Journal of Molecular Structure 1259 (2022) 132512

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Journal of Molecular Structure

journal homepage: www.elsevier.com/locate/molstr

Pyrazinederived 1,2,3-triazole linked silanes and their magnetic

nanoparticles for the colorimetric and fluorimetric dual sensing of
Cu2+ ions
Gurjaspreet Singh∗, Suman∗, Diksha, Sushma, Mohit, Priyanka, Jashandeep Kaur,
Anamika Saini, Anita Devi
Department of Chemistry, Centre of Advanced Studies, Panjab University, Chandigarh 160014, India

a r t i c l e i n f o a b s t r a c t

Article history: In this article, pyrazinebased 1,2,3-triazole linked organosilanes have been synthesized and character-
Received 6 November 2021 ized with techniques like 1 H, 13 C NMR spectroscopy and mass spectrometry. The synthesized compound
Revised 27 January 2022
6b (PTSR)could selectively and sensitively recognize Cu2+ ionswhereas the interference from other metal
Accepted 28 January 2022
ions was negligible. The 6(b) behaves as reversible sensor for Cu2+ ions. The fluorescence response of
Available online 2 February 2022
6(b) toward Cu2+ ions showed a significant fluorescence quenching. Further,the sensing application was
enhanced by the immobilization of organic moiety on the magnetic silicasurface. The adsorption of
organosilaneis confirmed by various techniques such as FT-IR, EDX, XRD, TGA, SEM, HR-TEM, DLS. The hy-
brid magnetic nanoparticles (HNPs)are found to be more sensitive for Cu2+ ions with low LOD as compare
to 6(b)i.e. 2.7 × 10− 6 M. In addition, the synthesized alkynes and silanes were examined fordrug-likeness
profile by using online available toolkits like MOLINSPIRATION and ADMET.
© 2022 Elsevier B.V. All rights reserved.

1. Introduction and the use of toxic solvents [8–10]. High selectivity, fast response,
and easy visualization of the optical sensor have much more at-
Pyrazine derivatives are low in toxicity that allows to use them traction in the recent field of sensing due to operational simplic-
as a pharmacophore in the development of drugs or prodrugs [1]. ity [11]. Organosilanes have attracted the interest of researchers in
Pyrazine-2-carboxamide was exhibiting various type of pharmaco- recent decades due to their various applications in organic syn-
logical activities like antibacterial, antifungal, antiviral, antioxidant, thesis, catalysis, drug design, analyte sensing, and medicinal sci-
and anti-cancer activities [2]. Copper is an important transition ence [12,13]. As 1,2,3-triazole group provides nitrogen donor site,
metal that is involved in a variety of physiological processes [3]. attachment of this group with trialkoxysilyl moiety is essential in
Excessive use of copper can result in serious side effects in human the field of chemosensor [14,15]. Furthermore, assimilation of these
such as growth retardation, melena, Wilson’s disease, hepatolen- two groups with an organic moiety such as pyrazine can provide
ticular degeneration, gastrointestinal disorders, liver injury, and so structural linkage that can be used in the development of ana-
on [4,5]. Conversely, insufficient consumption of this trace metal lyte sensors [16]. Core-shell nanoparticles, which are made up of
can result in diseases such as Menkes, low white blood cell count, a magnetically sensitive core and a silica shell, offer several ad-
weakness, brittle bones [6,7]. WHO limits the level of Cu2+ ions in vantages. First, the magnetic core allows them to be easily sepa-
drinking water to 15–25 μM. As a result, the development of col- rated and recycled from the matrix using an external magnet [17].
oro/fluorescent probes for the identification of Cu2+ ions in envi- Second, they are more biocompatible and stable [18]. Furthermore,
ronmental/biological samples is getting more interest and demand. they are easily synthesized using the sol-gel technique and may be
Various traditional methods for Cu2+ ion detection were available easily changed by a wide spectrum of functional groups [19,20]. In
in the literature including atomic absorption spectroscopy, electro- addition, iron oxides have undoubtedly been and continue to be
chemical analysis, adsorptive stripping voltammetry, liquid chro- the most intensively studied magnetic materialsdue to several fac-
matography, and spectrophotometry but they have some disadvan- tors including cancer treatment through hyperthermia [21], mag-
tages such as high operating costs, complicated sample handling, netic drug targeting delivery [22], magnetic resonance imaging as
contrast agents [23], and environmental and biomolecular separa-
tions [24,25]. Further, the adsorption of silica on magnetic oxide
∗
Corresponding authors.
provided a large surface area and high chemical stability [26].
E-mail addresses: [email protected] (G. Singh), [email protected] (Suman).

https://doi.org/10.1016/j.molstruc.2022.132512
0022-2860/© 2022 Elsevier B.V. All rights reserved.
G. Singh, Suman, Diksha et al. Journal of Molecular Structure 1259 (2022) 132512

Herein, we synthesized pyrazine-2-carbohydrazide tethered Pyrazine-2-carbohydrazide (0.50 g, 3.6 mmol), alkyne 4a

triazole-linkedorganosilanes and modified their activity by anchor- (0.68 g, 3.6 mmol)
ing them onto magnetic silica nanoparticles. To make these silyl 1 H NMR (500 MHz, CDCl ) δ 9.88 (s,-N=CH-,1H), δ 9.42 (s,
3
frameworks and hybrid magnetic nanoparticles more potent for Pz-H, 1H), δ 8.77 (d, Pz-H, 1H), δ 8.56 (d, Pz-H, 1H), δ 7.14–
various analytical applications, testing ability with Cu2+ ions is ex- 7.48(m, Ar-H, 4H), δ 5.99(s, -C-NH–N-,1H), δ 4.86(d, -O-CH2 -, 2H), δ
plored in this article. 3.95(s,-O-CH3 ,3H),δ 2.58(t, -C≡CH1H).13 C NMR (126 MHz, CDCl3 ),
δ 165.34(C7), 152.13(C16), 150.05(C2, C15), 147.53(C1), 144.60(C11),
2. Experimental section 144.17(C5), 142.76(C4),130.95(C12), 126.24(C13), 112.63(C14),
109.51(C17), 77.48(C20), 77.32(C21), 56.62(C19), 56.04(C23).
2.1. Chemical and reagents
2.3.2. General procedure for the synthesis of silanes (6a,6b)
Pyrazine-2-Carboxamide (Aldrich), Hydrazine hydrate (Avra), In a 2-neck round bottom flask (flame dried), pyrazine-2-
4- Hydroxybenzaldehyde (Avra), Vanillin (Avra), Propargyl Bro- carbohydrazide substituted alkyne (1 Equiv.) was mixed with
mide (Merck) were used directly as received. Organic solvents AzPTES (1 Equiv.) under nitrogen atmosphere using TEA/THF (1:1)
were dried according to the previous kind of literature. Metal as the solvent and 0.01 mmol of [CuBr(PPh3 )3 ] as a catalyst. The
chlorides were bought from S.D. fine chem. Ltd., India. 3- reaction mixture was refluxed for 9 h. at 60 °C. After refluxing,
azidopropyltriethoxysilane (AzPTES) was synthesized by previously the solution was cooled and the solvent was evaporated under re-
known methods [27]. ducedpressure. The left residue was extracted by adding dry ether.
The solid product was dried in a filtration unit under a nitrogen
2.2. Instrumentation atmosphere.

Synthesis of (E)-N’-(4-((1-(3-(triethoxysilyl)propyl)−1H-1,2,3-
Thermo-Scientific NICOLET IS50 spectrophotometer was used to
triazol-5-yl)methoxy)benzylidene)pyrazine-2-carbohydrazide
record infrared spectra. 1 H and 13 C NMR spectra were recorded
(6a)
using CDCl3 as an internal reference on Bruker Avance II 400 FT-
Light brown solid, yield:75%, melting point: 240–242 °C.
NMR spectrophotometer and tetramethylsilane (TMS) was used as
5a (0.20 g,0.71 mmol), AzPTES (0.17 g,0.71 mmol), 1 H NMR
a relative reference. Mass spectral analysis was carried out on a
(500 MHz, CDCl3 ): δ 9.85 (s, -N=CH-,1H), 9.43 (s, Pz-H, 1H),
WATERS, Q-TOF microMASS spectrometer (the ESI source having a
8.78 (s, Pz-H 2H), 8.56 (d, Ar-H, 4H), 7.42 (s, Tz-H, 1H), 5.92
capillary voltage of 30 0 0 V). Spectral measurements were done on
(s, -NH-,1H), 5.39 (s, -O-CH2 -Tz, 2H), 4.35 (q, -N-CH2– CH2 -, 2H),
Hitachi F-70 0 0 Fluorescence spectrophotometer. Mel Temp II de-
3.79 (q, -O-CH2– CH3 , 6H), 2.02 (dd, -CH2– CH2– CH2 -, 2H), 1.11
vice was used to measure melting points.Thermogravimetric anal-
– 1.42 (m, -O-CH2– CH3 , 9H), 0.59 (t, -Si-CH2– CH2 -, 2H). 13 C
ysis of the samples was done on SDT Q 600 V20.9 Build 20 using
NMR (126 MHz, CDCl3 ): δ 165.45(C7), 159.91(C15), 147.60(C11),
the TGA instrument. X-Ray Diffraction (XRD) was conducted using
146.70(C4), 144.67(C5), 144.23(C1), 142.83(C21), 136.56(C13,C17),
a PANalyticalX’pert PRO spectrophotometer with Cu-Ka radiation.
133.15(C14,C16), 128.63(C20), 120.81(C12), 77.29, 72.96, 58.50
Hitachi SU8010 ultra-high-resolution scanning electron microscope
(C32,C34,C36), 31.91(C26), 18.33(C33,C35,C37), 15.77(C27). MS: m/z
was used for FESEM analysis. UV–Vis analysis was done by using a
476.16 [M-2(OCH2CH3 )+Li]+ .
V-530 double-beam spectrophotometer.
Synthesis of (E)-N’-(3–methoxy-4-((1-(3-
2.3. Synthesis and characterization (triethoxysilyl)propyl)−1H-1,2,3-triazol-5-
yl)methoxy)benzylidene)pyrazine-2-carbohydrazide (6b)
2.3.1. General procedure for the synthesis of Light brown solid, yield: 80%, melting point: 260–262 °C.
pyrazine-2-carbohydrazide substituted alkynes (5a,5b) 5b (0.30 g,0.9 mmol), AzPTES (0.23 g, 0.9 mmol)1 H NMR
Pyrazine-2-carbohydrazide and alkynes were synthesized by (500 MHz, CDCl3 ) δ 9.85 (s, -N=CH-,1H), 9.42 (s, Pz-H, 1H), 8.78
the known procedure in literatures [28,29]. For the synthesis of (d, Pz-H,1H), 8.56 (d, Pz-H, 1H), 7.67 (d, Ar-H, 2H), 7.43 (s, Tz-
pyrazine-2-carbohydrazide substituted alkynes, compound (2) was H,1H), 7.24 (s, Ar-H,1H), 6.03 (s, -NH-, 1H), 5.39 (s, -O-CH2 -Tz, 2H),
added in a 100 mL round bottom flask (RBF) with 50 mL ethanol as 4.36 (t, -N-CH2– CH2 -, 2H),3.93(s,O–CH3 ,3H) 3.80 (q, -O-CH2– CH3 ,
solvent. The mixture was stirred for 10 min. to get a clear solution. 6H), 2.03 (dd, -CH2– CH2– CH2 -,2H), 1.20 (t, -O-CH2– CH3 , 9H), 0.59
After that, already synthesized alkyne (1 Equiv.) was added and the (t, -Si-CH2– CH2 -,2H). 13 C NMR (126 MHz, CDCl3 ) δ 165.35(C7),
reaction mixture was refluxed for 12 h. at 60 °C.After completion of 153.10(C15), 149.94(C11), 147.54(C4), 144.61(C1,C5), 144.18(C1),
the reaction, the solid product was filtered and recrystallized with 142.77(C21), 132.14(C17), 132.06 (C13), 130.57(C20), 126.78(C12),
MeOH/CH3 CN. 123.14(C14,C16), 63.01(C19), 58.54(C32,C34,C36), 56.01(C39),
Synthesis of (E)-N’-(4-(prop–2-yn-1- 52.57(C25), 24.19(C26), 18.28(C33,C35,C37), 7.48(C27). MS: m/z
yloxy)benzylidene)pyrazine-2-carbohydrazide (5a) 559.68 [M + 2H]+ .
Yellowish white solid, yield: 82%, melting point: 185–187 °C.
2.4. Synthesis of metal
Pyrazine-2-carbohydrazide (0.50 g, 3.6 mmol), alkyne 4a
(0.57 g, 3.6 mmol)
1 H NMR (500 MHz, CDCl ) δ 9.88 (s,-N=CH-,1H), δ 9.42 In a 50 mL round bottom flask, synthesized silane (6b) and cop-
3
per chloride were dissolved in 20 mL methanol. The reaction mix-
(s,Pz-H,1H), δ 8.77 (d,Pz-H, 1H), δ 8.56 (d,Pz-H, 1H), δ 7.14–
ture was stirred for 4 h. at room temperature. The solid product
7.48(m,Ar-H, 4H), δ 5.99(s, -C-NH–N-,1H),δ 4.86 (d, -O-CH2 -, 2H),
was filtered and washed with methanol. Color: Light green. Melt-
δ 2.58(t, -C≡CH, 1H). 13 C NMR (126 MHz, CDCl3 ) δ 165.34(C7),
ing Point: 250–255 °C.
152.13(C16), 150.05(C2, C15), 147.53(C1), 144.60(C11), 144.17(C5),
142.76(C4),130.95(C12), 126.24(C13), 112.63(C14), 109.51(C17),
2.5. Preparation of organosilane functionalized hybrid magnetic
77.48(C20), 77.32(C21), 56.62(C19).
nanoparticles
Synthesis of (E)-N’-(3–methoxy-4-(prop–2-yn-1-
yloxy)benzylidene)pyrazine-2-Carbohydrazide (5b) The systematic route for the synthesis of hybrid magnetic
Light brown solid, yield: 85%, melting point: 180–182 °C. nanoparticles is shown in Fig. 1. FeCl3 •6H2 O and FeCl2 •4H2 O were

2
G. Singh, Suman, Diksha et al. Journal of Molecular Structure 1259 (2022) 132512

Fig. 1. Synthesis of Fe3 O4 @SiO2 @silane.

taken in a 2:1 molar ratio and dissolved in 30 mL deionized water culated by using linear calibration curve, stern-Volmer, and Benesi-
in a 250 mL round bottom flask. After 30 min. of stirring 25% am- Hildebrand (B-H) plot respectively.
monium solution was added to the reaction. Instantly, black pre-
cipitates were formed and the solution was stirred for 1 hr. The 2.6.2. Job’s plot experiment
precipitates were washed with ethanol and dried in the oven to The stock solution of sensor and Cu2+ ions was prepared
obtain the desired Fe3 O4 nanoparticles. Since bare Fe3 O4 nanopar- of same concentration (10− 4 M). 2.0,1.8,1.6,1.4,1.2,1.0,0.8,0.6,0.4,0.2
ticles will aggregate and lose their magnetization, functionalization and 0 mL solution of sensor were taken in separate vials. Then, in
of Fe3 O4 nanoparticles was done by adsorption of SiO2 on their each vial 0,0.2,0.4,0.6,0.8,1.0,1.2,1.4,1.6,1.8 and 2.0 mL of Cu2+ ions
surface. For this Fe3 O4 (0.3 g) nanoparticles were stirred in 30 mL solutionswere added. Each vial has total 2 mL solution.The ab-
absolute ethanol for 30 min. followed by the addition of 20 mL sorbance spectra were recorded a few seconds after addition and
NH3 solution and 0.2 mL tetraethyl orthosilicate. The reaction mix- mixing of these two solutions.
ture was allowed to reflux for 4 h. After refluxing, brown-colored
Fe3 O4 @SiO2 nanoparticles were separated magnetically. In the fi- 2.6.3. Interference experiment
nal step, 0.2 g of Fe3 O4 @SiO2 and 0.3 g of silane (6b) were taken in The competitive experiment was performed by adding 10μL of
two neck round bottom flask (flame dried) with 20 mL dry toluene. each metal ion (10−2 M) in sensor solution(10− 3 M). 10μLsolutions
The reaction was refluxed for 24 h. at 80 °C. The hybrid nanopar- of Cu2+ ions was added to the previously mixed solution of sensor
ticles were separated with a magnet and washed with chloroform and different metal ions. The UV–Vis spectra of different metal ions
to remove excess silane. Then, nanoparticles were dried under re- mixed with sensor and Cu2+ solutions were recorded.
duced pressure for 6 h.
2.6.4. Determining the association constant (Ka )
The association constant was calculated by plotting 1 / (A -Ao )
2.6. Chromogenic sensing of Cu2+ ions and 1 / (F - Fo ) against 1 / [Cu2+ ] respectively. Where A and F are
the absorbance and emission intensity after the addition of Cu2+
2.6.1. UV–Vis and fluorescence spectra analysis ions in sensor solution and Ao and Fo are the absorbance and emis-
The stock solution sensor(10−3 M) was prepared in methanol sion intensity in the absence of Cu2+ ions or free sensor. The asso-
by dissolving 27.88 mg of the compound in 50 mL of methanol. ciation constant was obtained by intercept/slope of the line.
The stock solution of all the cations (Ag+ , Al3+ , Ba2+ , Cd2+ , Co2+ ,
Cs+ , Cu2+ , Fe3+ Hg2+ , Mn2+ , Ni2+ , Pb2+ , Rb+ , Sn2+ , Zn2+ )(10−2 M) 2.6.5. Determining the limit of detection (LOD)
were prepared in methanol. 10μL of each metal ion solution was The limit of detection was calculated by the formula: 3σ /S,
added to 2 mL of 10− 3 M solution of 6(b) and observation was where σ is the standard deviation and S is the slope. The standard
recorded on UV–Vis spectrometer. It was observed that compound deviation(σ ) and slope (S) were obtained by plotting absorbance
6(b) shows selectivity toward Cu2+ ions as shown in Figs. 2(a,b) and emission intensity against Cu2+ ion concentration.
and S11. For the fluorescence measurements (slit: 15.0 nm), exci-
tation was provided at 270 nm and emission was recorded from 2.7. Determination of physicochemical properties
420 to 450 nm. Fluorescence spectra was recorded with different
cations as shown in Fig. S12. A decrease in fluorescence intensity In the present work, web servers like PreADMET
of compound 6(b) was observed with the addition of Cu2+ ions so- (http://preadmet.bmdrc.org) and MOLINSPIRATION (http://
lution. The concentration of Cu2+ was varied from 10μL-120μL to www.molinspiration.com) were used tocalculate physicochem-
record the UV–Vis and fluorescence spectra. The limit of detection, ical propertiesof synthesized compounds 5(a,b) and 6(a,b). The
stern-Volmer constant (Ksv), and binding constant (Ka) were cal- ‘rule of five’ of Lipinski was used to demonstrate the drug-likeness

3
G. Singh, Suman, Diksha et al. Journal of Molecular Structure 1259 (2022) 132512

Fig. 2. Bar graph representation of colorimetric responses of PTSR upon addition of different metal ions at 268 nm using UV-Visible spectroscopy (a), Visual of color changes
of the PTSR after adding various metal ions under UV light at 268 nm (b).

Table 1
In silico physicochemical properties of synthesized compound 5(a,b) and 6(a,b).

Compound ID M.W miLogP TPSA n-ON Acceptor n-OHNH Donar nRotB Lipinski’s Violations

Rule <500 <5 <140 ≤10 ≤5 ≤15 ≤1

5a 280.29 1.85 76.48 6 1 5 0
5b 310.33 1.44 85.71 7 1 6 0
6a 527.66 2.73 135.90 12 1 16 3
6b 557.68 2.33 144.13 13 1 17 3

MW: molecular weight, mi Log P: logarithm of partition coefficient of compound between octanol and water, TPSA: topo-
logical polar surface area, n-ON acceptors: number of hydrogen bond acceptors, n-OHNH donors: number of hydrogen
bonds donors, n-ROTB: number of rotatable bonds.

Table 2 Madin–Darby canine kidney (MDCK), and plasma protein binding

The activity score of Kinase inhibitor, nuclear receptor, protease inhibitor and
(PPB) permeability were calculated using the Pre-ADMET tool for
enzyme inhibitor of the synthesized compounds 5(a,b) and 6(a,b).
compounds 5(a,b) and 6(a,b). Oral administration of a drug de-
Compound ID GPCR ICM KI NR PI EI pends on transportation through the Gastro-Intestinal tract (GIT).
5a −0.23 −0.93 −0.39 −0.56 −0.48 −0.30 The absorption feasibility of a drug through the small intestine is
5b −0.22 −0.92 −0.34 −0.55 −0.47 −0.31 measured by Human Intestine Absorption (HIA%). The other prop-
6a 0.06 −0.37 −0.12 −0.46 0.46 0.07 erty is the feasibility of a drug to cross the Blood-Brain Barrier
6b 0.03 −0.49 −0.15 −0.51 −0.11 0.01
(BBB) as this barrier restricts most of the compounds to cross
from blood to the brain. Plasma Protein Binding (PPB) predicts
the drug’s half-life as it denotes the binding of the drug in blood
with albumin. The stronger is the binding lesser would be the
of synthesized compounds. According to this rule, the molecule
action of the drug. The calculated physicochemical properties of
under observation should follow some basic molecular properties
the synthesized compounds 5(a,b) and 6(a,b) are given in Table 3.
like molecular weight <500, octanol-water partition coefficient
(logP) < 5,the number of hydrogen bond acceptors (HBA) < 10,
and the number of hydrogen bond donors (HBD) < 5. According 3. Results and discussions
to Veber et al., the compound with TPSA levels greater than140
and n-ROTB levels greater than 15 has poor oral bioavailability.We 3.1. Synthetic aspects and characterization
calculated these values given in Table 1 and found to be modest.
Bioactivity scores like kinase inhibitor, protease inhibitor, enzyme The synthetic route of pyrazine-2-carboxamide tethered
inhibitor, and nuclear receptor ligand of the compounds 5(a,b) triazole-linked silane receptor (PTSR) is shown in Scheme 1. The
and 6(a,b) were also calculated and depicted in Table 2. The alkyne(4a,4b) and pyrazine-2-carbohydrazide were synthesized
pharmacokinetics of a drug molecule is determined by ADMET according to the reported method22,23 . These two intermediates
(Absorption, Distribution, Metabolism, Excretion, and Toxicity) were used to synthesize new substituted pyrazine substituted
studies. Parameters such as Caco-2, blood-brain barrier (BBB), alkyne by condensation reaction. In the final step, alkynes (5a,5b)

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G. Singh, Suman, Diksha et al. Journal of Molecular Structure 1259 (2022) 132512

Table 3
Pharmacokinetic descriptors of synthesized compounds 5(a,b) and 6(a,b).

Compound Caco-2 Cell Permeability (nm s-1) BBB HIA MDCK (nm s-1) Plasma Protein Binding (%) Skin Permeability (logKp)

5a 4.426 0.057 96.172 151.894 87.153 −3.055

5b 0.046 15.306 96.152 101.392 84.189 −3.207
6a 21.491 0.090 98.478 0.385 89.837 −3.271
6b 21.526 0.070 98.352 0.123 88.329 −3.458

Scheme 1. Synthetic approach for the synthesis of pyrazine-2-carbohydrazide based sensor.

were clicked with 3-azidopropyltriethoxysilane to synthesize 1,2,3- (10− 3 M) in methanol at 264 nm (Fig. 4a). This pattern may have
triazole linked organosilanes (Fig. 3). The pyrazine substituted emerged as a result of an increase in conjugation during complex-
alkynes were characterized by1 H and 13 C NMR spectroscopy. ation with copper ions, which resulted in a decrease in the dif-
The propargylic group in alkyne is confirmed by one doublet ference in n–π ∗ transition energy. The intensity of the absorbance
and one triplet at 4.86 ppm, 2.58 ppm correspond to -C≡C–H band further increases with the addition of Cu2+ ion solution up
and-CH2– C≡C–H respectively. The 1 H NMR spectra give triplet to 120μL. The association constant of PTSR with Cu2+ ion was cal-
(1.10–1.20 ppm) and quartet (3.50–3.80 ppm) due to –CH2 CH3 culated from the B-H plot (Fig. 4b) and found to be 4.03 × 103
and –OCH2 - groups respectively. The signal at 5.10–5.20 ppm is of M−1 . Limit of detection (LOD) was calculated from a linear cali-
–OCH2 - group of alkyne linked to triazole ring further confirms bration curve (Fig. 4c) by using the Eq. (3) σ /S where σ is stan-
the formation of triazole ring. The synthesized silanes give a dard deviation and S is the slope of the curve. The value of LOD
peakin the range δ = 162–165 ppm corresponds to (-C = O) group was found to be 3.4 × 10−6 M. The high association constant
of pyrazine-2-carbohydrazide. The peak corresponds to the imine and low limit of detection suggested that the PTSR sensor showed
group of Schiff base is in the range δ =147–149 ppm. The peaks high selectivity towards Cu2+ ions.To predict the stoichiometry of
in the range δ =17–19 ppm, 57–59 ppm, 50–52 ppm are due to the complex job’s continuous variation method was used where
–CH2 CH3 , –OCH2 , and -NCH2 -respectively.The mass spectra of maxima at 0.5 suggested 1:1 stoichiometry for PTSR and Cu2+
6(a) and 6(b) give base peak at 476.16 [M-2(OCH2 CH3)+Li]+ and ion complex (Fig. 4d). The B-H plot between 1/A-Ao and [Cu2+ ]−1
559.18(M + 2H)+ respectively. was linear with high regression coefficient (R2 ) also suggested the
1:1 stoichiometry of the complex. The high selectivity of ligand
for Cu2+ ions was also tested with interference study with other
3.2. Benesi–Hildebrand plots, association constants and stoichiometry
metal ions (Fig. 4e). A similar hyperchromatic shift was observed
calculations for receptor–cation complexation
in the absorbance spectra of HNPs (5 × 10−5 M) with Cu2+ ions
(10−3 M) (Fig. 5a).The LOD and association constant of hybrid
The sensor PTSR exhibited a sharp absorbance band at 264 nm.
magnetic nanoparticles (HNPs) were also calculated (Fig. 5b, c)and
A hyperchromic shift was observed in the absorbance spectra of
found to be better than sensor PTSR with values 2.7 × 10− 6 M and
PTSR (10− 5 M) with the addition of 10μL of Cu2+ ion solution

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G. Singh, Suman, Diksha et al. Journal of Molecular Structure 1259 (2022) 132512

Fig. 3. Synthesized compounds (5a-5b) and (6a-6b).

Table 4 orescence quenching by Cu2+ ions might be attributed to factors

Association constant and LOD values of PTSR and HNPs.
like excitation energy transfer from fluorophore to metal d-orbital,
Sensor Association Constant (Ka) Limit of Detection (LOD) charge transfer from fluorophore to Cu2+ ion, and heavy metal ef-
6(b)/ PTSR 6.7 × 103 M−1 4.2 × 10−5 M fect. The quenching effect of PTSR can be rationalized with the
Fe3 O4 @SiO2 @6b 5.1 × 105 M−1 8.6 × 10−7 M Stern-Volmer equation.
F0 /F = 1 + KSV [Q] where F and F0 are the intensities of the
sensor in the presence and absence of Cu2+ ions respectively. Ksv
1.19 × 104 M−1 respectively(Table 4). This suggests that nanoparti- is the stern-Volmer constant and [Q] is the quencher concentra-
cles provide more surface area as compared to a ligand for the tion. By plotting the graph between F0 /F and concentration of Cu2+
binding of Cu2+ ions. The binding ability and limit of detection ions, we calculated the linear correlation coefficient, stern-Volmer
of the present chemosensors were compared with the recently re- constant, and LOD of sensor PTSR and found it to be 0.9953,
ported kinds of literature [30–33](Table 5). According to the data, 6.7 × 103 M−1 , and 4.2 × 10−5 M respectively (Fig. 6b). The lin-
the synthesized sensor has a better binding affinity and low limit ear graph is attributed to the static quenching of the sensor with
of detection than previously known colorimetric and fluorescent Cu2+ ions.
probes. Similarly, based on the absorbance spectra of HNPs, emis-
sion spectra were recorded with various concentrations of Cu2+
3.3. Reversibility study ion solution. Free HNPs show high fluorescence intensity got
considerably quenched upon the addition of Cu2+ ions solution
To know the reversible and irreversible binding responses of (Fig. 6c).Similarly, with the help of the graph between F0 /F and
PTSR with Cu2+ ions, we performed the reversibility test. The test concentration of Cu2+ ions, the linear correlation coefficient, stern-
was conducted by adding sodium salt of EDTA and Cu2+ ions sys- Volmer constant, and LOD of HNPs were calculated and values
tematically to the solution of PTSR+Cu2+ ions. It was observed that were found to be 0.9997, 5.1 × 105 M − 1 , 8.6 × 10−7 M respec-
with the addition of EDTA to the PTSR+Cu2+ ion complex solution, tively. (Fig. 6d)
the absorbance intensity decreased. This indicated that Cu2+ ions
form a strong bond with EDTA as compared to PTSR. It was regen- 3.5. Binding mode of the sensor with Cu2+ ions
erated after the addition of Cu2+ ions solution as shown Fig. S13.
This pattern was repeated several times and the sensor was regen- To predict the binding site of the sensor with a metal ion, we
erated every time as depicted in Fig. S14. This revival makes the synthesized a metal complex of PTSR with Cu2+ ions. The silane
sensor reusable and attractive for real-time applications to sense 6(b) was stirred with Copper (II) chloride in methanol for 4 h.
Cu2+ ions. The reaction mixture was filtered and dried under a vacuum to
get the solid product. The formation of the metal complex was ex-
3.4. Fluorescence emission spectral analysis amined with the help of NMR spectroscopy (Fig. S15). Significant
changes were observed in the NMR of complex w.r.t silane 6(b).
We measured the fluorescence emission spectra of PTSR There was a shift of signal at δ 3.93 ppm (s,3H, O–CH3 ). The signal
(10−3 M) with different concentrations of Cu2+ ions (10−2 M) as at δ 9.42 ppm (HN-C=O) disappeared. This suggests the role of ni-
shown in Fig. 6a. The emission intensity of PTSR gradually de- trogen of the amide group and oxygen of the methoxy group. The
creases with the gradual addition of Cu2+ ions solution.The flu- plausible mode of metal binding with silane is shown in Fig. 7.

6
G. Singh, Suman, Diksha et al. Journal of Molecular Structure 1259 (2022) 132512

Fig. 4. Absorption titration spectra of sensor PTSR (10−5 M) in the presence of increasing concentrations of Cu2+ ions (10−3 M) (a), B-H plot of 1/(A-A0 ) versus 1/ [Cu2+ ] for
PTSR titration with Cu2+ ions (b), Calibration plot for absorbance of PTSR 268 nm versus Cu2+ ion concentrations (c),Stoichiometric calculations of PTSR by Job’s plot at a
various mole fraction of Cu2+ ions (d), interference study of PTSR+ Cu2+ with different competitive ions using UV-Visible spectroscopy at 268 nm (e).

Table 5
Comparison of PTSR with few reported sensors for Cu2+ ions.

Sensors Measured Signals LOD(M−1 ) Reference

Naphthopyran-diaminomaleonitrile dyad Absorbance 5.39 × 10−4 30

Spherical Nanocellulose Absorbance 1.622 × 10−5 31
Bi-functional building block Fluorescence 17.5 × 10−6 32
1,2,3-triazole-decorated imino-phenol Fluorescence 9.06 × 10−6 33
PTSR-Nps Fluorescence 8.6 × 10−7 Present work

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G. Singh, Suman, Diksha et al. Journal of Molecular Structure 1259 (2022) 132512

Fig. 5. Absorption titration spectra of sensor HNPs in the presence of increasing concentrations of Cu2+ ions (a), B-H plot of 1/(A-Ao ) versus 1/ [Cu2+ ] for HNPs titration
with Cu2+ ions (b), Calibration plot for absorbance of HNPs 270 nm (c).

Fig. 6. Fluorescence response of PTSR on the addition of Cu2+ ions (a), Stern-volmer plot of PTSR with Cu2+ ions at 425 nm (b), Fluorescence response of HNPs on addition
of Cu2+ ions (c), Stern-volmer plot of HNPs with Cu2+ ions at 480 nm (d).

8
G. Singh, Suman, Diksha et al. Journal of Molecular Structure 1259 (2022) 132512

Fig. 7. Possible mode of interaction of PTSR with Cu2+ ions.

3.6. Characterization of magnetic nanoparticles

The IR spectra of Fe3O4, FE3O4@SiO2, and Fe3 O4 @SiO2 @6b are Fig. 9. Thermogravimetric curves of Fe3 O4 @SiO2 @6b and Silane (6b).
shown in Fig. S16. The additional peak at 1650cm−1 in IR spec-
tra of Fe3 O4 @SiO2 @6b is due to the -C=O bond of pyrazine car-
boxamide group confirmed the adsorption of organic moiety on 3.7. Thermogravimetric analysis
the surface magnetic silica nanoparticles. So, IR spectra give the
first insight into the characterization of functionalized nanoparti- The thermogravimetric graph could be divided into three
cles. The structural features of unfunctionalized and functionalized stages: moisture vaporization, oxidation, and complete combustion.
magnetic nanoparticlesare checked by XRD pattern. The peak cor- In the TGA graph of compound 6b and Fe3 O4 @SiO2 @6b, there is
responds to 2θ =20–25° is due to organic moiety on the surface moisture vaporization at a temperature between 50 and 200 °C in
of magnetic silica nanoparticles (Fig. S17). The compositional study the first stage. The second stage is the pyrolysis of biomass leads to
of Fe3 O4 @SiO2 @6b was done by EDX (Energy Dispersive X-Ray). mass loss of approximately 50–65%. In the final stage, there is pure
The elemental peaks of carbon, oxygen, nitrogen, iron, silicon fur- carbon combustion left with some residual ash. Organic weight
ther confirmed the coating of silane moiety on the surface of the loss is directly proportional to the amount of silane compound on
magnetic silica surface (Fig. S18). The surface topography of hybrid the Fe3 O4 surface. In compound 6b, the residual left was 22% (ap-
magnetic nanoparticles was explained by FESEM images at differ- prox.) correspond to SiO2 . But in the case of hybrid magnetic silica
ent magnifications as shown in Fig. 8. It was concluded from the nanoparticles, the residue left was 55% suggested organic moiety
images of Fe3 O4 @SiO2 @6b that the particles possess well defined adsorbed on magnetic silica nanoparticles shown in Fig. 9.
spherical surface, smooth texture, and well dispersed. Further, the
average diameter of HNPs is usually determined by HRTEM and 4. Conclusion
DLS. HR-TEM give the diameter of particles while DLS is the hy-
drodynamic size of the particles. From the images of HR-TEM (Fig. In summary, the current study reported the synthesis of a new
S19), the size of HNPs was found to be 123 nm. The average parti- pyrazine-2-carbohydrazide based chemosensor, PTSR, for the col-
cle size of HNPs was examined by DLS experiment is about 95 nm. orimetric and fluorimetric detection of Cu2+ ions. The sensor also
DLS graph of HNPs is shown in Fig. S20. showed the anti-interference ability with other metal ions. The

Fig. 8. FESEM images of HNPs at different magnifications.

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G. Singh, Suman, Diksha et al. Journal of Molecular Structure 1259 (2022) 132512

limit of detection and binding constant for PTSR complex with [11] Y.W. Li, J. Li, X.Y. Wan, D.F. Sheng, H. Yan, S.S. Zhang, H.Y. Ma, S.N. Wang,
Cu2+ ion was found to be 3.4 × 10−4 M and 4.03 × 103 M−1 re- D.C. Li, Z.Y. Gao, J.M. Dou, D. Sun, Nanocage-based N-rich metal–organic frame-
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sized sensor was adsorbed on the surface of magnetic nanoparti- [12] G. Singh, Suman, P. Satija, Shilpy A.Singh, D.G. Silvera, C.E. Ruiz, M.A. Este-
cles. Surface functionalization of magnetic nanoparticles shows a ban, S. C.Sahoo, Synthesis and X-ray characterization of antipyrine-tethered
organosilanes and their magnetic nanoparticles: potent anti-oxidants and re-
better limit of detection (LOD) compared to unfunctionalized coun- ceptors for Sn(II) ions, New J. Chem. 44 (2020) 15157–15168.
terpartsi.e. 2.7 × 10−6 M. Moreover, the Cu2+ ions added PTSR [13] G. Singh, Diksha, A. Singh, P. Satija, Pawan, Mohit, D.G. Silvera, C.E. Ruíz,
probe acts as a “turn-off” fluorescent sensor. The pharmacoki- M.A. Esteban, Organosilanes and their magnetic nanoparticles as naked eye
red emissive sensors for Ag+ ions and potent anti-oxidants, New J. Chem. 45
netic and physicochemical studies were performed on the prepared
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alkynes and organosilanes. Thus, the synthesis and designing of [14] A. Keivanloo, M. Fakharian, S. Sepehri, 1,2,3-triazoles based 3-substituted
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tal monitoring technologies.
[15] G. Singh, Mohit, Diksha, Suman, Priyanka, K.N. Singh, D.G. Silvera, C.E. Ruiz,
M.A. Esteban, Functionalized organosilanes and their magnetic nanoparticles
Declaration of Competing Interest as receptor for Sn (II) ions detection and potent antioxidants, J. Mol. Struct.
1247 (2022) 131297–131306.
[16] R.K. Cherie, E.F. Laura, G. Yury, T.H. Paula, C.H. Mark, E.N. Andre, M.P. Reginald,
There are no conflicts to declare. C.G. Willson, P.S. Weiss, Nano day: celebrating the next decade of nanoscience
and nanotechnology, ACS Nano 10 (2016) 9093–9103.
CRediT authorship contribution statement [17] J. Isaad, A. El-Achari, BODIPY modified silica coated magnetite nanoparticles
as fluorescent hybrid material for Cu (II) detection in aqueous medium, Dyes
Pigments 99 (2013) 878–886.
Gurjaspreet Singh: Supervision, Project administration. [18] J. Wang, S. Zheng, Y. Shao, J. Liu, Z. Xu, D. Zhu, Amino-functionalized
Suman: Conceptualization, Investigation. Diksha: Methodol- Fe3 O4 @SiO2 core-shell magnetic nanomaterial as a novel adsorbent for aque-
ous heavy metals removal, J. Colloid Interface Sci. 349 (2010) 293–299.
ogy. Sushma: Writing – review & editing. Mohit: Writing – [19] X. Yang, Q. Wei, H. Shao, X. Jiang, Multivalent amino-saccharide-based gold
original draft. Priyanka: Resources. Jashandeep Kaur: Data nanoparticles as narrow-spectrum antibiotics in vivo, ACS Appl. Mater. Inter-
curation. Anamika Saini: Validation. Anita Devi: Validation. faces 11 (2019) 7725–7730.
[20] A. Kumar, J. Young, L.H.S. Kim, Sensitive and selective fluorescence OFF-ON-OFF
sensor for cascade detection of Ga3+ cation and I- anion based on pyrenesulfon-
Acknowledgement amide-functionalized inorganic/organic hybrid nanoparticles, Sens. Actuators B
Chem. 239 (2017) 85–93.
[21] Y. Wang, S. Gong, S.J. Wang, X. Yang, Y. Ling, L.W. Yap, D. Dong, G.P. Simon,
The authors are grateful to the CSIR,UGC and DST-PURSE for
W. Cheng, Standing enokitake-like nanowire films for highly stretchable elas-
providing financial assistance. tronics, ACS Nano 12 (2018) 9742–9749.
[22] S.E. Sandler, B. Fellows, O.T. Mefford, Best practices for characterization of
Supplementary materials magnetic nanoparticles for biomedical applications, Anal. Chem. 91 (2019)
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found, in the online version, at doi:10.1016/j.molstruc.2022.132512. calized antimicrobial drug delivery, ACS Appl. Mater. Interfaces 8 (2016)
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