R. Hong, et al.
InorganicaChimicaActa507(2020)119583
studying, for the flexible mood to detect Cu2+ ion and practical re-
quirements for Cu2+ ion detection.
As the third most abundant transition metal ion only less than zinc
and iron in the human body, copper plays an essential role in various
biological processes. Living cells must maintain moderate concentration
of copper ions to keep the normal functioning of enzymes and in-
tracellular metabolic balance. The deficiency and excess of Cu2+
mainly results in severe diseases, such as myelodysplasia, anemia,
leukopenia (low white blood cell count), neutropenia and Wilson’s
disease, Alzheimer’s disease, Parkinson’s disease, etc. Cu2+ is also a
significant environmental pollutant due to its extensively applications
in industry, agriculture, and human daily life [26,27]. The ideal
fluorescent probes for Cu2+ ion should possess the merits of high sen-
sitivity and specificity, low cost, simple operation, good reproducibility
and non-destruction. Application of small organic molecules as che-
mosensor for transition and heavy metal ions have drawn continuous
interest, but the poor water solubility and toxicity is an important issue
that must be solved, they are still limited in practical applications to
some extent [13,28-33]. To further study for fluorescent probes, the
continuation of our previous work based on the ortho-amino azo-
benzene derivatives, we report in this full account the design, synthesis,
and metal-ion sensing behavior of a new compound, 2′-hydroxy-2,4-
diaminoazobenzene (L1-OH). The reaction mechanism of probe L1-OH
and the effect of –OH group on recognition were investigated system-
atically by instrumental analysis and chemical methods. The practical
application of L1-OH on real water sample and HepG2 cell has been
reported to pave path for more reliable and sophisticated chemosensors
based on ortho amino azobenzene.
Fig. 1. UV absorption spectra of L1-OH (16.7 µM) in the presence of 1.0 equiv.
of different cations (Na+, K+, Ca2+, Ag+, Pb2+, Zn2+, Mn2+, Mg2+, Al3+
,
Ni2+, Fe2+, Fe3+, Co2+, Cd2+ and Cu2+).
possible. The limit of detection (LOD) in UV–Vis is estimated to 8.33 µM
for Cu2+ ion, which is much lower than the World Health Organization
(WHO) recommended level (below 30 µM) for safe drinking (Fig. 2a).
The binding constant of the complex was determined from the intensity
data according to Benesi–Hildebrand equation [24]. Accordingly, the
measured absorbance 1/[A-A0] varied as a function of 1/[Cu2+] in a
linear relationship, supporting 1:1 stoichiometry between L1-OH and
Cu2+ with binding constant of 4.157 × 104 (Fig. 2b). The pattern of
absorption spectra remained unchanged when more than 1.0 eq of
Cu2+ was added, corroborated with the formation of a 1:1 (M:L)
complex, and the same conclusion was obtained using the continuous-
variation plot (Job’s plot) (Fig. 3a) [34-36]. To further prove the co-
ordination ability of L1-OH to Cu2+, the addition of EDTA to L1-OH
and Cu2+ system showed that the process of titrating L1-OH with EDTA
was reversible at 5–6 times, reflecting the chelation Cu2+ with L1-OH
and repeatability of the L1-OH as colorimetric sensor (Fig. 3b).
2. Results and discussion
2.1. Colorimetric study of L1-OH to Cu2+ ion
The UV absorbance spectrum of L1-OH was determined in EtOH
solution at a concentration in range of 6.67 ∼ 33.33 µM, revealing the
strong absorption peak at 465 nm, a linear relationship was observed
between the absorption intensity and concentration of L1-OH with
molar absorption coefficient ε = 2.403 × 104 L·mol−1·cm−1 (Fig S1).
The relative big value of ε can make colorimetric response between L1-
OH and Cu2+ more sensitive. Initially, the sensing ability of L1-OH
Most of the probes are performed only in the solution phase and
hence this restricts their practical applications. Therefore, to evaluate
the potential use of the sensor L1-OH to determine Cu2+ ions in real
water samples, L1-OH loaded test paper was prepared. The target test
papers were simply produced by immersing filter papers into EtOH
solution of L1-OH (5 × 10−4 M) and then drying in air for 20 min. To
investigate whether L1-OH could be applicative to real samples under
laboratory condition, the experiments were performed in
Quanyangquan drinking mineral water samples (Major Components:
H2SiO3 25.0–35.0 mg/L, K+ 0.8–3.0 mg/L, Na+ 1.7–5.8 mg/L, Ca2+
3.1–5.9 mg/L, Mg2+ 1.6–7.1 mg/L), which is sourced from Changbai
Mountain 5A Natural Scenic Reserve in Jilin Province, China. Without
any spectroscopic instrumentation, the test paper dyed with L1-OH was
soaked in aqueous solution to be measured (Adding a certain range
concentration of copper ion in advance: ≥10 µM), the marked color
change from yellow to light pink was observed immediately (Fig. 4).
The rapid and obvious color change of the test paper reflects the
practical application of L1-OH for water quality monitoring. To eval-
uate the selectivity of L1-OH for Cu2+ (5 × 10−4 M), a variety of metal
toward various metal cations (Na+, K+, Ca2+, Ag+, Pb2+, Zn2+
,
Mn2+, Mg2+, Al3+, Ni2+, Fe2+, Fe3+, Co2+, Cd2+,and Cu2+) has been
investigated qualitatively by visual examination of the cation-induced
color changes of the receptor. As shown in Fig. 1, upon the addition of
1.0 equiv. of Cu2+ ions to the solution of L1-OH (16.7 µM), the obvious
color change was observed instantly from yellow to light pink. By
contrast, the addition of other surveyed metal ions failed to cause any
vivid color change, which reflects the capability of L1-OH to response
Cu2+ ion selectively. To provide fundamental insights into the suit-
ability of L1-OH, the Cu2+ ion recognition behavior of the sensor was
explored using UV–Vis spectroscopy upon the addition of various con-
centrations of cations to ligand solution. The UV–Vis spectrum of the
free ligand exhibited an intense band centered at 465 nm which is at-
tributed to the π-π* transition. Upon the addition of various metal ions,
only Cu2+ was able to have a profound effect on the electronic spec-
trum of L1-OH. Addition of aqueous solution of Cu2+ ion to the L1-OH
solution led to the emergence of a new band with absorption maxima at
505 nm with a simultaneous decrease in the absorbance at 465 nm.
However, no such apparent spectral changes were induced by the ad-
dition of other metal ions. To further assess the binding characteristics
of L1-OH, the UV–Vis spectroscopic titration of L1-OH (16.7 µM) were
performed with Cu2+ ion. With increasing Cu2+ concentration, the
isosbestic points at about 482 nm can be observed explained by the
form of “Cu2+ + L1-OH” complex. A plot of absorbance at 505 nm of
L1 vs Cu2+ concentration (8.33 ∼ 16.7 µM) showed a linear relation,
hence the quantitative detection of Cu2+ via UV–Vis absorption is
ions with same concentration including Na+, K+, Ca2+, Ag+, Pb2+
,
Zn2+, Mn2+, Mg2+, Ni2+, Fe2+, Fe3+, Co2+, Cd2+ and Al3+ were
examined under the same condition as Cu2+, the fast and sharp color
change of the test paper can also be seen, indicating the sensor is
promising for the analysis of environmental samples. It is noted that the
test paper can be regenerated by immersing in EDTA (5 × 10−3 M)
solution for around 5 min. The pH scope of application is 2.0 ∼ 12.0,
which is determined by adding various pH buffer solution to mixture to
2