Fluorescence quenching detection of UO₂ ²⁺ in aqueous solution based on an organic molecule probe of 6-chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide

2.1 Materials

The chemicals used in the tests were of analytical grade with no further purification. 6-Chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide (L) was purchased from Sigma-Aldrich. Uranyl nitrate hexahydrate, UO₂(NO₃)₂·6H₂O, was manufactured by Mallinckrodt Company. All the other chemicals used in this study were purchased from Alpha Company.

2.2 Instruments

Fluorescence emission spectra were measured using Meslo-PN (Lumina fluorescence Spectrometer; Thermo Scientific, USA). Both emission and excitation slits were at 5.0 nm. The absorption spectra were measured using UV-Vis Evolution 300 (Thermo Fisher Scientific Company, UK). As a reference, UO₂ ²⁺ was determined by inductively coupled plasma optical emission spectrometry (ICP-OES) (iCAP 6500 ICP-OES; Thermo Fisher Scientific, UK). Experiments were carried out in safeguards destructive analysis lab (ETZ-, KMP-I), Nuclear and Radiological Regulatory Authority.

2.3 Analytical procedures

In order to assess the UO₂ ²⁺ concentration by L sensor, a stock solution of UO₂ ²⁺ (100 µM) was prepared by dissolving an accurately weighed amount of uranyl nitrate hexhydrate (UO₂(NO₃)₂·6H₂O) in deionized water containing few drops of concentrated sulphuric acid and standardized by a known method [24]. All working solutions were prepared by diluting the stock solution suitably with deionized water to give various concentrations of UO₂ ²⁺ from 0.08 to 5.0 µM. These various concentrations of UO₂ ²⁺ were added to L (1.0 mL, 100 µM). The solutions have been mixed with 10 mL of methanol. Measurements of the fluorescence emission intensities were carried out at λ _ex = 340 nm, λ _em = 380 nm, and 1 min (to reach the equilibrium). The linear range of the fluorescence emission intensity of L sensor was observed between 0.08 and 5.0 µM of UO₂ ²⁺ concentrations.

3.1 Preliminary studies

6-Chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide (L) belongs to the thiazide class. Clearly, it emitted fluorescence as it possessed conjugated functional groups. In addition, its molar absorption is a factor deciding the selection of L to be studied as a fluorescence sensor. High molar absorption of L is favoured because at a specified wavelength it enhances the absorption of light, so as it interacts with the target analyte, the sensitivity of L enhances. The molar absorptivity of L is 2.0 × 10⁴ L/mol cm according to Jothieswari et al. [25].

The variations in the absorption and fluorescence emission spectra that appear after the addition of UO₂ ²⁺ to L (10 μM) have been examined in order to determine that L can be used as a sensitive fluorescent sensor for UO₂ ²⁺.

The absorbance spectra of L and UO₂/L are shown in Figure 1. The L shows three absorption bands, at 230, 270, and 318 nm. This finding was similar to the findings of the literature. When adding UO₂ ²⁺ to L, the absorption band intensity increased at 230 nm and the absorption band intensity decreased at 318 nm, which is a possible indicator of the formation of UO₂/L species.

Figure 1

Absorbance spectra of the L sensor in the presence of different concentrations of UO₂ ²⁺.

Fluorescence emission spectra of L in the presence of various concentrations of UO₂ ²⁺ (8.0 × 10⁻⁸, 3.0 × 10⁻⁷, 6.0 × 10⁻⁷, 9.5 × 10⁻⁷, 1.5 × 10⁻⁶, 2.3 × 10⁻⁶, 2.8 × 10⁻⁶, 3.5 × 10⁻⁶, and 5.0 × 10⁻⁶ M, respectively) are shown in Figure 2. At an excitation wavelength 340 nm, the L sensor shows a characteristic fluorescent emission band at 380 nm.

Figure 2

Fluorescence emission spectra of the L sensor in the presence of different concentrations of UO₂ ²⁺ at λ _ex/λ _em = 340/380 nm.

The experimental results show that with the increase in the UO₂ ²⁺ concentration, the fluorescence emission intensity decreases and is fully quenched at a UO₂ ²⁺ concentration of 60.0 μM. This indicates that the fluorescence quenching ability of L sensor is good in the presence of UO₂ ²⁺. In addition, in the UO₂ ²⁺ concentration range of 0.08–5.0 µM, the intensity of the fluorescence emission is linearly quenched. The UO₂ ²⁺ and L species interact through electrostatic interaction between positively charged UO₂ ²⁺ and negatively charged nitrogen atom of the sulfonamide group of L. It reveals that it is possible to use the chelating reaction between UO₂ ²⁺ and L to develop a fluorescence sensor for UO₂ ²⁺ determination. Since the absorbance intensity of L at λ = 318 nm decreased, and fluorescence emission intensity of L at λ _ex/λ _em = 340/380 nm decreased as a function of UO₂ ²⁺ concentration, both indicated the prevalent formation of a UO₂/L species.

3.2 Mechanism

Scheme 1 shows the mechanism of UO₂ ²⁺ assessment using L as a fluorescent probe. Initially, when L was free in aqueous solution, they showed strong fluorescence intensity at λ _ex/λ _em = 340/380 nm. In the presence of UO₂ ²⁺, the fluorescence of L was quenched significantly. The results indicate that UO₂ ²⁺ induced fluorescence quenching of L by the chelation of positively charged UO₂ ²⁺ with negatively charged nitrogen atom of the sulfonamide group of L, thus facilitating the non-radiative recombination of UO₂ ²⁺ and L through the charge transfer or electron transfer processes and leading to the fluorescence quenching of L. Job’s method of continuous variation was used to study stoichiometry. The plot of absorbance against the UO₂ ²⁺ mole fraction has shown that the complex formation between UO₂ ²⁺ and L is with a molar ratio of 1:2. Also, the reaction between UO₂ ²⁺ and L was studied by spectrofluorometry at different temperatures (293 and 303 K) (Figure 3) with the Stern–Völmer equation to understand the fluorescence quenching mechanism [26,27]:

where F 0 and F are the fluorescence emission intensities of L in the absence and presence of UO₂ ²⁺, respectively, Q is the quencher concentration of UO₂ ²⁺, and K sv is the constant of Stern–Völmer. Stern–Völmer’s plot of F ₀/F versus [Q] at different temperatures (293 and 303 K) is shown in Figure 3. And the calculated K _sv are 4.8 × 10⁵ and 4.6 × 10⁵ mol⁻¹ at 293 and 303 K, respectively. This result indicates that there is a good agreement between quenching process and the Stern–Völmer equation, which is an indicator for static quenching mechanism because K _sv decreases with increase in temperature.

Scheme 1

A schematic representation of the proposed quenching mechanism of the L sensor with UO₂ ²⁺.

Figure 3

Stern–Völmer’s plot of F ₀/F versus [Q] at two different temperatures (293 and 303 K) (values are mean of five replicates), and all the RSD values are less than 2.0%.

3.3 Inner filter effect (INF)

In fluorescence spectroscopy, the INF is a significant issue that especially affects spectral measurements. UO₂ ²⁺ is a fluorescent species and has absorption and fluorescence spectra. The steps that are being carried out in the experimental work to avoid INF are as follows: (1) choose sample with very low concentration to avoid primary INF and (2) select a particular excitation wavelength to reduce the absorption of the sample. The excitation is decreased to 80 nm (λ _ex = 340) below the absorption maximum of UO₂ ²⁺ (420 nm) to avoid secondary INFs.

3.4 Parameter study

3.4.1 Solvent effect

The L sensor showed the fluorescence emission spectra at λ _ex = 340 nm in different solvents. The influences of the solvent on the fluorescence emission intensity of L sensor are shown in Figure 4. The results showed that the fluorescence emission intensity of the L sensor is much higher in methanol than in other solvents. These could be due to various influence factors of solvent such as dielectric constant, polarity, etc. The dielectric constant and polarity of methanol (ε = 33, polarity = 0.762), acetonitrile (ε = 37.5, polarity = 0.46), DMF (ε = 36.71, polarity = 0.386), and DMSO (ε = 47.24, polarity = 0.444), respectively, indicate that the fluorescence quantum yield of L is increasing with the decrease in the solvent dielectric constant and with the increase in solvent polarity. In addition, L is more soluble in methanol. Thus, the high fluorescence quantum yields in methanol could be anticipated, and methanol was selected as the best solvent.

Figure 4

Fluorescence emission spectra of the L sensor in the presence of different solvents at λ _ex/λ _em = 340/380 nm.

3.5 Analytical figures of merit

The analytical parameters are measured in order to apply the fluorescent L sensor for UO₂ ²⁺ assessment. As shown in Figure 3, a good linear relation was obtained between F ₀/F versus UO₂ ²⁺ concentration at 293 K. The regression equation was F ₀/F = 1.0326 + 4.8 × 10⁵ [UO₂ ²⁺] (R ² = 0.9992) within a concentration range of 0.08–5.0 µM. The limit of detection (LOD) is defined as the lowest amount of analyte in a sample, which can be detected but not necessarily quantitated as an exact value. The limit of quantification (LOQ) is defined as the lowest amount of analyte in a sample, which can be quantitatively determined with suitable precision and accuracy. In order to show the sensor constraint, LOD and LOQ were also calculated. According to the ICH guidance, LOD = 3.3S/b and LOQ = 10S/b (where S is the standard deviation and b is the slope) are tabulated in Table 1. These results suggested that the L sensor could be used as a tool for the assessment of UO₂ ²⁺ at very low concentration levels.

3.6 Application

In order to investigate the validity of the L sensor to various real aqueous samples, the various R&D samples are assessed by the L sensor and compared with the ICP-OES analysis. The various R&D samples spiked with UO₂ ²⁺ at different concentrations (0.10, 0.30, and 0.50 µM). The L sensor was added to the sample and mixed with 10 mL of methanol and then left for 1 min before measurement of the intensity of fluorescence. Table 2 shows the resulting data obtained. It was indicated that the recovery values are in the range of 95.6–105%. Also, the various R&D samples and their spiked samples are measured by ICP-OES analysis.

The results reveal that the UO₂ ²⁺ concentration detected from the L sensor was in a good agreement with the concentration results using the ICP-OES analysis. To check the accuracy of L sensor, the recovery results and the results from ICP-OES are used. The precision of the L sensor was measured and the three measurements were recorded as relative standard deviation (RSD%) values. The results of RSD% were lesser than 2.5%. These findings indicate that the procedure is precise and can be used for UO₂ ²⁺ assessment.

Further study is carried out by comparing the analytical parameters of the L sensor with some of other sensors developed, as shown in Table 3 [17,18,28,29,30,31,32,33]. The table reveals that, with adequate performance, the L sensor has impressive features, including a broader linear range and a lower detection limit, good selectivity, and remarkable simplicity.