Rhodamine-Based Fluorescent Probe for Highly Selective Determination of Hg²⁺

Abstract

The determination of mercuric ions (Hg²⁺) in environmental and biological samples has attracted the attention of researchers lately. In the present work, a novel turn-on Hg²⁺ fluorescent probe utilizing a rhodamine derivative had been constructed and prepared. The probe could highly sensitively and selectively sense Hg²⁺. In the presence of excessive Hg²⁺, the probe displayed about 52-fold fluorescence enhancement in 50% H₂O/CH₃CH₂OH (pH, 7.24). In the meantime, the colorless solution of the probe turned pink upon adding Hg²⁺. Upon adding mercuric ions, the probe interacted with Hg²⁺ and formed a 1:1 coordination complex, which had been the basis for recognizing Hg²⁺. The probe displayed reversible dual colorimetric and fluorescence sensing of Hg²⁺ because rhodamine’s spirolactam ring opened upon adding Hg²⁺. The analytical performances of the probe for sensing Hg²⁺ were also studied. When the Hg²⁺ concentration was altered in the range of 8.0 × 10^–8 to 1.0 × 10^–5 mol L^–1, the fluorescence intensity showed an excellent linear correlation with Hg²⁺ concentration. A detection limit of 3.0 × 10^–8 mol L^–1 had been achieved. Moreover, Hg²⁺ in the water environment and A549 cells could be successfully sensed by the proposed probe.

Introduction

Recently, developing highly selective and sensitive sensors for toxic metal ions have been in demand in environmental and biological studies.¹⁻³ Heavy metal mercury is toxic and bio-accumulating, can be generated by both natural resources and anthropogenic activities, and ubiquitously exists in the global environment. Mercury includes elemental mercury, inorganic mercury, and organic mercury. Mercuric ions (Hg²⁺) are much more common than mercurous ions (Hg⁺), and low-dose Hg²⁺ in the body could cause long-standing irreversible harm to the human health.^4,5 Methylmercury is a dominant form of organic mercury and could be produced through microbiological transformation of Hg²⁺ in the aquatic environment. Moreover, methylmercury could bio-accumulate in the human body by biological food chain and result in a variety of illnesses including cardiovascular diseases, Minamata disease, growth retardation, dyskinesia, and so forth.⁶⁻⁹ The maximum amount of Hg²⁺ allowed in drinking water is 2 ppb (10^–8 mol L^–1) according to the US Environmental Protection Agency.¹⁰ Thus, sensing Hg²⁺ has high significance in environmental and medical science.

To date, many analytic approaches have been employed for sensing Hg²⁺ such as high-performance liquid chromatography–inductively coupled plasma mass spectrometry (HPLC–ICP-MS),¹¹ HPLC coupled with atomic fluorescence spectrometry (HPLC–AFS),¹² ICP-atomic emission spectrometry (ICP-AES),¹³ atomic absorption spectrometry (AAS),¹⁴ ultraviolet–visible absorption spectrometry (UV–vis),¹⁵ fluorometry,^16,17 electrochemical methods,^18,19 and so on. Among all the methods developed, fluorometry possess remarkable advantages because of its high sensitivity, inherent simplicity, instrument operability, in situ detection, and bioimaging analysis in vivo.^16,17 Hitherto, many fluorescent probes had been constructed to determine Hg²⁺.²⁰⁻³⁰ However, some analytical performances of these probes are unsatisfactory, including the slow response time,²⁰⁻²³ cross interference,^24,25 poor sensitivity,²⁶⁻²⁹ and so forth. Therefore, novel Hg²⁺ fluorescent probes are still desirable, which possess fast response speed, excellent selectivity, and sensitivity.

Rhodamine and rhodamine derivatives have been applied widely as fluorescent probes owing to their remarkable optical characteristics including a high fluorescence quantum yield (Φ), a large molar extinction coefficient (ε), and longer excitation and emission wavelengths.³¹ So far, many fluorescent probes utilizing rhodamine derivatives had been utilized to sense different cations including Cu²⁺,³² Pb²⁺,³³ Cr³⁺,³⁴ Fe³⁺,³⁵ Al³⁺,³⁶ Zn²⁺,³⁷ and so forth. The sensing mechanism of these probes for cations is based on the transformation from a spirocyclic form into an open cyclic structure. In the absence of cations, these probes displayed no fluorescence and colorlessness because of their spirocyclic form. After adding cations, these probes emitted a powerful fluorescence and showed a pink color because the cyclic structure of the spirolactam or spirolactone was opened through a reversible complexation or nonreversible chemical interaction. The above sensing mechanism has also been applied to construct rhodamine-based Hg²⁺ fluorescent probes.³⁸⁻⁴⁷ However, there are still more or less limitations for some of these Hg²⁺ fluorescent probes including the delayed response,³⁸ a narrow pH range,³⁹ and cross interference.⁴⁰⁻⁴⁴ Thus, developing unusual rhodamine-based fluorescent probes for Hg²⁺ is still very important.

Herein, a new Hg²⁺ fluorescent probe 1 (Scheme 1) had been constructed, which chose rhodamine as the fluorophore. Because sulfur had strong affinity for Hg²⁺,^39,48 we introduced a sulfur-based functional unit to the probe in the present article. To prepare an optical chemical sensor (optode) for Hg²⁺ in the next work, a terminal double bond was included in probe 1 to allow the probe to covalently immobilize on the activated surface of glass slides with the double bond by UV irradiation. When Hg²⁺ was absent, the probe existed in a spirocyclic form and exhibited no fluorescence and colorlessness. When Hg²⁺ was present, the probe emitted yellowish-red fluorescence, and the solution color changed to pink. The Hg²⁺ probe exhibited excellent sensing performances including a fast response time, excellent sensitivity and selectivity, a broad pH working range, and so forth. In addition, nearly no cytotoxic reaction was found, and the probe could be successfully utilized to image Hg²⁺ in A549 cells. Besides, the developed probe was also magnificently utilized to monitor Hg²⁺ in water environments.

Scheme 1. Preparation of Fluorescent Probe 1: (a) Hydrazine Monohydrate (85%), CH₃CH₂OH, Reflux, 12 h, 86%; (b) Lawesson’s Reagent, Toluene, Reflux, 4 h, 17%; (c) Allyl Bromide, Sodium Bicarbonate, Dormyl Dimethylamine, 70 °C, 48 h, 61%; and (d) CH₃CH₂OH, Reflux, 12 h, 75%.

Results and Discussion

Spectral Characteristics of Probe 1

Figure 1 shows a graph depicting the variation in the fluorescence spectrum of probe 1 after adding different concentrations of mercury ions in Tris–HCl buffer (CH₃CH₂OH/H₂O, 1:1, v/v, pH 7.24). As illustrated in Figure 1, probe 1 emitted a very weak fluorescence when Hg²⁺ was not added. As the concentration of Hg²⁺ was increased, probe 1 exhibited enhanced fluorescence. When excess Hg²⁺ was added, the probe demonstrated a 52 times fluorescence increase at 586 nm. The experiment was based on these results to determine the concentration of mercury ions.

Figure 1.

Fluorescence spectra of 5.0 μM probe 1 in the presence of different concentrations of Hg²⁺: 0, 0.03, 0.05, 0.07, 0.08, 0.10, 0.20, 0.40, 0.50, 0.60, 0.80, 1.0, 2.0, 4.0, 5.0, 6.0, 8.0, 10, 20, 40, 50, 60, 80, and 100 μM from 1 to 24. Inset: variation of fluorescence intensity of 5.0 μM probe 1 with Hg²⁺ concentration (λ_ex = 520 nm; λ_em = 586 nm).

In order to explore the sensing mechanism of probe 1 toward Hg²⁺, the variation of the absorption spectrum of probe 1 with the gradual addition of mercury ions was recorded (Figure 2). As demonstrated in Figure 2 the UV–visible absorption spectrum of probe 1 showed a weak absorbance at 565 nm before the addition of mercury ions, which could be ascribed to the presence of the partial ring-opened structure of the spirolactam unit of probe 1. With the increment of Hg²⁺, probe 1 showed a gradual increase in absorption at 565 nm. When the Hg²⁺ concentration was 50 μM, the absorbance reached the maximum value. Meanwhile, the solution color change of probe 1 from colorless to pink after the addition of Hg²⁺ could be seen by the naked eye. The possible reason for absorption intensity enhancement at 565 nm was that the ring of the spirolactam structure of probe 1 opened upon the addition of mercury ions. The UV–visible absorption spectroscopy results also indicated that probe 1 interacted with Hg²⁺. Furthermore, a chemical shift of 64.23 ppm in the ¹³C NMR spectrum of probe 1 corresponded to the characteristic chemical shift value of the spirocyclic carbon, which also indicated that probe 1 existed in the spirolactam form before the addition of Hg²⁺.

Figure 2.

UV–vis spectra of 5.0 μM probe 1 with the gradual addition of Hg²⁺: 0, 0.08, 0.10, 0.20, 0.40, 0.50, 0.60, 0.80, 1.0, 2.0, 4.0, 5.0, 6.0, 8.0, 10, 20, 40, 50, 60, 80, and 100 μM from 1 to 21. Inset: the variation of absorbance of 5.0 μM probe 1 with Hg²⁺ concentration.

Principle of Operation

To explore the linear correlation between the fluorescence intensity of probe 1 and mercury ions, the fluorescence intensity of probe 1 was studied after the addition of various concentrations of Hg²⁺. The fluorescence intensity of probe 1 at 580 nm linearly depended on the Hg²⁺ concentration when the Hg²⁺ concentration was varied from 8.0 × 10^–8 to 1.0 × 10^–5 mol L^–1 (Figure 3). The linear regression equation used was F = 25.1293 + 25.1931 × 10⁶ × C (r = 0.9949); here, F is the fluorescence intensity, C represents the concentration of Hg²⁺, and r is the linear correlation coefficient. The detection limit was estimated to be 3.0 × 10^–8 M based on 3S_B/m (where S_B is the standard deviation of 10 blank measurements and m is the slope of the linear regression equation),^49,50 which was more sensitive than that of formerly developed Hg²⁺ fluorescent probes.²⁶⁻²⁹ Moreover, according to the UV–vis titration profile in Figure 2, the absorbance of probe 1 at 565 nm increased linearly with the change of the Hg²⁺ concentration of 8.0 × 10^–8 to 2.0 × 10^–5 mol L^–1 (Figure S1, Supporting Information). The linear regression equation used was A = 0.0569 + 0.1253 × 10⁶ × C (r = 0.9975), where A is the absorbance, C represents the concentration of Hg²⁺, and r is the linear correlation coefficient. Moreover, the detection limit was found to be 4.8 × 10^–8 mol L^–1 based on 3S_B/m (where S_B is the standard deviation of 10 blank measurements and m is the slope of the linear regression equation).³⁹

Figure 3.

Calibration curve between the fluorescence intensity of 5.0 μM probe 1 and the concentration of mercury ions. Inset: plot of fluorescence intensity of 5.0 μM probe 1 as a function of Hg²⁺ concentration in the range of 0.08–1.0 μM.

In addition, we also confirmed the stoichiometry of probe 1 with mercury ions by using Job’s plot (Figure 4). When the molar fraction of the amount of Hg²⁺ was close to 0.5, the absorbance of probe 1 reached a maximum, indicating that probe 1 coordinated with Hg²⁺ through a 1:1 stoichiometry. Based on the alteration in the absorption spectrum of probe 1 in the absence and presence of mercury ions and the coordination stoichiometry between probe 1 and Hg²⁺, we proposed a possible structural model for the formation of complexes of probe 1 and mercury ions (Scheme 2). To further support the hypothesis, the NMR titration of probe 1 in the presence of Hg²⁺ was carried out (Figure S2, Supporting Information). Hg²⁺ is a heavy metal ion and could influence the proton signals near the Hg²⁺ binding site.⁵¹ From Figure S2, the ¹H NMR signal of H_a belonging to probe 1 was shifted to the upfield in the presence of Hg²⁺, which demonstrated the binding between the oxygen atom of the hydroxyl group and Hg²⁺. As displayed in Figure S2, the ¹H NMR signal of H_b in probe 1 also shifted upfield after the addition of Hg²⁺, which illustrated the coordination between the nitrogen atom of the group (−CH=N−) and Hg²⁺. The result of NMR titration experiment further confirmed the coordination mode between the probe and Hg²⁺.

Figure 4.

Job’s plot for confirming the stoichiometry of probe 1 and Hg²⁺ in the CH₃CH₂OH/H₂O (1:1, v/v) solution. The whole concentration of probe 1 and Hg²⁺ was 40 μM.

Scheme 2. Likely Complexation Style between the Proposed Probe and Hg²⁺.

Effect of pH

For obtaining information about the pH effect, the fluorescence intensity changes of probe 1 before and after adding Hg²⁺ are investigated in various pH conditions (Figure 5). As exhibited in Figure 5, the fluorescence intensity of probe 1 is substantially unchanged in the range of pH 4.50 to 12.00. However, in the presence of Hg²⁺, the probe exhibited a strong fluorescence in the range of 4.50–8.50. The above results demonstrated that the fluorescent probe was not affected in the pH range of 4.50–8.50 and thus could be exploited to sense Hg²⁺ in the actual samples. When the pH was less than 4.50, probe 1 emitted enhanced fluorescence with reduced pH values, which could be attributed to the ring-opened structure of probe 1 under acidic conditions. Considering the response speed, sensitivity, and practical application, the pH 7.24 Tris–HCl buffer was used in this experiment.

Figure 5.

Fluorescence intensity variation of 5.0 μM probe 1 with pH before (open circles) and after (solid circles) adding Hg²⁺.

Selectivity

In order to ensure that probe 1 could be used in a wide variety of environments, we examined the selectivity of probe 1 for Hg²⁺ (Figure 6). It can be found from the black histogram in Figure 6 that the solution of Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Fe³⁺, Al³⁺, Cd²⁺, Zn²⁺, Ag⁺, Mg²⁺, Pb²⁺, Ba²⁺, and Cr³⁺ ions had almost no effect on the fluorescence intensity of probe 1 at a concentration of 10 times the Hg²⁺ concentration. In addition, when the concentrations of Cu²⁺, Co²⁺, Ni²⁺, Mn²⁺, and Hg²⁺ were equal, probe 1 displayed a notable fluorescence increase only upon adding Hg²⁺. The color change, fluorescence change, and UV–vis response of the probe in the absence and presence of those different ions mentioned above were also studied (Figure S3, Supporting Information). From Figures 6 and S3, the probe showed high selectivity for Hg²⁺ by fluorometry. However, the detection of Hg²⁺ was interfered by Cu²⁺ by ultraviolet–visible absorption spectrometry. Moreover, competition experiments were executed in which the developed probe was used to sense Hg²⁺ (1.0 × 10^–5 mol L^–1) in the coexistence solution of Hg²⁺ and other metal ions (white histogram in Figure 6). The experimental results showed that the relative error of usual interference including alkaline earth, alkali, and transition-metal ions was lower than ±5%, which was thought to be acceptable. These experiments demonstrated that the coexistence of different metal cations and mercury ions did not affect the determination of Hg²⁺, making the probe more likely to be used to determine actual samples.

Figure 6.

Metal-ion selectivity of 5.0 μM probe 1. The concentration of ions was 1.0 × 10^–5 M for Hg²⁺, Co²⁺, Cu²⁺, Ni²⁺, and Mn²⁺ and 1.0 × 10^–4 M for all other ions. Black bars: various metal ions were provided. White bars: various metal ions in the presence of Hg²⁺ were provided.

For comparison, the complexation study with Hg²⁺ had been carried out using compound 2 (Figure S4, Supporting Information) or compound 3 (Figure S5, Supporting Information) in Tris–HCl buffer (CH₃CH₂OH/H₂O, 1:1, v/v, pH 7.24). As demonstrated in Figure S4, in Tris–HCl buffer (CH₃CH₂OH/H₂O, 1:1, v/v, pH 7.24), compound 2 showed fluorescence enhancement only in the presence of Cu²⁺. From Figure S5, compound 3 demonstrated a strong fluorescence increase and a pink color only in the presence of Cu²⁺ or Hg²⁺ (Figure S5 in the revised Supporting Information). Thus, probe 1 possessed higher selectivity for Hg²⁺ compared with compound 2 and compound 3.

Reversibility and Response Time

It was apparent to all that reversibility is a significant factor to prepare an excellent chemical sensor. We tested the reversibility of probe 1 by adding an EDTA solution (Figure 7). As demonstrated in Figure 7, the fluorescence intensity of probe 1 drastically increased after providing mercury ions, and the solution color transformed from colorlessness to pink. However, after the EDTA solution was added to the above pink solution, the fluorescence intensity decreased rapidly, and the solution color was altered from pink to colorlessness. These experimental consequences explained that the complexation process of probe 1 and Hg²⁺ was reversible.

Figure 7.

Reversibility of probe 1 for Hg²⁺ in Tris–HCl buffer (CH₃CH₂OH/H₂O, 1:1, v/v, pH 7.24). ···:5.0 μM probe 1; −:5.0 μM probe 1 with 10 μM Hg²⁺; —:5.0 μM probe 1 with 10 μM Hg²⁺; and following the addition of 40 μM EDTA 2Na.

At the same time, we also studied the time response of probes to mercury ions (Figure 8). Hg²⁺ (1.0 × 10^–5 mol L^–1) were added to probe 1 solution, and the change in fluorescence intensity from 0.0 to 5.0 min was recorded to examine the time response of probe 1 for Hg²⁺. From Figure 8, it can be observed that the complexation speed of probe 1 and Hg²⁺ was very rapid, and the fluorescence intensity could reach the maximum within 60 s. The response time of the proposed probe for Hg²⁺ was shorter than that of formerly reported fluorescent probes for Hg²⁺.²⁰⁻²³ Therefore, the fluorescent probe would be utilized for real-time Hg²⁺ detection.

Figure 8.

Time response of probe 1 (5.0 μM) for 10 μM Hg²⁺. Time points are 0, 30, 60, 90, 120, 150, 180, 210, 240, 270, and 300 s; λ_ex = 520 nm. Inset: visual fluorescence color of probe 1 (5.0 μM) in the absence (left) and presence (right) of Hg²⁺ for 60 s (UV lamp, 365 nm).

Compared with other probes for Hg²⁺ based on rhodamine derivatives (as displayed in Table S1, Supporting Information), this probe possessed many advantages including high sensitivity and specificity, fast response time, a wide pH working range, and so forth.

Preliminary Analytical Application

For verifying the application of probe 1 in actual sample analysis, probe 1 was utilized for the sensing of mercury ions in tap water and river water samples. The river water and tap water were directly used after being filtered by a 0.45 μm filter. The river water and tap water were measured by probe 1 for mercury ion content, which contained no Hg²⁺, and then the standard solutions of different concentrations of Hg²⁺ were separately added for the determination of the recovery rate (Table 1). As found from Table 1, the fluorescent probe has a satisfactory determination result of Hg²⁺ recovery rate in the tap water and river water. Thus, the probe could be effectively used to sense Hg²⁺ in the actual samples.

Table 1. Sensing Hg²⁺ in Tap and River Water Samples with Probe 1.

	sample	Hg2+ spiked (mol L–1)	Hg2+ recovered (mol L–1)	recovery (%)
river water	1	0	not detected
	2	5.00 × 10–6	(5.15a± 0.10b)	103.0
	3	1.00 × 10–5	×10–6	102.0
			(1.02a± 0.02b) × 10–5
tap water	1	0	not detected
	2	5.00 × 10–6	(5.18a± 0.12b)	103.6
	3	1.00 × 10–5	×10–6	98.0
			(0.98a± 0.03b) × 10–5

^aMean values of three determinations.

^bStandard deviation.

We also conducted a bio-imaging application research of probe 1. To assess the biocompatibility of probe 1, we performed a cytotoxicity assay using the MTT colorimetric assay (Figure S6, Supporting Information). From Figure S1, the cell viability of A549 cells was found to be higher than 90% when probe 1 was present, indicating that probe 1 was almost not cytotoxic. Next, we verified whether probe 1 could be employed for sensing Hg²⁺ in living cells by laser confocal fluorescence imaging experiments (Figure 9). As can be seen from Figure 9a, after culturing A549 cells in medium containing probe 1 (5.0 μM) for 30 min, the cells showed substantially no fluorescence. However, when A549 cells were cultured for 30 min with 1.0 μM Hg²⁺ under the same conditions, the cells exhibited a strong fluorescence (Figure 9d). According to the research results, probe 1 could be applied to fluorescence imaging of Hg²⁺ in living cells.

Figure 9.

Images of A549 cells incubated with the developed probe 1. (a) Fluorescence image of A549 cells attached to 5.0 μM probe 1 for 30 min at 37 °C; (b) bright-field transmission image of cells demonstrated in (a); (c) overlap image of (a,b); (d) fluorescence image of A549 cells attached to 5.0 μM probe 1 for 30 min, washed three times, and further treated with 1.0 μM Hg²⁺ for 30 min; (e) bright-field transmission image of cells displayed in (d); and (f) overlap image of (d,e).

Conclusions

On the whole, a new fluorescent probe has been planned and prepared to quantify Hg²⁺, which used rhodamine as the fluorophore. After providing Hg²⁺, the probe displayed a strong fluorescence emission and a pink color due to its open-cycle structure of the corresponding spirolactam via a reversible coordination. The fluorescent probe exhibited excellent sensing abilities, including excellent sensitivity and specificity, rapid response, a wide pH working range, and so forth. The fluorescent probe had been applied to sense Hg²⁺ in both tap and river water samples and demonstrated acceptable consequences. Furthermore, the probe also demonstrated superior biocompatibility, which permitted us to obtain fluorescence imaging of Hg²⁺ in A549 cells.

Experimental Section

Materials and Instruments

Lawesson’s reagent (97%) and allyl bromide were purchased from Aldrich. Rhodamine B and hydrazine monohydrate (85%) were bought from Shanghai Sinopharm Group Company. Before toluene was utilized, it was freshly distilled through adding sodium. Unless otherwise stated, other chemical reagents used in this work were of analytical grade and could be applied directly. Water used in all assays was ultrapure water.

UV–vis and fluorescence spectra were measured on a UV-2600 spectrometer and a Hitachi F-7000 spectrophotometer, respectively. A Bruker DRX-500 NMR spectrometer was utilized to measure the NMR spectra. Fluorescence images of living cells were acquired through an Olympus FV1200-MPE multiphoton laser scanning confocal microscope with a 40× objective lens. The pH of the solution was measured with a Mettler Toledo Delta 320 pH meter. SigmaPlot software was used to perform the data processing.

Syntheses

The method of preparation of fluorescent probe 1 is demonstrated in Scheme 1. During the process, product 2 was obtained through the interaction of hydrazine monohydrate (85%) and rhodamine B, as reported in the previous literature.⁵² Compound 3 was obtained by the chemical reaction between Lawesson’s reagent and compound 2 following an earlier developed method.⁵² 4-Formyl-3-hydroxyphenyl allyl ether (compound 4) was synthesized from 2,4-dihydroxybenzaldehyde and allyl bromide following a formerly developed procedure.⁵³

Synthesis of Compound 1

Compound 3 (0.19 g, 0.40 mmol) and compound 4 (0.096 g, 0.54 mmol) were put in 50 mL CH₃CH₂OH. The mixture was warmed to refluxing for 12 h. After the solvents were cleaned in decompression, purification of the crude product was done through column chromatography (CH₃COOCH₂CH₃/petroleum ether = 1:10, v/v) to produce probe 1 (0.19 g, 75%) as a pale yellow solid. ¹H NMR (500 MHz, CDCl₃): δ(ppm) 11.50 (1H, s), 8.62 (1H, s), 8.09 (1H, dd, J = 5.8 Hz, 3.2 Hz), 7.41 (2H, dd, J = 5.8 Hz, 3.2 Hz), 7.19 (1H, d, J = 8.5 Hz), 7.13–7.11 (1H, m), 6.74 (2H, d, J = 8.8 Hz), 6.49–6.42 (2H, m), 6.32–6.27 (4H, m), 6.05–5.97 (1H, m), 5.39 (1H, dd, J = 17.2 Hz, 1.4 Hz), 5.27 (1H, dd, J = 10.5 Hz, 1.2 Hz), 4.51 (2H, d, J = 5.3 Hz), 3.31 (8H, q, J = 7.0 Hz), 1.14 (12H, t, J = 7.0 Hz) (Figure S7, Supporting Information). ¹³C NMR (125 MHz, CDCl₃): δ(ppm) 170.13, 162.38, 161.97, 161.07, 155.13, 151.93, 148.33, 135.19, 133.02, 132.67, 132.16, 130.11, 127.91, 127.12, 122.21, 117.99, 111.75, 110.17, 108.28, 107.63, 101.92, 97.53, 68.83, 64.23, 44.37, 12.60 (Figure S8, Supporting Information). MS (ESI) m/z: 633.2902 (M + H)⁺ (Figure S9, Supporting Information).

Measurement Procedures of Fluorescence Intensity

The preparation of 1.0 × 10^–5 mol L^–1 probe 1 was done by adding the needed quantity of probe 1 to CH₃CH₂OH. 8 × 10^–7–1.0 × 10^–3 mol L^–1 Hg²⁺ stock solution was obtained by gradually diluting 1.0 × 10^–2 mol L^–1 mercury nitrate solution with 0.05 mol L^–1 Tris–HCl buffer (pH, 7.24). The 0.05 mol L^–1 Tris–HCl solution was adjusted by adding HCl or NaOH solution to obtain a range of pH buffer solutions. 12.50 mL of 1.0 × 10^–5 mol L^–1 probe 1 and 2.50 mL of different concentrations of Hg²⁺ solution were put into a 25 mL volumetric flask and then it was made up to 25 mL using 0.05 mol L^–1 Tris–HCl buffer. A solution containing 5 × 10^–6 mol L^–1 probe 1 and 8 × 10^–8–1×10^–4 mol L^–1 Hg²⁺ was obtained. The same procedure without adding Hg²⁺ was used to obtain the blank solution of probe 1.

All solutions were kept at 4 °C in the dark. The excited wavelength was 520 nm, and both the entrance and exit slits were of 2.5 nm, and the fluorescence intensity was recorded in the range of 540–650 nm.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c03336.

• ¹H NMR spectra of probe 1 before and after adding Hg²⁺; absorbance of probe 1 at 565 nm with different metal ions; fluorescence and color changes of probe 1 with different metal ions; absorbance and fluorescence of compound 2 at 586 nm with different metal ions; fluorescence and color changes of compound 2 with different metal ions; absorbance and fluorescence of compound 3 at 586 nm with different metal ions; fluorescence and color changes of compound 3 with different metal ions; comparison of fluorescent probes for determination of Hg²⁺ based on rhodamine derivatives; assay of A549 cells in the presence of different concentrations of probe 1 (0, 2, 4, 8, and 16 μM) for 24 h at 37 °C; ¹H and ¹³C NMR spectra of compound 1; and ESI–MS spectra of compounds 1 (PDF)

The authors declare no competing financial interest.