Development of an amperometric assay for phosphate ions in urine based on a chemically modified screen-printed carbon electrode

An amperometric assay for the determination of inorganic phosphate (Pi) in urine has been developed without the need for sample preparation. A screen-printed carbon electrode modified with the electrocatalyst cobalt phthalocyanine (CoPC–SPCE) and covered with a cellulose acetate membrane (CAM) serves as the sensor. The sensor detects hydrogen peroxide (H₂O₂), which is produced as a result of the oxidative decarboxylation of pyruvate, catalyzed by pyruvate oxidase (PyOd), in the presence of Pi, oxygen, and cofactors. Following optimization of solution conditions, and in the presence of a urine sample, a linear range was found to exist between the rate of current increase and phosphate concentration over the range of 2.27 × 10⁻⁵ to 1.81 × 10⁻⁴ M, and the limit of detection was found to be 4.27 × 10⁻⁶ M. The assay was applied to the determination of phosphate ions in the urine of a normal subject, and the mean concentration in unspiked urine was found to be 3.40 × 10⁻⁵ M with a coefficient of variation of 8.0% (n = 5). The mean recovery of phosphate added to urine samples was 98.7% with a coefficient of variation of 5.5% (n = 3). To the authors’ knowledge, this is the first report of an amperometric assay for Pi that incorporates a CoPC–SPCE as the sensing device.     

Amperometric choline biosensor based on multiwalled carbon nanotubes/zirconium oxide nanoparticles electrodeposited on glassy carbon electrode

Perturbation of the tubulin/microtubule dynamic in cells is perhaps the single most important mode of action of anticancer drugs. Standard methods for identifying and evaluating compounds for their ability to alter tubulin polymerization are low throughput, labor intensive, expensive, or make their assessment in vitro. Here we report a method to rapidly quantify the extent of tubulin polymerization in whole cells using flow cytometry, and we use this technique to evaluate compounds that stabilize and destabilize microtubule formation. This facile method is useful for conveniently, quantitatively, and cost-effectively comparing small molecules that perturb tubulin polymerization.     

Simultaneous voltammetric determination of norepinephrine, ascorbic acid and uric acid on polycalconcarboxylic acid modified glassy carbon electrode

A novel polycalconcarboxylic acid (CCA) modified glassy carbon electrode (GCE) was fabricated by electropolymerization and then successfully used to simultaneously determine ascorbic acid (AA), norepinephrine (NE) and uric acid (UA). The characterization of electrochemically synthesized Poly-CCA film was investigated by atomic force microscopy (AFM), electrochemical impedance spectroscopy (EIS) and voltammetric methods. It was found that the electrochemical behavior of the polymer-modified electrode depended on film thickness, i.e., the electropylmyerization time. Based on the electrochemical data, the charge transfer coefficient (alpha) and the surface coverage (Gamma) were calculated. This poly-CCA modified GCE could reduce the overpotential of ascorbic acid (AA), norepinephrine (NE) and uric acid (UA) oxidation in phosphate buffer solution (pH 6.0), while it increases the peak current significantly. The current peak separations of AA/NE, NE/UA and AA/UA on this modified electrode are 91mV, 256mV and 390mV in CV at 100mVs(-1), respectively. Therefore, the voltammetric responses of these three compounds can be well resolved on the polymer-modified electrode, and simultaneously determination of these three compounds can be achieved. In addition, this modified electrode can be successfully applied to determine AA and NE in injection and UA in urine samples without interferences.     

Ultrasensitive DNA sensor based on gold nanoparticles/reduced graphene oxide/glassy carbon electrode

We have designed a simple and novel electrochemical biosensor based on glassy carbon electrode (GCE) for DNA detection. GCE was modified with reduced graphene oxide (RGO) and gold nanoparticles (AuNPs) by the electrochemical method, which is helpful for immobilization of thiolated bioreceptors. The electrode modification processes were characterized by scanning electron microscopy (SEM) and electrochemical methods. Then a single-stranded DNA (ssDNA) probe for BRCA1 5382 insC mutation detection was immobilized on the modified electrode for a specific time. The experimental conditions, such as probe immobilization time and target DNA (complementary DNA) hybridization time and temperature with probe DNA, were optimized using electrochemical methods. The electrochemical response for DNA hybridization and synthesis was measured using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) methods. The calibration graph contains two linear ranges; the first part is in the range of 3.0 × 10⁻²⁰ to 1.0 × 10⁻¹² M, and the second segment part is in the range of 1.0 × 10⁻¹² to 1.0 × 10⁻⁷ M. The biosensor showed excellent selectivity for the detection of the complementary sequences from noncomplementary sequences, so it can be used for detection of breast cancer.     

Amperometric and impedimetric characterization of a glutamate biosensor based on Nafion and a methyl viologen modified glassy carbon electrode

An electrochemical biosensor based on a glassy carbon (GC) electrode chemically modified with the perfluorinated cation-exchange polymer Nafion and methyl viologen (MV) is described. The enzyme was immobilized by cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA), methyl viologen and Nafion. Operating variables such as the enzyme/BSA ratio, cross-linking time in glutaraldehyde vapor, methyl viologen and Nafion percentages were investigated with regard to their influence on the biosensor sensitivity by using glucose oxidase as the enzyme model due to its high stability and low cost. The glutamate biosensor was elaborated by using optimized parameters and its electrochemical properties were investigated by cyclic voltammetry, amperometry and by electrochemical impedance spectroscopy. The glutamate biosensor shows a detection limit of 20 microM and a linear range extended to 0.75 mM. Its selectivity was tested with 15 different amino acids, each with a concentration of 20 microM, 25 microM acetaminophen, 20 microM uric acid and 200 microM ascorbic acid. No amperometric response was observed for the interfering species. This good selectivity allows glutamate detection in biological media without previous separation of the analyte.     

An electrochemical sensor for detection of laccase activities from Penicillium simplicissimum in compost based on carbon nanotubes modified glassy carbon electrode

An electrochemical sensor for detection of the activity of laccase from Penicillium simplicissimum isolated from the composting has been developed. The sensor is based on glassy carbon electrode modified with multi-wall carbon nanotubes (CNTs). The introduction of CNTs into this system can greatly enhance the electrochemical signal in this assay more sensitively, selectively and rapidly than that in conventional spectrophotometric assays. It was found that the optimal pH value of the electrolyte was 5.6. The results showed a good linear correlation between the current and the concentration of laccase activities measured by spectrophotometry, where the current slope was measured by chronoamperometry with a coefficient of 0.9835. Therefore, this electrochemical sensor can be used for rapid detection of laccase activity from P. simplicissimum. Furthermore, it may be potentially used for rapid quantification of P. simplicissimum according to the relationship between the laccase activities and the biomass.     

Fabrication of chronocoulometric DNA sensor based on gold nanoparticles/poly(l-lysine) modified glassy carbon electrode

In this work, we fabricated a sensitivity chronocoulometric DNA sensor (CDS) based on gold nanoparticles (AuNPs)/poly(l-lysine) complex film modified glassy carbon electrode. Hexaammineruthenium(III) chloride ([Ru(NH₃)₆]³⁺) was used as the electroactive indicator. The assembled process was investigated by cyclic voltammetry (CV) and chronocoulometry (CC). CC is used to monitor the DNA hybridization event by measurement of electrostatic binding [Ru(NH₃)₆]³⁺. Under the optimal conditions, the signal of [Ru(NH₃)₆]³⁺ was linear with the logarithm of the concentration of the complementary oligonucleotides from 1.0 × 10⁻¹³ to 1.0 × 10⁻¹¹ M, and the detection limit is 3.5 × 10⁻¹⁴ M.     

An electrochemical sensor for determination of calcium dobesilate based on PoPD/MWNTs composite film modified glassy carbon electrode

A poly-o-phenylenediamine and multi-wall carbon nanotubes composite (PoPD/MWNTs) modified glassy carbon electrode (GCE) was prepared by in situ electropolymerization using an ionic surfactant as the supporting electrolyte. The morphology of the resulting PoPD/MWNTs composite was characterized by TEM and the electrochemical properties of the modified electrode were characterized by cyclic voltammetry. The electrochemical behavior of calcium dobesilate on PoPD/MWNTs modified electrode was also investigated. The large current response of calcium dobesilate on PoPD/MWNTs modified electrode is probably caused by the synergistic effect of the electrocatalytic property of PoPD and MWNTs. The reductive peak current increased linearly with the concentration of calcium dobesilate in the range of 0.1–1.0 μmol/L and 4.0–400 μmol/L by square wave adsorptive stripping voltammetry, respectively. The detection limit (three times the signal blank/slope) was 0.035 μmol/L. The modified electrode could eliminate the interference of dopamine, norepinephrine and epinephrine at 100-, 90- and 70-fold concentration of 1.0 μmol/L calcium dobesilate, respectively. The proposed modified electrode provides a new promising and alternative way to detect calcium dobesilate.     

Optical ascorbic acid sensor based on the fluorescence quenching of silver nanoparticles

A sensitive and selective fluorimetric sensor for the assay of ascorbic acid (AA) using silver nanoparticles as emission reagent was investigated. In this study, silver nanoparticles were prepared based on aqueous–gaseous phase reaction of silver nitrate solution and ammonia gas. The nanoparticles were water‐soluble, stable and had a narrow emission band. They were used as a fluorescence probe for the assay of ascorbic acid on its quenching effect on the emission of silver nanoparticles. The principal reason for quenching is likely to be a complexation between ascorbic acid and silver nanoparticles. The quenching mechanism was established by Stern–Volmer law. Under the optimum conditions, the quenched fluorescence intensity was linear with the concentration of ascorbic acid in the range of 4.1 × 10^?6 to 1.0 ×10^?4 m (r = 0.9985) with a detection limit of 1.0 × 10^?7 m . The RSD for repeatability of the sensor for the assay of ascorbic acid concentration of 3.0 × 10^?5 and 4.0 × 10^?6 m was found to be 1.5 and 1.3%, respectively. The proposed method was applied to the determination of ascorbic acid in vegetables and vitamin C tablets. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of DNA electrochemical biosensor based on immobilization of ssDNA on the surface of nickel oxide nanoparticles modified glassy carbon electrode

A sensitive electrochemical method for DNA hybridization based on immobilization of DNA probe and [Ru(NH₃)₅Cl]PF₆ complex onto nickel oxide nanomaterials (NiOx_np) modified glassy carbon electrode was developed. Due to strong affinity of NiOx_np for phosphate groups, oligonucleotides probe with a terminal 5′-phosphate group was attached to the surface of the modified electrode. DNA immobilization and hybridization were characterized by electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry using K₃Fe(CN)₆/K₄Fe(CN)₆ and [Ru(NH₃)₅Cl]PF₆ as probe and indicator, respectively. The Ru-complex current response indicates only the complementary sequence showing an obvious current signal in comparison to non-complementary and three or single point mismatched sequences. The fabricated biosensor possessed good selectivity and sensitivity for complementary probe, taxon: 32630 tumor necrosis factor (TNF). The linear dynamic range, sensitivity and detection limit of the proposed biosensor were 4 × 10⁻¹⁰ M to 1 × 10⁻⁸ M, 34.32 nA nM⁻¹ and 6.8 × 10⁻¹¹ M, respectively. Excellent reproducibility and stability, quite simple and inexpensive preparation are the other advantages of proposed biosensor.     

Selective electrochemical sensor for folic acid at physiological pH using ultrathin electropolymerized film of functionalized thiadiazole modified glassy carbon electrode

This paper demonstrated the selective determination of folic acid (FA) in the presence of important physiological interferents, ascorbic acid (AA) and uric acid (UA) at physiological pH using electropolymerized film of 5-amino-2-mercapto-1,3,4-thiadiazole (p-AMT) modified glassy carbon (GC) electrode. Bare GC electrode fails to determine the concentration of FA in the presence of AA and UA due to the surface fouling caused by the oxidized products of AA and FA. However, the p-AMT film modified electrode not only separates the voltammetric signals of AA, UA and FA with potential differences of 170 and 410 mV between AA–UA and UA–FA, respectively but also shows higher oxidation current for these analytes. The p-AMT film modified electrode displays an excellent selectivity towards the determination of FA even in the presence of 200-fold AA and 100-fold UA. Using amperometric method, we achieved the lowest detection of 75 nM UA and 100 nM each AA and FA. The amperometric current response was increased linearly with increasing FA concentration in the range of 1.0 × 10⁻⁷–8.0 × 10⁻⁴ M and the detection limit was found to be 2.3 × 10⁻¹⁰ M (S/N = 3). The practical application of the present modified electrode was successfully demonstrated by determining the concentration of FA in human blood serum samples.     

Amperometric hydrogen peroxide biosensor based on the immobilization of HRP on nano-Au/Thi/poly (p-aminobenzene sulfonic acid)-modified glassy carbon electrode

A novel hydrogen peroxide biosensor was fabricated for the determination of H(2)O(2). The precursor film was first electropolymerized on the glassy carbon electrode with p-aminobenzene sulfonic acid (p-ABSA) by cyclic voltammetry (CV). Then thionine (Thi) was adsorbed to the film to form a composite membrane, which yielded an interface containing amine groups to assemble gold nanoparticles (nano-Au) layer for immobilization of horseradish peroxidase (HRP). The electrochemical characteristics of the biosensor were studied by CV and chronoamperometry. The factors influencing the performance of the resulting biosensor were studied in detail. The biosensor responded to H(2)O(2) in the linear range from 2.6 x 10(-6) mol/L to 8.8 x 10(-3) mol/L with a detection limit of 6.4 x 10(-7) mol/L. Moreover, the studied biosensor exhibited good accuracy and high sensitivity. The proposed method was economical and efficient, making it potentially attractive for the application to real sample analysis.     

Amperometric hydrogen peroxide biosensor based on the immobilization of HRP on nano-Au/Thi/poly (p-aminobenzene sulfonic acid)-modified glassy carbon electrode

A novel hydrogen peroxide biosensor was fabricated for the determination of H₂O₂. The precursor film was first electropolymerized on the glassy carbon electrode with p-aminobenzene sulfonic acid (p-ABSA) by cyclic voltammetry (CV). Then thionine (Thi) was adsorbed to the film to form a composite membrane, which yielded an interface containing amine groups to assemble gold nanoparticles (nano-Au) layer for immobilization of horseradish peroxidase (HRP). The electrochemical characteristics of the biosensor were studied by CV and chronoamperometry. The factors influencing the performance of the resulting biosensor were studied in detail. The biosensor responded to H₂O₂ in the linear range from 2.6 × 10^− 6 mol/L to 8.8 × 10^− 3 mol/L with a detection limit of 6.4 × 10^− 7 mol/L. Moreover, the studied biosensor exhibited good accuracy and high sensitivity. The proposed method was economical and efficient, making it potentially attractive for the application to real sample analysis.     

A new scheme of hybridization based on the Au(nano)-DNA modified glassy carbon electrode

DNA hybridization on the Au(nano)-DNA modified glassy carbon electrode (GCE) was investigated. The thiol modified probe oligonucleotides (SH-ssDNA) at the 5' phosphate end were assembled on the Au(nano)-DNA modified GCE surface. The electrochemical response of the probe immobilization and hybridization with target DNA was measured by differential pulse voltammetry (DPV) using methylene blue (MB) as the electroactive indicator. Gold nanoparticles can be dispersed effectively on the GCE surface in the presence of calf thymus DNA. Au(nano)-DNA modified GCE could greatly increase the active sites and enhance the response signal during immobilization and hybridization. The hybridization amount of target DNA could be greatly increased. The linear detection range of Au(nano)-DNA electrode for the complementary 21-mer oligonucleotide (cDNA) was achieved from 1.52 x 10(-10) to 4.05 x 10(-8) mol L(-1). The detection limit could reach the concentration of 10(-10) mol/L.     

Glucose biosensor based on immobilization of glucose oxidase in poly(o-aminophenol) film on polypyrrole-Pt nanocomposite modified glassy carbon electrode

Novel Pt nanoclusters embedded polypyrrole nanowires (PPy-Pt) composite was electrosynthesized on a glassy carbon electrode, denoted as PPy-Pt/GCE. A glucose biosensor was further fabricated based on immobilization of glucose oxidase (GOD) in an electropolymerized non-conducting poly(o-aminophenol) (POAP) film that was deposited on the PPy-Pt/GCE. The morphologies of the PPy nanowires and PPy-Pt nanocomposite were characterized by field emission scanning electron microscope (FE-SEM). Effect of experimental conditions involving the cycle numbers for POAP deposition and Pt nanoclusters deposition, applied potential used in glucose determination, temperature and pH value of the detection solution were investigated for optimization. The biosensor exhibited an excellent current response to glucose over a wide linear range from 1.5 × 10⁻⁶ to 1.3 × 10⁻² M (r = 0.9982) with a detection limit of 4.5 × 10⁻⁷ M (s/n = 3). Based on the combination of permselectivity of the POAP and the PPy films, the sensor had good anti-interference ability to ascorbic acid (AA), uric acid (UA) and acetaminophen. The apparent Michaelis–Menten constant (K_m) and the maximum current density (I_m) were estimated to be 23.9 mM and 378 μA/cm², respectively. In addition, the biosensor had also good sensitivity, stability and reproducibility.     

Electrochemical DNA biosensor based on silver nanoparticles/poly(3-(3-pyridyl) acrylic acid)/carbon nanotubes modified electrode

In this work, we present an electrochemical DNA sensor based on silver nanoparticles/poly(trans-3-(3-pyridyl) acrylic acid) (PPAA)/multiwalled carbon nanotubes with carboxyl groups (MWCNTs-COOH) modified glassy carbon electrode (GCE). The polymer film was electropolymerized onto MWCNTs-COOH modified electrode by cyclic voltammetry (CV), and then silver nanoparticles were electrodeposited on the surface of PPAA/MWCNTs-COOH composite film. Thiol group end single-stranded DNA (HS-ssDNA) probe was easily covalently linked onto the surface of silver nanoparticles through a 5′ thiol linker. The DNA hybridization events were monitored based on the signal of the intercalated adriamycin by differential pulse voltammetry (DPV). Based on the response of adriamycin, only the complementary oligonucleotides gave an obvious current signal compared with the three-base mismatched and noncomplementary oligonucleotides. Under the optimal conditions, the increase of reduction peak current of adriamycin was linear with the logarithm of the concentration of the complementary oligonucleotides from 9.0 × 10⁻¹² to 9.0 × 10⁻⁹ M with a detection limit of 3.2 × 10⁻¹² M. In addition, this DNA sensor exhibited an excellent reproducibility and stability during DNA hybridization assay.     

Amperometric glucose biosensor based on multilayer films via layer-by-layer self-assembly of multi-wall carbon nanotubes, gold nanoparticles and glucose oxidase on the Pt electrode

A novel amperometric glucose biosensor based on the nine layers of multilayer films composed of multi-wall carbon nanotubes (MWCNTs), gold nanoparticles (GNp) and glucose oxidase (GOD) was developed for the specific detection of glucose. MWCNTs were chemically modified with the H₂SO₄–HNO₃ pretreatment to introduce carboxyl groups which were used to interact with the amino groups of poly(allylamine) (PAA) and cysteamine via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide cross-linking reaction, respectively. A cleaned Pt electrode was immersed in PAA, MWCNTs, cysteamine and GNp, respectively, followed by the adsorption of GOD, assembling the one layer of multilayer films on the surface of Pt electrode (GOD/GNp/MWCNTs/Pt electrode). Repeating the above process could assemble different layers of multilayer films on the Pt electrode. PBS washing was applied at the end of each assembly deposition for dissociating the weak adsorption. Film assembling and characterization were studied by transmission electron microscopy and quartz crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The marked electrocatalytic activity of Pt electrode based on multilayer films toward H₂O₂ produced during GOD enzymatic reactions with glucose permitted effective low-potential amperometric measurement of glucose. Taking the sensitivity and selectivity into consideration, the applied potential of 0.35 V versus Ag/AgCl was chosen for the oxidation detection of H₂O₂ in this work. Among the resulting glucose biosensors, the biosensor based on nine layers of multilayer films was best. It showed a wide linear range of 0.1–10 mM glucose, with a remarkable sensitivity of 2.527 μA/mM, a detection limit of 6.7 μM estimated at a signal-to-noise ratio of 3 and fast response time (within 7 s). Moreover, it exhibited good reproducibility, long-term stability and the negligible interferences of ascorbic acid, uric acid and acetaminophen. The study can provide a feasible approach on developing new kinds of oxidase-based amperometric biosensors, and can be used as an illustration for constructing various hybrid structures.     

Amperometric immunosensor based on polypyrrole/poly(m-pheylenediamine) multilayer on glassy carbon electrode for cytokinin N6-(Delta2-isopentenyl) adenosine assay

A novel immunosensor based on a multilayer-coated glassy carbon electrode was designed to determine isopentenyl adenosine (iPA) in plants. The multilayer consists of polypyrrole and poly(m-phenylenediamine) with K4Fe(CN)6 and horseradish peroxidase (HRP) entrapped during electropolymerization. The ferrocyanide doped in polypyrrole functions as the mediator. The glucose oxidase bound on the immunosensor by the competitive immunoreaction involving iPA catalyzed the oxidation of the added glucose with the formation of H2O2, which is in turn reduced in the presence of HRP entrapped in poly(m-phenylenediamine). The current of the oxidized production of ferrocyanide reduced at -50 mV is inversely proportional to the concentration of iPA in the competitive immunoreaction. This immunosensor is able to be used about 40 times; after that its surface can be regenerated for a new immunosensor assembly by washing with 0.1M citrate-phosphate buffer (pH 4.6). The percentage of current response reduction (CR%) (y) is linearly related to the logarithm of the concentration of iPA (x) in the 5-300 microg/ml range, with a regression equation of the form y = 42.13x - 27.79 and a correlation coefficient of 0.9861. Five hybrid rice grain samples were analyzed with results in satisfactory agreement to those obtained by high-performance liquid chromatography.     

Glucose biosensor based on immobilization of glucose oxidase in poly(o-aminophenol) film on polypyrrole-Pt nanocomposite modified glassy carbon electrode

Novel Pt nanoclusters embedded polypyrrole nanowires (PPy-Pt) composite was electrosynthesized on a glassy carbon electrode, denoted as PPy-Pt/GCE. A glucose biosensor was further fabricated based on immobilization of glucose oxidase (GOD) in an electropolymerized non-conducting poly(o-aminophenol) (POAP) film that was deposited on the PPy-Pt/GCE. The morphologies of the PPy nanowires and PPy-Pt nanocomposite were characterized by field emission scanning electron microscope (FE-SEM). Effect of experimental conditions involving the cycle numbers for POAP deposition and Pt nanoclusters deposition, applied potential used in glucose determination, temperature and pH value of the detection solution were investigated for optimization. The biosensor exhibited an excellent current response to glucose over a wide linear range from 1.5 × 10⁻⁶ to 1.3 × 10⁻² M (r = 0.9982) with a detection limit of 4.5 × 10⁻⁷ M (s/n = 3). Based on the combination of permselectivity of the POAP and the PPy films, the sensor had good anti-interference ability to ascorbic acid (AA), uric acid (UA) and acetaminophen. The apparent Michaelis–Menten constant (K_m) and the maximum current density (I_m) were estimated to be 23.9 mM and 378 μA/cm², respectively. In addition, the biosensor had also good sensitivity, stability and reproducibility.     

An amperometric biosensor based on horseradish peroxidase immobilized onto maize tassel-multi-walled carbon nanotubes modified glassy carbon electrode for determination of heavy metal ions in aqueous solution

A biosensor for trace metal ions based on horseradish peroxidase (HRP) immobilized on maize tassel-multiwalled carbon nanotube (MT-MWCNT) through electrostatic interactions is described herein. The biosensor was characterized using Fourier transform infrared (FTIR), UV–vis spectrometry, voltammetric and amperometric methods. The FTIR and UV–vis results inferred that HRP was not denatured during its immobilization on MT-MWCNT composite. The biosensing principle was based on the determination of the cathodic responses of the immobilized HRP to H₂O₂, before and after incubation in trace metal standard solutions. Under optimum conditions, the inhibition rates of trace metals were proportional to their concentrations in the range of 0.092–0.55 mg L⁻¹, 0.068–2 mg L⁻¹ for Pb²⁺ and Cu²⁺ respectively. The limits of detection were 2.5 μg L⁻¹ for Pb²⁺ and 4.2 μg L⁻¹ for Cu²⁺. Representative Dixon and Cornish-Bowden plots were used to deduce the mode of inhibition induced by the trace metal ions. The inhibition was reversible and mixed for both metal ions. Furthermore, the biosensor showed good stability, selectivity, repeatability and reproducibility.