A Colorimetric Distinct Color Change Cu(II) 4-{[1-(2,5-dihydroxyphenyl)ethylidene]amino}-1,5-dimethyl-2-phenyl-1,2-dihydro-3H-pyrazol-3-one Chemosensor and its Application as a Paper Test Kit

In the current research work “4-{[1-(2,5-dihydroxyphenyl)ethylidene]amino}-1,5-dimethyl-2-phenyl-1,2-dihydro-3H-pyrazol-3-one” chemosensor (C1) synthesized by condensation reaction using “4-amino-1,2-dihydro-1,5-dimethyl-2-phenylpyrazol-3-one” and “2,5-dihydroxy actophenone” was used as the effective sensor of metal ion. The C1 shows absorption peak at 326 nm due to the C = C bond (π-π* transition), while the absorption peak at 364 nm is caused by the C = O bond (n-π* transition). In the presence of copper, C1 only demonstrated a redshift in absorption peak from 364 to 425 nm. Even in the presence of other competing metal ions, the hypsochromic shift of the absorption band and the quenching of the fluorescence emission intensity were different for detecting Cu2+, in CH3OH-H2O (v/v = 6:4). The capacity of the C1 to bind with Cu2+ was further proved using DFT simulations. The complex C1 + Cu2+ has a HOMO–LUMO energy gap of 2.8002 eV, which is lesser than C1 (2.9991 eV) showing improvement in the stability of the C1 + Cu2+ complex. Using the Benesi-Hildebrand and Scatchard plots, calculated Kb values were to be 47,340 and 48369 M−1 respectively, showing the creation of stable complexation between Cu2+ and C1 with 1:1 stoichiometry. The limit of detection (LOD) for Cu2+ ion was 649 nM. Strip sheets were also built and tested to detect varying amounts of Cu2+ in aqueous solution, and their color change suggested that they might be used for on-site Cu2+ detection in polluted water.


Introduction
The present rise of chemosensors development has sparked a lot of interest in contemporary research communities to create single organic molecules with the capability to detect a variety of metal ions using a variety of analytical methodologies. Such sensing compounds may become critically important since they play an important function in biological systems and have a high hazardous effect on the environment [1][2][3][4][5][6].
Over the last several years, inexpensive chemical sensors have played an essential role in monitoring a wide range of physiologically and environmental important species, such as heavy metal ions [7][8][9]. Metal ion detection with both fluorescence emission sub units and metal binding are highly sought after. In the case of fluorescent chemosensors for tiny compounds, poor selectivity and fluorescence quenching qualities are common [10,11]. Although considerable progress has been achieved in this field, new methods for the detection of metal ions are still needed. Organic sensors have a number of advantages over traditional identification techniques, including simplicity of synthesis, high sensitivity, selectivity, and low cost [12][13][14][15].
Molecular sensors are very effective tools for recognizing medicinal, biological, chemical, and environmental species in particular. Despite the fact that chromatography and other analytical methods are widely used, ICP-MS, GFAAS, ICP-AES, AMS, NAA, ETAAS, LAMMA, and so on are available, fluorescence chemosensing is thought to be the best instrument since it is selective, sensitive, rapid, and low-cost technology that provides significant advantages over other tactics [16,17].
Cu is a necessary trace component for all living things, and it participates in a wide range of biological functions including enzyme catalysis, gene expression, and protein synthesis. Cu 2+ levels in blood and drinking water are allowed to be between 15.7 to 23.6 mM under normal circumstances [18,19]. A modest amount of Cu 2+ is necessary for the body's regular operation, but too much Cu 2+ may lead to a variety of neurological problems, including Wilson's disease and Alzheimer's disease [20][21][22]. As a result, developing a simple, quick, and sensitive analytical approach for detecting Cu 2+ is critical. Some fluorescent sensors can selectively and colorimetrically identify metal ions and offer a number of benefits, including high sensitivity and selectivity, cost effectiveness, and ease of process, making them appealing for Cu 2+ detection [23][24][25][26].
In the current study, we synthesized C1 and explored its absorption and emission properties toward various metal ions. Only Cu exhibits a distinguishing color change due to a hypsochromic shift in the absorption band and a quenching of the fluorescence emission intensity. It possessed a 1:1 Cu 2+ binding ratio and a LOD of 649 nM, making it suitable for detecting Cu 2+ in real environmental samples. MASS and FTIR were used to confirm the structures of C1 and C1 + Cu 2+ and furthermore it was established by the DFT design. Furthermore, the probe was utilized to make the test paper, which changed from yellow to orange-brown in the presence of ion.

Materials and Instrumentation
Analytical reagent-grade organic solvents were used throughout. All of the cations utilized had high-quality nitrate and chloride salts and were employed without purification.
Stock solutions for C1 (1 mM) and nitrate and chloride salts of various metals were produced in CH 3 OH-H 2 O (v/v 6:4). Absorption and fluorescence titrations were carried out on 10.0 µM C1, with aliquots of newly generated standard aqueous metal ion solutions added to record the absorption and fluorescence spectra.
Absorption IR spectra were taken on "UV-1800 UV-Vis spectrophotometer", Shimadzu and "Brukar IR" spectrophotometer, respectively with an excitation slit of 15.0 nm and an emission slit of 7.0 nm. 1 H-NMR spectra were acquired from Bruker DTX-400 spectrometer in DMSO. Mass spectra were obtained by using micrOTOF-Q II mass spectrometer.

Synthesis of C1 Complex with Copper
The C1 (0.337 g, 1 mmol) was added to 30 mL of CH 3 OH and then 1 mmol of CuNO 3 was added while stirring. The entire mixture was refluxed for 3 h with constant stirring. A dark brown solid (C1 + Cu 2+ complex) developed after cooling, was filtered and refined further by recrystallization in ethanol.

Photophysical Studies
The working solution of C1 was scanned for both UV-visible and fluorescence spectrometers first, and then the different metal ions solutions were added one by one in aqueous methanol for cation selectivity on both devices. Metal ions with distinguishing spectrum shifts from other metal ions were used 1 3 for titration studies. Photometric titrations between C1 and the selected metal ions were used to determine the binding constant for comprehensive research.

DFT Calculation
The geometry conformation and optimization calculations of the highly functionalized sensors C1 and C1 + Cu 2+ have been carried out using the "Gaussian 09 program". The calculation of the density functional theory (DFT-B3LYP) was performed by defining the basis function system 6-311 + + G(d,p) for all atoms.

Limit of Detection Calculation
The standard deviation of blank data was calculated by measuring the emission intensity and absorbance of C1 ten times.
The following calculation was used to compute the detection limit: where δ slope of the intensity versus Cu 2+ concentration and S Standard deviation of the blank measurement

Result and Discussion
On the basis of IR, 1 H, and mass spectroscopic techniques, the purity of C1 has been determined. 1 3 . The spectral examination data were compatible with the C1 configuration ( Fig. S1 to S3 in supplement file) [29][30][31][32].
UV-vis spectroscopies of C1 in an aqueous solution were recorded to examine their optical characteristics. Two peaks of absorption at 326 and 364 nm can clearly seen in the UV-vis spectrums. The absorption peak at 326 nm is due to the C = C bond (π-π* transition), whereas the C = O bond absorption peak is at 364 nm due to n-π* transition [31,33]. However, after adding 1 equivalent of different metal ions such as Gd(III), Cr(III), Nd(III), Al(III), Mg(II), Hg(II), Mn(II), Cd(II), Pb(II), Co(II), Cu(II), Na(I), Ag(I), K(I), and Li(I) the absorption spectra showed no discernible alterations. Only in the presence of copper, C1 showed a change in absorption peak wavelength to a higher wavelength (red shift) from 364 to 425 nm respectively. The C1 + Cu(II) complex changed from bright yellow to dark brown colour with hypsochromic shift appearance of a new peak at 425 nm (Fig. 1). Absorbance titrations were used to test the sensitivity of C1. When the concentrations of Cu(II) (0-1 equiv.) in C1 were increased, a progressive rise in the absorbance band at 425 and decrease at 364 nm (Fig. 2), due to creation of C1 + Cu(II) complex with high stability and new absorption band indicate ligand to metal charge transfer mechanism. The R 2 = 0.9547 on the linear plot obtained from the titration reveals that C1 binds linearly to the Cu 2+ ion (Fig. 3). The ratiometric graph found from the selectivity study demonstrated that the only addition of Cu 2+ in C1 showed appreciable discriminating absorption shift (Fig. 4). The C1 absorption sensitivity was checked toward the Cu 2+ in the presence of various major ions under the same   (Fig. 5) [34,35]. When Cu 2+ ion solution was added to C1, the emission (λ exc = 425 nm) of C1 was investigated. In aqueous methanol, the emission spectrum of C1 exhibits a very faint fluorescence band at 465 nm. The incorporation of the Cu 2+ ion to C1 resulted in a significant quench in fluorescence (Fig. 6a). Increased fluorescence effect due to Cu 2+ ion-induced chelation is reflected from, the fluorescence enhancement about 5 times larger than the individual receptor. The rise in emission intensity is most likely due to the complexation of the probe with Cu 2+ through imine nitrogen and thiol SH, this reduces the accessibility of imine nitrogen atom lone pairs, shutting off PET (photoinduced electron transfer) and activating fluorescence [36][37][38]. In this instance, the substantial CHEF (Chelation-Enhanced Fluorescence) effect stiffens the chemosensor framework, and inhibiting "isomerization of the C = N double bond of the C1 in an excited state may also result in a considerable increase in the C1 fluorescence intensity" [39]. Fluorescence quenching refers to the absence of fluorescence. The C1 in aqueous methanol quenches the emission maxima and becomes "TURN-OFF" in this circumstance. When the Cu 2+ ion was introduced to the C1, the emission maxima was dramatically increased, resulting in fluorescence ON (Fig. 6b).
Fluorescence titration studies were carried out in order to have a better grasp of the sensing behaviour of C1 to Cu 2+ . The fluorescence intensity fell progressively as the quantity of Cu 2+ increased, as seen in Fig. 6b. A considerable quenching of fluorescence was seen with the addition of 10 equiv. of Cu 2+ , [(I 0 I)/I 0 100 percent]. At roughly 0.5 mol fractions, a minimum was seen in the Job plots using fluorescence titrations, showing that C1 formed 1: 1 combination with Cu 2+ (Scheme 1).

HOMO-LUMO Analysis
The difference in energy between HOMO and LUMO is an important parameter for determining the excitability of molecules. In general, the frontier molecular orbital (FMO) diagram is a vital one to differentiate between the effect of metal binding with the sensors and its electronic characteristics. In this piece of research, to distinguish the same (i.e., effect of binding of Cu 2+ with highly functionalized sensor C1) and its electronic characteristics, FMOs of Cu 2+ with highly functionalized sensor C1 and highly functionalized sensor C1 alone have been assessed and the results provided interesting insights into the relevant LUMO/HOMO energy levels and electron distribution. Pictorial representation of electron distribution in LUMO and HOMO of the highly functionalized sensor C1 and its metal coordinated one (sensor C1 + Cu 2+ ) are depicted in Fig. 7 and the associated energy values are furnished in Table 1. In the present case, the energy difference between the LUMO and HOMO of the highly functionalized sensor C1 is noted to be 3.00 eV which is higher than that of the distinction in energy between HOMO and LUMO of the sensor C1 + Cu 2+ (2.80 eV). These results imply that the firmness of the highly functionalized C1 + Cu 2+ complex is superior as a result of the explicit binding of highly functionalized sensor C1 with Cu 2+ which meritoriously decreases the HOMO-LUMO energy difference (i.e., precise binding of highly functionalized sensor C1 to metal Cu 2+ increases the stability of the sensor C1 + Cu 2+ complex). In the HOMO of the highly functionalized sensor C1, the electron density is localized on the pyrazole unit and aryl moiety integrated at the N-position of pyrazole structural motif while in LUMO of the same, the electron density is localized mainly on the imino structural motif bridging between pyrazole and dihydroxyaryl scaffolds [40][41][42]. On the other hand, in the HOMO of the highly functionalized sensor C1 + Cu 2+ , the electron density is denser on the dihydroxyaryl moiety, imino

Absorbance
Metal ions functionality as well as the pyrazole structural motif along with metal whereas in the LUMO of the same, the electron density is denser over the aryl moiety at the N-of the pyrazole unit along with pyrazole structural motif. These observations imply that electron transfer from one part to another takes place within the molecules as for as HOMO and LUMO are concerned. Overall, the DFT results are in good harmony with the experimental outcomes of the metal binding. Broadbands were found in the FTIR spectra of pure ligand at 3238 and 3062 cm −1 , respectively, which correspond to the -OH and -NH stretching frequencies. At 1625 and 1498 cm −1 , respectively, carbonyl and imine stretching frequency characteristic bands developed clearly. In the FTIR spectra of the matching C1-Cu 2+ combination (Fig. S4 in supplement file), the unique bands associated with -OH and -NH are diminished/ decreased, and the bands for carbonyl and imine stretching are relocated to a lower wavelength in the complex at 1662 and 1492 cm −1 , respectively [43,44]. According to the above facts, the ligand's amide functionality must have been subjected to amido-imidol tautomerism for the imidol structure   [45][46][47]. Ka was calculated using "Benesi-Hildebrand" and "Scatchard Plot" at 47,340 and 48369 M −1 respectively (Figs. 8 and 9), showing the formation of stable complexation between Cu 2+ and C1 with 1:1 stoichiometry [48][49][50][51][52]. From the equation LOD = 3 s/m, it was discovered that the detection limit was 649 nM. Job's receptor plot revealed peaks corresponding maximum at 0.5 mol fraction for C1-Cu 2+ complex formation, indicating 1:1 complex formation (Fig. 10). To assess the binding stoichiometry between C1 and Cu 2+ , a Job's plot for C1 with metal ions was built [53][54][55]. It reached a climax at a mole ratio fraction of 0.5, showing that the binding mode of C1 with Cu 2+ is 1:1 stoichiometry. Figure 10 displays a jobs plot of the host's absorbance H/([H] + [G]) at various concentrations. Where H stands for host and G for guest Cu 2+ metal ions [56,57]. The finding indicates that a 1:1 stoichiometry combination formed between C1 and Cu 2+ [58]. The visible color shift that occurs when Cu 2+ is added to the C1 is an important component of this work, which looked at onsite Cu 2+ detection in water samples and the reversibility test with the addition of EDTA, and successful formation of EDTA + Cu and C1 (Figs. 11 and 12) [59][60][61].

Application as a Test Kit
We made test strips by dipping filter papers (3 × 1 cm 2 ) in the CH 3 OH-H 2 O (v/v = 6:4) solution of C1 (1 × 10 -3 M) followed by drying them in air to see whether a "dip-stick" approach for the detection of Cu 2+ , similar to that typically used for pH testing, was suitable [62][63][64]. The evident color shift from yellow to Orange was noticed when the test  strips coated with C1 (1 × 10 -3 M) were submerged in pure water solutions of Cu 2+ with varied concentrations (Fig. 13). The creation of a "dip-sticks" method was very appealing for "in-the-field" measurements that did not need any extra equipment [65,66]. As a result, the C1 test strips offer a high applicability value for detecting Cu 2+ .

Conclusions
A Schiff-base "4-{[1-(2,5-dihydroxyphenyl)ethylidene] amino}-1,5-dimethyl-2-phenyl-1,2-dihydro-3H-pyrazol-3-one" as colorimetric and fluorescence chemosensor C1 for Cu 2+ was developed. UV-vis spectrum shows two peaks of absorption at 326 nm and 364 nm. Only in the presence of copper, from 364 to 425 nm, C1's absorption peak shifted to a higher wavelength (red shift). Even in the presence of other competing metal ions, the hypsochromic shift of the absorption band and the quenching of the fluorescence emission intensity were unique for the detection of Cu 2+ , in "CH 3 OH-H 2 O (v/v = 6:4)". The capacity of the C1 to bind Cu 2+ was further proven using DFT simulations. The complex C1 + Cu 2+ has a HOMO-LUMO energy gap of 2.8002 eV, which is lesser than C1 (2.9991 eV). From these results, it can be seen that the specific binding of C1 to Cu 2+ improves the stability of the C1 + Cu 2+ complex and effectively reduces the HOMO-LUMO energy gap. Using the Benesi-Hildebrand and Scatchard plots, the K b value were calculated and found to be 47,340 and 48369 M −1 respectively, showing the formation of stable complexation between Cu 2+ and C1 with 1:1 Stoichiometry with C1 detection limit for Cu 2+ ion analysis was found to be 649 nM.