Spectrofluorimetric determination of Hg²⁺ and Pb²⁺ using acetylcholinesterase (AChE)-based formation of silver nanoparticles

Abstract

We herein report a novel fluorimetric detection method for Hg²⁺ and Pb²⁺ based on in situ formation of AgNPs, where thiocholine (TCh), a product obtained by the hydrolysis of acetylcholine (ACh) by acetylcholinesterase (AChE), can act as a stabilizing agent. Uniformly dispersed AgNPs were obtained in the control sample (in the presence of optimized concentrations of AChE, ATCh, AgNO₃ and NaBH₄) without addition of any inhibitors (Hg²⁺ or Pb²⁺), resulting in decreased fluorescence intensity. However, the addition of Hg²⁺ and Pb²⁺ to the reaction mixture tends to decrease the vibrational and rotational speed with an increase in the aggregate diameter of particles, and thus increases the emission intensity of AgNPs at 423 nm. The increase in the emission intensity can be well correlated to the increase in concentration of metal ions. The limit of detection (LOD) was found to be 1.47 × 10⁻¹⁴ M for Hg²⁺ and 0.324 × 10⁻¹⁰ M for Pb²⁺, and the proposed method was successfully used for the determination of Hg²⁺ and Pb²⁺ in environmental samples. Further, this sensor model was also employed to perform the concurrent detection of Hg²⁺ in the presence of Pb²⁺ or vice versa in aqueous solutions.

---

1. Introduction

The prominent discharge of metal-contaminated wastewater to the environment by industrial effluents causes serious health effects. The major impact of heavy metals is on soils, plants, and human beings. The microbial and enzymatic activities of microorganisms in the soils are significantly inhibited by heavy metals due to soil contamination,¹ and the cellular uptake of various heavy metal contaminates by plants exceeds their tolerance and can cause plants to die.² When human beings are exposed over a long time to low levels of heavy metals through food, water, air and commercial products, it causes several health problems. Heavy metal toxicity may cause damage to the central nervous system (CNS), cardiovascular system, gastrointestinal system, organs, and reduced growth and development.³ These heavy metals indirectly inhibit the active site of acetylcholinesterase (AChE), which breaks down acetylcholine (ACh) at cholinergic synapses.⁴

Heavy metals like mercury (Hg), cadmium (Cd), lead (Pb), chromium (Cr), arsenic (As) etc., are widely used in many industries as commercial products.² Mercury, which exists in metallic, organic, and inorganic forms, has attracted a lot of attention due to its high toxicity. Hg²⁺ is more stable in the form of inorganic mercury due to its high solubility in aqueous solutions.⁵ Elemental mercury is widely used in fluorescent lamps, batteries, mercury vapor lamps, electrical switches, thermometers, rectifiers, electrodes, and in medical fields as dental fillings, which are the major sources of Hg²⁺ discharge into the environment.⁶ Similarly, lead (Pb) also exists in different forms in natural sources. Pb²⁺ is found to exhibit acute toxicity in human beings, and a long-term exposure of Pb²⁺ can have harmful effects on the biological system, and it does not undergo biodegradation.⁷ The major sources of Pb²⁺ discharge are batteries, paint and fertilizer industries, and petrol additives.⁸ According to USEPA, the permissible limits for Hg and Pb ions in drinking water are 10 × 10⁻⁹ M and 72 × 10⁻⁹ M, respectively.

Though heavy metals are useful for various applications, they are considered as high density chemical components, and hence, are toxic even at low concentrations. Although varying amounts of some heavy metals like iron (Fe), zinc (Zn), copper (Cu), manganese (Mn), and cobalt (Co) are required by living organisms in lower quantities, even these metals are toxic at higher concentrations.⁹ Among all the heavy metals, mercury and lead are considered hazardous due to their ability to show high toxicity even at lower concentrations. Thus, the determination of heavy metals like Hg²⁺ and Pb²⁺ in environmental samples with high sensitivity and selectivity, without any interference from other metal ions, is essential.

There are some techniques like cold-vapor atomic absorption spectroscopy (CVAA),¹⁰ cold-vapor atomic fluorescence spectroscopy (CVAF),¹¹ inductively coupled plasma mass spectrometry (ICP-MS),¹² voltammetry,¹³ colorimetry,¹⁴ and spectrophotometry,¹⁵ which have been used to measure heavy metals, especially Hg²⁺ and Pb²⁺. Though many of these techniques are modern and have advantages to detect different quantities of heavy metals from any sample matrix, they also have some disadvantages as they are time consuming, consume lots of energy, have low sensitivity and selectivity, and require high-cost and advanced instruments.¹⁶ Nowadays, nanomaterials in the field of biosensors attract more attention towards the determination of heavy metals due to their high sensitivity in chemical and biological sensing.¹⁷

To date, various sensor methods that have been used to determine Hg²⁺ based on organic compounds,^18,19 oligonucleotides,²⁰ conjugated polymers,^21,22 DNAzyme,^23,24 nanoparticles,^25,26 and nanorods,^27,28 have been reported. Similarly, different spectrophotometric methods have been reported for the detection of Pb²⁺ (ref. 14, 17, 29 and 30) using DNAzyme-directed assembly of AuNPs^31,32 and other modified NPs,³³ peptides,³⁴ and proteins.³⁵ Though earlier reported methods could successfully determine Hg²⁺ and Pb²⁺, NP modification and the complicated process for NP preparation, time-consuming sample preparation and preconcentration procedures limit their applications. In addition, these methods could not achieve the detection of Hg²⁺ and Pb²⁺ at trace levels with very high sensitivity and selectivity.

However, some of these methods could achieve Hg²⁺ and Pb²⁺ detection with high selectivity and sensitivity. Recently, Li et al. demonstrated the detection of Hg²⁺ and cysteine (Cys) using graphene quantum dots (GQDs) as a fluorescent probe. The GQDs can get quenched by fluorescence charge transfer when Hg²⁺ is added to the system, and their fluorescence was enhanced and restored when Cys combined with Hg²⁺ (due to a strong metal–thiol bond formation). The limit of detection (LOD) for Hg²⁺ and Cys were 0.439 × 10⁻⁹ M and 4.5 × 10⁻⁹ M, respectively. However, the preparation of GQDs is quite complicated and time consuming.³⁶ Similarly, Lee et al. reported the use of rhodamine 6G (R6G) and 3-mercaptopropionic acid (MPA)-modified AuNPs (R6G/MPA-AuNPs) for sensing Hg²⁺, and bovine serum albumin (BSA)-capped AuNPs in the presence of thiosulfate (S₂O₃²⁻) and 2-mercaptoethanol (2-ME) for the detection of Pb²⁺ in highly saline media. These processes are complicated and require more time to prepare and modify the NPs. The systems could attain an LOD of ca. 2.0 × 10⁻⁹ M for Hg²⁺ and ca. 5.0 × 10⁻⁹ M for Pb²⁺.²⁹ Guo et al. developed a rapid colorimetric detection technique for Hg²⁺, Pb²⁺ and Cu²⁺ using papain-functionalized gold nanoparticles (P-AuNPs). The developed system can detect Hg²⁺, Pb²⁺ or Cu²⁺ as low as 200 × 10⁻⁹ M using P-AuNPs, but it is very time consuming as it requires about 24 h to functionalize the papain on AuNPs.¹⁴

The detection of Hg²⁺ and Pb²⁺ using these existing methods mainly requires fluorophores, organic dyes, or functionalized NPs to improve the selectivity and sensitivity of the system. Recently, in our previous work we reported the sensing of organophosphorus pesticides like trichlorfon and malathion in aqueous solution by an enzyme-based method using the modulated synthesis of AgNPs that can be prepared using sodium citrate as a capping and stabilizing agent.³⁷ In the current work, we describe a novel approach to detect Hg²⁺ and Pb²⁺ using fluorescence spectroscopy by thiol-induced formation of AgNPs through an enzyme-based method. In the present work, there is no external stabilizing or capping agent added, and a mild reducing agent is sufficient to form nanoparticles using an enzymatic reaction. The principle of the system is mainly based on the formation of stable AgNPs by the enzymatic generation of thiol (–SH) groups, which act as a stabilizing agent and can also control the particle size of AgNPs. Hg²⁺ and Pb²⁺ were found to highly inhibit the enzyme activity among all the other heavy metals. The addition of these heavy metals (Hg²⁺ and Pb²⁺) to the system can destabilize the NPs by hindering the enzyme activity, which can cause an increase in the fluorescence emission intensity of AgNPs at 423 nm.

To the best of our knowledge the present work demonstrated for the first time that the fluorimetric detection of Hg²⁺ and Pb²⁺ using thiocholine-mediated stabilization can be achieved by an enzymatic reaction during the synthesis of AgNPs. This method was able to achieve the lowest detection level so far for Hg²⁺ (1.47 × 10⁻¹⁴ M) and Pb²⁺ (0.324 × 10⁻¹⁰ M) compared with earlier detection methods that have employed fluorescent carbon nanoparticles, silver and gold clusters, carbon nanodots, AuNPs and DNA (Table 1 and 2). The detection of Hg²⁺ was performed during the synthesis of AgNPs, unlike the other studies reported, which may have resulted in the enhanced sensitivity. The use of biological recognition molecules (enzymes) having high specificity towards interactions with the heavy metals, also played a major role in ultrasensitive detection. The system was also able to concurrently determine the concentrations of both Hg²⁺ and Pb²⁺ present in aqueous solution. The application of the developed method was successful for the determination of Hg²⁺ and Pb²⁺ in tap and lake water samples.

Table 1 Comparison of Hg²⁺ detection limits in the present study with earlier reported methods

Reagent	LOD^a (M)	Ref.
a Limit of detection.b System included AgNO3, NaBH4, AChE, and ATCh.
Au nanoclusters	0.5 × 10^−9	47
Structure-switching DNA	3.2 × 10^−9	48
Ag clusters	10 × 10^−9	49
Carbon NPs	0.23 × 10^−9	50
Carbon nanodots	4.2 × 10^−9	51
Thrombin-binding aptamer (TBA) probe	5.0 × 10^−9	52
During synthesis of AgNPs^b	1.47 × 10^−14	Present work

---

Table 2 Comparison of Pb²⁺ detection limits in the present study with earlier reported methods

Reagent	LOD^a (M)	Ref.
a Limit of detection.b Cadmium tellurium quantum dots.c 2-Mercaptoethanol (2-ME)–thiosulfate (S2O3^2−)–AuNPs.d System included AgNO3, NaBH4, AChE, and ATC.
DNAzyme and AuNPs	3.0 × 10^−9	32
Gallic acid-capped AuNPs	10.0 × 10^−9	33
Thrombin-binding aptamer (TBA) probe	0.3 × 10^−9	52
CdTe QDs^b	2.7 × 10^−7	53
2-ME/S2O3^2−–AuNPs^c	0.5 × 10^−9	54
During synthesis of AgNPs^d	0.32 × 10^−10	Present work

---

2. Experimental

2.1. Materials

Acetylthiocholine iodide (ATChI), acetylcholinesterase (from Electrophorus electricus), and mercury(II) chloride (Hg²⁺) were obtained from Sigma-Aldrich, India. Silver nitrate (AgNO₃) and sodium borohydride (NaBH₄) were purchased from SRL Pvt. Ltd (India). Tris(hydroxymethyl) aminomethane and the heavy metal salts Mn²⁺, Fe²⁺, Fe³⁺, Pb²⁺, and Cr⁶⁺ were obtained from SRL Pvt. Ltd (India), Ni²⁺, Cr³⁺, Zn²⁺, Cd²⁺, and Mg²⁺ were from Sigma-Aldrich, India.

The stock solutions for all the heavy metals (1 mM) were prepared freshly in Milli-Q water, and the heavy metals were further diluted to the appropriate concentration for further experimental use and spectroscopic studies. An AChE stock solution (100 mU mL⁻¹) was made by using tris buffer (10 mM) and further, required concentrations were diluted with tris buffer and stored at 4 °C when not in use. The ATChI stock solution (10 mM) was also prepared using tris buffer (10 mM). The iodide ions in the ATChI solution were removed by our previous method to enhance the limit of detection.³⁸ Analytical grade chemical reagents were used throughout the experiments without further purification, and all the solutions were prepared using Milli-Q water unless stated otherwise.

2.2. Instrumentation

UV absorption spectra were recorded on a UV-2600 spectrophotometer (Shimadzu, Tokyo, Japan). All the measurements were made in the spectral range from 200 to 800 nm. Fluorescence emission spectra were obtained using a Cary eclipse fluorescence spectrophotometer (Agilent Technologies, Model-G9800A). The emission wavelength at 423 nm corresponding to the excitation at 340 nm was recorded, and the spectral wavelength range studied was from 400 to 450 nm. Additional tests were carried out to confirm that the obtained emission peak of AgNPs at 423 nm was not due to a Rayleigh scattering phenomenon by varying the excitation value from 300 to 380 nm, and the excitation peak of AgNPs was constantly obtained at 340 nm (data not shown). The surface morphological studies of AgNPs and the particle size of the dispersed and aggregated AgNPs were obtained using transmission electron microscopy (TEM, FEI Company Tecnai™, G² 20 Twin) at an accelerating voltage of around 200 kV. The particle size distribution of the AgNPs before and after addition of Hg²⁺ and Pb²⁺ in the presence of AChE and ATCh was obtained using dynamic light scattering (DLS) analysis (NanoBrook 90Plus PALS Particle Size Analyzer, Brookhaven Instruments Corporation, USA).

2.3. Detection of Hg²⁺

The fluorimetric determination of Hg²⁺ concentration was measured by studying the spectra of AgNPs with an excitation wavelength of 340 nm, and their emission spectra were recorded from 400 to 450 nm. A maximum emission intensity was obtained at 423 nm, which corresponds to the SPR band of AgNPs formed during the synthesis process. Initially, the formation of AgNPs (control sample without addition of Hg²⁺) was followed by the addition of 40 μL of AChE (4 mU), 80 μL of ATCh (1 mM), 400 μL of AgNO₃ (0.1 mM) and 80 μL of NaBH₄ (1 mM) respectively, and the reaction mixtures were diluted up to 800 μL with Milli-Q water. A pale yellow color was observed during the formation of NPs (details are provided in ESI Fig. S1A†). Subsequently, 50 μL of each concentration of Hg²⁺ from 1 to 100 × 10⁻¹³ M was added to the reaction mixture in each vial. Then, the reaction mixture of each vial was incubated for 15 min at 37 ± 2 °C. After incubation, the color of the reaction mixture changed from pale yellow to grey (details are provided in ESI Fig. S1B†), and concomitantly the increase in fluorescence emission intensity was recorded at 423 nm for each sample. As the concentration of Hg²⁺ increases, an increase in the size of NPs occurs, and the reaction mixture without Hg²⁺ addition indicated the formation of stable NPs in the presence of AChE and ATCh. All the experiments were performed in triplicate to acquire reproducible data.

2.4. Detection of Pb²⁺

The different concentrations of Pb²⁺ from 0.1 to 10 × 10⁻⁹ M (50 μL each concentration) were incubated with the reaction mixtures, which contained 40 μL of AChE (4 mU), 80 μL of ATCh (1 mM), 400 μL of AgNO₃ (0.1 mM) and 80 μL of NaBH₄ (1 mM) respectively, and the final volume of the solution was adjusted to 800 μL with Milli-Q water. Then, the reaction mixture of each concentration of Pb²⁺ was incubated for 20 min at 37 ± 2 °C. The florescence spectrum of each solution was recorded immediately at 423 nm, and then the measured spectral changes in the emission intensity were used to plot the calibration curve for Pb²⁺. In this system, the duration of incubation time was observed to be higher because the binding capacity and degree of inhibition efficiency of Pb²⁺ is much lower compared with Hg²⁺. After incubation, the color of the solution changed from pale yellow to grey when Pb²⁺ was added during the formation of AgNPs.

2.5. Concurrent detection of both Hg²⁺ and Pb²⁺

The detection of both heavy metal ions, Hg²⁺ and Pb²⁺, was ascertained by maintaining a fixed concentration of one metal and their spectral changes were studied by fluorescence spectroscopy. Different concentrations of Hg²⁺ (1 to 100 × 10⁻¹³ M) were added in the presence of a fixed concentration of Pb²⁺ (2 × 10⁻⁹ M), and 50 μL of both metal ion mixtures was added to the system containing 40 μL of AChE (4 mU), 80 μL of ATCh (1 mM), 400 μL of AgNO₃ (0.1 mM), and 80 μL of NaBH₄ (1 mM). The reaction volume was adjusted to 800 μL with Milli-Q water. Then, the reaction mixture was incubated for 20 min at 37 ± 2 °C. The spectral changes in emission intensity at 423 nm was recorded immediately after the reaction was complete. Similarly, when changing the concentration of Pb²⁺ (0.1 to 10 × 10⁻⁹ M), maintaining a constant concentration of Hg²⁺ (1 × 10⁻¹³ M) and incubating the resulting mixtures for 20 min, a significant increase in the emission intensity at 423 nm was noticed, and the obtained spectral measurements were used to plot the linear calibration graph for Pb²⁺ in the presence of Hg²⁺.

2.6. Real sample analysis

The practical application of the developed method was estimated by determining the Hg²⁺ and Pb²⁺ in real samples. The real samples used were tap and lake water, wherein the lake water samples were collected from the Vellore Institute of Technology Lake (VIT University, Vellore, India) and the tap water samples were collected in our laboratory. The collected lake water was filtered through a 0.22 μm membrane filter to remove foreign residue materials. The pH of both tap (pH 7.1 ± 0.1) and lake (pH 6.9 ± 0.1) water was recorded before spiking the metal ions, and there was no significant change in the pH even after completion of the analysis. The stock solutions were prepared by spiking higher concentrations of both metal ions separately. From the stock solutions, three different working solutions were prepared for Hg²⁺ (5, 10, and 60 × 10⁻¹³ M) and Pb²⁺ (0.5, 1, and 5 × 10⁻⁹ M), respectively. The control sample was also prepared using the same filtration procedure, but it did not contain either metal ions. When the real samples alone were added to the system (in the absence of both metal ions), they did not cause any increase in the fluorescence intensity.

3. Results and discussion

3.1. Developing a sensor model for the detection of heavy metals (Hg²⁺ and Pb²⁺) using in situ enzymatic formation of AgNPs

We have developed a sensor model for the detection of Hg²⁺ and Pb²⁺ using AgNPs, wherein thiocholine (TCh) (a product that is obtained by the hydrolysis of ATCh using AChE) can act as a stabilizer during the formation of stable AgNPs.

Initially, the formation of AgNPs involves the optimization of AgNO₃ and NaBH₄ concentrations, and this was studied by using fluorescence spectroscopy. The emission intensity of AgNPs was recorded at 423 nm with an excitation wavelength at 340 nm. The changes in fluorescence signal with different concentrations of AgNO₃ and NaBH₄ have been studied by varying the concentration of AgNO₃ (0.1–0.001 mM) and fixing the concentration of NaBH₄ (1 mM) without the addition of AChE and ATCh. As shown in Fig. 1A, the emission intensity of AgNPs at 423 nm decreased as the concentration of AgNO₃ increased while using a fixed concentration of NaBH₄ (1 mM). Similarly, as the concentration of NaBH₄ (1–0.01 mM) increases the emission intensity decreased slightly with a fixed concentration of AgNO₃ (0.1 mM), as shown in Fig. 1B. However, at a fixed concentrations of AgNO₃ (0.1 mM) and NaBH₄ (1 mM), there is slight decrease in emission intensity at 423 nM as shown in Fig. 1A and B (as compared to other concentrations of AgNO₃ and NaBH₄).

Fig. 1 Emission spectra for the optimization of AgNPs formed during the addition of (A) different concentrations of AgNO₃ (0.1, 0.01, and 0.001 mM), and a fixed concentration of NaBH₄ (1 mM). (B) Different concentrations of NaBH₄ (1.0, 0.1, and 0.001 mM) and a fixed concentration of AgNO₃ (0.1 mM). The control has AgNO₃ (0.1 mM), NaBH₄ (1 mM), AChE (4 mU), and ATCh (1 mM).

On the addition of AgNO₃ and NaBH₄, the as-formed AgNPs were found to aggregate immediately and thus formed a less stable colloidal solution owing to the absence of a stabilizing agent. This was further confirmed by transmission electron microscopy (TEM); Fig. 2A shows the AgNPs (0.1 mM of AgNO₃, 1 mM of NaBH₄), but the particles are unstable and accumulate due to a lack of stabilizing agent. The interaction of AChE and ATCh produces a trace amount of TCh, which can act as a stabilizing agent during the formation of AgNPs. Therefore, the formation of more stable AgNPs was achieved when AChE, ATCh (under the optimized conditions of AChE 4 mU, and ATCh 1 mM), AgNO₃ and NaBH₄ are all used. Fig. 1A and B show a sudden decrease in the emission intensity of AgNPs at 423 nm in the presence of AChE, ATCh, AgNO₃ and NaBH₄ (control). The decrease in emission intensity was attributed to the increased stability of AgNPs in the presence of an enzyme–substrate complex compared with using AgNO₃ and NaBH₄ alone, and the color of the solution was found to change to pale yellow. Further, the addition of AChE and ATCh to the system produces more stable AgNPs with controlled particle size. The formation of AgNPs was corroborated by TEM. Fig. 2B suggests that the formation of AgNPs was stabilized by trace amounts of a thiol-bearing compound, TCh, and their average particle diameter was found to be 9 ± 1 nm.

Fig. 2 TEM images for AgNPs formed in the presence of (A) AgNO₃ (0.1 mM) and NaBH₄ (1 mM) and (B) AgNO₃ (0.1 mM), NaBH₄ (1 mM), AChE (4 mU), and ATCh (1 mM).

The particle size distribution of AgNPs before and after addition of AChE and ATCh with Hg²⁺ and Pb²⁺ was further confirmed using dynamic light scattering (DLS) analysis. The mean hydrodynamic size of AgNPs (AgNO₃ and NaBH₄) was found to be around 100 ± 2 nm (for details see ESI Fig. S2A†). The addition of AChE and ATCh to AgNO₃ and NaBH₄ during the formation of AgNPs caused the particle size of AgNPs to decrease to 66 ± 1 nm (for details see ESI Fig. S2B†). The diameter of AgNPs in the presence of AChE and ATCh is observed to be larger than the core diameter measured by TEM (9 ± 1 nm), because during DLS measurement, the forward angle is more heavily biased towards the aggregated molecules than the smaller molecules, and thus, the analysis captures the hydrodynamic diameter.^39,40 The addition of the heavy metal, either Hg²⁺ (10 × 10⁻¹³ M) or Pb²⁺ (1 × 10⁻⁹ M), to the system during the synthesis of AgNPs results in the accumulation of AgNPs, and the particle size of AgNPs was found to increase to 125 ± 1 nm for Hg²⁺ and 120 ± 0.5 nm for Pb²⁺ (for details see ESI Fig. S2C and S2D†), respectively.

The formation of AgNPs was further confirmed by UV-visible spectroscopy using the absorption spectral changes during the addition of AgNO₃ and NaBH₄. Similarly, experiments were repeated to investigate the changes in absorption intensity of AgNPs with different concentrations of AgNO₃ and NaBH₄. The absorption peak intensity at 420 nm increased slightly as the concentration of AgNO₃ (0.1–0.001 mM) increased by keeping the concentration of NaBH₄ fixed (1 mM) Fig. S3A (ESI†). Similarly, as the concentration of NaBH₄ (1–0.01 mM) increases, the intensity at 420 nm increased slightly when a fixed concentration of AgNO₃ (0.1 mM) was used, as shown in Fig. S3B.† In the control sample, a sudden increase in the intensity of AgNPs was observed at 420 nm as shown in both Fig. S3A and S3B (ESI†). The increase in intensity can again be attributed to the stability of AgNPs in the presence of the enzyme–substrate complex when compared with the usage of AgNO₃ and NaBH₄ alone. The details of all these experiments are provided in the ESI.†

To find out the minimum concentration of AChE and ATCh required to produce stable AgNPs another series of tests were carried out. The fluorescence response was investigated by using different concentrations of ATCh and fixing the amount of AChE. As the concentration of ATCh increases (0 to 1 mM), the response of the fluorescence signal decreases, and the system showed maximum stability in the formation of AgNPs at 1 mM of ATCh with 4 mU of AChE, as shown in Fig. 3A. As the concentration of ATCh increased, the stability of the AgNPs increased, and the system reached saturation after 1 mM of ATCh. In the same manner, the effect of AChE concentration was studied by fixing the amount of ATCh (1 mM). Similarly, as the concentration of AChE increases (0 to 10 mU), there was a gradual decrease in fluorescence as shown in Fig. 3B. Maximum stability of AgNPs was achieved when the AChE concentration was 4 mU. A further increase in the concentration of AChE above 4 mU, resulted in the saturation of the system. For the rest of the experiments, the concentrations of AChE (4 mU) and ATCh (1 mM) were fixed in order to obtain more stable AgNPs.

Fig. 3 Emission spectra of AgNPs formed during the addition of AgNO₃ (0.1 mM) and NaBH₄ (1 mM) with (A) different concentrations of ATCh: (a) 0 mM; (b) 0.05 mM; (c) 0.1 mM; (d) 0.2 mM; (e) 0.4 mM; (f) 0.6 mM; (g) 0.8 mM and (h) 1.0 mM, with fixed AChE concentration (4 mU), (B) different concentrations of AChE (a) 0 mU; (b) 1.0 mU; (c) 2.0 mU; (d) 4.0 mU; (e) 8.0 mU and (f) 10.0 mU with fixed ATCh concentration (1 mM).

Heavy metals can act as indirect inhibitors for acetylcholinesterase and can reduce the enzyme activity by inhibiting the active sites of AChE. In this work, the enzyme–substrate complex formation was found to yield more stable AgNPs, and the addition of different concentrations of heavy metals (Hg²⁺ or Pb²⁺) to this system could cause an increase in the size of AgNPs and a decrease in the stability of NPs gradually as the concentration of heavy metal was increased. Based on this concept, a sensor model can be developed for the indirect determination of heavy metals using thiol-stabilized AgNPs.

3.2. Mechanism for fluorescent enhancement

The observed increase in emission intensity can be due to the reduction in the vibrational and rotational speed with increasing aggregate diameter of particles, and this is indicated by the decrease in the rate of nonradiative decay.⁴¹ The instability in Brownian movement and decrease in colloidal probability between the AgNPs results in the increase in external energy transfer and quantum yield, which can also lead to fluorescence enhancement.^42,43

On the other hand, uniformly dispersed AgNPs were observed in the control sample (in the presence of optimized concentrations of AChE, ATCh, AgNO₃ and NaBH₄) without the addition of any inhibitor (Hg²⁺ or Pb²⁺), which resulted in a decrease in emission intensity at 423 nm. This decrease may be due to the lower fluorescence quantum yield of intersystem crossing to a triplet state, which causes energy transfer to the metal, and thereby is attributed to the heavy atom effect of NPs.^44,45 Stable AgNPs can induce their vibrational and rotational speed in colloidal solution and increase their Brownian motion, which increases the repulsive force between the NPs that can lead to a decrease in the external energy transfer rate.⁴⁶

3.3. Fluorimetric determination of Hg²⁺

The spectral change in the fluorescence intensity of AgNPs has been observed in the reaction mixture (0.1 mM of AgNO₃ and 1 mM of NaBH₄), in the presence and absence of AChE, ATCh, or Hg²⁺ alone. In Fig. 4A (curve A), the addition of AgNO₃ and NaBH₄ alone could not form stable NPs and similarly, the addition of the AChE (curve B), ATCh (curve C), or Hg²⁺ (curve E) alone to the reaction mixture resulted in the formation of less stable NPs compared to the more stable AgNPs (curve E) formed in the presence of both AChE and ATCh. However, the addition of Hg²⁺ (1 × 10⁻⁹ M) to the reaction mixture in the presence of AChE and ATCh resulted in an increase in fluorescence intensity (curve F). The addition of Hg²⁺ to the system causes changes in the morphology of NPs, and the formed spherical NPs were observed to come closer by decreasing the enzymatic generation of thiol groups, which act as the stabilizing agent, and thereby the nano-sized particles tended to clump together, as shown in Fig. 4B.

Fig. 4 Emission spectra of (A) AgNPs formed in the presence and absence of AgNO₃ (0.1 mM), NaBH₄ (1 mM), AChE (4 mU), ATCh (1.0 mM) and Hg²⁺ (1 × 10⁻⁹ M) at λ_em = 423 nm. (B) TEM image of AgNPs after the addition of Hg²⁺ (10 × 10⁻¹³ M).

The detection of Hg²⁺ was carried out under optimized conditions during the formation of AgNPs in the presence of both AChE and ATCh. The emission intensity of AgNPs increases with the increase in concentration of Hg²⁺ (0 to 100 × 10⁻¹³ M). The increase in emission intensity is proportional to the increase in the size of AgNPs and increase in concentration of Hg²⁺, which inhibits the active sites of AChE. Compared to the control sample (absence of Hg²⁺ in the system), the addition of Hg²⁺ significantly increases the emission intensities of AgNPs at 423 nm, as shown in Fig. 5A. This is because the increase in the concentration of Hg²⁺ from 10 to 100 × 10⁻¹³ M caused an increase in the size of NP diameter by hindering the stabilizing force on the surface of AgNPs (Fig. 4B). A good linearity (R² = 0.9729) was found between different concentrations of Hg²⁺ (0 to 100 × 10⁻¹³ M) and emission intensity of AgNPs at 423 nm as shown in Fig. 5B, and the limit of detection (LOD) was found to be 1.47 × 10⁻¹⁴ M (S/N = 3). The obtained LOD for Hg²⁺ in the present work is compared with the earlier reported papers, and is given in Table 1. It can be seen from Table 1 that the lowest detection limit was attained in the present study for Hg²⁺ detection when compared with other previous methods. The increase in fluorescence intensity for respective Hg²⁺ concentrations in the range (0 to 100 × 10⁻¹³ M) was examined for statistical analysis using one-way ANOVA (analysis of variance), and a p-value <0.005 was found to ensure the sensitivity of the system.

Fig. 5 (A) Emission spectra of AgNPs formed during the addition of AgNO₃ (0.1 mM), NaBH₄ (1 mM), AChE (4 mU), ATCh (1.0 mM), and different concentrations of Hg²⁺ (a) 0; (b) 1.0; (c) 5; (d) 10; (e) 15; (f) 20; (g) 30; (h) 40; (i) 50; (j) 70 and (k) 100 × 10⁻¹³ M. (B) Calibration curve for the detection of Hg²⁺ (λ_em = 423 nm).

One of the principal objectives of the present study was to determine trace amounts of Hg²⁺ in aqueous solution with other common interfering substances. Fig. 6 shows the emission spectra of AgNPs formed during the addition of AChE and ATCh to the system in the presence of 1 × 10⁻⁹ M of different metal ions, namely, Mn²⁺, Fe³⁺, Fe²⁺, Pb²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Mg²⁺, Cr³⁺, Cr²⁺, and Hg²⁺ (1 × 10⁻¹¹ M). The emitted fluorescence intensity of AgNPs can be enhanced with other common interfering substances by resonance energy transfer, salt-induced aggregation, and charge transfer. Such phenomena may affect the specificity of this sensor for Hg²⁺ when compared with other metal ions. However, the presence of other metal ions in the system does not result in a significant increase in the fluorescence intensity compared to Hg²⁺. These results indicated that the developed sensor has a significant selectivity for Hg²⁺. The specificity of the current work by an enzyme-based inhibition method demonstrated greater specificity for the detection of heavy metal ions. The enzyme selectively binds with Hg²⁺ in comparison to other heavy metals (Hg, Pb, Fe, Ni, and Cr) as understood by comparing the degrees of aggregation of nanoparticles based on the fluorescence emission intensities. This is due to the fact that Hg²⁺ shows more thiophilic attraction towards the enzyme than other metal ions.¹⁴ In other words, among all the heavy metals except Hg²⁺, Pb²⁺ also showed a slight increase in fluorescence emission intensity. Based on this concept, a sensor model for the detection of Pb²⁺ during the synthesis of AgNPs in the presence of AChE and ATCh can be developed.

Fig. 6 Emission response of Hg²⁺ in the system containing AgNO₃ (0.1 mM), NaBH₄ (1 mM), AChE (4 mU), ATCh (1.0 mM) with Hg²⁺ (10⁻¹¹ M), other different interfering heavy metals (10⁻⁹ M), and the control (AgNO₃ 0.1 mM, NaBH₄ 1 mM, AChE 4 mU, ATCh 1.0 mM).

3.4. Fluorimetric determination of Pb²⁺

The fluorimetric detection of Pb²⁺ was performed in a similar manner to that of Hg²⁺ detection. The fluorescence increase in Pb²⁺ was observed in the system containing AgNO₃, NaBH₄, AChE, and ATCh, and the gradual increase in fluorescence intensity at 423 nm was observed in the system during the addition of different concentrations of Pb²⁺. Increasing concentrations of Pb²⁺ in the system lead to an increase in the diameter of the formed AgNPs in the presence of AChE and ATCh, and it was found that Pb²⁺ requires a higher duration of incubation time than Hg²⁺. In addition, the sensitivity of the system for Pb²⁺ is very low compared with the sensitivity for Hg²⁺. This is because the enzymatic activity of AChE in the system is extremely hindered by Hg²⁺ as it has a very high affinity to the thiol group (–SH) compared to Pb²⁺. The reaction mixtures with each concentration of Pb²⁺ (0.1 to 10 × 10⁻⁹ M) were incubated for 20 min at 37 ± 2 °C. Afterward, the emission intensity of each sample was recorded, as shown in Fig. 7A. A linear calibration curve (R² = 0.9819) was observed when a graph was plotted of the emission intensity of AgNPs at 423 nm and different concentrations of Pb²⁺ in the range from 0.1 to 10 × 10⁻⁹ M, as shown in Fig. 7B. The limit of detection (LOD) for Pb²⁺ was calculated to be 0.324 × 10⁻¹⁰ M with a signal-to-noise ratio of 3. The addition of Pb²⁺ (1 × 10⁻⁹ M) to the system can also trigger the formation of nano-sized aggregates of AgNPs (Fig. 7C).

Fig. 7 (A) Emission spectra of AgNPs formed during the addition of AgNO₃ (0.1 mM), NaBH₄ (1 mM), AChE (4 mU), ATCh (1.0 mM) and different concentrations of Pb²⁺ (a) 0; (b) 0.1; (c) 0.4; (d) 0.8; (e) 1.0; (f) 4.0; (g) 8.0 and (h) 10.0 × 10⁻⁹ M. (B) Calibration curve for the detection of Pb²⁺ (λ_em = 423 nm). (C) TEM image of AgNPs after addition of Pb²⁺ (1 × 10⁻⁹ M).

3.5. Detection of Hg²⁺ and Pb²⁺ separately in real samples

The practical application of the developed sensor was estimated by determining the concentrations of Hg²⁺ and Pb²⁺ in tap and lake water samples, respectively. The detection of Hg²⁺ in both tap and lake water was performed by analyzing the Hg²⁺ content with the proposed method after spiking three different concentrations, 5, 10, and 60 × 10⁻¹³ M, of Hg²⁺. The increase in emission intensity at 423 nm has confirmed the presence of Hg²⁺ in tap and lake water (Table 3). Similarly, various concentrations of Pb²⁺ (0.5, 1, and 5 × 10⁻⁹ M) spiked in real samples were examined, and the increase in emission intensity at 423 nm confirmed the presence of Pb²⁺ in tap and lake water (Table 4). The spiked and measured values were found to be satisfactory for both Hg²⁺ and Pb²⁺. The recovery and relative standard deviation (RSD) values for both spiked Hg²⁺ and Pb²⁺ in tap water and lake water are given in Tables 3 and 4, respectively. The RSD and recovery values suggested that the present method can be used for the detection of Hg²⁺ or Pb²⁺ alone in real samples.

Table 3 Detection of Hg²⁺ in spiked real samples

Sample	Added (10^−13 M)	Found (10^−13 M)	Recovery (%)	RSD (%)
Tap water	5	4.91	98.20	0.18
	10	9.95	99.50	0.64
	60	58.91	98.18	1.57
Lake water	5	4.79	95.80	0.57
	10	9.75	97.50	0.42
	60	55.51	92.52	2.89

---

Table 4 Detection of Pb²⁺ in spiked real samples

Sample	Added (10^−9 M)	Found (10^−9 M)	Recovery (%)	RSD (%)
Tap water	0.5	0.52	104	0.66
	1	1	100	0.63
	5	4.86	97.20	0.46
Lake water	0.5	0.51	102	0.65
	1	1.01	101	0.38
	5	4.56	91.20	0.47

---

3.6. Concurrent detection of Hg²⁺ and Pb²⁺

In the above method, the fluorescence detection of both Hg²⁺ and Pb²⁺ can be carried out during the synthesis of AgNPs with AChE and ATCh. The addition of Hg²⁺ or Pb²⁺ alone to the thiol-stabilized AgNPs results in an increase in the fluorescence intensity due to increase in the size of NPs during synthesis, and this will be proportional to the inhibition of AChE. This method can lead to the significant development of a sensor for the quantitative analysis of Hg²⁺ and Pb²⁺ in aqueous samples.

The concurrent detection of Hg²⁺ was performed by keeping a fixed concentration of Pb²⁺ (2 × 10⁻⁹ M) throughout the experiment. The change in the fluorescence emission intensity was observed upon addition of different concentrations of Hg²⁺ (0 to 100 × 10⁻¹³ M) in the reaction mixtures, containing Pb²⁺, AgNO₃, NaBH₄, AChE, and ATCh. Fig. 8A reveals that the presence of Pb²⁺ along with various concentrations of Hg²⁺ can also enhance the fluorescence emission intensity at 423 nm. This could happen either because Hg²⁺ ions have greater tendency to adsorb on the surface of the thiol-stabilized AgNPs¹⁴ or it could be due to the higher efficiency of Hg²⁺ to inhibit the active site of AChE compared to Pb²⁺ metal ions. This process could induce the aggregation of AgNPs by increasing the electrostatic interaction due to the formation of the Hg–SR bond and by decreasing the repulsive force between thiol-stabilized AgNPs. Therefore, there is an increase in the external energy transfer rate as the concentration of Hg²⁺ increases, which leads to a gradual increase in the fluorescence emission intensity of AgNPs. Thus, a good linear calibration curve (R² = 0.9585) was obtained when the emission intensity at 423 nm was plotted against different concentrations of Hg²⁺ with a fixed concentration of Pb²⁺ as shown in Fig. 8B.

Fig. 8 (A) Emission spectra of AgNPs during the addition of AgNO₃ (0.1 mM), NaBH₄ (1 mM), AChE (4 mU), ATCh (1.0 mM), and Pb²⁺ (2 × 10⁻⁹ M) with different concentrations of Hg²⁺ (a) 0; (b) 1.0; (c) 5; (d) 10; (e) 15; (f) 20; (g) 30; (h) 40; (i) 50; (j) 70 and (k) 100 × 10⁻¹³ M. (B) Linear calibration curve for Hg²⁺ in the presence of Pb²⁺.

Similarly, the concurrent detection of Pb²⁺ was measured by keeping a fixed concentration of Hg²⁺ (1 × 10⁻¹³ M) throughout the experiment. Fig. 9A shows an increase in the fluorescence emission intensity at 423 nm in the presence of Hg²⁺ (fixed) with different concentrations of Pb²⁺ (0 to 10 × 10⁻⁹ M) in the system. Here, the probability of AChE inhibition by Hg²⁺ is much less when a lower concentration of Hg²⁺ is used when compared with Pb²⁺ concentration in the system. It was observed that the fluorescence of AgNPs was enhanced due to an increase in the size of AgNPs in the presence of both Pb²⁺ and Hg²⁺ also. The fluorescence intensity at 423 nm was found to increase with increasing concentrations of Pb²⁺ while keeping a fixed concentration of Hg²⁺ (Fig. 9A). A linear calibration curve (R² = 0.9419) was plotted for fluorescence emission intensity of AgNPs versus different concentrations of Pb²⁺ with a fixed concentration of Hg²⁺, as shown in Fig. 9B.

Fig. 9 (A) Emission spectra of AgNPs during the addition of AgNO₃ (0.1 mM), NaBH₄ (1 mM), AChE (4 mU), ATCh (1.0 mM), and Hg²⁺ (1 × 10⁻¹³ M) with different concentrations of Pb²⁺ (a) 0; (b) 0.1; (c) 0.4; (d) 0.8; (e) 1.0; (f) 4.0; (g) 8.0 and (h) 10.0 × 10⁻⁹ M. (B) Linear calibration curve for Pb²⁺ in the presence of Hg²⁺.

4. Conclusion

The present work demonstrated that the enzymatic reaction-based stabilization of silver nanoparticles can serve as a fluorimetric probe, which allows the sensitive and selective detection of Hg²⁺ and Pb²⁺ in aqueous solutions as well as in real samples. Using fluorescence spectroscopy, the developed method can determine the inhibition in the enzymatic reaction in the presence of Hg²⁺ or Pb²⁺, which can lead to an increase in the emission intensity of silver nanoparticles at 423 nm. The determination of Hg²⁺ and Pb²⁺ was carried out based on the emission intensity of AgNPs with an inexpensive and simple assay.

This method permits very high sensitivity compared with the other reported methods and can measure low concentrations of Hg²⁺ and Pb²⁺ with detection limits of 1.47 × 10⁻¹⁴ M and 0.324 × 10⁻¹⁰ M, respectively. The use of a biological recognition molecule (enzyme) enables enhanced specificity towards the interaction with the heavy metals. The developed method is simple and requires less time for the detection of Hg²⁺ and Pb²⁺. The nanoparticles can be synthesized directly without the requirement of any external stabilizing agent in the presence of an enzyme-based reaction. Further, this concept can also be used to perform the concurrent detection of Hg²⁺ in the presence of Pb²⁺ or vice versa. However, the method has its limitations also. The enzyme is expensive and it requires specific storage condition for better enzyme activity. Additionally, the potential practical applications of the concurrent detection method would require a more detailed study into overcoming the possible effects of co-existing Hg²⁺ and Pb²⁺ in the real samples, on the enzymatic interactions during the synthesis of AgNPs. The proposed sensing strategy could also be used for the detection of other inhibitor heavy metals using the enzymatic-based formation of silver nanoparticles.

Acknowledgements

The authors would like to acknowledge funding from the Defence Research & Development Organization (ERIP/ER/1103964/M/01/1485), Ministry of Defence, Government of India. We also acknowledge the VIT University for providing TEM facility.

References

1. F. Kandeler, C. Kampichler and O. Horak, Biol. Fertil. Soils, 1996, 23, 299–306.
2. C. Su, L. Jiang and W. Zhang, Environmental Skeptics and Critics, 2014, 3, 24–38.
3. M. A. Barakat, Arabian J. Chem., 2011, 4, 361–377.
4. F. Arduini, F. Ricci, I. Bourais, A. Amine, D. Moscone and G. Palleschi, Anal. Lett., 2005, 38, 1703–1719.
5. T. W. Clarkson, L. Magos and G. J. Myers, N. Engl. J. Med., 2003, 349, 1731–1737.
6. J. Wang, B. Deng, H. Chen, X. Wang and J. Zheng, Environ. Sci. Technol., 2009, 43, 5223–5228.
7. B. V. Tangahu, S. R. Sheikh Abdullah, H. Basri, M. Idris, N. Anuar and M. Mukhlisin, Int. J. Chem. Eng., 2011, 2011, 1–31.
8. S. M. Maliyekkal, K. P. Lisha and T. Pradeep, J. Hazard. Mater., 2010, 181, 986–995.
9. R. Singh, N. Gautam, A. Mishra and R. Gupta, Indian J. Pharmacol., 2011, 43, 246–253.
10. M. Ghaedi, M. Reza Fathi, A. Shokrollahi and F. Shajarat, Anal. Lett., 2006, 39, 1171–1185.
11. L. Rahman, W. Corns, D. Bryce and P. Stockwell, Talanta, 2000, 52, 833–843.
12. C. Huang and B. Hu, Spectrochim. Acta, Part B, 2008, 63, 437–444.
13. W. Yantasee, Y. Lin, T. S. Zemanian and G. E. Fryxell, Analyst, 2003, 128, 467–472.
14. Y. Guo, Z. Wang, W. Qu, H. Shao and X. Jiang, Biosens. Bioelectron., 2011, 26, 4064–4069.
15. A. Hamza, A. Bashammakh, A. Al-Sibaai, H. Al-Saidi and M. El-Shahawi, J. Hazard. Mater., 2010, 178, 287–292.
16. A. Fan, Y. Ling, C. Lau and J. Lu, Talanta, 2010, 82, 687–692.
17. H. N. Kim, W. X. Ren, J. S. Kim and J. Yoon, Chem. Soc. Rev., 2012, 41, 3210–3244.
18. C.-C. Huang and H.-T. Chang, Anal. Chem., 2006, 78, 8332–8338.
19. H. Lu, Y. Tang, W. Xu, D. Zhang, S. Wang and D. Zhu, Macromol. Rapid Commun., 2008, 29, 1467–1471.
20. S.-J. Liu, H.-G. Nie, J.-H. Jiang, G.-L. Shen and R.-Q. Yu, Anal. Chem., 2009, 81, 5724–5730.
21. I.-B. Kim and U. H. Bunz, J. Am. Chem. Soc., 2006, 128, 2818–2819.
22. X. Liu, Y. Tang, L. Wang, J. Zhang, S. Song, C. Fan and S. Wang, Adv. Mater., 2007, 19, 1471–1474.
23. Y. Xiao, A. A. Rowe and K. W. Plaxco, J. Am. Chem. Soc., 2007, 129, 262–263.
24. J. Liu and Y. Lu, Angew. Chem., 2007, 119, 7731–7734.
25. C. C. Huang, Z. Yang, K. H. Lee and H. T. Chang, Angew. Chem., 2007, 119, 6948–6952.
26. A. Zheng, J. Chen, G. Wu, H. Wei, C. He, X. Kai, G. Wu and Y. Chen, Microchim. Acta, 2009, 164, 17–27.
27. C. J. Murphy, A. M. Gole, S. E. Hunyadi, J. W. Stone, P. N. Sisco, A. Alkilany, B. E. Kinard and P. Hankins, Chem. Commun., 2008, 544–557.
28. M. Rex, F. E. Hernandez and A. D. Campiglia, Anal. Chem., 2006, 78, 445–451.
29. Y.-F. Lee, F.-H. Nan, M.-J. Chen, H.-Y. Wu, C.-W. Ho, Y.-Y. Chen and C.-C. Huang, Anal. Methods, 2012, 4, 1709–1717.
30. N. Ratnarathorn, O. Chailapakul and W. Dungchai, Talanta, 2015, 132, 613–618.
31. J. Liu and Y. Lu, J. Am. Chem. Soc., 2003, 125, 6642–6643.
32. Z. Wang, J. H. Lee and Y. Lu, Adv. Mater., 2008, 20, 3263–3267.
33. K.-W. Huang, C.-J. Yu and W.-L. Tseng, Biosens. Bioelectron., 2010, 25, 984–989.
34. S. Deo and H. A. Godwin, J. Am. Chem. Soc., 2000, 122, 174–175.
35. E. Mohamed Ali, Y. Zheng, H.-h. Yu and J. Y. Ying, Anal. Chem., 2007, 79, 9452–9458.
36. Z. Li, Y. Wang, Y. Ni and S. Kokot, Sens. Actuators, B, 2015, 207, 490–497.
37. D. N. Kumar, A. Rajeshwari, S. Alex, M. Sahu, A. Raichur, N. Chandrasekaran and A. Mukherjee, RSC Adv., 2015, 5, 61998–62006.
38. D. N. Kumar, A. Rajeshwari, S. A. Alex, N. Chandrasekaran and A. Mukherjee, New J. Chem., 2015, 39, 1172–1178.
39. O. C. Compton and F. E. Osterloh, J. Am. Chem. Soc., 2007, 129, 7793–7798.
40. H. Jans, X. Liu, L. Austin, G. Maes and Q. Huo, Anal. Chem., 2009, 81, 9425–9432.
41. S. A. McFarland and N. S. Finney, J. Am. Chem. Soc., 2002, 124, 1178–1179.
42. D. Xiang, G. Zeng, K. Zhai, L. Li and Z. He, Analyst, 2011, 136, 2837–2844.
43. S. Shankar and S. A. John, Sens. Actuators, B, 2015, 221, 1202–1208.
44. P. Khoza and T. Nyokong, J. Mol. Catal. A: Chem., 2014, 395, 34–41.
45. L. Balan, R. Schneider, C. Turck, D. Lougnot and F. Morlet-Savary, J. Nanomater., 2012, 2012, 1.
46. A. Menbari, A. A. Alemrajabi and Y. Ghayeb, Energy Convers. Manage., 2016, 108, 501–510.
47. J. Xie, Y. Zheng and J. Y. Ying, Chem. Commun., 2010, 46, 961–963.
48. Z. Wang, J. H. Lee and Y. Lu, Chem. Commun., 2008, 6005–6007.
49. C. Guo and J. Irudayaraj, Anal. Chem., 2011, 83, 2883–2889.
50. W. Lu, X. Qin, S. Liu, G. Chang, Y. Zhang, Y. Luo, A. M. Asiri, A. O. Al-Youbi and X. Sun, Anal. Chem., 2012, 84, 5351–5357.
51. L. Zhou, Y. Lin, Z. Huang, J. Ren and X. Qu, Chem. Commun., 2012, 48, 1147–1149.
52. C.-W. Liu, C.-C. Huang and H.-T. Chang, Anal. Chem., 2009, 81, 2383–2387.
53. H. Wu, J. Liang and H. Han, Microchim. Acta, 2008, 161, 81–86.
54. Y.-Y. Chen, H.-T. Chang, Y.-C. Shiang, Y.-L. Hung, C.-K. Chiang and C.-C. Huang, Anal. Chem., 2009, 81, 9433–9439.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra00193a

---