Design and development of amperometric biosensor for the detection of lead and mercury ions in water matrix—a permeability approach

Abstract

Intake of water contaminated with lead (Pb²⁺) and mercury (Hg²⁺) ions leads to various toxic effects and health issues. In this context, an amperometric urease inhibition-based biosensor was developed to detect Pb²⁺ and Hg²⁺ ions in water matrix. The modified Pt/CeO₂/urease electrode was fabricated by immobilizing CeO₂ nanoparticles and urease using a semi-permeable adsorption layer of nafion. With urea as a substrate, urease catalytic activity was examined through cyclic voltammetry. Further, maximum amperometric inhibitive response of the modified Pt/CeO₂/urease electrode was observed in the presence of Pb²⁺ and Hg²⁺ ions due to the urease inhibition at specific potentials of −0.03 and 0 V, respectively. The developed sensor exhibited a detection limit of 0.019 ± 0.001 μM with a sensitivity of 89.2 × 10⁻³ μA μM⁻¹ for Pb²⁺ ions. A detection limit of 0.018 ± 0.003 with a sensitivity of 94.1 × 10⁻³ μA μM⁻¹ was achieved in detecting Hg²⁺ ions. The developed biosensor showed a fast response time (<1 s) with a linear range of 0.5–2.2 and 0.02–0.8 μM for Pb²⁺ and Hg²⁺ ions, respectively. The modified electrode offered a good stability for 20 days with a good repeatability and reproducibility. The developed sensor was used to detect Pb²⁺ and Hg²⁺ ions contaminating Cauvery river water and the observed results were in good co-ordination with atomic absorption spectroscopic data.

Introduction

Heavy metal ions such as lead (Pb²⁺) and mercury (Hg²⁺) are the common toxicants [1,2,3], highly persistent in nature and their unique properties such as malleability, ductility, softness, resistance to corrosion and low melting point [4] have enhanced their usage for various industries like automobiles, ceramics, paints and plastics [5]. These metal ions are non-degradable [6] and are considered as potential hazardous substances by the Agency for Toxic Substances and Disease Registry (ATSDR) [7, 8]. Consumption of heavy metal ions contaminated water leads to acute and chronic poisoning with symptoms such as muscle pain, fatigue, abdominal pain, headache, convulsions, vomiting, coma and lethargy [9,10,11]. In view of these toxic effects, the World Health Organization (WHO), International Agency for Research and Cancer (IARC) and Bureau of Indian Standards (BIS) have limited the permissible limit of Pb²⁺ and Hg²⁺ ions as 50 and 1 μg L⁻¹ respectively [12].

Conventional methods such as atomic absorption spectroscopy (AAS) [13, 14], ion chromatography [15], chemiluminescence and inductively coupled plasma mass spectroscopy (ICP-MS) are being used to detect Pb²⁺ and Hg²⁺ ions contamination in water. All these techniques are limited due to expensive instrumentation, time-consuming process and requirement of a highly skilled man power [16,17,18]. To overcome these limitations, electrochemical biosensor is used due to its rapidity, portability, sensitivity, and ease of analysis [19,20,21,22].

To detect Pb²⁺ ions in water matrix, Illangovan [23] developed an urease inhibition-based biosensor with a linear range of 0.1–1 mM, but with a limited stability [23]. Shen et al. [24] have developed an electrochemical biosensor to detect Pb²⁺ ions using the modified gold electrode by attaching the oligonucleotides to gold nanoparticles. In spite of its lower detection limit (LOD) of 1 nM, background current lead to direct oxidation of nucleotides, which resulted in inaccurate estimation. Apart from urease, enzymes like invertase [25], horse radish peroxidase (HRP), tyrosinase, acetyl cholinesterase (AChE) and glucose oxidase (GOD) [26,27,28] were also utilized to develop biosensors to detect Pb²⁺ and Hg²⁺ ions. Various interfaces such as poly-o-phenylenediamine (PPD) [29], ZnO nanoparticles [26], poly neutral red (PNR)-modified carbon film, etc. were employed in various electrochemical biosensors. But, majority of these approaches have limitations in terms of response time, stability and precision. Hence, it is important to design and develop biosensor for the selective detection of Pb²⁺ and Hg²⁺ ions in water matrix with lower detection limit and high stability.

In this work, an electrochemical urease inhibition-based biosensor has been developed to detect Pb²⁺ and Hg²⁺ ions in water using ceria (CeO₂) nano-interface. The higher differences in isoelectric points (IEP) of urease (IEP = 4.9) and CeO₂ (IEP = 9.2) provided strong electrostatic interaction between enzyme and nano-interface [30]. The usage of CeO₂ nano-interface helped in enhancing direct electron transfer kinetics owing to its unique properties such as high surface to volume ratio, surface reactivity, adsorption rate and catalytic activity. Tuning potential window for selective detection of Pb²⁺ and Hg²⁺ ions at lower potentials is the major highlight of this work. Reliability of the developed Pt/CeO₂/urease electrode was proved by optimizing various experimental conditions for the selective amperometric detection of Pb²⁺ and Hg²⁺ ions in contaminated water samples.

Experimental

Chemicals and reagents

Urease (E.C.) from Canavalia ensiformis (E.C. 3.5.1.5, activity: 15 U mg⁻¹), cerium acetate dehydrate, sodium chloride, hydrosulfuric acid, sodium citrate, citric acid, lead nitrate, cadmium acetate, cobalt acetate, zinc chloride, mercuric sulphate, arsenic (III) iodide, urea, ethylenediaminetetraacetic acid (EDTA), nickel nitrate, iron chloride, calcium carbide, potassium acetate and nafion were procured from Sigma-Aldrich, India. All the studies were carried out at room temperature using deionized water of conductivity <1 μ mho, pH 5.6. Citrate buffer of 0.1 M with pH ranging from 4 to 10 were prepared by dissolving appropriate amount of sodium citrate and citric acid in 100 mL of deionized water.

Details on apparatus, synthesis and characterization of CeO₂ nanoparticles are included in the Electronic Supplementary Material (ESM) in sections 1 and 2.

Fabrication of Pt-modified working electrode

Pt electrode was polished using alumina powder and subsequently cleaned electrochemically. During this cleaning process, Pt electrode was scanned in the potential range of 0 to 0.8 V vs Ag/AgCl in 0.1 M H₂SO₄ at 0.1 Vs⁻¹. For the fabrication of modified Pt electrode, CeO₂ nanoparticles (0.1 mg mL⁻¹) were dispersed in deionized water and sonicated for 15 min. Then, 3 μL of CeO₂ dispersed solution was drop casted on to the Pt electrode. Subsequently, 100 μL of urease (0.1 U mL⁻¹) was dispersed along with 5 μL of nafion (0.5 (v/v)) solution and this mixture was coated on to the CeO₂embedded Pt electrode surface and air dried for 30 min (Fig. 1).

Fig. 1

Schematic representation of Pt electrode modification and inhibition of urease in the presence of Pb²⁺ or Hg²⁺ ions

Procedure for urea determination

The steady state of urea interaction with urease was measured in terms of current response recorded in the presence of 0.1 M citrate buffer (pH 6) in the range of −0.2 to 0.8 V vs Ag/AgCl. The concentration studies were performed by increasing the urea levels till saturation.

Electrochemical estimation of Pb²⁺ and Hg²⁺ ions

At the saturating concentration of urea, inhibitive response of Pb²⁺ and Hg²⁺ ions was evaluated based on the percentage inhibition (Eq. (1)). Besides, the analytical parameters of the developed sensor such as LOD, sensitivity and linear range were estimated. Reusability of the developed sensing element was studied in terms of reactivation (Eq. (2)) using 0.1 M EDTA. Finally, the fabricated electrode was used to detect Pb²⁺ and Hg²⁺ ions in Cauvery river water (filtered using a filter paper of 0.45 mm pore size) and the observed results were validated using AAS.

$$ \mathrm{Inhibition}\ \left( I\%\right)=\frac{\left({I}_0-{I}_i\right)}{I_0}\times 100 $$

(1)

where I ₀ and I _i represent the current response of urea on Pt/CeO₂/urease electrode without and with Pb²⁺ and Hg²⁺ ions.

$$ \mathrm{Reactivation}\ \left( R\%\right)=\frac{I_r-{I}_{p, \exp }}{I_{p,\mathrm{control}}-{I}_{p, \exp }}\times 100 $$

(2)

where I _r is the current response of urea on Pt/CeO₂/urease electrode with EDTA reactivation, I _p,exp. is the current response of urea on Pt/CeO₂/urease electrode with metal ion inhibition and I _p,control is the current response of urea on Pt/CeO₂/urease electrode.

Results and discussion

Electrochemical behaviour of the modified electrodes

The initial cyclic voltammetric experiments were conducted using 0.1 M citrate buffer solution with pH 7 as electrolyte. The responses of bare Pt (black), Pt/CeO₂ (red), Pt/urease (green) and Pt/CeO₂/urease (blue) electrodes in the presence of 1.0 μM of urea at a scan rate of 0.1 Vs⁻¹ are shown in Fig. 2a. Bare Pt electrode showed no redox peak and the modified electrodes exhibited an oxidation peak at 0 V vs Ag/AgCl, which corresponds to the decomposition of urea to ammonia, carbon dioxide and hydrazine. The reaction mechanism is as follows:

(3)

Fig. 2

a Cyclic voltammograms of bare and various modified Pt electrode in 0.1 M citrate buffer (pH 7), scan rate of 0.1 Vs⁻¹ containing 1.0 μM of urea and b CVs of Pt/CeO₂/urease in the presence of 0.1 M citrate buffer (pH 6), scan rate of 0.08 Vs⁻¹ with varying concentrations of urea

The oxidation current responses of various modified working electrodes were calculated and represented in the ESM in Table S1. Pt/urease, Pt/CeO₂ and Pt/CeO₂/urease electrodes vs Ag/AgCl exhibited anodic peak potential (E_pa) of −0.03, 0.01 and 0.05 V, respectively. Pt/urease, Pt/CeO₂ and Pt/CeO₂/urease electrodes showed a maximum anodic peak current (I_pa) of 3.51, 3.63 and 3.96 μA, respectively. This has confirmed the catalytic activity of modified electrode. Pt/CeO₂ also exhibited an oxidation peak owing to the dual reversible oxidation state of cerium Ce(III)/Ce(IV) redox couple and hence, urea get oxidized to ammonia, carbon dioxide and hydrazine. (Mechanism is given in the ESM in section 3). The lower electron transfer rate of Pt/CeO₂ was confirmed by its large full width half maximum (FWHM = 62.5 mV/nαA, where n is the number of electrons transferred (n = 1), α is the electron transfer coefficient (α = 0.5) and A is the area of Pt working electrode (0.0314 cm²)). The calculated electron transfer rate constant (K _s = I _pa/Q, where I _pa is the anodic peak current and Q is the amount of charge consumed) is given in Table S1 (see ESM). Considering increased current response, enhanced electron transfer of Pt/CeO₂/urease over Pt/urease and Pt/CeO₂, Pt/CeO₂/urease electrode was used for further electrochemical studies. The experimental conditions like electrolyte, pH, scan rate, urease loading efficiency and urea concentration were optimized and the related information is given in the ESM in section 4.

Determination of Pb²⁺ and Hg²⁺ ions by an inhibition process

The response of biosensor was recorded in 0.1 M citrate buffer (pH 6) at a scan rate of 0.08 Vs⁻¹. With an increase in urea concentration ranging from 0.1 to 1.8 μM, an increase in current response was observed at 0 V vs Ag/AgCl and reached saturation at 1.5 μM (Fig. 2b). To the saturated concentration of urea, addition of Pb²⁺ and Hg²⁺ ions of various concentrations (0.05, 0.08, 0.1, 0.5, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0 μM) lead to a decrease in current response (Fig. 3a). This trend might be due to the inhibition of active sites of urease by metal ions (see ESM, section 5). This inhibition is facilitated by the natural affinity between Ni metallic center of urease and soft base target ions (Pb²⁺ and Hg²⁺). As both the metal ions are selective towards urease (see ESM, Fig. S3A-D), the ability of the active sites to discriminate the particular metal ion from a mixture of metal ions has become crucial. Hence, to selectively detect the target ions, amperometric studies were carried out for the varying concentrations of Pb²⁺ (0.05 to 3 μM) and Hg²⁺ (0.05 to 3 μM) ions at various oxidation potentials (0, −0.03, 0.03, −0.05 and 0.05 V vs Ag/AgCl) in the presence of 1.5 μM of urea, 0.1 M of citrate buffer (pH 6). Similar to voltammetric studies, amperometric studies also exhibited a decreased current response with an increase in concentration of Pb²⁺ and Hg²⁺ ions. It was evident from Fig. 3b that amperogram at −0.03 V vs Ag/AgCl exhibited a 95.2% inhibition for Pb²⁺ and 97.1% for Hg²⁺ at 0 V vs Ag/AgCl (Fig. 3c). This variation in potentials was greatly influenced by the permeability of metal ions. Hence, the permeability of Ni²⁺, Fe³⁺, Co²⁺, Ca²⁺, Zn²⁺, K⁺, Cd²⁺, Cr³⁺, Cu²⁺, Pb²⁺, Hg²⁺ and As³⁺ ions (0.5 μM) was calculated using Eqs. (4) and (5) and presented in Fig. 3d.

$$ P\ \left[ S\right]\%\kern0.5em =\kern0.5em \frac{\mathrm{Slope}\ M\ at\ Pt/{CeO}_2/\mathrm{urease}}{\mathrm{Slope}\ M\ at\ bare\ Pt}\times 100 $$

(4)

Fig. 3

a Cyclic voltammograms of Pt/CeO₂/urease electrode in the presence of 1.5 μM of urea, citrate buffer (pH 7) by varying Pb²⁺ and Hg²⁺ ion concentrations; amperometric determination of b Pb²⁺ and c Hg²⁺ ions at various oxidation potentials, d permeability, e selectivity of Hg²⁺ and Pb²⁺ ions and f mechanism of urea and urease interaction in the absence and presence of Pb²⁺ and Hg²⁺ ions 

where M  =  Pb ²⁺ or Hg ²⁺ions

$$ P\ \left[ I\right]\%=\frac{I_{\mathrm{interfernt}\ }\left(0.5\;\upmu \mathrm{M}\right)\ \mathrm{at}\ Pt/{CeO}_2/\mathrm{urease}\kern0.5em }{I_{\mathrm{interferent}\ }\left(0.5\;\upmu \mathrm{M}\right)\ \mathrm{at}\ \mathrm{bare}\ Pt}\times 100 $$

(5)

Permeabilities of Pb²⁺ and Hg²⁺ ions were observed to be 4 and 1.2 times greater than that of Fe³⁺, Co²⁺, Ca²⁺, Zn²⁺, K⁺, Cd²⁺, Cr³⁺ and As³⁺ ions at −0.03 and 0 V vs Ag/AgCl, respectively. Also, amperometric selectivity coefficient (Eq. (6)) and response ratio (Eq. (7)) of mixed metal ion solutions (Table 1) were evaluated at −0.03 and 0 V vs Ag/AgCl.

$$ \mathrm{Amperometric}\ \mathrm{selectivity}\ \mathrm{coefficient}\left({K}_{i, j}^{amp}\right)\kern0.5em =\kern0.5em \frac{\Delta {I}_j}{\Delta {I}_t-\Delta {I}_j}\kern0.5em \times \kern0.75em \frac{\Delta {C}_i}{\Delta {C}_j} $$

(6)

$$ \mathrm{Response}\ \mathrm{ratio}\ \left({R}_{i, j}\right)=\frac{\Delta {I}_j}{\Delta {I}_i} $$

(7)

Table 1 Interference studies of mixed and separate solution response

where △I _i is the amperometric current response of analyte solution, △I _j is the amperometric current response of interfering cation solution, △I _t is the amperometric current response of the mixed solution, C _i is the concentration of analyte of interest and C _j is the concentration of interfering cation.

The lower values of amperometric selectivity coefficient and response ratio of Pt/CeO₂/urease electrode at −0.03 V vs Ag/AgCl in the presence of interferents confirmed the selectivity towards Pb²⁺ ions. Similarly at 0 V vs Ag/AgCl, the amperometric selectivity coefficient and response ratio of Pt/CeO₂/urease electrode were observed to be very low including Pb²⁺ ions. The estimated analytical parameters of Pb²⁺ and Hg²⁺ ion biosensor are presented in Table 2.

Table 2 Response characteristics of Pt/CeO₂/urease biosensor

Interferent study

Selective nature of Pb²⁺ and Hg²⁺ towards Pt/CeO₂/urease electrode was tested in the presence of other metals namely Fe³⁺, Co²⁺, Ca²⁺, Zn²⁺, K⁺, Cd²⁺, Cr³⁺, As³⁺ and Ni²⁺ (0.5 μM) (Fig. 3e). All these metal ions showed an inhibition of less than 30%, which confirmed the selectivity of the developed sensor towards Pb²⁺ (2.2 μM) and Hg²⁺ (0.8 μM) ions. The ultra-low potential of 0 and −0.03 V vs Ag/AgCl offered by Pt/CeO₂/urease electrode helped in avoiding cross reactivity. Besides, amperometric selective coefficients obtained for both the Pb²⁺ and Hg²⁺ ions are in the magnitudes of 10⁻³ demonstrated anti-interferent nature of the Pt/CeO₂/urease electrode.

Mechanism of inhibition and kinetics

Urease is a metallo enzyme rich in cysteine residues with α-subunit containing a binuclear nickel center [31]. While urease mobile flap is in open conformation, urea binds at the active site and changes to a closed conformation. In this case, when urea comes closer to urease, carbon atom of urea comes into close proximity to nickel-binding ligand forming nucleophiles by polarizing C-O and C-NH₂ bonds. In closed conformation, histidine at hydrogen bonding distance forms nascent ammonia and breaks C-N bond which resulted in open conformation [32] (Fig. 3f).

The binding of Pb²⁺ and Hg²⁺ ions at the active sites of urease is based on the hard and soft acids and bases concept (HSAB). These ions belong to class B prefers specifically binding to soft donors such as sulphur, nitrogen and oxygen in a preferential order S > N > O. Once Pb²⁺ or Hg²⁺ binds at the active site, the open flap changes its conformation to closed conformation by blocking urea [1] and hence a decreased current response was observed with an increase in Pb²⁺ and Hg²⁺ concentration.

Since the concentration of urea, Pb²⁺ and Hg²⁺ has a great influence on sensor performance, inhibition kinetics of urease in the presence of Pb²⁺ as well as Hg²⁺ were evaluated. The decreased activity of urease was a result of competitive, mixed inhibition and the same was confirmed from the Lineweaver-Burk plot for Pb²⁺ (Fig. 4a) and Hg²⁺ (Fig. 4b) respectively. Varying concentrations of Pb²⁺ showed a linear trend over the inverse of anodic peak current and the extrapolated linear plot intersected on Y-axis, which revealed the value of I _max. The K _M values for the concentrations of 0, 0.05, 0.08 and 1.0 μM were found to be 2.419, 2.6, 3.125 and 3.3 μM respectively. This trend proved the competitive inhibition between Pb²⁺ and urea. In the case of Hg²⁺, extrapolated linear plot intersected at the negative co-ordinates of X- and Y-axes with decreased I _max and increased K _M values revealed the mixed inhibition by Hg²⁺ ions.

Fig. 4

Lineweaver-Burk plot of a Pb²⁺ indicating the competitive inhibition, b Hg²⁺ indicating mixed inhibition, c Dixon plot of Pb²⁺ inhibition, d Dixon plot of Hg²⁺ inhibition and e operational stability of Pt/CeO₂/urease in the presence of Pb²⁺ and Hg²⁺ ions

The Dixon plot (1/I _pa vs lead concentration) at various urea concentrations (0.1, 0.5, 1 μM) showed a linear regression with R ² = 0.99 and inhibition constant (K _i) of 0.58 and 0.72 μM, respectively, for Pb²⁺ (Fig. 4c) and Hg²⁺ ions (Fig. 4d) (Table 2). This lower inhibition constant revealed the stronger binding force between the target ions and urease-urea complex. The estimated ‘n’ value (>1) from Hill plot confirmed the positive co-operativity for both Pb²⁺ and Hg²⁺ ions with R ² = 0.98.

Evaluation of Pt/CeO₂/urease-modified electrode

• (a) Reactivation studies: To carry out the reactivation studies, Pt/CeO₂/urease electrode was immersed in 0.1 M EDTA reactivator for 10 min to remove metal ions from the active sites of urease. The EDTA-treated Pt/CeO₂/urease electrode showed a reactivation of 97.25 and 95.02% towards Pb²⁺ and Hg²⁺ respectively.

• (b) Repeatability and reproducibility (precision): To evaluate repeatability and reproducibility, five modified Pt/CeO₂/urease electrodes were fabricated to detect Pb²⁺ and Hg²⁺ ions in 0.1 M citrate buffer (pH 6) containing 1.5 μM of urea. Intra-assay (n = 5) of Pt/CeO₂/urease electrode showed an RSD of 0.02 and 0.123% for Pb²⁺ and Hg²⁺ ions, respectively. Inter-assay (n = 5) showed an RSD of 0.057 and 0.283% Pb²⁺ and Hg²⁺ ions respectively. Since RSD was <5%, it can be concluded that the fabricated Pt/CeO₂/urease electrode can be reproduced and deployed for water sample analysis.

• (c) Accuracy: accuracy of Pt/CeO₂/urease electrode was tested for various concentrations of Pb²⁺ ions (0.5, 0.8, 1.2, 1.8 and 2.2 μM). The predicted Pb²⁺ concentration ± relative error (n = 5) was calculated as 0.5 ± 0.038, 0.8 ± 0.023, 1.2 ± 0.015, 1.8 ± 0.010 and 2.2 ± 0.008 μM respectively. Similarly for Hg²⁺ ions (0.2, 0.35, 0.5, 0.65 and 0.8 μM), the predictive values were estimated as 0.2 ± 0.09, 0.35 ± 0.05, 0.5 ± 0.036, 0.65 ± 0.027 and 0.8 ± 0.022 μM respectively. The detailed accuracy estimation is presented in the ESM in section 6.

• (d) Stability of Pt/CeO₂/urease electrode: the operational stability (Eq. ((8)) of the developed Pt/CeO₂/urease electrode in the presence of 1.5 μM of urea, 1 μM of Pb²⁺ and Hg²⁺ ions was examined for a period of 20 days at room temperature (Fig. 4e). It exhibited an operational stability of 92.94 and 86.7% for Pb²⁺ and Hg²⁺ ions respectively.

$$ \%\mathrm{Stability}\kern0.5em =\kern0.5em 100\kern0.5em \left[\frac{I_n-{I}_0}{I_0}\right]\times 100 $$

(8)

where I ₀ is the current measured on the first day and I _n on ‘nth’ day.

Real sample analysis and validation

In India, Cauvery river is considered to be one of the fresh water bodies covering an area of around 72,000 km² and has many tributaries. But, in recent years, due to release of untreated effluent from industries and waste disposal, the metal ion concentration has increased [33, 34].

Towards the detection of Pb²⁺ and Hg²⁺ ions in Cauvery river water (TDS = 318 ppm, pH 6.2 and conductivity of 5.5 × 10⁻⁶ S m⁻¹) (Tables 3 and 4), standard addition method was adopted. The acceptable ranges of 91.63–107.09 and 90.00–103.00% were obtained for Pb²⁺ and Hg²⁺ ions respectively (Tables 3 and 4). The validation study was carried out using AAS and the results are given in Table 5.

Table 3 Recovery studies of Pb²⁺ Cauvery river water

Table 4 Recovery studies of Hg²⁺ in Cauvery river water

Table 5 Determination of Pb²⁺ and Hg²⁺ in Cauvery river water using the developed sensor and AAS

As the amount of Pb²⁺ and Hg²⁺ ions were present in 100 μL of 100-fold diluted metal ion samples, the concentration of Pb²⁺ and Hg²⁺ ions were estimated using the following equations

$$ \mathrm{Metal}\ \mathrm{ion}\ \mathrm{concentration}\ \left(\upmu \mathrm{g}/\upmu \mathrm{L}\right)\kern0.5em =\kern0.5em \frac{\mathrm{Current}\ \left(\upmu \mathrm{A}\right)\times \mathrm{dilution}\ \mathrm{factor}}{\mathrm{slope}\left(\frac{\upmu \mathrm{A}}{\upmu \mathrm{g}}\right)\times \mathrm{volume}\ \mathrm{of}\ \mathrm{diluted}\ \mathrm{metal}\ \mathrm{ion}\ \mathrm{used}\ \mathrm{for}\ \mathrm{the}\ \mathrm{assay}\left(\upmu \mathrm{L}\right)} $$

(9)

$$ \mathrm{Recovery}\ \left(\%\right)\kern0.5em =\kern0.5em \frac{\mathrm{Mean}\ \mathrm{metal}\ \mathrm{ion}\ \mathrm{detected}}{\mathrm{Metal}\ \mathrm{ion}\ \mathrm{added}\ \mathrm{for}\ \mathrm{the}\ \mathrm{assay}}\kern0.5em \times \kern0.5em 100 $$

(10)

where dilution factor is 100 and volume of diluted metal ion used for the assay is 100 μL.

Also, the observed results were in good agreement with AAS data. The performance comparison of the developed sensor with the available sensors (Table 6) highlights the superior performance in terms of detection limit, linear range and response time of Pt/CeO₂/urease electrode.

Table 6 Comparison of analytical characteristics of the developed Pt/CeO₂/urease sensor with existing biosensors in detecting Pb²⁺ and Hg²⁺ ions

Conclusion

This work highlights the fabrication of Pt/CeO₂/urease electrode for the selective detection of Pb²⁺ and Hg²⁺ ions using amperometry. First time, the permeability of Pb²⁺ and Hg²⁺ ions with respect to the applied potential was measured, which could enable a specific potential based discrimination of Pb²⁺ and Hg²⁺ ions in a mixed solution. The ability of the developed electrode to work at lower potentials enabled it to be anti-interferent. The detection limit of the developed sensor towards Pb²⁺ and Hg²⁺ ions is less than WHO limit. In addition, lower response time has proved Pt/CeO₂/urease to be a reliable sensing platform to detect Pb²⁺ and Hg²⁺ ions. The results obtained in quantification of Pb²⁺ and Hg²⁺ ions using the developed Pt/CeO₂/urease electrode are in good agreement with the AAS data. Hence, the developed electrode can be deployed to detect Pb²⁺ and Hg²⁺ ions in water bodies.