Tannic Acid-Capped Gold Nanoparticles as a Novel Nanozyme for Colorimetric Determination of Pb²⁺ Ions

Abstract

In this study, tannic acid-modified gold nanoparticles were found to have superior nanozyme activity and catalyze the oxidation reaction of 3,3′,5,5′-tetramethylbenzidine in the presence of hydrogen peroxide. Enhancing the catalytic activity of the nanozyme by Pb²⁺ ions caused by selectively binding metal ions by the tannic acid-capped surface of gold nanoparticles makes them an ideal colorimetric probe for Pb²⁺. The parameters of the reaction, including pH, incubation time, and concentration of components, were optimized to reach maximal sensitivity of Pb²⁺ detection. The absorption change is directly proportional to the Pb²⁺ concentration and allows the determination of Pb²⁺ ions within 10 min. The colorimetric sensor is characterized by a wide linear range from 25 to 500 ng×mL⁻¹ with a low limit of detection of 11.3 ng×mL⁻¹. The highly sensitive and selective Pb²⁺ detection in tap, drinking, and spring water revealed the feasibility and applicability of the developed colorimetric sensor.

1. Introduction

The widespread use of toxic metals in the manufacture of batteries, pigments, paints, ammunition, toys, and in other applications has led to environmental pollution and public health problems [1]. Pb²⁺ is one of the most dangerous pollutants as its high levels cause serious damage to the nervous system, renal, kidney, and brain. Moreover, prolonged exposure to low metal concentrations can lead to multiple adverse health effects [2]. In accordance with the World Health Organization guidelines, the level of Pb²⁺ in drinking water should not exceed 10 µg·L⁻¹ [3]. Common methods for the determination of Pb²⁺ include flame atomic absorption spectroscopy [4], atomic emission spectroscopy [5], X-ray fluorescence [6], inductively coupled plasma mass spectrometry [7], and electrochemical methods [8,9]. However, implementation of these methods requires high-cost complex instrumentation and time-consuming procedures, which complicates on-site monitoring of target analyte.

The alternative approach is colorimetric methods that provide direct and simple detection of Pb²⁺ ions. The possibility to exclude sophisticated instruments staffed by highly trained personnel enables extensive use of colorimetric methods for the detection of Pb²⁺. The most widely applied analytic technique relies on the use of noble metal nanoparticles (most often, silver and gold nanoparticles) as carriers of various receptor molecules that specifically bind target metal ions. In this case, the surface modification of nanoparticles with various chelating ligands [10,11,12,13] and oligonucleotides [14,15,16,17] provides highly specific detection of Pb²⁺ [18]. In the presence of detectable heavy metal ions, aggregation of nanoparticles occurs, followed by a concentration-dependent color change, which is observed with the naked eye or recorded photometrically [19,20]. However, obtaining functionalized nanoparticles increases the complexity and cost of the analysis.

Recently, a great interest has been attracted by nanomaterials with enzyme-like catalytic properties, namely, nanozymes due to their ease of preparation, controlled activity, high stability, and low cost [21]. In the presence of metal ions, the catalytic activity of nanozymes may change, which enables the concentration-dependent colorimetric sensing of target ions [22,23]. Various metallic nanoparticles and nanohybrid materials were found to exhibit intrinsic peroxidase-mimicking activity and produce a colored product by catalyzing the oxidation of the enzyme substrate [21]. Thus, Tang et al. described the use of two-dimensional layered tungsten disulfide (WS₂) nanosheets that possess peroxidase-like activity to design colorimetric sensor for Pb²⁺ detection in environmental and biological samples [24]. It was found that Pb²⁺ ions inhibit the catalytic activity of WS₂ nanosheets, the proposed mechanism of which is blocking the electron transfer from the nanosheets to hydrogen peroxide and, as a result, the lack of (•OH) radicals for the oxidation of the enzyme substrate 3,3′,5,5′-tetramethylbenzidine (TMB). Among a variety of peroxidase-mimic nanomaterials, gold nanoparticles (AuNPs) are of particular interest because they are easily synthesized and functionalized by various receptor molecules. The ability of metal ions to affect the catalytic activity of AuNPs is used in a number of colorimetric sensors [25,26]. Thus, Lien et al. reported colorimetric analysis of Hg²⁺ and Pb²⁺ ions using peroxidase-mimic activity of AuNPs in the presence of Pt⁴⁺ and Bi³⁺ ions allowing to detect the target ions in nanomolar concentrations [27]. It should be noted that due to the mechanism of formation of metal-gold alloys, the colorimetric signal could be influenced by other interfering metal ions present in the sample. In this case, the surface modification of AuNPs makes it possible to improve the selectivity of the colorimetric sensor for detection of a target metal ion [28]. Citrate-stabilized gold nanoparticles were earlier proposed for aggregation analysis of different analytes, including heavy metal ions [29,30]. However, the use of unmodified gold nanoparticles for the determination of lead does not allow reaching a sensitivity higher than 18 μM [29]. It is explained by the absence of a specific recognizing molecule on the particle surface. Since the carboxyl groups of organic acids are capable to interact with many metal ions [31], a coating with functional groups is required to selectively recognize specific ions. Earlier, we developed a colorimetric technique for the determination of Pb²⁺ ions using tannic acid as a modifying agent [20]. The developed aggregation analysis made it possible to detect lead ions with a detection limit of 310 ng/mL. Changing sample/reactants volume ratio reduced the detection limit to 60 ng/mL. The use of catechin as an alternative tanning compound demonstrated the presence of a peroxidase-like activity in modified gold nanoparticles for the specific determination of lead in the H₂O₂-mediated oxidation of Amplex UltraRed [32]. The work of Yoosaf et al. explained the formation of the shell with necessary functional groups available for interaction with Pb²⁺ ions when synthesizing nanoparticles with the use of different tannins as reducing and stabilizing agents [33].

Herein, AuNPs modified by tannic acid (TA) are proposed as a selective sensing probe for nanozyme-based colorimetric detection of Pb²⁺ in water. The implementation of TA as a capping agent showed a peroxidase-like activity of AuNPs for substrate containing H₂O₂ and TMB. The addition of Pb²⁺ ions further stimulated the catalytic activity of the nanoparticles, resulting in a faster formation of a colored product. The Pb²⁺-promoted nanozyme activity was investigated under different conditions (pH, concentrations of reactants). The practicability and reliability of nanozyme-based colorimetric sensor was validated through the analysis of Pb²⁺ in drinking, spring and tap water. The proposed approach allows for rapid, sensitive, and easy-to-use tool for monitoring of Pb²⁺ levels without sample pre-treatment.

2. Materials and Methods

2.1. Chemicals and Instruments

All of the heavy metal salts were purchased from the Center of Reference Materials and High-Purity Substances, LTD. (Saint-Petersburg, Russia) and used as stock solutions. Chloroauric acid (HAuCl₄), sodium citrate, potassium carbonate, 3, 3, 5, 5-tetramethylbenzidine (TMB), dimethyl sulfoxide (DMSO) and tannic acid (TA) were obtained from Sigma Aldrich (St. Louis, MO, USA). 33% hydrogen peroxide was supplied by Fluka (St. Louis, MO, USA). All chemicals were of analytical grade. Ultrapure water (18.5 MΩ·cm at 22 °C; Millipore, Bedford, MA) was applied for the preparation of aqueous solutions. The colorimetric analysis was carried out in 96-well transparent Costar 9018 polystyrene microplates (Costar, Tewksbury, MA, USA).

Absorption spectra of gold nanoparticles were recorded using a UV-2450 spectrophotometer (Shimadzu Kyoto, Japan). The transmission electron microscopy (TEM) images were obtained by JEM CX-100 electron microscope (Jeol, Tokyo, Japan) with an accelerating voltage of 80 kV. The samples were placed onto 300-mesh grids (Pelco International, Redding, CA, USA) coated with formvar film. Subsequent image analysis was performed using Image Tool software (University of Texas Health Science Center, San Antonio, TX, USA).

2.2. Synthesis of TA-Capped AuNPs

The stock solution of TA-capped AuNPs was prepared according to our previous study [31]. Briefly, the solution 1 was prepared by adding 100 µL of 5% HAuCl₄ to 39.5 mL of water. The solution 2 was prepared by mixing 2 mL of 1% sodium citrate, 250 µL of 1% TA, and 250 µL of 0.025 M potassium carbonate in 7.5 mL of ultrapure water. Both solutions were heated to 60 °C, followed by pouring solution 2 into solution 1, boiling for 4–5 min and cooling to room temperature. The concentration of prepared TA-capped AuNPs was 10⁻⁸ M. The solution was stored at 4–6 °C prior to use.

2.3. Comparison of Catalytic Activity of TA-Capped AuNPs in the Absence/Presence of Pb²⁺

The peroxidase-mimic activity of TA-capped AuNPs was investigated by assessing the ability of nanozyme to oxidize a colorless peroxidase substrate (TMB) into a colored product in the presence of H₂O₂. The assay was performed at a fixed concentration of TA-capped AuNPs using 500 μM freshly prepared TMB (1% TMB prepared in DMSO) and 500 mM H₂O₂ in sodium citrate buffer (pH 4.0). The kinetic analysis was performed by varying the concentration of H₂O₂ (0.1–0.6 M) at a fixed concentration of TA-capped AuNPs and TMB (500 μM). Similarly, the TMB-H₂O₂ catalytic reaction was carried out by varying the concentration of TMB (0.2–1 mM) with 500 mM H₂O₂. The reaction was started by adding an appropriate amount of H₂O₂, and the optical density at 650 nm corresponding to the maximum of TMB oxidation product was recorded using an EnSpire Multi-mode Plate Reader (PerkinElmer, USA). The initial reaction rates were determined from initial linear segment of “A₆₅₀-time” curve, reflecting growing concentration of the TMB oxidation product. To calculate the initial reaction rates, the molar absorption coefficient ɛ (650 nm), equal to 39,000 M⁻¹cm⁻¹ was used. To determine Michaelis-Menten constant (K_m) and maximum reaction velocity (V_max), kinetic dependences were plotted in the Lineweaver-Burk coordinates and the following equation was used:

$$\frac{1}{V_0}=\frac{K_m}{V_{max}}\times \frac{1}{{\left[S\right]}_0}+\frac{1}{V_{max}},$$

where $V_0$—initial reaction velocity, $V_{max}$—maximum reaction velocity, $S_0$—initial substrate concentration, $K_m$—Michaelis-Menten constant.

2.4. Colorimetric Detection of Pb²⁺ in Aqueous Solutions

Briefly, 100 µL of different concentrations of Pb²⁺ solutions were added to 20 µL of TA-capped AuNPs. After incubation for 5 min at room temperature, 50 µL of TMB solution containing 6% H₂O₂ was added. Immediately, 50 µL of 1M H₂SO₄ was added to stop the reaction. To quantify the concentration of Pb²⁺ the absorbance at 450 nm (A₄₅₀) was recorded. Each experiment was repeated three times. Dynamic range was determined by plotting A₄₅₀ nm with increasing Pb²⁺ concentration. The limit of detection (LoD) was defined using the equation $3\times \sigma$, where $\sigma$ is the standard deviation of the colorimetric signal of the blank sample.

2.5. Specificity of the Analysis

The specificity of the proposed colorimetric sensor was evaluated by testing samples of 13 metal and metalloid ions, namely Hg²⁺, Zn²⁺, Cu²⁺, Co²⁺, Sr²⁺, Mn²⁺, Fe³⁺, Sn⁴⁺, Cr³⁺, Cd²⁺, As²⁺, Sb³⁺, and Ba²⁺. 100 μL of the listed ions at a concentration of 100 ng·mL⁻¹ were added to 15 μL of TA-capped AuNPs. Furthermore, the experiment was carried out as described in Section 2.4.

2.6. Analysis of Real Water Samples

To verify the practical feasibility of the developed sensor, drinking, spring, and tap water samples were analyzed. The container was rinsed with water to be collected at least three times prior to sampling. The samples were purified using a membrane filter with a pore size of 0.22 μm, after which concentrated HNO₃ was added to preserve the sample and adjust pH to 4.5–5.0. Water samples were stored in a freezer until analysis. A series of spiked water samples, containing Pb²⁺ at concentrations 25, 50, and 100 ng×mL⁻¹, were prepared by diluting Pb²⁺ stock solution (0.1 mg·mL⁻¹ in deionized water). The processing of obtained data was performed using Excel and OriginPro9 (Origin Lab, Northampton, MA, USA).

3. Results

3.1. Characterization of TA-Capped AuNPs

TA-capped AuNPs were prepared by reduction of chloroauric acid by citric and tannic acids, with the latter also providing a stabilizing effect. The as-prepared gold nanoparticles were characterized by wine-red color with an absorption peak at 515 nm (Figure 1a). The morphology of the nanoparticles was observed by TEM (Figure 1b) and revealed monodisperse nanoparticles with the particle size distribution of 6.8 ± 0.9 nm (the counted number of particles was 94, Figure 1c).

The data on the reproducibility of the synthesis were evaluated in experiments with the determination of the optical density of nanoparticles. Thus, during the synthesis of nanoparticles, the optical density was estimated at 515 nm, which after five repeated syntheses was 0.98, 1.0, 0.98, 0.99, and 0.98, respectively. In addition, the value of the average error of detection was estimated when interacting with lead ions, which was no more than 5.8%.

3.2. Principle of the Nanozyme-Based Colorimetric Pb²⁺ Detection

The principle of proposed colorimetric sensor is illustrated in Figure 2 For Pb²⁺ sensing, TA-capped AuNPs were prepared and applied. Figure 2 clearly demonstrates the mechanism of reaction, absorbance peaks as well as visually observed changes occurred under interaction of nanoparticles with lead ions.

The scheme of the reactants’ interaction and catalytic oxidation of TMB by TA-capped AuNPs is presented on Figure 2a. In the absence of Pb²⁺ ions AuNPs had weak catalytic activity toward TMB oxidation. Due to this, low absorbance at 650 nm was observed, and visually confirmed by the weak color development (Figure 2b). In the presence of lead, tannic acid residues chelated Pb²⁺ ions to form the complexes indicated at Figure 2a. This interaction promoted the catalytic activity of the AuNPs as nanozymes and resulted in increasing of absorbance peak at 650 nm related to oxidated product of TMB transformation (Figure 3c), the color of solution became bright blue. After adding the sulfuric acid stop solution, the blue color turned to yellow and thus absorbance peak at 450 nm increased (Figure 3d). The addition of a TMB solution to a solution of lead ions and tannic acid did not lead to any catalytic activity in the absence of AuNPs.

It was previously shown that, in addition to the reducing properties of tannic compounds, they form complexes with metal ions [34,35,36]. The proposed mechanism of the analytical use of such tannic acid-containing nanoparticles is as follows. AuNPs synthesized with TA as a reducing and stabilizing agent have carbonyl groups on their surface and able to hydrolysis of phenolic compounds. They, in turn, specifically bind to Pb²⁺ ions, providing the formation of bridges that unite several nearby modified nanoparticles into clusters. This arrangement and the presence of free electron orbitals of Pb²⁺cations provide a free transition of an electron participating in the oxidation reaction of the substrate (TMB). The proposed principle of Pb²⁺ colorimetric detection corresponds to the previously described “turn-on” mechanism based on the enhancement of the catalytic activity of noble metal nanoparticles in the presence of metal ions [22].

The mechanism of oxidation of TMB catalyzed by AuNPs as a peroxidase-like nanozyme has been considered in earlier [28,37]. The catalytic process is similar to the Fenton reaction [38] and includes the following stages: (1) the formation of a hydroxyl radical (OH˙) from hydrogen peroxide in the presence of nanozyme, (2) the oxidation of TMB by the hydroxyl radical with the formation of the blue product TMB ⁺. The high affinity of gold nanoparticles for TMB accelerates the excited electron transfer, which complicates the recombination of electron-hole pairs and, in turn, promotes the formation of OH˙. Thus, the peroxidase-like activity is increased. In addition, the presence of lead ions stimulates the formation of hydroxyl radicals [39].

3.3. Optimization of the Experimental Conditions for Colorimetric Pb²⁺ Detection

As shown in Figure 3a, the reaction of TMB oxidation by H₂O₂ catalyzed by TA-capped AuNPs reaches a plateau after 120 s. This incubation time was used as optimal in further experiments. To select the optimal pH, the catalytic activity of Pb²⁺-promoted TA-capped AuNPs was studied in a wide pH range from 3.5 to 7.5. As follows from Figure 3b, the highest signal after adding the stopping solution (A₄₅₀) is recorded in the pH range 4.5–5. Furthermore, the ratios of the components in the colorimetric analysis were optimized by varying concentrations of TA-capped AuNPs, TMB, and H₂O₂. Figure 3c,d shows that the highest colorimetric signal was observed when 11.8×10⁻¹⁰ M TA-capped AuNPs and 0.5 mM TMB were applied. The peroxide-like activity of nanozymes also depends on the concentration of H₂O₂, since the latter forms hydroxyl radicals that oxidize TMB [40]. In this regard, the influence of the concentration of H₂O₂ on the catalytic activity of Pb²⁺-promoted TA-capped AuNPs was investigated (Figure 3e). The growth of the H₂O₂ concentration up to 0.53M gives rise to the signal enhancement.

3.4. Colorimetric Technique for Determining the Catalytic Activity of TA-Capped AuNPs

The peroxidase-mimic activity of bare TA-capped AuNPs was investigated using TMB. TMB is a common peroxidase substrate that is oxidized in the presence of H₂O₂ forming a blue product. The mixture of TMB with H₂O₂ is initially colorless; however, when TA-capped AuNPs are added, the mixture turns blue, which indicates the peroxidase-like activity. Nevertheless, the addition of Pb²⁺ to the mixture increases the catalytic activity and the solution instantly acquires a deep blue color (see Figure 2d).

The Pb²⁺-promoted catalytic activity was investigated comparing the nanoparticles with and without Pb²⁺ and plotting the time dependence of absorbance at 650 nm (stop solution was not added in these studies). As can be seen from the Figure 4, the presence of Pb²⁺ leads to a sharp increase of absorbance. A linear segment of the curve up to 120 s was used to calculate the initial velocity. Thus, the highest rate (3 × 10⁻³ s⁻¹) was observed for the nanozyme in the presence of Pb²⁺, being three times the value of 1 × 10⁻³ s⁻¹, observed in the absence of Pb²⁺. The estimated data confirm Pb²⁺-promoted catalytic activity of TA-capped AuNPs, which can be applied for detection of Pb²⁺ ions.

Furthermore, the stability of TA-capped AuNPs was evaluated. The TA-capped AuNPs were stored at 4 °C and Pb²⁺-promoted catalytic activity of the nanozyme was measured every month to investigate long-term stability. As shown in Figure 4b, the nanozyme displays stable high catalytic activity for 4 months, and after this period, it decreases slightly.

To investigate the catalytic properties of the TA-capped AuNPs the kinetic parameters were defined using by varying the concentration of both TMB and H₂O₂ and data processing by the Michaelis-Menten model as described in [41]. The Michaelis-Menten constants (K_m) and maximum initial reaction rates (ʋ_max) were calculated by fitting the Lineweaver-Burk plots (Figure 5b,d). As shown in

Table 1. Comparison of kinetic parameters (K_m and ʋ_max) of the oxidation reaction catalyzed by HRP and the proposed colorimetric probe.

Sensing probe	[E] (M)	Substrate	Km (mM)	ʋmax (M s−1)
TA-capped AuNPs	11.8 × 10−10	TMB	0.2	6.7 × 10−8
		H2O2	190	3.8 × 10−8
Pb2+-TA-capped AuNPs	11.8 × 10−10	TMB	0.09	1.4 × 10−7
		H2O2	100	3.3 × 10−7
HRP [43]	6.2 × 10−11	TMB	0.43	10 × 10−8
		H2O2	3.7	8.7 × 10−8

, the TA-capped AuNPs exhibited a lower K_m value when using TMB as a substrate compared to horseradish peroxidase (HRP) (0.2 mM versus 0.43 mM) indicating the stronger affinity of this nanozyme toward substrate. Pb²⁺ ions enhanced the catalytic activity of TA-capped AuNPs, which is reflected in a decrease of the K_m to 0.09 mM. In contrast, the K_m determination for H₂O₂ as a substrate revealed much higher values than that of HRP (190 mM versus 3.7 mM, see Table 1), highlighting the need for H₂O₂ concentrations to ensure maximum catalytic activity of the nanozyme. As previously shown [42], strong affinity of HRP for H₂O₂ is due to the presence of amino acids in the active centre of the natural enzyme. To sum up, these results demonstrated Pb²⁺-promoted nanozyme activity, and TA-capped AuNP is an appropriate sensing probe for Pb²⁺ detection.

3.5. Quantitative Pb²⁺ Detection Using Colorimetric Sensor

According to the TA-capped AuNPs-based colorimetric sensor design format, the amount of Pb²⁺ ions determine the peroxide-mimicking activity of the sensing probe. Consequently, the sensing responses of Pb²⁺ ions with different concentrations were investigated under optimal conditions. To assess the sensitivity and dynamic linear range of the developed sensor, concentrations of Pb^{2 +} from 1 to 1000 ng×mL⁻¹ were tested, and A₄₅₀ was measured. As shown in Figure 6, with an increase in the Pb²⁺ concentration, A₄₅₀ increases, accompanied by a color change from light blue to dark blue. The dynamic linear range for Pb²⁺ detection is 25–500 ng×mL⁻¹ with the linear regression equation of y = 0.406 − 0.03x and correlation coefficient (R²) of 0.994 (Figure 6b). The detection limit is calculated to be 11.3 ng×mL⁻¹ using the signal-to-noise ratio (S/N = 3), which is roughly in line with WHO drinking water guideline of 10 ng×mL⁻¹. The linearity and Pb²⁺ detection limit of the developed sensor was mapped to previously reported colorimetric sensors and summarized in

Table 2. Comparison of the developed analysis for Pb²⁺ with previously reported colorimetric methods.

Sensing Material	Sensing Mechanism	LOD (ng×mL−1)	Linear Range (ng×mL−1)	Analysis Time (min)	Ref
Rapid assays
TA-capped AuNPs	Pb2+-promoted nanozyme activity of TA-capped AuNPs	11.3	25–500	10	This work
1-(2-mercaptoethyl)-1,3,5-triazinane-2,4,6-trione functionalized silver nanoparticles (MTT-AgNPs)	aggregation of the MTT−AgNPs in the presence of Pb2+ ions	19.8	103–600	3	[44]
N-decanoyltromethamine(NDTM)-capped AuNPs	Pb2+-induced aggregation of NDTM-AuNPs	72.4	0–6200	<1	[13]
Oligonucleotide functionalized AuNPs	Change in guanine-rich ssDNA conformation into a rigid G-quartet structure in the presence of Pb2+ and NaCl-induced aggregation of unmodified AuNPs	1035	20.7–2070	5	[45]
AuNPs	Aggregation of the as-synthesized AuNPs in the presence of Pb2+	3730	0–20.7 × 103	<5	[29]
Valine-capped AuNPs	Pb2+-induced aggregation of valine-capped AuNPs	6300	0–289 × 103	5	[46]
Time consuming assays
Catechin synthesized AuNPs	Pb2+-promoted nanozyme activity of catechin modified AuNPs in the H2O2-mediated oxidation of Amplex UltraRed	0.3	2–207	60	[32]
Gold core-platinum shell nanohybrids (Au@PtNPs)	Pb2+-S2O32− ions-inhibited nanozyme activity of Au@PtNP	0.6	4–166	40	[47]
Sodium thiosulfate and hexadecyl trimethyl ammonium bromide (CTAB) modified AuNPs	leaching of CTAB-capped Au NPs induced by Na2S2O3 and Pb2+	8.3	207–1200	30	[48]

. The proposed colorimetric sensor allows the detection of Pb²⁺ ions in the nanogram range without additional sample pretreatment and demonstrates comparable or superior performance compared to other colorimetric analyses reported in the literature [13,41,43,44].

3.6. Selectivity

To confirm the specificity of the nanozyme-based colorimetric sensor, along with Pb²⁺, the contribution of other metals to the colorimetric response was studied. The concentration of all tested ions was 100 ng×mL⁻¹. As shown in Figure 7, the reaction solution containing Pb²⁺ showed bright yellow while competing metal ions were almost colorless. All interfering metal ions demonstrated almost negligible changes in A₄₅₀ after stopping the oxidation reaction (Figure 7). Thus, the results demonstrate excellent selectivity of TA-capped AuNPs toward Pb²⁺ ions among all interfering metal ions. The results obtained in this study are in accordance with previously reported data on strong adsorption capacity of tannin compounds toward Pb²⁺ ions in comparison to other metal [49,50].

3.7. Evaluation of Nanozyme-Based Colorimetric Sensor

To evaluate the practicability of the developed approach, the detection of Pb²⁺ was performed in drinking, spring, and tap water. It was found that there were no Pb²⁺ ions in spring, bottled drinking and tap water samples, therefore known concentrations of Pb²⁺ (25, 50, and 100 ng×mL⁻¹) were added to them.

Table 3. Colorimetric detection of Pb²⁺ in spiked water samples.

Sample	Added (ng×mL−1)	Found (ng×mL−1)	Recovery (%)	RSD (%, n = 3)
Drinking water	25	33.3	133.4	2.8
	50	54.5	108.9	3.2
	100	128.2	128.2	5.6
Tap water	25	27.7	110.9	2.9
	50	56.5	112.9	7.7
	100	106.6	106.6	3.8
Spring water	25	29.7	118.7	1.9
	50	56.8	113.6	2.9
	100	106.8	106.8	6.3

shows the results of Pb²⁺ detection in spiked water samples. The obtained recoveries were from 106.6% to 133.4% with relative standard deviation less than 8%. These results indicate good precision and accuracy of the Pb²⁺ detection in real water samples.

4. Conclusions

To sum up, a novel colorimetric sensor for Pb²⁺ detection in water was developed based on peroxidase-like properties of small-sized TA-capped AuNPs. It was found that TA-capped AuNPs catalyze the oxidation of TMB by H₂O₂ under slightly acidic environment, thus providing a simple colorimetric detection of Pb²⁺ ions. The detection limit of Pb²⁺ ions was 11.3 ng×mL⁻¹, which is close to the value established by the WHO guidelines of drinking water quality. The developed sensor was successfully applied to determine Pb²⁺ in real water samples from different sources with good recovery and RSD. Compared to conventional methods for the determination of Pb²⁺ in water, the proposed sensor has a number of tangible advantages, such as: (1) simplicity of the nanozyme preparation without an additional stage of gold nanoparticle modification; (2) excellent analytical performance for Pb²⁺ detection among other interfering metal ions; (3) analysis time takes 10 min. Taken together, these benefits enable to consider the developed colorimetric sensor as one of the approaches for the determination of Pb²⁺ ions in aqueous media, which can be further extended for practical use.