Bathophenanthroline as Turn-off Fluorescence Sensors for Selective and Sensitive Tetection of Fe(II)

Bathophenanthroline (BPhen) is a versatile bidentate ligand for transition metals, also frequently used as a universal colorimetric probe for Fe(II). The effect of pH on the uorescence intensity of BPhen and quenching constant of Fe(II) were studied at different pH values using working solutions of 50% ethanol buffered with acetate buffer. Fluorescence intensity of a 3.0 μM Bphen solution in 50% ethanol buffered at pH 6.6 with acetate buffer was selectively quenched by Fe(II) at 25 °C. The LOD of 19 nM at 3.3 σ and the linear range of 63 nM – 224 nM with R 2 = 0.9919 revealed that this method is more sensitive than the colorimetric method (detection range =1.8 μM - 18 μM). Quenching of the BPhen-Fe(II) complex is temperature-dependent, which may be due to the increased stability of the formed complex with temperature, supporting a static quenching mechanism. Interference from foreign ions on the uorescence of the BPhen-iron(II) complex was also studied. A tolerance limit exceeding 5% was observed and recorded. The interference from cations, Ni(II), Co(II), and Cu(II), was relatively higher, while the most common anions showed only a little to no interference. In response time analysis, the uorescence intensity, which remains constant after 10 minutes, reveals that the system approached equilibrium. This method would be quicker and more accurate than a colorimetric method due to the absence of a solvent extraction step.


Introduction
Several methods, such as atomic absorption spectroscopy (AAS) [1], inductively coupled plasma mass spectrometry (ICP-MS) [2], and inductively coupled plasma atomic emission spectroscopy (ICP-AES) [3] are available to detect metal ions in trace amounts. Although these techniques provide sensitive and accurate results, signi cant drawbacks like non-portability, time-consuming sample processing, high cost, and sophisticated operation [4] have led to researchers' search for alternative methods.
The development of uorescence sensors is an increasing research interest among researchers as these sensors have unique features such as high sensitivity and simplicity. Any process that results in a change in uorescence intensity, wavelength, anisotropy, or lifetime can be used for sensing studies. A typical uorescence probe has two moieties; a receptor (the recognition site) and a signaling unit ( uorophore) capable of absorbing and emitting light. Thus, the development of cost-effective, susceptible uorescent sensors with low detection limits has gained much attention in recent years. [5] A decrease in uorescence intensity is called uorescence quenching, and it can be static or dynamic.
Dynamic quenching occurs due to collisions of the quencher with the uorophore. Static quenching is caused by the interactions of the ground state of the uorophore with a quencher. Static quenching, quencher concentration, and formation constants are essential factors, while excited state lifetime is critical for collisional quenching. [6][7] Fluorescence quenching is temperature dependant in accordance with the mechanism of quenching. [8][9][10][11] Iron is a crucial trace metal in living systems. It participates in various metabolic processes, including oxygen transportation, DNA synthesis, chlorophyll synthesis, electron transportation, and photosynthesis. [12][13] Iron exists as the Fe(II) or Fe(III) state, and depending on the oxidation state, it shows a unique reactivity. Disorders in iron metabolism cause a broad spectrum of diseases in humans, ranging from anemia to Parkinson's disease. Labile iron, which is not bound to proteins, produces reactive oxygen species, causing potential cell damage and various iron-related disorders. The form of endogenous labile iron is Fe(II) rather than Fe(III). [14] When considering the oxidative degradation of cellulose by Fe(II), monitoring Fe(II) in trace amounts has a signi cant role in biomedical studies, water quality assessments, and several other aspects like paper conservation and the textile industry. Furthermore, Fe(II) impurities are common in reagents, and is determining these levels is necessary for analytical applications. [15][16][17] 1,10-Phenanthroline and its derivatives are chemically versatile ligands due to their promising structural and chemical properties. Phenanthroline plays an important role in coordination chemistry due to its unique features like planarity, rigidity, hydrophobicity, aromaticity, and chelating capability [18]. 1,10-Phenanthroline (λ ex = 266 nm, λ em = 366), is a uorescent molecule. A non-uorescent complex with Fe(II) formation makes 1,10-Phenanthroline a sensitive uorescence probe for the iron with a LOD of 24 nM at 3σ. (Linear range 0.5 µM-20µM). [19] The compound 4,7-diphenyl-1,10-phenanthroline, known as Bathophenanthroline (BPhen), [A], is one of the most common phenanthroline derivatives. BPhen can form a stable 1:3 red coloured complex (B) selectively with Fe(II) but not with Fe(III), known as the ferroin reaction. [20,21]. This reaction can be used to detect extremely low concentrations of Fe(II) ions in aqueous solutions. The use of BPhen as a sensitive chromophore to detect iron was initially described by the Smith method. After this, various modi cations have been made to increase the effectiveness of the Fe(III) to Fe(II) conversion in the original method described by Smith et al. [22]. These colorimetric-based procedures to detect Fe(II) with BPhen include an organic extraction step, as Fe(II) is generally an aqueous medium. The use of BPhen (molar absorptivity = 22,400) which gives a detection range of 1.8-18 µM, is a signi cant improvement over 1, 10-phenanthroline (molar absorptivity = 11,100) for the colorimetric determination of Fe(II) [15], [23][24].
In this study, a novel turn-off uorescence probe based on BPhen (A) was developed successfully to quantify Fe(II) in a 50% ethanol solvent system. Compared to 1,10-phenanthroline, improved reproducibility solubility and selectivity were observed with the novel probe. The improved detection limit of this probe is 19 nM.

Apparatus
All the uorescence studies were carried out by using a Hitachi F7000 uorescence spectrophotometer equipped with a 150 W xenon lamp using a 1 × 1 cm 2 quartz uorescence cuvette with a 1.0 cm quartz cell holder and a thermostat bath. The excitation and the emission slit widths were set to 5 nm. The absorbance studies were done on a Labomed Inc. (USA) double beam UV-Visible spectrophotometer.
Homogenization of solutions was done using a sonicator. Precisa XB 120 analytical balance was utilized for weighing purposes, and the pH was determined using a pHS-2C pH-meter (Shanghai DaPu Instruments Co., Ltd, Shanghai, China) at ambient temperature.

Reagents
Analytical grade purity solvents and reagents were used without any further puri cation. A Sigma Aldrich (USA) 99% Bathophenanthroline was used to prepare the BPhen stock solutions. Fe(II) solutions were made by using an analytical grade (NH 4 ) 2 Fe(SO 4 ) 2 .6H 2 O from Merck Specialities Private Limited (India).
Analytical grade CH 3 COONa (Daejung chemicals and metals, South Korea) and Sigma Aldrich 99.8% acetic acid were used to prepare the buffer solution. AR grade absolute ethanol (VWR Chemicals, France) was used to make ethanol solutions, and doubly distilled deionized water was used wherever dilution was needed. All glassware was washed with diluted sulphuric acid along with deionized water and oven-dried before each experiment.

BPhen stock solution (3.0 mM)
A mass of 10.0 mg of BPhen was dissolved in 100% ethanol in a 10 ml volumetric ask using a sonicator and made up to the mark. The working solution of BPhen was freshly prepared by diluting the appropriate volume of the stock solution using the solvent system.

Preparation of Fe(II) stock solution (5.0 mM) in water
A mass of 20.0 mg of analytical grade (NH 4 ) 2 Fe(SO 4 ) 2 .6H 2 O was dissolved in double-distilled deionized water in a 10 ml volumetric ask using a sonicator and made up to the mark. Fe(II)'s working solution was freshly prepared by diluting the appropriate volume from the stock solution using the solvent system.

2.3.3
Preparation of working solutions of pH 4.5, pH 5.5, and pH 6.6 using acetate buffer Acetate buffers are used to prepare the working solutions at pH 4.5, pH 5.5, and pH 6.6.

BPhen in 50% ethanol (3.0 µM)
A volume of 10 µl of the 1.5 ´ 10 -3 M BPhen in 50% ethanol was added to a 5 ml volumetric ask and made up to the mark with 50% ethanol, without buffer. Another set of working solutions of pH 4.5, pH 5.5, and pH 6.6 (made using the acetate buffer) were prepared separately with 3.0 µM BPhen in 50% ethanol.

Excitation and emission spectra of BPhen
In UV-Visible absorption studies, the uorophore's stock solution was diluted enough to avoid the molecules' aggregation. In the absorption and the emission spectra of BPhen in 50% ethanol (Fig. 1), two distinct peaks were observed at 224 nm and 272 nm. They were assumed to be due to π→π* transitions of phenyl and phenanthroline groups of BPhen, respectively. Computational studies revealed no n→π* transition is possible in the absorption spectrum of the phenanthroline derivative. [25,26]. The emission spectrum of BPhen (λ ex = 272 nm) is a broad single peak with λ max at 385 nm.

Effect of the concentration of BPhen on uorescence
The uorescence intensity, as well as the degree of uorescence quenching, depends on the uorophore concentration. Two different solvent systems, 50% ethanol and 100% ethanol, were used to optimize the appropriate solvent system for further studies.
The uorescence intensity of BPhen was higher in 50% ethanol than in absolute ethanol. In 50% ethanol, the maximum intensity was observed at the concentration of 4.5 µM of BPhen. After this point, the intensity gradually decreases due to self-quenching. The BPhen concentration of 3.0 µM in a 50% ethanol solvent system was selected to avoid any possibility of self-quenching in further studies.

pH effect on BPhen uorescence intensity
The medium's pH intensely affects the uorescence intensity of BPhen and quenching constants of Fe(II). The emission spectra of a series of 3.0 µM BPhen solutions containing 0.10 µM of Fe(II) were recorded at different pH values. The pH of the solutions was adjusted using 98% conc. H 2 SO 4 and a saturated NaOH solution. When the emission maximum at 385 nm (I 385 ) was recorded against the solution pH, a reasonably linear range was obtained between pH 3.5 and 11, as illustrated in Fig. 3. At higher pH, the decrease in free Fe(II) in the system due to hydroxide formation lowers the freely available Fe(II) ions, decreasing the quenching response. When a 3.0 µM BPhen solution was titrated with H + , a gradual decrease in pH resulted, and the intensity of the peak at 385 nm was also reduced. Another peak was observed at 444 nm, as shown in Fig. 4, indicating the formation of a uorescent protonated BPhen complex.
3.4 Quenching effect of Fe(II) on BPhen uorescence 3.4.1 When the system is not buffered (50% ethanol without buffer) The uorescence intensity of a 3.0 µM BPhen solution in 50% ethanol with increasing Fe(II) concentration was studied. Fluorescence intensity at 385 nm reduces, and no other uorescence signals were observed upon the addition of Fe(II). The intensity of the BPhen uorescence peak at 385 nm was plotted against the Fe(II) concentration and is shown in Fig. 5. This observation suggests that Fe(II) effectively quenches the uorescence of BPhen and forms a non-uorescent complex with BPhen. According to the equation, since the gradient is equal to the quenching constant K in Fig. 5 (b), the quenching constant is 0.0072 nM − 1 .

When the system is buffered (50% ethanol with the acetate buffer)
Although the calibration curve plotted without a buffer in the system gave good linearity, a working solution buffered with acetate buffer was used in this study. Working solutions buffered with acetate buffer at three different pH values were used to study the difference in quenching responses to select an appropriate buffer system for the probe. Three working solutions were prepared, and in all working solutions, the concentration of BPhen was maintained as 3.0 µM, whereas the concentration of Fe(II) was increased from 0 nM to 40 nM. Figure 6 illustrates the uorescence emission spectra, calibration plot, and Stern Volmer plot obtained when titrating 3.0 µM BPhen in pH 6.6 working solution with increasing Fe(II) concentration. Table 1 summarises the analytical data obtained for pH = 4.5 and pH = 5.5 working solutions buffered with acetate buffer.
The LOD, LOQ standard deviation of the intercept and the slope obtained for probe BPhen are summarized in Table 1. LOD and LOQ values were calculated using the following equations [27], LOD = 3.3 σ/S, LOQ = 10 σ/S, where σ is the standard deviation of the regression intercept line. Since the gradient of the Stern -Volmer plot is equal to the quenching constant K, the highest quenching constant was observed in pH 5.5 and pH 6.6. The steepest slope in the calibration plot was obtained for the pH 6.6 system, indicating the highest sensitivity. Its R 2 value implied the highest correlation between the uorescence intensity and Fe(II) concentration. Based on the LOD and LOQ values and the linear range, it was concluded that the pH 6.6 working solution was the most appropriate of these three buffered solutions.

The blank study that corresponds to the addition of Fe(II) in pH 6.6 working solution
This study was carried out to clarify if quenching is due to the Fe(II) solution or a pH change in the system. In Sect. 3.4.2, the quenching of 3.0 µM BPhen in pH 6.6 working solution with increasing Fe(II) concentration was studied by spiking 25 µM Fe(II) in 50% ethanol with 0.2 µl increments as in Fig. 6 (b). A blank study was performed by spiking a 50% ethanol solution to a 3.0 µM BPhen in pH 6.6 working solution.
A plot of uorescence intensity vs the volume of added 50% ethanol is shown in Fig. 7(a). Similarly, 25 µM Fe(II) in 50% ethanol was spiked with 0.2 µl increments to a solution of 3.0 µM BPhen in pH 6.6 working solution and plotted in Fig. 7(b).
It was observed that the change of uorescence intensity due to the addition of 50% ethanol up to the volume of 8 µl (0.2⋅40 µl) was insigni cant. However, when Fe(II) was added, the intensity decreased without any signi cant pH change. Therefore, a pH 6.6 working solution was selected for further studies.
The LOQ value reported for the uorescence probe based on (A) was 7.5×10 − 10 M (4.2×10 − 5 ppm). A selective uorescence sensor for Fe(II) determination, based on pristine 1,10-phenanthroline, reported a LOD of 24 nM at 3.3 σ in a previous study [28]. The calculated LOD value for (A) through this study was lower than that, indicating a higher sensitivity that has not been reported so far.

Temperature studies
3.5.1 The temperature effect on the quenching of BPhen by Fe(II).
When a particular concentration of Fe(II) ions is added to the solution at once, the quenching is lower than when it is added gradually to the solution. Therefore it is essential to clarify whether this phenomenon can be affected by temperature. The effect of temperature on the quenching process is also a key parameter for determining the quenching mechanism [29]. Thus, a temperature study was carried out to determine whether there is a temperature effect for the quenching by Fe(II) ions. The temperature of 3.0 µM BPhen in pH 6.6 working solution was increased from room temperature 25 0 C to 60 0 C in the presence of 20 nM Fe(II) solution. Quenching of the BPhen-Fe(II) complex was increased with temperature. Figure 8 depicts that the uorescence decreases with increasing temperature in the presence of 20 nM Fe(II).
Even though the temperature studies suggest that the quenching mechanism may be dynamic, previously reported studies to indicate that the phenanthroline-Fe(II) complex formation is static. Further con rm the effect of temperature on the quenching of Fe(II) ions, the ratio between uorescence intensity at room temperature (I 0 ) and uorescence intensity at a particular temperature (I) were plotted against temperature as in Fig. 9. The plot I 0 /I vs.
[Q] also resulted in a straight line, which can be taken as evidence for static quenching [30].
After adding BPhen, the solutions were kept for one hour to ensure complete solubility of BPhen, and then Fe(II) was added. According to these studies, the quenching of BPhen by Fe(II) ions was increased with increasing temperature due to the increased stability constant of the formed complex with temperature, which supported the static quenching mechanism. However, these studies were done at an average room temperature of 25 0 C.

Response time with a higher Fe(II) concentration
The uorescence intensity of a 3.0 µM BPhen solution buffered at pH = 6.6 with and without 0.10 µM of Fe(II) reduced with time, as shown in Fig. 10. However, after 10 minutes, the intensity remained constant, indicating that an equilibrium [31] was reached. Hence in further studies, emission intensity was measured after 10 minutes.

Calibration curve
Based on the optimum conditions obtained, using a 3.0 µM BPhen solution in pH 6.6 working solution and measuring the uorescence intensity 10 minutes after Fe(II) was spiked gave the calibration curve given in Fig. 11 [32]. Moreover, the interference of metal ions commonly present in actual analytical samples such as Na(I), Li(I), Ca(II), Mg(II), Zn(II), Pb(II), Mn(II), and Al(III) was also investigated. In this study, a 3.0 µM solution of BPhen containing 0.10 µM concentration of Fe(II) was titrated with different interfering metal ions. The tolerance limits [19] for the interfering ions were calculated and are given in Table 2. The tolerance limit is the maximum concentration in which there is less than a 5% effect on uorescence intensity. The higher the molar ratio, the less the ion's interference in the determination of Fe(II) [11], [33]. Sulphonate (BPS), the water-soluble analogous of BPhen can also be used for colorimetric determination of Fe(II) without an extraction step; both analogs' sensitivity is similar [1]. This method proposed that aqueous Fe(II) solutions in 50% ethanol can be detected directly using only a 3.0 μM solution of BPhen in 50% ethanol buffered at pH 6.6. LOD of 19 and the linear range of 63 nM -224 nM of the new method reveals the capability of BPhen to detect trace Fe(II) amounts using uorescence more than colorimetry.
Therefore, BPhen can be used as a turn-off uorescence sensor to determine nano-level Fe(II) concentrations with minimum interference from other common foreign ions. Co(II), Ni(II), and Cu(II) were identi ed as the foreign ions, which bring a signi cant interference for the selective determination of Fe(II). However, it is generally observed when using phenanthroline-derived compounds as uorescence sensors. Fe(II) quenching was increased with increasing temperature, possibly due to the formed complex's increased stability constant. According to the interference study, the blank study, and the temperature studies, the quenching process of BPhen involved a combination of both static and dynamic quenching.

Declarations
Funding No Funding received. Department of Chemistry supported the necessary Chemicals since this work was carriedout as the B. Sc special degree student project.

Con icts of interest/Competing interests
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.    The emission spectra of a 3.0 µM BPhen solution in 50% ethanol spiking with an 18.0 µM H+ solution     Change in uorescence intensity in pH 6.6 working solution of 3.0 µM BPhen with increasing temperature from 25 0C to 60 0C on the 20 nM Fe(II) ions.

Figure 9
The ratio between uorescence intensity at room temperature (I0) and uorescence intensity at the particular temperature (I), with increasing temperature.