2,6-bis(E)-4-methylbenzylidine)-cyclohexan-1-one as a Fluorescent-on Sensor for Ultra Selective Detection of Chromium Ion in Aqueous Media

The ligand 2,6-bis(E)-4-methylbenzylidine)-cyclohexan-1-one sensor has been synthesized as a fluorescence-on sensor/probe for the trace level detection of chromium III ion. The synthesized ligand was characterized by FTIR, 1H-NMR spectroscopy, and fluorimetery. The sensor exhibited an ultra-selective response to chromium among the tested heavy metal ions. Different parameters were optimized like pH, effect of concentration of sensor C, metal ion and contact time. The binding stoichiometry of C:Cr3+ was calculated to be 2:1 (Job’s plot) with a significantly low detection limit of 2.3 × 10− 9 M. Sensor C were practically employed for detection of chromium in spiked water samples.


Introduction
Chromium is considered one of the major toxicant due to its high solubility and mobility in water. Most of the pollutants in environment are degraded gradually by living organisms into simpler harmless substances; but some pollutants including heavy metal ions like chromium are non-degradable [1,2]. Chromium is mainly added to water bodies either from natural sources; like volcanic eruption, rock weathering or man-made activities; including mining operations, metal extraction, and production of alloys, steel industries, and pigment producing factories, improper waste disposal, electroplating, synthetic fertilizers and other fields. In environment chromium readily gets dissolved in water and is persistent for longer period of time within the bodies of organisms [3,4], causing a number of disorders in organisms like tumors, gastrointestinal infections, ulceration, dermal irritation, lung cancer and anemia. Several analytical methods such as absorption spectrometry [5], plasma mass spectroscopy [6], chemical reduction [7], adsorption [8], electrocoagulation [9], chemical precipitation [10], flotation [11], ion exchange [12], electrochemical [13] and amperometric analysis [14] have been applied for detection of chromium ions, but due to their time-consuming nature, complex operation, expensive instrumentation, sludge production, requirement of large amount of chemicals, and consequent risk of secondary pollutants, these techniques are not preferred for the trace detection of chromium. In the last few years, the development of optical sensors capable of sensing heavy metal ions is greatly demanded.
Among the metal ion sensors, fluorescence sensors have gained attention due to their high sensitivity, lower cost, instantaneous response, excellent photo-physical properties, wide use, subsequent impact on the environment and nature, the easy operability and availability and possibility of real time monitoring [15]. The process of fluorescence is based on light emission by fluorophore as soon as they are excited [16]. A number of fluorescent sensors have already been reported and have been efficiently employed for selective metal ion recognition yielding promising results in terms of specificity [17,18]. But the design and synthesis of fluorescence based receptors for chromium ion detection in environmental samples is still demanding research topic, as the determination of chromium by spectrofluorimetry has been rarely studied [19].
Curcumin has very exciting photo physical and fluorescence properties. Apart from their application in biological field, the chemically synthesized pi-extended derivatives of curcumin analogues, possessing variety of chemical sites can be used as a potential sensor for metal ion determination. In current study, we report the development of curcumin derivative as a potential fluorochrome possessing efficient optical properties and binding capability. Due to the presence of electron donor atoms in the chemical structure of curcumin, it can easily form stable complex with cations [20].

Required Materials
All the chemicals and reagents used in the current study were used as received.

Instrumentation
F l u o r e s c e n c e a n a lys e s we r e p e r fo r m e d w i t h spectrof luorophotometer RF-5301PC (Shimadzu, Japan). Ultraviolet-Visible spectroscopic analysis was accomplished with double-beam UV-Visible spectrophotometer (Shimadzu, Japan) model 1601. For 1 H-NMR and IR spectra characterization Bruker Advance 400 MHz spectrometer and FTIR spectrophotometer Pretige 21 (Shimadzu, Japan) was used respectively.

Solutions Preparation for Spectroscopic Measurements
Stock solution of sensor C in acetonitrile, and metal ions solutions using respective salts of nickel, cadmium, lead, cerium, copper, mercury, zinc, manganese and chromium were prepared in distilled water. Each time a fresh working solution of both sensor C and metal ion was prepared by appropriate dilution form stock solution.

Sensor C Synthesis and Characterization
The sensor C [2,6-bis(E)-4-methylbenzylidine)-cyclohexan-1-one] was synthesized with slight modification of already existing method in literature [21] by reacting aldehyde and ketone in specific ration i.e. 2: 1. To a 12 mmol 4-methyl benzaldehyde (0.26 g in 12 mL ethanol) was added 6 mmol of cyclohexanone with continuous addition of sodium hydroxide (40%). The resulted mixture was refluxed for 4 h. By monitoring reaction progress with TLC, the final products was processed in ice with yellow colored pure crystals formed having melting point of 159 °C (yield 78%) and is given in (Scheme 1). 1

Spectroscopic Studies
For exploring optical properties and sensing capability of sensor C for chromium detection, UV-Vis and fluorescence spectrometry were used. The fluorescence and absorption spectra were taken in acetonitrile due to its freely solubility in it. In all of the spectroscopic experiments distilled water was used for metal solution and their spectra were recorded accordingly. For Uv/vis analysis, test solutions containing a fixed amount (8 µM) of sensor C while chromium (16 µM) concentration range were mixed and recorded. The fluorescence response of sensor C was monitored before and after mixing with different metal ions in distilled water. The mixtures were equilibrated at Scheme 1 Synthesis of sensor C room temperature before measuring the fluorescence response. In addition, the practical applicability of sensor C for chromium ion determination in the vicinity of co-existing of similar metal cations in aqueous media was also investigated.

Quantum Yield Calculation
Rhodamine 6G dye was used as a standard possessing quantum yield (φ) 95% for enumerating quantum yield of the sensor C. For doing the experiment, Rhodamine 6G solution was prepared and its spectroscopic analysis (UV-Visible and fluorescence) were performed. The quantum yield was determined with the help of the given formula where "C" and "R" represent sensor C and dye respectively. "I" represent the integrated fluorescence intensity, "η" correspond to refractive index of solvent and "A" correspond to absorbance.

Analytical Sensitivity
Stern Volmer plot analysis were performed for detection limit (δ) and quantification limit (β) determination using Eqs. 2 and 3 respectively where δ represent the standard deviation, and S being the slope of fluorescence response versus concentration curve. The respective experiment was performed seven times to examine the replicability of the system.

Computation of Binding Constant
To calculate approximately the binding constant of sensor C (Benesi-Hilderbrand plot) was drawn between fluorescence intensity and reciprocal of chromium concentration. The binding constant was attained using the given Eq. 4.

Spiking of Natural Water Damples
Fluorescence spectroscopy was used for detecting the binding of sensor C towards the ion of interest, Chromium, in Intercept Slope real water samples. Therefore, the selected water samples (river water, Tap water) were used for spiking of different concentration of chromium (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) µM in the presence of (8 µM) sensor C and the fluorescence response was measured at specific wavelengths of excitation and emission. For verification of the test results, all analysis were repeated four times.

Ultraviolet-Visible study
For the evaluation of the photophysical properties of sensor C and its chromium complex UV-Visible analysis was initially used. For considerable solubility and prospective applicability of sensor C, acetonitrile was selected as a solvent for sensor C in the current study. Ultraviolet-Visible spectrum of sensor C was taken in the presence and absence of different concentration of chromium ions. The Uv/vis spectrum for sensor C showed characteristic main absorption band (λmax) at360 nm.Upon addition of chromium solution bathochromic shift occurred and maximum absorption band appeared at 420 nm, confirming the formation of chromium complex with sensor C (Fig. 1). Each time baseline was established by putting respective blanks in the reference slot.

Fluorescence-on Studies by Cr 3+ Ion
In our ongoing detection for a better understanding and a feasible intonation of the sensing ability of curcumin based fluorochrome, we have tagged curcumin based sensor with metal ion. In order to realize the photo physical properties of sensor C prior to metal ions recognition, the fluorescence response of free sensor C was taken. According to results obtained, sensor C displayed a very weak emission peak at 560 nm (λem). To investigate metal ions sensing by sensor C, different heavy metal ions Ni 2+ , Ce 3+ , Cu 2+ , Zn 2+ , Cd 2+ , Mn 2+ , Cr 3+ , Pb 2+ , Co 2+ (200 μM) were added to sensor C solution separately. The respective mixtures were allowed for 3 min to equilibrate, and were transferred into quartz cuvette separately by monitoring fluorescence intensity in the range of 400-700 nm. The spectra obtained for all the studied metal was compared with the spectrum of sensor C. In our case, a prominent enhancement in fluorescence emission intensity was observed at λem 560 nm upon the addition of aqueous chromium ion (200 µM) to sensor C solution. While no apparent enhancement in intensity was spotted for the rest of metal ions (Fig. 2). So among the studied metal ions only chromium forms complex with sensor C. This specific fluorescence-on response could be accredited to restriction of ICT phenomena. In the absence of chromium ions, due to ICT phenomena, a weak fluorescene was observed for sensor C. while in the presence of chromium ions ICT process is negligible and fluorescence intensity was extensively enhanced as the result of formation of a stable complex [24].

Wavelength (nm)
Fluorescent sensor C C + Mn 2+ C + Ni 2+ C + Co 2+ C + Cu 2+ C + Hg 2+ C + Ce 3+ C + Cd 2+ C + Pb 2+ C + Cr 3+ As discussed, we postulated that the apparent fluorescence enhancement of sensor C could be ascribed to a distinctive chemical resemblance for chromium ion. It is presented in already reported articles, fluorescent molecules like sensor C retain certain preference for chromium ion, thus allow a superficial prejudice from its close neighbor Fe 3+ along with other similar co-existing ions through fluorescence enhancement [25].

pH Study
In the interests of avoiding interference in chromium ion recognition by protons, we focused on pH analysis on the fluorescence retaliation. The pH value of the test system was set to acquired value by HCl and NaOH solution. The sensor C (8 μM) did not change its fluorescent intensity from pH = 1to 14, indicating its consistency. The weak fluorescent response of sensor C may be owing to intra-molecular photo-induced electron transfer. However, sensor C complex with (16 μM) chromium showed strong fluorescence at pH 8, on account of its binding with Cr 3+ leading to intra-molecular photoinduced electron transfer process inhibition. The chromium complex was unstable at lower pH thus displaying quenching in intensity as a result of protonation carbonyl oxygen that acts as binding site. By gradually increasing the pH, the fluorescence intensity strappingly enhanced (Fig. 3). At pH 8, high stability was displayed by the chromium complex and maximum enhancement in intensity was observed at λem 560 nm. While at pH > 8, a gradual decrease in intensity occurred due to formation of hydroxyl complexes that had poor solubility in water solution. From the results obtained, it can be assume that the synthesized sensor C can successfully be used for chromium ion determination at pH 8 in aqueous samples [26].

Sensor C Concentration Effect
The determination of concentration effect of sensor Con fluorescence response of the sensor C-Cr 3+ complex is one of the important parameters in fluorimetric analyses. Working solution for analysis were prepared by keeping the chromium concentration constant (16 μM), and varying the sensor C concentration in the range of 5-40 μM. The fluorescence emission intensity of each sample was monitored in 440-700 nm range (Fig. 4). With increasing concentration of sensor C, a linear enhancement in the emission intensity was observed. At higher concentration, the fluorescence intensity remains constant.

Quantitative Study for Cr 3+
For the confirmation of sensitivity of sensor C toward chromium ion, fluorescence titration experiments were carried out. During these titrations, the sensor C concentration was kept constant (8 µM) while changing the chromium ions concentration from 1-15 µM. A linear enhancement in the fluorescence intensity of chromium complex was noted at 560 nm (λem) upon increasing chromium concentration (Fig. 5). Thus showing that sensor C can be used for quantitative determination of Cr 3+.

Binding Mode Study
The binding stoichiometry between sensor C and chromium was investigated using a Job's plot, A Job's plot between the different mole fraction of sensor C-Cr 3+ complex versus respective emission intensities showed a maxima at 0.7 mol fraction indicating a 2:1 stoichiometric ratio between sensor C and Cr 3+ (Fig. 6).
The association constant was determined with the help of Benesi-Helderbrand plot. For determination of association constant the given Eq. (5) was used.

The Detecting Mechanism of Fluorescent Sensor C for Cr 3+
The detecting mechanism of sensor C for Cr 3+ ion was suggested using the fluorescence spectra. The fluorescent response of the sensor C towards Cr 3+ ion may be allocated to intra molecular charge transfer (ICT). Before being coordinated with Cr 3+ ions , sensor C displays a weak fluorescence emission spectrum due to lone electron pair of oxygen atom, which result in intra molecular charge transfer. Moreover, the lone electron pair of the oxygen atom give rise to a non-radiative process by the n-π* transition, as a result quenching in fluorescence intensity takes place. On the other hand, after sensor C was coordinated to Cr 3+ ion , radiation process was primarily viaπ-π*transitiions and the coordination complex was more rigid thus the ICT process was restricted upon addition of Cr 3+ ionat the receptor site.
On the other hand, sensor C act as monodentate ligand as there is one potential coordination cite, the carbonyl oxygen. The coordination sphere of d-block elements like Cr is either 6 or 2 and complexes of chromium with both these coordination spheres have been reported [27][28][29][30]. Since the bulk of our sensor C is high so the bonding with chromium is expected to be strong and possible structures of sensor C-Cr 3+ complex have been proposed in the (Scheme 2) ( Table 1).

Time Study
Rapid response time is highly preferred in flurorimetric studies, therefore the time effect on emission response of sensor C and its complex with chromium was studied in time range of 0-7 min for different concentrations of chromium. During the assay, the fluorescence emission response of sensor C remained unaffected with increasing time, whereas the complex showed a linear response in fluorescence intensity with time and maximum fluorescence response was observed at 2 min and then remained constant (Fig. 8). These results indicate effectivity of sensor C for real time assay of chromium detection in environmental samples.

Effect of Competitive Metal Cations
Competitive binding experiments were taken for evaluation of efficacy of the synthesized sensor C as a selective chromium ion sensor as sensor C should have the potential to detect chromium ions in the presence of coexisting metal cations. For this purpose, different commonly existing metal ions in water were selected like Cu 2+ ,Hg 2+ ,Zn 2+ ,Cd 2+ ,Pb 2+ ,Mn 2+ ,Co 2+ ,Ni 2+ including the  (Fig. 9). Thus, the obtained results may indisputably provide the reported sensor C is particularly valuable in efficiently selective chromium ion determination from its chemically similar cations like cerium and other commonly found interfering ions. Therefore, it can be realistically incidental that the synthesized sensor C exhibits an ultra-sensitive and highly selective fluorescence-on response toward chromium ion in aqueous samples.

Reversibility of Sensor C
To determine whether the metal complex formation process is reversible or not, fluorescence emission titration experiment was performed using the sensor-Cr 3+ complex with EDTA.
The reversibility of the designed fluorescence-based sensor is curricula for its economy and many times usage. In this regard, EDTA is added into sensor C combined with Cr 3+ solution to confirm the effect of Cr 3+ ions on the fluorescence response. When EDTA is added to the aqueous solution, the fluorescence spectrum is returned to its original state. The interaction between sensor C and Cr 3+ ions is prevented by the formation of Cr 3+ -EDTA chelate. After the addition of EDTA, the fluorescence of sensor C is recovered. The fluorescence-on sensor C is proved to be reversible using turn off-on mechanism based on the fluorescence emission intensity measurement (Fig. 10). The experiments were repeated 4 times by getting the same results. Furthermore, since the fluorescence emission intensity was restored upon addition of EDTA, the sensing process was considered to be reversible rather than an ion-catalyzed reaction. sensor C C + Ni 2+ + Cr 3+ C + Hg 2+ + Cr 3+ C + Pb 2+ + Cr 3+ C + Mn 2+ + Cr 3+ C + Cd 2+ + Cr 3+ C + Ce 3+ + Cr 3+ C + Cu 2+ + Cr 3+ C + Zn 2+ + Cr 3+ C + Co 2+ + Cr 3+

Quantitative Detection of Cr Ion in Aqueous Samples
To study in deep the suitability and on-field applicability of prepared sensor C for ultra-selective detection of chromium ion, the different water samples were spiked by adding chromium to have sample solutions in (1-10) µM conc. range. Accordingly, the results attained displayed a significant recovery of chromium ions from different environmental water samples (Fig. 11). Thus, we presented a cost efficient method for highly selective determination of Cr 3+ ion in aqueous samples.

Significance of Current Work
As compared to other chromium ion detection methods (Table 2), the present methodology has the compensation in terms of linearity and ultra-low sensitivity. In addition, it display higher potential for chromium detection.

Conclusion
In summary, we have developed a sensor C for a possible Cr 3+ ion-selective detection in aqueous samples. The sensor C exhibited remarkable sensing properties in terms of fluorescence response, sensitivity, selectivity and detection limit. Specially, sensor C showed a highly selective response for chromium among other interfering metal ions with limit of detection and limit of quantification 2.3 × 10 −9 M and 3.14 × 10 −8 M respectively. Considering the photophysical properties and the chemosensing capability, the highly emissive curcumin-based sensor C, [2,6-bis(E)-4methylbenzylidine)-cyclohexan-1-one] can be considered as one of the very promising candidate for chemosensing applications. Validity of the present method was effectively realized by real sample assay.

Supplementary Information
The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s10895-021-02806-0. Funding There has been no significant financial support for conduction of current research work that could have influenced its outcomes.

Data Availability
All the data associated with this research has been presented in this paper.

Declarations
Ethics Declarations In the current study the procedure adopted is safe both for animals, humans and their environment.

Consent to Participate
All authors have participated in the current research work.

Consent for Publication
All authors have given their consent for publishing of this article in the said journal.

Conflicts of Interest
The authors declare that they have no conflicts of interest.