2,6-Pyridinedicarbohydrazide-Salicylal hydrazone-base derivative with High detection limit and binding Constant for emissive ion chemosensing in aqueous solution

Article abstract of DOI:10.1016/j.jphotochem.2019.112344

A new tridentate luminescent molecule, N²,N⁶-bis(salicylidene)pyridine-2,6-dicarbohydrazide (BSPDH), was introduced and facilely eco-synthesized with high yield. It was then characterized through Fourier transform infrared, Hydrogen-

Full text of DOI:10.1016/j.jphotochem.2019.112344

Journal Pre-proof
2,6-Pyridinedicarbohydrazide-Salicylal Hydrazone-base Derivative with
High Detection Limit and Binding Constant for Emissive Ion
Chemosensing in Aqueous Solution
Abbas Amini (Supervision) (Conceptualization), Marjan Rahimi,
Hossein Behmadi, Marziyeh Nazari, Veronika Benson, Chun
Cheng, Bijan Samali
PII:
S1010-6030(19)31713-7
DOI:
https://doi.org/10.1016/j.jphotochem.2019.112344
JPC 112344
Reference:
To appear in:
Journal of Photochemistry & Photobiology, A: Chemistry
Received Date:
Revised Date:
Accepted Date:
7 October 2019
12 December 2019
24 December 2019
Please cite this article as: Amini A, Rahimi M, Behmadi H, Nazari M, Benson V, Cheng C,
Samali B, 2,6-Pyridinedicarbohydrazide-Salicylal Hydrazone-base Derivative with High
Detection Limit and Binding Constant for Emissive Ion Chemosensing in Aqueous Solution,
Journal of Photochemistry and amp; Photobiology, A: Chemistry (2020),
doi: https://doi.org/10.1016/j.jphotochem.2019.112344

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2,6-Pyridinedicarbohydrazide-Salicylal Hydrazone-base Derivative with High Detection
Limit and Binding Constant for Emissive Ion Chemosensing in Aqueous Solution
Abbas Aminia,b*, Marjan Rahimic, Hossein Behmadic,*, Marziyeh Nazarid, Veronika Bensone,
Chun Chengf, Bijan Samalia
a Centre for Infrastructure Engineering, Western Sydney University, Penrith, NSW 2751,
Australia
b Department of Mechanical Engineering, Australian College of Kuwait, Safat 13015, Kuwait
c Department of Chemistry, Mashhad Branch, Islamic Azad University, Mashhad, Iran
d Department of Mathematics, Australian College of Kuwait, Safat 13015, Kuwait
e Institute of Microbiology, Czech Academy of Sciences, Videnska, Czech Republic
f Department of Materials Science and Engineering, South University of Science and
Technology, Shenzhen, China
* Corresponding authors.
E-mail address: a.amini@westernsydney.edu.au (A. Amini), behmadi@mshdiau.ac.ir (H. Behmadi)
Highlights





Developed new tridentate acylhydrazone-base chemosensor with large Stokes shift
Enhanced emission of the ligand in solution and solid-state due to aggregation
Fast turn-off Cu2+ detection by probe in the aqueous medium at pH=7.4
High detection limit and binding constants of 3.11×10−5 M and 1.978×105 M-1
DFT Computational results confirmed the experimental sensory behavior of ligand
1

Abstract
A new tridentate luminescent molecule, N2,N6-bis(salicylidene)pyridine-2,6-dicarbohydrazide
(BSPDH), was introduced and facilely eco-synthesized with high yield. It was then characterized
through Fourier transform infrared, Hydrogen-1 nuclear magnetic resonance, Carbon-13 nuclear
magnetic resonance, mass and photo-luminescence spectroscopy, X-Ray Diffraction and
Ultraviolet-visible absorption methods. BSPDH showed an enhanced aggregation-induced
emission as well as highly fast-response “turn-off” selectivity and sensitivity behaviors towards
Cu2+ ions in aqueous solutions at physiological pH. Density Functional Theory calculations
confirmed the formation of strong BSPDH-Cu2+ complex with lower energy gap compared to
BSPDH. Among PDH-base fluorescent sensors reported so far, BSPDH is the first
tautomerization-base luminescence molecular chemo-sensor with the high detection limit of
3.11×10−5 M and binding constant of 1.978×105 M-1 at the concentration of 10 μM of BSPDH for
detecting Cu2+ ions without any interference from other metal cations in the aqueous solution at
the physiological pH.
Abbreviations
BSPDH, N2,N6-bis(salicylidene)pyridine-2,6-dicarbohydrazide; DMSO, dimethyl sulfoxide;
ESIPT, excited-state intramolecular proton transfer; Δλ, Stokes shift; PL, photo-luminescence;
DFT, Density Functional Theory; DL, Detection limit; Kb, Binding constant; THF,
Tetrahydrofuran;
pH=7.4,
Physiological
pH;
HEPES,
(4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid) buffer; DMF, Dimethyformamide; FT-IR, Fourier transform
infrared spectra; XRD, X-Ray Diffraction; TMS, tetramethylsilane; AIEE, Aggregation induced
enhanced emission; fw, water fraction; SSBs, salicylidene Schiff-base derivatives; ACQ,
2

aggregation caused quenching; PDH-base, pyridine-2,6-dicarbohydrazide base; ACN, acetonitrile;
MeOH, Methanol; HEPES buffer, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer;
EtOH, Ethanol; PDA, Pyridine-2,6-dicarboxylic acid; SOCl2, Thionyl chloride; DMAC, N,N-
dimethylacetamide; ICT, intramolecular charge transfer; and, CHEF, chelation-enhanced
fluorescence.
Key words: Molecular fluorescent chemo-sensor, Salicylaldehyde derivative, AIEE-activity,
diacylhydrazone, tridentate ligand
Introduction
A variety of π-conjugated organic molecules, including salicylidene Schiff-base derivatives
(SSBs), has been utilized for various optoelectronic applications, e.g., fluorescent sensors [1, 2],
optical memories [3], OLEDs [4, 5] and photo-switches [6]. SSBs structures have excellent photo-
physical and photo-chromism properties, due to the tautomerism of keto/enol through
intramolecular hydrogen bond formed between the phenol hydrogen atoms of salicylaldehyde
derivatives and imine-N. This setup constructs a six-member loop acting as chelating ligands to
combine with metal ions where the intramolecular interactions result in pH dependent properties
[7-9]. SSBs have been extensively used as model systems to study fundamental photophysical
processes, such as excited-state intramolecular proton transfer (ESIPT) [10], solvatochromism
[11], mechanochromism [12] and thermochromism [13]. As such, SSBs are potential multi-
responsive materials with significant photo-physical property and thermal stability to meet the
versatile needs of modern technology [14].
3

According to a discovery by Tang et al., the “aggregation induced emission enhancement (AIEE)”
phenomenon is occurred in some luminescent molecules which are weak or non-emissive when
dissolved in good solvents as molecules, and highly luminescent when clustered in poor solvents
or aggregated in solid-state [15]. By then, AIEE-active materials expanded with diverse
functionalities for various fields, such as optoelectronic materials [16], chemical sensors [17] and
biomedical probes [18]. The emission of such luminophore solutions is quenched by increasing
the concentration of luminophore.
After iron and zinc, copper (Cu) is the third most abundant metal ions, which is well-known for its
vital role in numerous physiological organisms and human health, including enzyme function,
blood formation, respiration, and transcriptional events in biological processes. Deficiency of
copper causes failure in growth and deterioration of nerve system. On the other hand, Copper is a
significant metal pollutant due to its widespread use in life science, medicine, chemistry and
biotechnology. Excessive Cu content is highly toxic and may cause unregulated problems in
humans including Alzheimer, Parkinson, Menke and Wilson diseases [19], due to its involvement
in the generation of reactive oxygen species. Therefore, it is important to develop a fast-response
and sensitive probe to detect the balance of copper ions in aqueous media and living systems. So
far, some selective and sensitive classical chemo-sensors have been used to monitor Cu2+ ions
through atomic absorption spectrometry [20], ICP-OES (Inductively Coupled Plasma Optical
Emission Spectrometry) [21], anodic stripping voltammetry (ASV) [22], and total reflection X-
Ray fluorescence (TXRF) spectrometry [23]. Unfortunately, most of these sensors have various
drawbacks, including the requirement to complicated instrumentations and data analysis,
operational difficulties, delayed response time, serious interferences from other transition metal
ions, etc.
4

Among various probes, fluorescent sensors are demanded due to their cost-effective analytical
configurations. They work on the basis of light-controlled molecular data processing through
molecular changes and intramolecular interactions induced by light, photons, different solvents,
pHs and metal ion complexes, as well as easy adjustment/tune-up of the molecular structures.
These characteristics facilitate them with a fast response, portable and easy manipulation,
operational simplicity, high selectivity and sensitivity, and detectability by naked eye for
medicinal, biological and environmental purposes. Despite considerable efforts devoted to
fluorescent-based probes for detecting Cu2+ ions [24-27], there is no reliable design for the
selective recognition of Cu2+ in aqueous solutions. Most of the reported probes either work in
organic toxic solvents [28, 29] or in the mixtures at non-physiological pHs [30-32] (Table S1,
Supplementary Information).
It is well known that Schiff-base compounds have the advantages of relatively simple synthetic
procedure and easily coordination with metal ions. So, herein, a new Schiff-base multi-responsive
“turn off” fluorescent chemo-sensor was designed and synthesized through a facile one-step eco-
reaction by incorporating SSB and 2,6-pyridinedicarbohydrazide (PDH) units linked with
hydrazone bonding to detect Cu2+ ions in aquatic media. After synthesizing the N2,N6-
bis(salicylidene)pyridine-2,6-dicarbohydrazide (BSPDH) molecule, the structural-induced photo-
physical properties were thoroughly investigated, which include AIEE-activity, pH-dependency,
and the selectivity and sensitivity towards Cu2+. As far as we know, BSPDH is the first high-
efficient luminescent PDH-base fluorescent sensor so far for detecting Cu2+ ions in aqueous
solutions at the physiological pH range.
5

Scheme 1. Synthesis route of BSPDH.
Scheme 2. Optimized spatial view of BSPDH.
Experimental Section
Material and methods
Pyridine-2,6-dicarboxylic acid (PDA), Thionyl chloride (SOCl2), Hydrazine hydrate 85%,
Salicylaldehyde and solvents were obtained from MERCK Company, Germany, and used without
further purifications. Phosphate buffer solution (PBS) (0.05 M, pH=7.4) was prepared using the
proper amount of Na2HPO4 and NaH2PO4 under the controlling adjustment by a pH meter.
Aqueous solutions were prepared from double distilled deionized water. Ultraviolet-Visible (UV-
6

Vis) absorption measurements were acquired from a CARY50, VARIAN Spectrometer, Australia,
within a wavelength range of 200-800 nm. Fluorescence measurements were performed on a
Varian Cary Eclipse fluorescence spectrophotometer using 1 cm path quartz cell. Emission spectra
were recorded from 370 to 800 nm. All test solutions were excited at 360 nm using the emission
slit of 5 nm and excitation filter of 250-395 nm and emission filter of auto. All spectral
investigations were carried out at room temperature. Melting points were recorded through a Stuart
SMP3 melting point apparatus. Fourier transform infrared (FT-IR) spectra were recorded by a
Tensor 27 spectrophotometer, Germany, using the KBr pellet mode in the region of 4000-400
cm−1. The X-Ray Diffraction (XRD) patterns were recorded by a diffractometer, PANanalytical,
Netherland, using Ni-filtered Cu Kα radiation (k=1.5418 Å). Hydrogen-1 nuclear magnetic
resonance (1H NMR) 300 MHz and Carbon-13 nuclear magnetic resonance (13C NMR) 75 MHz
spectra were obtained from a Bruker Avance DRX-300 spectrophotometer with tetramethylsilane
(TMS) as an internal standard using CDCl3, and DMSO-d6 and D2O as deuterated solvents. The
following abbreviations were used to describe spin multiplicities in 1H NMR spectra: s = singlet;
d = doublet; t = triplet; m = multiplet. Mass spectrum (MS) was obtained from a Varian Mat, CH-
7, Germany, at 70 eV.
Spectroscopic measurements
AIEE measurements
Due to the insolubility in water, initially a stock solution of BSPDH (10-3 M) was prepared in
Dimethylsulfoxide (DMSO). To furnish 10 μM solution at a fixed concentration, 50 μL stock
solution of BSPDH was poured into a 5 mL volumetric flask, and diluted by adding DMSO/water
mixtures which contained different fractions of water (fw, varied from 0 to 98%, v/v). The
7

absorption and emission measurements of resultant solutions were carried out immediately under
identical conditions.
pH measurements
pH values were measured using a pH-meter 827 pH lab, Metrohm, Switzerland, equipped with a
glass electrode (Thermo Scientific Orion 9157BNMD Triode). The machine was calibrated with a
standard commercial buffer solution at pH 7 where the desired pH was adjusted by adding 0.1 M
NaOH and 0.1 M HCl. Fresh stock solution of BSPDH (10-3 M) was prepared in DMSO. The test
solutions (20 μM) were prepared by diluting 100 μL stock solution with 5 mL of 0.05 M PBS.
Cation sensing measurements
Stock solutions of metal ions (1.0×10−2 M), including Al3+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+,
Fe3+, Hg2+, K+, Li+, Mg2+, Mn2+, Ni2+, Pb2+, Sn2+, (VO)2+, and Zn2+ were prepared from their nitrate
and chloride salts, except [VO]2+ which was obtained from its oxide salt (VO2) in deionized double
distilled water. Test samples were prepared by adding 50 μL stock solution of BSPDH and 100 μL
stock solution of metal cations in a 5-mL volumetric flask and diluted by adding a solution mixture
of DMSO/PBS, 0.05 M (1:9, v/v, pH=7.4). Titration of Cu2+ ions was carried out and the
fluorescent intensity (I0/I) was plotted versus the concentration of Cu2+ ([Cu2+]) to determine the
slope (k). I and I0 represent the emission intensity of BSPDH in the presence and absence of Cu2+,
respectively. Ksv (=Kb, binding constant) was calculated from the slope of the Stern-Volmer
correlation as follows: (I0/I)=1+Ksv[Cu2+]. Detection limit (DL) was calculated using the equation
of DL=3σ/k. The fluorescence intensity of BSPDH without metal ions (blank) was measured ten
consecutive times through fluorescence spectra to determine the standard deviation (σ) of the blank
sample.
8

Synthesis process and characterization
Synthesis of PDH
N,N-dimethylacetamide, DMAC (20 mL) and PDA (1.6 g, 8.2 mmol) were mixed in a 100-ml
round-bottom flask, and cooled in a salt/ice bath. While stirring magnetically, SOCl2 (3 mL, 41.35
mmol) was added drop-wise from a funnel and stirred for 30 minutes. The color of reaction
gradually turned to light yellow. In the next stage, a mixture of hydrazine hydrate 85% (5 mL) and
DMAC (5 mL) was added drop-wise to the above mixture; white precipitates were formed. The
mixture was stirred overnight at ambient temperature and filtered through a Büchner funnel,
washed with ethanol, recrystallized with ethanol/H2O (1:1), and air-dried. (C7H9N5O2): white
needles, yield: 4.68 g (47.06 %); m.p.: 248°C. FT-IR (cm-1, refer to Fig. S1): 3243 cm-1 υ (NH2
stretching), 3148 cm-1 υ (NH stretching), 2982, 2698, 2580 cm-1 υ (fermi resonance), 1980, 1630
cm-1 υ (C=O, amide stretching), 1577, 1496 cm-1 υ (Ar-CH), 1411 1240, 1115 cm-1 υ (C=N,
pyridine ring stretching), 966 cm-1 υ (N-N), 619, 500. 1H NMR (300 MHz, DMSO-d6, TMS); δ
(ppm): 7.295 (s, broad/overlapped peaks, 3H, Ar-CH and 6H, NH-NH2), (Fig. S2). MS (m/z) for
C7H9N5O2: 195.6 (M+), 197.6 (M++2H) (calculated: 195.182) (Fig. S3).
Synthesis of BSPDH
In a round-bottom flask (100 mL) equipped with a reflux condenser and magnet, a mixture of
Salicylaldehyde (2.08 mL, 20 mmol), PDH (1.95 g, 10 mmol) and glacial acetic acid (25 mL) was
refluxed after which the completion of reaction was confirmed by thin layer chromatography
(TLC). The mixture was cooled down to ambient temperature and the content of flask was filtered
through a Büchner funnel, washed with ethanol and dried. (C21H17N5O4): yellow crystals, yield:
2.7612 g (68.45%); m.p. 218-223°C. FT-IR (cm-1, Fig. S4): 3433 cm-1 υ (Phenolic OH stretching),
3041, 1623 cm-1 υ (C=N, imine stretching), 1571, 1486, 1387, 1273, 1201, 1107 cm-1 υ (C=N,
9

pyridine ring stretching). 1H NMR (300 MHz, DMSO-d6, TMS); δ (ppm): 11.1 (s, 2H, NH), 9.0
(s, 2H, OH), 8.35 (s, 2H, CH=N), 7.72 (dd, 2H, Ar-CH), 7.40 (td, 2H, Ar-CH), 7.20 (m, 3H,
pyridine ring), 7.05 (dd, 2H, Ar-CH), 6.95 (td, 2H, Ar-CH) (Fig. S5). 13C NMR (75 MHz, DMSO-
d6); δ (ppm): 163, 159, 157, 133, 131, 128, 120, 119, 118, 117, and 116 (Fig. S6). MS (m/z) for
C21H17N5O4: 405 (M+ +2H), 406 (M+ +3H) (calculated: 403.398) (Fig. S7).
Results and discussion
Schiff-base compound BSPDH was eco-synthesized through a facile one-step condensation
reaction between two -CO-NH-NH2 functional hydrazide groups of PDH and two equivalent (eq.)
salicylaldehyde (Scheme 1). They were refluxed at the molar ratio of 2:1 in acidic solvent with
continuous stirring over oil bath till the completion of reaction. Pure symmetric crystalline
hydrazone-linked Schiff-base compound was finally obtained with a sharp yellow color and high
yield. The molecular structures of PDH and BSPDH were well-characterized and their
corresponding spectral analyses were given in Figs. S1-8; the characterization analysis results
clearly supported the assigned structure in Scheme 1. As shown in the 1H NMR spectrum of PDH
(Fig. S2), the first signal at 2.52 ppm was basically related to the peak of solvent, DMSO. In spite
of obtaining white needle crystalline product after crystallization, three minor signals were
appeared perhaps due to unwanted impurities in PDH and/or the trace amount of water content in
DMSO and/or in PDH; the major broad overlapped peak at 7.295 was attributed to 3H of Ar-CH
in pyridine ring and 6H of NH-NH2 (two hydrazide groups). In fact, the solvent was very polar and
aprotic capable in forming hydrogen bonding with both PDH and water molecules appearing a
broad peak for NH2 and NH- protons when solved in DMSO. The present NMR analysis was
supported by the mass spectra analysis. According to XRD data (Fig. S8), the diffraction pattern
10

exhibited multiple strong and sharp diffraction peaks, implying that the sample was in a
polymorphous microcrystalline state [33].
UV-Vis absorption and fluorescence emission characterizations
BSPDH was characterized in solution and in solid-state using absorption and fluorescence
spectroscopic methods. Photo-physical properties of ligand BSPDH (5×10-6 M) were investigated
in various solvents with different polarities; Dicholoroethane (DCE), n-Hexane (n-Hex), DMSO,
Dimethylformamide (DMF), Tetrahydrofuran (THF), Acetonitrile (ACN), Methanol (Meth), and
water (Fig. 1). The results of photo-physical properties of BSPDH are summarized in Table S2.
Two summits of absorption (λabs) for BSPDH in DMSO appeared at 288 and 357 nm. The
absorption bands in the UV region, ranging from 250 to 350 nm, corresponded to π→π* transition
of salicylaldehyde groups and intramolecular charge transfer (ICT) of BSPDH ligand [13].
Phenolic hydrogen atom of salicylaldehyde derivative formed an intramolecular hydrogen bond
with the imine-N atom and six-member ring in BSPDH molecule (Scheme 1). The molar extinction
coefficient (ε) of compound in diluted buffer was measured as ~6000 Lmol-1 cm-1 (Fig. 1a). As
shown in Fig. 1b, BSPDH exhibited different fluorescence emission trends in protic and aprotic
solvents. Generally, the emission intensity change in nonpolar/aprotic and protic solvents supports
the ESIPT behavior for tautomer band. In fact, using protic solvents can lead to the disruption of
intramolecular hydrogen bonding. Further, the emission spectra of BSPDH was evaluated in
ACN/PBS (pH=7, 0.1 M) (1:9, v/v) at various excitation wavelengths (λex= 273, 299, 360, 370,
384 and 392 nm) (Fig. 1c, Table S2). An increase in the excitation wavelength was concomitant
with the increment of emission band intensity. Also, Fig. 1d exhibited the emission spectra of
BSPDH at varied concentrations (4-18 µM) in ACN/H2O (1:9, v/v) at the excitation wavelength
of 360 nm (λex= 360 nm).
11

Figure 1. Photo-physical properties of BSPDH (5 μM) at room temperature: (a) absorption spectra
in different solvents with various polarities; Dicholoroethane (DCE), n-Hexane (n-Hex),
Dimethylsulphoxide (DMSO), Dimethylformamide (DMF), Tetrahydrofuran (THF), Acetonitrile
(ACN), Methanol (Meth), and water, (b) emission spectra in different solvents with various
polarities; DCE, n-Hex, DMSO, DMF, THF, ACN, Meth, and water at λex= 360 nm (excitation
filter: 250-395 nm), (c) emission spectra in ACN/PBS (pH=7, 0.1 M) (1:9, v/v) at different
excitation wavelengths (273, 299, 360, 370, 384 and 392 nm), and (d) emission spectra at varied
concentrations (4-18 µM) in ACN/H2O (1:9, v/v), λex= 360 nm (excitation filter of 250-395 nm).
12

AIEE behavior and solid-state structure of BSPDH
The compound BSPDH dissolved readily in common organic solvents, such as chloroform,
toluene, THF, DMF, acetone, methylacetate, ACN and DMSO (Lewis-base), but was insoluble in
water. BSPDH emitted strong yellow fluorescence light in its solid-state; this suggested the
potential AIEE-active property [34]. To further verify this, we probed the absorption and emission
behavior of BSPDH (10 μM) in ACN/water (as a solvent/non-solvent) binary mixtures. When the
percentage of water content (fw) increased from 0 to 70% (fw ≤ 70%), some slight shifts were
observed in the absorption spectra of BSPDH solution between 294-298 nm and 354-358 nm (Fig.
2a), while BSPDH was nearly non-emissive (Fig. 2b). This property was due to the presence of a
free rotatable N−N single bond in BSPDH which obliterated the excited state by non-radiative
emission, and led to weak or non-emissive behavior. At fw ≤ 70%, the solutions emitted bright
yellow-green light and exhibited weak fluorescence peak at the emission wavelength ~536 nm.
As shown in Fig. 2b, at fw = 80%, a small increment in the fluorescence intensity appeared; at fw ≥
80%, the fluorescence intensity increased quickly and reached to the maximum at fw = 90%. The
~116-fold increment in the emission intensity, compared to that in pure ACN solvent, confirmed
BSPDH as an excellent AIEE-active molecule. In addition, BSPDH showed an excellent AIEE
behavior in DMSO/H2O binary mixture (Fig. S10).
13

Figure 2. AIEE behavior of BSPDH (10 μM) in ACN/H2O mixture by increasing water fraction
(fw) from 0 to 98%, (v/v): (a) absorption spectra, and (b) fluorescence spectra, at λex=360 nm,
(excitation filter of 250-395 nm).
The fluorescent behavior of BSPDH in ACN/H2O mixture originated from the formation of
aggregates in water that restricted the intramolecular rotations of single N−N bond [35], and
rigidified the molecular conformation. The ESIPT property of salicylidene moiety was responsible
for reducing the intermolecular interactions in aggregate or in the solid-state, and brought difficulty
to the cis-trans isomerization of C=N group [36]. The aggregation directly affected the molecular
arrangement, conformational flexibility and intermolecular interactions, thus, the novel solid-state
properties of fluorophores were correlated to the AIEE phenomenon [37]. As confirmed by PL
spectra of BSPDH in Fig. S11, fluorescence emissions under 250 and 280 nm excitation
wavelengths were seen for the powder solid-state form. This was occurred by the formation of
emissive aggregates as a result of effectively tightened molecular packing in the wide spectral
emission range with broad summits at 375 and 414-422 nm [38].
14

pH-dependent property
The chemical property of ligand BSPDH can be induced by varying pHs due to the
protonation/deprotonation of -OH functional group in the SSBs moieties which reduces/enhances
the ESIPT process [39]. So, for evaluating the sensitivity of BSPDH to pH variations, a series of
solutions at different pHs (2–11) were prepared in diluted PBS. As shown in Fig. 3, the emission
spectra of BSPDH exhibited a significant pH dependent trend. Four major fluorescence intensities
are at pH=4, 5, 7, 8 which are demanded for biological probes. The decrease of fluorescence
intensity was recorded by increasing the pH value from pH=8 to 11. This was due to the
deprotonation of NH group that resulted in a negative charge on the nitrogen and, thus, the
repulsion from the neighboring negative nitrogen atoms. The repulsion was alleviated only after
the conversion of benzoid form to quinoid which was responsible for decreasing the fluorescence
emission at pH 9, 10, 11 [28]. Interestingly, the solutions were colorless before pH=8, but upon
increasing the pH to non-emissive level (11), the intensifying yellow-green color was seen by
naked eyes.
Figure 3. pH dependency of fluorescence emission of BSPDH (20 μM) in PBS (0.05 M) at λex=360
nm (excitation filter: 250-395 nm), and (b) Maximum fluorescence intensity (Max. FI) vs. pH.
15

Cation Sensing
The cation selectivity was examined through measuring the fluorescence response of BSPDH (10
μM) to various metal ions (200 μM) including: Al3+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Fe3+, Hg2+,
K+, Li+, Mg2+, Mn2+, Ni2+, Pb2+, Sn2+, (VO)2+, and Zn2+ in DMSO/PBS, 0.05 M (1:9, v/v, pH=7.4),
λex = 360 nm. Fig. 4a shows the emission spectral changes of BSPDH induced by various metal
ions (20 eq.). Among the metal cations tested, only Cu2+ ion showed quenching in the fluorescence
signal of BSPDH at emission wavelength of 538 nm (λem=538 nm). No response was seen by the
addition of 20 eq. of other metal ions in the BSPDH solution. To corroborate the practical
applicability of BSPDH as a selective fluorescent sensor for detecting Cu2+, BSPDH was mixed
with other competing metal ions (20 eq.) in the presence of Cu2+ (20 eq.). As shown in Fig. 4b,
there was no significant variation in the fluorescence intensity of Copper ions in the presence of
other 17 metal cations; no interference identified. These results confirmed that BSPDH possessed
a good selectivity property as a fluoro-sensor for targeting Cu2+ in aqueous solution without any
effect from other metal cations.
16

Figure 4. (a) Fluorescence spectral response of BSPDH (10 μM) to various metal cations (2×10-4
M), and (b) Max. FI of BSPDH against various metal ions in the absence (colorful bars) and
presence (sky blue bars) of Cu2+ in DMSO/PBS, 0.05 M (1:9, v/v, pH=7.4), λex=360 nm (excitation
filter of 250-395 nm).
For further insight into the sensing behavior of BSPDH (10 μM) towards Cu2+, fluorescence
titration experiments were carried out, where the fluorescence intensity decreased gradually by
increasing the concentration of Cu2+ (0-8.6 μM) (Fig. 5a). A perfect linear relationship was
obtained from the fluorescence intensity (I0/I) vs. [Cu2+] in the Stern-Volmer plot (Fig. 5b). The
parameter Kb was calculated as 1.978×105 M-1. The correlation coefficient (R2=0.99409) and
DL=3.11×10−5 M. The obtained Kb illustrated a strong binding tendency of Cu2+ for the probe.
The detection selectivity of Cu2+ by BSPDH was due to the inhibition of ESIPT; the cis-trans
isomerization around the CH=N group was prohibited because of interacting BSPDH with Cu2+
ions. In addition, two -CONH-, CH=N group, and phenolic -OH of salicylaldehyde were involved
in the coordination process. So, the moiety structure of SSBs setup was locked by Cu2+ as a result
of increasing the molecular rigidity of ligand [19]. This phenomenon reduced the energy loss by
17

thermal vibrational decay (confirmed by DFT in Fig. 6) and led to the chelation mechanism. Beside
high selectivity, the chemo-sensor BSPDH responded to Cu2+ fast because of strong Kb (observed
fast changes in the fluorescence intensity). In fact, strong interactions between BSPDH ligand and
Cu2+ formed a stable complex BSPDH-Cu2+ in the solution.
Figure 5. (a) Trend of fluorescence spectra of BSPDH (10 μM) upon the addition of Cu2+ (200
μM) at various concentrations (0-8.6 μM) in DMSO/PBS, 0.05 M (1:9, v/v, pH=7.4), λex=360 nm
(excitation filter of 250-395 nm), and (b) Stern-Volmer plot, I0/I intensity vs. increasing
concentration of Cu2+, obtained Kb (=Ksv): 1.978×105 M-1, and calculated DL: 3.11×10−5 M (the
correlation coefficient, R2=0.99409).
Theoretical Calculations
DFT calculations were carried out to further confirm the electronic structure, the geometry and
energy difference between frontier orbitals of BSPDH and BSPDH-Cu2+ complex, as well as the
Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital
(LUMO). The molecular structures of BSPDH and BSPDH-Cu2+ were optimized by Gaussian 09
quantum chemistry package using DFT/B3LYP/6–31G (d,p) and DFT/B3LYP/LANL2DZ sets,
18

respectively. The output results from GaussView 5, and some of the optimized geometrical
parameters of functional groups of ligand BSPDH and BSPDH-Cu2+ complex, such as bond
lengths and bond angles, are presented in Table 1. As seen, the increment of C=O bond length in
BSPDH (Scheme 2, O14-C13, 1.22278 Å) indicated its involvement in the chelation with Cu2+ in
BSPDH-Cu2+ complex (O14-C13, 1.55723 Å) (Fig. 7). The elongation of imine bond size revealed
its donation to the metal center of Cu2+ in the present case. The optimized geometry and HOMO-
LUMO energy gap of frontier orbitals of fluorescent ligand in BSPDH and its BSPDH-Cu2+
complex is presented in Fig. 6. The calculated energy gap (ΔE, eV) for both molecules, BSPDH
and BSPDH-Cu2+, were from HOMO and LUMO energy eigen values found as 2.7125 (eV) and
1.1451 (eV), respectively (Eq. S1 and S2). Large energy gap of 2.7125 (eV) in BSPDH was
reduced to 1.1451 (eV) for BSPDH-Cu2+ complex, confirming the remarkable stability of BSPDH-
Cu2+ complex compared to BSPDH, as well as corroborating the observed experimental Kb [40].
19

Figure 6. Optimized geometry and HOMO-LUMO energy gap of BSPDH and BSPDH-Cu2+ using
DFT/B3LYP/6–31G(d,p) and DFT/B3LYP/LANL2DZ sets, respectively, by Gaussian 09
software.
The Molecular Electrostatic Potential (MEP) maps of BSPDH and its corresponding complex,
BSPDH-Cu2+, are exhibited in Fig. 7. The nucleophilic regions in BSPDH (electron rich sites are
the red zones around oxygen and nitrogen atoms) are involved in the formation of BSPDH-Cu2+.
The nucleophilic sites of BSPDH are susceptible for electrophilic attack of Cu2+ ions during the
sensory process of Cu2+ by BSPDH [41].
20

Figure 7. Molecular Electrostatic Potential (MEP) maps of BSPDH (left) and BSPDH-Cu2+ (right),
using DFT/B3LYP/6–31G (d, p) and DFT/B3LYP/LANL2DZ sets, respectively by Gaussian 09
software.
Table 1. Bond lengths and bond angles in BSPDH and BSPDH-Cu2+.
BSPDH
Bond
BSPDH-Cu2+
Bond
Bond length
(Å)
Bond angle
(degree)
Bond
Bond length
Bond
Bond angle
(degree)
(Å)
C5-C13
C13-N15
N15-N16
N16-C17
C17-C18
O14-C13
C23-O29
1.51057
1.37271
1.35932
1.29155
1.45012
1.22278
1.34614
C5-C13-N15
C13-N15-N16
N15-N16-C17
N16-C17-C18
C18-C23-O29
O14-C13-N15
C17-C18-C23
113.85733
120.64694
118.31976
121.64943
122.85834
124.18423
121.99282
C5-C13
C13-N15
N15-N16
N16-C17
C17-C18
O14-C13
C23-O29
1.56869
1.55200
1.45676
1.49297
1.56518
1.55723
1.53693
C5-C13-N15
C13-N15-N16
N15-N16-C17
N16-C17-C18
C18-C23-O29
O14-C13-N15
C17-C18-C23
112.34187
115.69942
130.34743
120.49284
126.29393
121.97283
126.44805
Conclusion
A new tridentate fast-response turn-off fluorescent sensor towards Cu2+ ions was designed and
facilely eco-synthesized with high yield on the basis of a new PDH-salicylidene hydrazone
21

derivative. The photo-physical analysis showed an enhanced emission in the aggregate state with
an excellent AIEE property in both ACN/H2O and DMSO/H2O solutions and solid-state.
Tautomerization of keto/enol or hydrogen bonding interactions in the molecule originated large
Stokes shifts which is useful for biological photonic devices. BSPDH performed as a highly
selective and sensitive PDH-base fluorescent sensor for detecting Cu2+ ions in aqueous solution at
physiological pH (pH=7.4). Chelation effect outreached a good detection limit as low as 3.11×10−5
M and strong Kb of 1.978×105 M-1 with the sensing unit of BSPDH (10 μM) without any
interferences from other metal cations co-existed in the solution. DFT calculations confirmed the
higher stability of BSPDH-Cu2+ complex compared to BSPDH with a lower energy gap,
supporting the experimental sensory results. By emitting light in the yellow-green region, BSPDH
showed an excellent AIEE-activity in both ACN/H2O (1:9, v/v) and DMSO/H2O (1:9, v/v) solution
mixtures and maximum fluorescence response at pH=4-8 which introduced it as a biological probe.
As far as we know, BSPDH with the above specifications is the first multi-responsive luminescent
molecule among PDH-base fluorescent molecule sensors for detecting Cu2+ ions in aqueous
solution at the physiological pH.
Author statement
Abbas Amini: Supervision, data analysis, conceptualization, design,
preparation, discussion Marjan Rahimi: Experimental performance, data
analysis, discussion, preparation, design Hoseein Behmadi: Discussion,
Supervision, lab allowance, data analysis Marziyeh Nazari: Preparation,
discussion Veronika Benson: Preparation, discussion Chun Cheng:
Discussion Bijan Samali: Discussion
22

Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that
could have appeared to influence the work reported in this paper.
Acknowledgments
The authors express their gratitude to Islamic Azad University, Mashhad Branch, for the financial
support. The Australian College of Kuwait is highly acknowledged for providing the Research
Grants No: IRC-2018/2019-SOE-ME-PR10 and R11.
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