Colorimetric determination of lead(II) or mercury(II) based on target induced switching of the enzyme-like activity of metallothionein-stabilized copper nanoclusters

Abstract

It is shown that metallothionein-stabilized copper nanoclusters (MT-CuNCs) display catalase-like activity. In the presence of either lead(II) or mercury(II), the catalase-like activity is converted to a peroxidase-like activity. On addition of Pb(II) or Hg(II), the inhibitory effect of MT-CuNCs on the chromogenic reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) with H₂O₂ is weakened. On the other hand, the catalytic effect of the nanoclusters on the chromogenic reaction is increased. The system MT-CuNCs-Pb(II)/Hg(II) exhibits high affinity for the substrates TMB and H₂O₂. Their catalytic behavior follows Michaelis-Menten kinetics. Based on these findings, a method was developed for visual detection (via the blue coloration formed) and spectrophotometric determination (at 450 nm) of Pb(II) and Hg(II). The linear range for Pb(II) extends from 0.7 to 96 μM, and the linear ranges for Hg(II) from 97 nM to 2.3 μM and from 3.1 μM to 15.6 μM. The detection limits are 142 nM for Pb(II) and 43.8 nM for Hg(II).

Introduction

Catalase is an antioxidant enzyme that catalyzes the decomposition of H₂O₂ into O₂ and H₂O. Catalase can effectively scavenge H₂O₂ in vivo so that H₂O₂ cannot react with O₂ under the action of iron chelates to produce highly oxidative hydroxyl radicals (·OH) [1]. Natural peroxidase is an enzyme that catalyzes the oxidation of specific substrates by providing electrons for H₂O₂ (electron acceptor). Natural enzymes catalyze reactions under very mild biological conditions, which limits their applications [2]. Compared to natural enzymes, many artificial nanozymes, such as carbon nanotubes, magnetic nanomaterials [3, 4], noble metal-graphene complexes [5], and metal nanoclusters [6, 7], have the advantages of simple manufacturing, low cost, high catalytic activity, good stability, etc. [8]. In particular, metal nanoclusters have been widely applied [9].

Lead and mercury ions can pollute the environment and endanger health. In order to quantify the concentration of Pb(II) and Hg(II) in water samples, analytical methods such as inductively coupled plasma mass spectrometry (ICP-MS), atomic absorption spectroscopy (AAS) and electrochemical analysis have been developed. The disadvansatages of these methods are bulky instruments, high cost, the need for professionally operators [10, 11]. Therefore, it is still highly desirable to develop a rapid and convenient method for detecting Pb(II) and Hg(II).

Pb(II) and Hg(II) can inhibit or enhance the activity of artificial nanozymes under certain conditions thus establishing a new method for detecting Pb(II) or Hg(II).^.Zhu [12] et al. found that Hg²⁺ inhibited the peroxidase-like activity of bovine serum albumin-capped AuNCs via Au⁺-Hg²⁺ interaction^. Liao [13] et al. developed a Pb²⁺ assay due to the ability of Pb²⁺ to induce the aggregation of glutathione (GSH) stablized AuNCs and enhance their peroxidase-like activity. Tseng [14] et al. prepared bimetallic Pt/Au NPs and found that Hg²⁺ transformed this peroxidase-like activity into a catalase-like activity. Yuan [15] et al. researched that the peroxidase-like activity of molybdenum disulfide nanosheets (MoS₂-NSs) was stimulated by Hg²⁺. Most of the platforms in related reports are based on the use of the Hg²⁺-Au⁺ reaction to change the enzyme activity of gold-containing nanozymes. By contrast, a study on the modfication of copper nanoclusters with enzyme-mimicking activity based on the heavy metal ions Pb²⁺ and Hg²⁺ has not been reported.

Herein, the effects of the heavy metal ions Pb²⁺ and Hg²⁺ on the enzyme-like activity of MT-CuNCs were explored, and Pb²⁺ and Hg²⁺ assay strategies were established. It is found that MT-CuNCs have catalase-like activity and that Pb²⁺/Hg²⁺ can induce the conversion of this catalase-like activity to peroxidase-like activity. Under certain conditions, H₂O₂ can self-oxidize to produce a small number of hydroxyl radicals (·OH), thus oxidizing TMB to 3,3′,5,5′-tetramethylbenzidine diimine (oxTMB). Correspondingly, the solution changes from colorless to light blue (Scheme 1a). The MT-CuNCs have catalase-like activity, which can decompose H₂O₂ and inhibit the oxidation of TMB. The solution color consequently become lighter (Scheme 1b). In the presence of Pb²⁺ or Hg²⁺, the catalase-like activity of the MT-CuNCs can be converted into peroxidase-like activity, thus catalyzing the oxidation and chromogenic reaction of TMB mediated by H₂O₂. Therefore, the solution changes from colorless to dark blue (Scheme 1c). Accordingly, a detection of Pb²⁺ or Hg²⁺ based on colorimetry and spectrophotometry was established. This detection method is selective,and feasible and has a wide linear range. The development of this method is informative for the progress of enzyme mimics.

Scheme 1

Schematic presentation of the colorimetric assay for Pb²⁺/Hg²⁺ based on target induced switching of enzyme-like activity of MT-CuNCs

Experimental section

Reagents and materials

Pb²⁺ and Hg²⁺ standard solutions were supplied by the Chinese Academy of Metrology (Beijing, China, www.nim.ac.cn). MT (induced by Zn²⁺ with a purity higher than 99%) was acquired from Yongkang Jiaxin Biological Technology Co., Ltd. (Beijing, China, www.jiaxintool.cn.globalimporter.net). CuCl₂, catalase (CAT), TMB, phosphate buffered saline (PBS), 2′-diamino-bis-3-ethylbenzothiazolin-6-sulfonic acid (ABTS) and absolute ethanol were supplied by McLean Biochemical Technology Co., Ltd. (Shanghai, China, www.macklin.company.com). NaOAc, H₂O₂ and H₂SO₄ were obtained from Beijing Chemicals Reagent Company (Beijing, China, www.bicr.com). The electrical resistivity of the ultrapure water used was 18.25 MΩ·cm.

Apparatus

All ultraviolet-visible absorbance spectra obtained using a Shimadzu UV-2450 spectrophotometer (Kyoto, Japan, www.shimadzu.com). A THERMO-SHAKER thermostat mixer (Heros, China) was utilized for the synthesis of the MT-CuNCs. The fluorescence spectra were acquired on a Lumina spectrofluorometer (Thermo Fisher Scientific, USA, www.thermofisher.com). A Titan G2 60–300 transmission electron microscope (FEI, USA, www.fei.com), a K-Alpha 1063 X-ray photoelectron spectrometer (Thermo Fisher Scientific, USA, www.thermofisher.com) and a Shimadzu IR Prestige-21 Fourier transform infrared spectrometer (Kyoto, Japan, www.shimadzu.com) were used to characterize the MT-CuNCs mixed with Pb²⁺ or Hg²⁺.

Preparation of MT-CuNCs

Briefly, MT solution (600 μL, 2.25 mg·mL⁻¹) and CuCl₂ solution (100 μL, 3 mM) were mixed at 55 °C. NaOH solution (200 μL, 1.0 M) was added 15 min later. The reaction was proceeded with shaking at 800 rpm for 12 h at 55 °C. After filtering through a 0.22 μm filter membrane, the light purple solution was stored at 4 °C.

Determination of Pb²⁺ and Hg²⁺

In a 2 mL EP tube, NaOAc-HOAc buffer (pH 4.0, 80 μL, 200 mM), MT-CuNCs solution (30 μL), TMB solution (40 μL, 5 mM), H₂O₂ solution (60 μL, 10 mM), and a certain amount of Pb²⁺ standard solution were mixed. The reaction took place for 40 min at 40 °C in a thermostatic mixer. Then, H₂SO₄ solution (100 μL, 0.2 M) was introduced, and the measurement was performed by colorimetry. Moreover, the absorbance value of this system at 450 nm was measured.

Likewise, in a 2 mL EP tube, NaOAc-HOAc buffer (pH 3.25, 60 μL, 200 mM), MT-CuNCs solution (20 μL), TMB solution (50 μL, 5 mM), H₂O₂ solution (60 μL, 10 mM), and a certain amount of Hg²⁺ standard solution were mixed. The reaction took place for 40 min at 40 °C in a thermostatic mixer. Then, H₂SO₄ solution (100 μL, 0.2 M) was introduced, and the absorbance of the system at 450 nm was measured.

The absorbance change of the system was calculated as follows: ΔA = A - A₀. A and A₀ represent the absorbance values in the presence and absence of Pb²⁺/Hg²⁺, respectively.

Results and discussion

Transformation of the enzymatic activity of MT-CuNCs induced by Pb²⁺ or Hg²⁺

The catalase-like activity of MT-CuNCs was verified by monitoring the absorbance of H₂O₂ at 240 nm, because catalase can catalyze the decomposition of H₂O₂ [16]. As shown in Fig. S1, the MT-CuNCs have high catalase-like activity and that their relative activity is 2.8 times that of the positive control group (1.5 mg·mL⁻¹ CAT).

The enzyme activity transition of MT-CuNCs after the addition of Pb²⁺/Hg²⁺ was verified by UV-Vis spectroscopy (Fig. 1a). Both TMB (a curve) and the MT-CuNCs-TMB system (b curve) have no absorption peak at 450 nm. The A₄₅₀ of the TMB solution (c curve) increases significantly after reacting with H₂O₂, indicating that H₂O₂ may yield ·OH through self-oxidation under these conditions. The ·OH species oxidizes TMB into oxTMB. After the reaction was terminated with H₂SO₄, the solution appeared light yellow and exhibited an absorption peak at 450 nm. In the MT-CuNCs system (d curve), the TMB-H₂O₂ chromogenic reaction is suppressed, and the A₄₅₀ value decreases. This effect may occur because MT-CuNCs with catalase-like activity catalyze the decomposition of H₂O₂ and inhibit the production of ·OH. When Pb²⁺ or Hg²⁺ was added, the A₄₅₀ values of the MT-CuNCs-Pb²⁺/Hg²⁺-TMB-H₂O₂ system increased gradually with increasing Pb²⁺ or Hg²⁺ concentration, suggesting that Pb²⁺ or Hg²⁺ can convert the catalase-like activity of MT-CuNCs into peroxidase-like activity. At this time, H₂O₂ may be adsorbed on the surface of the MT-CuNCs and turned into highly active ·OH via O-O bond cleavage, thus generating the catalytic activity of the nanoclusters [17]. The kinetics of each system within 60 min were further investigated (Fig. 1b). The absorbance of the TMB-H₂O₂ system (a curve) at 652 nm increases over time. After the addition of MT-CuNCs (b curve), the absorbance value from 0 to 1700 s increases very slowly, and the slope of the kinetic curve after 1700 s is still lower than that of the TMB-H₂O₂ system. These results indicate that MT-CuNCs show catalase-like activity, which inhibites the chromogenic reaction of TMB. In the presence of Pb²⁺/Hg²⁺ and MT-CuNCs (c, d curve), the A₆₅₂ value increases slowly from 0 to 1200 s. Later, the slope of the kinetic curve is larger than that of the TMB-H₂O₂ system. These results confirm our hypothesis that Pb²⁺ or Hg²⁺ can reduce and transform the catalase-like activity of MTs-CuNCs into peroxidase-like activity, The latter catalyzes the oxidation reaction of TMB.

Fig. 1

Switching effects of Pb²⁺/Hg²⁺ on enzyme-like activity of MT-CuNCs a Absorption spectrum of the systems with acid termination. (a) TMB. (b) TMB-MT-CuNCs. (c) TMB-H₂O₂. (d) TMB-H₂O₂-MT-CuNCs. (e) TMB-H₂O₂-MT-CuNCs-Hg²⁺. (f) TMB-H₂O₂-MT-CuNCs-Pb²⁺. b Absorbance-time curves (at 652 nm) of the systems. (a) TMB-H₂O_2。 (b) TMB-H₂O₂-MT-CuNCs. (c) TMB-H₂O₂-MT-CuNCs-Pb²⁺. (d) TMB-H₂O₂-MT-CuNCs-Hg²⁺

Mechanistic studies

Because surface chemistry is significant in regulating electron transfer and substrate adsorption, it affects the catalytic activity of nanozymes. Liu et al. found that fluoride capping enhanced the oxidase activity of cerium oxide NPs by more than 100 times [18], while DNA increased the peroxidase activity of Fe₃O₄ NPs [19]. The mechanism of these effects was F⁻ and DNA regulating the surface charges of nanozymes, thereby enhancing the electron transfer and substrate adsorption on the surface of the nanozymes. In this study, the positive charges on the surface of the MT-CuNCs increased after their interaction with Pb²⁺ or Hg²⁺, which might cause charge repulsion with positively charged TMB and weaken the catalytic activity of the MT-CuNCs in the oxidation of TMB. However, this hypothesis is not consistent with the enhancement of peroxidase activity in the experimental phenomenon. Hence, negatively charged ABTS was used as the chromatic substrate for validation (Fig. S2). The results are similar to that obtained when TMB is used as a substrate. Therefore, the transformation function of Pb²⁺/Hg²⁺ in the enzymatic activity of MT-CuNCs should not be attributed to the regulation of surface charges.

Changes in the morphology and size of nanomaterials may affect their intrinsic properties. Liao [13] et al. found that Pb²⁺ increased the peroxidase-like activity of GSH-AuNCs and confirmed that the reason was aggregation-induced enzyme activity enhancement, similar to that of aggregation-induced luminescence. TEM images (Fig. S3) show that both Pb²⁺ and Hg²⁺ can induce the aggregation of MT-CuNCs and an increase in the size of the MT-CuNCs. With the addition of Pb²⁺ or Hg²⁺, the photoluminescence property of the MT-CuNCs is decreased, and aggregation-induced luminescence enhancement is not observed. For further verification, the effect of Ag⁺ on the size and enzyme activity of MT-CuNCs was investigated. The size of MT-CuNCs is increased, but the absorption of oxTMB at 450 nm hardly enhance (Fig. S4). These results reveal that the enzymatic activity transformation of MT-CuNCs is not caused by the change of the morphology and size.

The catalytic activity of metal NCs and metal NPs is correlated with the valences of metal ions/complexes formed on their surfaces. Lien [20] investigated the influences of various metal ions on the oxidase activity, peroxidase-like activity and catalase-like activity, of AuNPs. They found that the formation of a metal-Au alloy over AuNPs greatly affected their catalytic performance. Therefore, in this study, the modulating function of Pb²⁺ or Hg²⁺ on the enzymatic activity of MT-CuNCs may be ascribed to the generation of Pb-Cu/Hg-Cu alloys, which was further verified by XPS (Fig. 2). The Cu 2p spectrum of MT-CuNCs show the characteristic peaks at 932.79 and 952.54 eV without the satellite peak of Cu(II) at approximately 942 eV, indicating the existence of Cu(0) and Cu(I) rather than Cu(II) (a) [21, 22]. After the interaction between MT-CuNCs and Pb²⁺, the Cu 2p spectrum did not change significantly. The characteristic Pb 4f peaks of Pb(II) and Pb(0) are observed at 138.34/143.29 eV and 136.29/141.35 eV, respectively (b) [23]. In a pH range of 3.5–4.5, the thiolate ligands (Zn²⁺-MT bonds) in MT were partially dissociated [24],and Pb²⁺ diffused into the α domains of MT and was reduced into Pb(0), which would be deposited on the surface of the MT-CuNCs to form a Pb-Cu alloy. In the Cu 2p spectrum of the MT-CuNCs-Hg²⁺ system (c), the characteristic satellite peak of Cu(II) is observed at 942.46 eV. Hg²⁺ might react with the Cu⁺ on the surface of the MT-CuNCs to generate Hg(0). The characteristic Hg4f peaks of Hg(II) and Hg(0) is observed at 100.86/104.91 eV and 99.54/103.59 eV, respectively (d) [25]. Hg²⁺ competed with Zn²⁺ to bind the sites in the α domains of MT, and a portion of Hg²⁺ was reduced to Hg(0) by MT. Consequently, the Pb-Cu/Hg-Cu alloy formed on the surface of the MT-CuNCs transformed their catalase-like activity into peroxidase-like activity.

Fig. 2

XPS spectra of the elements (Cu, Pb, Hg) in the absence and presence of Pb²⁺/Hg²⁺. a Cu 2p curve of MT-CuNCs. b Pb 4f curve in MT-CuNCs-Pb²⁺ system. c Cu 2p curve in MT-CuNCs-Hg²⁺ system. d Hg4f curve in MTs-CuNCs-Hg system. The solutions of MT-CuNcs and MT-CuNCs-Pb²⁺/Hg²⁺ were prepared respectively. XPS measurement were performed after vacuum drying at low temperature. c_Pb(II) = 10 μM, c_Hg(II) = 2.0 μM

The structural changes in MT-CuNCs after the addition of Pb²⁺/Hg²⁺ (Fig. 3) were analyzed with FTIR. After the reaction with Pb²⁺_, the number of hydrogen bonds (at 3000–3600 cm⁻¹) in the MT-CuNCs system is increased, and the asymmetric stretching vibration of ⁺N-H···O⁻ (at 2110 cm⁻¹) is intensified [26], along with the production of the N-H stretching peak (amide II bonds, at 1552 cm⁻¹). Likewise, after the interaction between Hg²⁺ and MT-CuNCs, the transmittance values of the bands corresponding to C-N/C-C (amide III bonds, at 1398 cm⁻¹), C=O/N-H (amide I bonds, at 1641 cm⁻¹) and N-H/O-H decrease significantly, and bands corresponding to amide II bonds appear [27]. All these indicate that the interactions among Pb²⁺/Hg²⁺, Cu/Cu⁺ and MT change the coordination bonds in the MT-CuNCs.

Fig. 3

Fourier transform infrared spectrum of MT-CuNCs in (a) the absence and presence of (b) Pb²⁺ or (c) Hg²⁺. The solutions of the systems (MT-CuNCs, MT-CuNCs-Pb²⁺ and MT-CuNCs-Hg²⁺) were prepared respectively. The solutions were dropped onto KBr flakes for determination. c_Pb(II) = 10 μM, c_Hg(II) = 2.0 μM

Steady-state kinetic analysis

The affinity of MT-CuNCs-Pb²⁺/Hg²⁺ for H₂O₂ and TMB was investigated via steady-state kinetic analysis [28]. The results (Figs. S5 and S6) show that after the reaction between the MT-CuNCs and Pb²⁺/Hg²⁺, the process of catalyzing the TMB-H₂O₂ reaction follow the typical Michaelis-Menten model (a, b). Using the Lineweaver-Burk double-reciprocal method, it is found that there is a positive correlation between the reciprocal of the initial rate (1/v) and the reciprocal of the TMB concentration (1/[S]) (c, d) [29]. After linear fitting, the values of maximum reaction rate (v_max) and the Michaelis constant (K_m) were received. The affinities of MT-CuNCs-Pb²⁺/Hg²⁺ (K_m = 1.08/3.45) for H₂O₂ is higher than that of HRP for H₂O₂ (K_m = 3.70), showing that only a small amount of H₂O₂ is enough to commence the catalytic oxidation reaction in the MT-CuNC-Pb²⁺/Hg²⁺ systems (Table S1).

Detection of Pb²⁺ and Hg²⁺

In order to improve analysis performance, the reaction conditions were optimized (Figs. S7 - S12). Under the selected reaction conditions, the selectivity of this method for Pb²⁺ was investigated with a 10 μM Pb²⁺ solution as a reference. According to the experimental results (Fig. S12a), within a relative deviation of ±5%, species at 500 times (Zn²⁺ or Na⁺), 250 times (Mg²⁺, K⁺, or Cd²⁺), 100 times (Ca²⁺ or Mn²⁺), 50 times (Al³⁺), 25 times (Ag⁺ or Cu²⁺), and 10 times (Hg²⁺ or Fe³⁺) greater concentrations do not interfere with the determination of Pb²⁺. Moreover, in the system for Hg²⁺ determination, the influence of several likely interfering components on the determination of Hg²⁺ was tested with a 2 μM Hg²⁺ solution as a reference. Within a relative deviation of ±5%, species at 500 times (K⁺ or Ca²⁺), 250 times (Na⁺ or Cd²⁺), 100 times (Mg²⁺, Zn²⁺, Mn²⁺ or Pb²⁺), 50 times (Al³⁺), 25 times (Ag⁺ or Cu²⁺), and 10 times (Fe³⁺) greater concentrations do not interfere with the determination of Hg²⁺ (Fig. S12b). In other words, this approach has great selectivity performance.

The calibration plot for the determination of Pb²⁺ and Hg²⁺ were plotted under the optimized experimental conditions. Within the concentration range c_Pb(II) of 707 nM-96 μM, a great linear relationship is observed (Fig. 4). The equation of linear regression was ΔA = 0.0113c_Pb(II) (μM)-0.034 with a correlation coefficient (r) of 0.9987. The detection limit (LOD) was calculated to be 142 nM based on the equation LOD = 3S_b/k. Precision experiments (n = 11) were carried out for three standard solutions with Pb²⁺ concentrations of 1.5, 60, and 96 μM. The relative standard deviations were 1.65%, 0.5% and 3.11%, respectively. These results show that this method for detecting Pb²⁺ has high accuracy. In the c_Hg(II) ranges of 97 nM-2.325 μM and 3.10 μM–15.59 μM, the ΔA value and Hg²⁺ concentration are in a good linear relationship (Fig. 5). The regression equations were ΔA = 0.0413c_Hg(II) (μM) + 0.0231 (r = 0.9950) and ΔA = 0.0157c_Hg(II) (μM) + 0.0159 (r = 0.9961), respectively. LOD of this method was 43.8 nM. Three standard Hg²⁺ solutions with concentrations of 0.775, 2.325, and 12.46 μM were each measured 11 times, and the relative standard deviations were 3.02%, 1.62%, and 2.32%, respectively. These demonstrate that the precision of this method for detecting Hg²⁺ is good. In comparison with other analytical methods for Pb²⁺/Hg²⁺ determination, the data in the Table 1 show that the method for detecting Pb²⁺/Hg²⁺ has a wide linear range and relatively good sensitivity.

Fig. 4

a Absorption spectra (at 450 nm) of the TMB-H₂O₂-MT-CuNCs system in the absance and presence of different Pb²⁺ concentrations (from a to q: 0, 0.75, 1.5, 3, 6, 9, 12, 15, 18, 24, 36, 42, 48, 54, 60, 72, and 96 μM). Insert: the chromogenic photograph of different Pb²⁺ concentrations (from left to right: 0, 1.5, 6, 12, 18, 24, 36, 42, 48, 54, 60, 72, 96 μM). The photograph was obtained without performing acid termination, because blue is easier to distinguish than yellow. b Calibration plot of Pb²⁺ detection (concentration is consistent with that in the absorption spectra)

Fig. 5

Absorption spectra (at 450 nm) of the TMB-H₂O₂-MT-CuNCs system in the absance and presence of different Hg²⁺ concentrations. Insert: the chromogenic photograph of different Hg²⁺ concentrations. The photograph was obtained without performing acid termination. a (from a to h: 0, 0.097, 0.195, 0.39, 0.775, 1.55, and 2.325 μM). Insert: (from left to right: 0, 0.097, 0.39, 0.775, 1.55, and 2.325 μM). c (from a to i: 3.1, 3.875, 4.672, 6.23, 7.788, 9.345, 10.903, 12.46, and 15.56 μM). Insert: (from left to right: 3.1, 4.672, 7.788, 9.345, 10.903, 12.46, and 15.56 μM). b, d Calibration plot of Hg²⁺ detection (concentration is consistent with that in the absorption spectra)

Table 1 Comparison of this approach with other methods for detecting Pb²⁺/Hg²⁺

Also, the method was employed to determine the ion concentrations in environmental water samples. After filtering twice with quantitative filter paper, all environmental water samples (collected from tap water, the pond water, Xiangjiang River,) were heated and boiled for 10 min. After cooling to room temperature, the solution was filtered through a 0.22 μm filter membrane for subsequent analysis. The recovery test of spiked samples were performed. The results (Tables S2,S3) demonstrate obviously that this method can be supplied for environmental water samples.

Conclusions

The relative catalase-like activity of MT-CuNCs is 2.8 times that of 1.5 mg·mL⁻¹ CAT. Due to the generation of the Pb-Cu/Hg-Cu alloy, Pb²⁺/Hg²⁺ induced the conversion of the catalase-like enzymatic activity of MT-CuNCs to peroxidase-like enzymatic activity. The latter catalyzed TMB-H₂O₂ oxidation according to a typical Michaelis-Menten model, and exhibited a higher affinity for H₂O₂ than HRP. Furthermore, a reliable colorimetry/spectrophotometry method for detecting Pb²⁺/Hg²⁺ was established.. which had great selectivity, wide linear ranges and high sensitivity. The assay displays potential application for monitoring Pb²⁺/Hg²⁺ concentration in environmental water sample.