and the results were calculated in percentage with
respect to untreated sample called control.
4.3 | General procedure for the synthesis of ligand 3
Rhodamine B Schiff base ligand 3 was synthesized from the
rhodamine B hydrazide 2 (1 eq) in the presence of 2-formyl-
5-picolyl (1 eq) and a catalytic amount of glacial acetic acid.
The rhodamine B hydrazide 2 was synthesized upon treat-
ment of rhodamine B acid chloride with hydrazine under
reflux conditions. The rhodamine B acid chloride was pre-
pared by the reaction of rhodamine B and phosphorus oxy-
chloride in the presence of methylene chloride as a solvent.
The final product was purified by column chromatography
and utilized for the analysis.
4.3.1
|
30,60-Bis(diethylamino)-2-(([5-methylpyridin-2-yl]
5
| CONCLUSIONS
methylene)amino)spiro[isoindoline-1,90-xanthen]-3-one (3)
Light yellow powder; yield: 78%; 1H NMR (500 MHz,
DMSO-d6) δ 8.55 (aromatic, 1H, s), 7.81–7.78 (aromatic, 3H,
m), 7.67 (aromatic, 1H, t, J = 7 Hz), 7.55–7.46 (aromatic,
2H, m), 7.00–6.98 (aromatic, 1H, m), 6.31(aliphatic imine,
In summary, a simple, economic, and efficient rhodamine
Schiff base derivative 30,60-Bis(diethylamino)-
2-(([5-methylpyridin-2-yl]methylene)amino)spiro[isoindo-
line-1,90-xanthen]-3-one (3) was synthesized by treatment
B

6
HANIF ET AL.
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CONFLICTS OF INTEREST
The authors declare no potential conflict of interests.
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ACKNOWLEDGMENTS
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This work was supported by the University of Sargodha.
The project was initiated and designed in the University of
Sargodha, Pakistan.
ORCID
Muhammad Ramzan Saeed Ashraf Janjua
https://orcid.org/0000-0002-
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4323-1736
SUPPORTING INFORMATION
Additional supporting information may be found online in
the Supporting Information section at the end of the article.
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