Multienzyme electrochemical array sensor for determination of phenols and pesticides

The screen-printed four-electrode system was used as the amperometric transducer for determination of phenols and pesticides using immobilised tyrosinase, peroxidase, acetylcholinesterase and butyrylcholinesterase. Acetylthiocholine chloride was chosen as substrate for cholinesterases to measure inhibition by pesticides, hydrogen peroxide served as co-substrate for peroxidase to measure phenols. The compatibility of hydrolases and oxidoreductases working in the same array was studied. The detection of p-cresol, catechol and phenol as well as of pesticides including carbaryl, heptenophos and fenitrothion was carried out in flow-through and steady state arrangements. In addition, the effects of heavy metals (Cu²⁺, Cd²⁺, Fe³⁺), fluoride (NaF), benzene and dimethylsulphoxide on cholinesterase activities were evaluated. It was demonstrated that electrodes modified with hydrolases and oxidoreductases can function in the same array.The achieved R.S.D. values obtained for the flow system were below 4% for the same sensor and less than 10% within a group of five sensors. For the steady state system, R.S.D.s were approximately twice higher. One assay was completed in less than 6 min. The limit of detection for catechol using tyrosinase was equal to 0.35 and 1.7 μM in the flow and steady state systems, respectively. On the contrary, lower limits of detection for pesticides were achieved in the steady state system—carbaryl 26 nM, heptenophos 14 nM and fenitrothion 0.58 μM.     

Amperometric biosensing of carbamate and organophosphate pesticides utilizing screen-printed tyrosinase-modified electrodes

A tyrosinase (Tyr) screen-printed biosensor based on the electroreduction of enzymatically generated quinoid products was electrochemically characterized and optimized for determination of carbamates and organophosphorus pesticides. A composite electrode prepared by screen-printing a cobalt (II) phthalocyanine (CoPc) modified cellulose-graphite composite on a polycarbonate support was employed as electrochemical transducer. The Tyr biosensor was prepared by immobilization of enzyme on the composite electrode surface by cross-linking with glutaraldehyde and bovine serum albumin. Parameters affecting the biosensor response such as response time, enzyme loading, concentration and pH of the buffer solution were optimized utilizing catechol as substrate. The maximum response for o-quinone enzymatically generated was obtained after 2 min of reaction. A good reproducibility and high operational stability were found for Tyr biosensor (60 units) at 50 mM phosphate buffer, pH 6.50. Under these conditions, the useful lifetime of biosensor was 10 days. After 15 days, the biosensor could be used with 20% of the initial value. Inhibition studies on the o-quinone steady-state current (at −0.20 V versus Ag/AgCl) were performed to investigate the inhibition kinetics of the pesticides in the enzymatic activity of mushroom tyrosinase. The results shown that the methyl parathion and carbofuran can lead to competitive inhibition process of the enzyme, while diazinon and carbaryl act as mixed inhibitors. Linear relationships were found for methyl parathion (6-100 ppb), diazinon (19-50 ppb), carbofuran (5-90 ppb) and carbaryl (10-50 ppb). Analysis of natural river water samples spiked with 30 ppb of each pesticide showed recoveries between 92.50% and 98.50% and relative standard deviations of 2%.     

Screen-printed multienzyme arrays for use in amperometric batch and flow systems

Screen-printing technology for electrode fabrication enables construction of amperometric devices suitable for combination of several enzyme electrodes. To develop a biosensor array for characterisation of wastewaters, tyrosinase and horseradish peroxidase (HRP) or cholinesterase-modified electrodes were combined on the same array. The behaviour of the tyrosinase-modified electrode in the presence of hydrogen peroxide (required co-substrate for the HRP-modified electrode) and acetylthiocholine chloride (required co-substrate for cholinesterase) was studied. Performance of bi-enzyme biosensor arrays in the batch mode and in the flow-injection system are discussed.     

Amperometric Flow‐Injection Determination of Phenolic Compounds Using a Biosensor with Immobilized Laccase,Peroxidase and Tyrosinase

A screen‐printed four‐electrode sensor based on immobilization of laccase (Coriolus hirsutus), peroxidase (horseradish) and tyrosinase (mushroom) in the same array was developed for monitoring of phenols. The enzymes were immobilized onto a self‐assembled monolayer (4‐mercapto‐1‐butanol) modified gold surface via covalent attachment by epichlorohydrin coupling. The experimental conditions for simultaneous operation of the three enzymes were optimized based on catechol determination. The sensors were further applied for the amperometric detection of several substituted phenolic compounds, carried out using a single line flow‐injection system. Hydrogen peroxide served as co‐substrate for peroxidase. The limits of detection for phenols in aqueous solutions were in the micromolar range, one assay was completed in less than 5 min. The preliminary studies showed that the compatibility of the above mentioned enzyme array enabled the multielectrode biosensor to be applied to real samples including industrial wastewaters and surface waters.     

A mediator-free screen-printed amperometric biosensor for screening of organophosphorus pesticides with flow-injection analysis (FIA) system

A mediator-free amperometric biosensor for screening organophosphorus pesticides (OPs) in flow-injection analysis (FIA) system based on anticholinesterase activity of OPs to immobilized acetylcholinesterase enzyme (AChE) has been developed. The enzyme biosensor is prepared by entrapping AChE in Al₂O₃ sol-gel matrix screen-printed on an integrated 3-electrode plastic chip. This strategy is found not only increase the stability of the embedded AChE, but also effectively catalyze the oxidative reaction of thiocholine, making the Al₂O₃-AChE biosensor detects the substrate at 0.25 V (versus Ag/AgCl), hundreds mini-volt lower than other reported mediator-free ones. The Al₂O₃-AChE biosensor is thus coupled to FIA system to build up a simple and low-cost FIA-EC system for screening OPs in real samples. A wide linear inhibition response for dichlorvos, typical OP, is observed in the range of 0.1-80 μM, corresponding to 7.91-84.94% inhibition for AChE. The detection limit for dichlorvos is achieved at 10 nM in the simulated seawater for 15 min inhibiting time, which allows the biosensor quantitatively detects the ecotoxicological effect of the real samples from the seaports in eastern China, where the OPs pollution is confirmed by GC-MS.     

Amperometric lactate biosensor for flow injection analysis based on a screen-printed carbon electrode containing Meldola's Blue-Reinecke salt, coated with lactate dehydrogenase and NAD

A biosensor for the measurement of lactate in serum has been developed, which is based on a screen-printed carbon electrode, modified with Meldola's Blue-Reinecke Salt (MBRS-SPCE), coated with the enzyme lactate dehydrogenase NAD⁺ dependent (from Porcine heart), and NAD⁺. A cellulose acetate layer was deposited on the top of the device to act as a permselective membrane. The biosensor was incorporated into a commercially available, thin-layer, amperometric flow cell operated at a potential of only +0.05 V vs. Ag/AgCl. The mobile phase consisted of 0.2 M phosphate buffer pH 10 containing 0.1 M potassium chloride solution; a flow rate of 0.8 ml min⁻¹ was used throughout the investigation. The biosensor response was linear over the range 0.55-10 mM lactate; the former represents the detection limit. The precision of the system was determined by carrying out 10 repeat injections of 10 mM l(+)lactic acid standard; the calculated coefficient of variation was 4.28%. It was demonstrated that this biosensor system could be applied to the direct measurement of lactate in serum without pre-treatment; therefore, this would allow high throughput-analysis, at low cost, for this clinically important analyte.     

Facilitated Transfer of Alkali and Alkaline‐Earth Metal Ions by a Calix[4]arene Derivative Across Water/1,2‐Dichloroethane Microinterface: Amperometric Detection of Ca2+

The host–guest complexation reactions between 5,11,17,23‐tetra‐tert‐butyl‐25,27‐diethoxycarbonylmethoxy‐26,28‐dimethoxy calix[4]arene (BDDC4) and alkali and alkaline‐earth metal ions were investigated by facilitated ion transfer processes across water/1,2‐dichloroethane microinterface by using steady‐state cyclic voltammetry and differential pulse voltammetry. The obtained facilitated transfers for Li⁺, Na⁺, K⁺, Rb⁺ and Ca²⁺ were evaluated under the different experimental conditions, at the excess concentrations of metal ions with respect to BDDC4 and vice versa. The association constants having 1 : 1 stoichiometry for Li⁺, Na⁺, K⁺ and Rb⁺ in 1,2‐DCE were determined. Also, we demonstrated that BDDC4 can play an important role for the development of highly selective chemical sensor for Ca²⁺ among alkaline‐metal ions in the concentration range of 0.1–1.0 mM in aqueous solution.     

Sensitive and Rapid Flow Injection Amperometric Hydrazine Sensor using an Electrodeposited Gold Nanoparticle Graphite Pencil Electrode

A gold (Au) nanoparticle-modified graphite pencil electrode was prepared by an electrodeposition procedure for the sensitive and rapid flow injection amperometric determination of hydrazine (N₂H₄). The electrodeposited Au nanoparticles on the pretreated graphite pencil electrode surface were characterized by scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray diffraction spectroscopy, and electrochemical impedance spectroscopy. Cyclic voltammograms showed that the Au nanoparticle-modified pretreated graphite pencil electrode exhibits excellent electrocatalytic activity toward oxidation of hydrazine because the highly irreversibly and broadly observed oxidation peak at +600?mV at the pretreated graphite pencil electrode shifted to ?167?mV at the Au nanoparticle pretreated graphite pencil electrode; in addition, a significant enhancement in the oxidation peak current was obtained. Thus, the flow-injection (FI) amperometric hydrazine sensor was constructed based on its electrocatalytic oxidation at the Au nanoparticle-modified pretreated graphite pencil electrode. The Au nanoparticle-modified pretreated graphite pencil electrode exhibits a linear calibration curve between the flow injection amperometric current and hydrazine concentration within the concentration range from 0.01 to 100?µM with a detection limit of 0.002?µM. The flow injection amperometric sensor has been successfully used for the determination of N₂H₄ in water samples with good accuracy and precision.     

Influence of Analyte Flow Rate on Signal and Response Time of the Amperometric Gas Sensor with Nafion Membrane

This paper presents results of an investigation on influence of volumetric flow rate on the signal and response time of a prototype of sulfur dioxide gas sensor with Nafion membrane. The sensors differing in type of working electrode and composition of internal electrolyte were compared. We used Au and Pt working electrodes obtained via vacuum sublimation deposition on a Nafion membrane surface. The electrolytes were aqueous solutions of sulfuric acid of the summary concentration 5 mol dm^?3 (electrolyte A). The electrolyte B contained an addition of dimethylsulfoxide (DMSO); the water/DMSO molar ratio was 2 : 1. Based on a proposed equation, which takes diffusion resistance into account, the obtained sensor signals were analyzed for the flow rate within a range of 0–100 cm³ min^?1. The sensor response time was also determined for the above flow rate range.     

Amperometric sensor for nitrite using a glassy carbon electrode modified with alternating layers of iron(III) tetra-(N-methyl-4-pyridyl)-porphyrin and cobalt(II) tetrasulfonated phthalocyanine

The development of a highly sensitive amperometric sensor for nitrite using a glassy carbon electrode modified with alternated layers of iron(III) tetra-(N-methyl-4-pyridyl)-porphyrin (FeT4MPyP) and cobalt(II) tetrasulfonated phthalocyanine (CoTSPc) is described. The modified electrode showed an excellent catalytic activity and stability for the nitrite oxidation decreasing the peak potentials about 200 mV toward less positive values and presenting much higher peak currents than those obtained on the bare GC electrode. A linear response range of 0.2-8.6 μmol l⁻¹, with a sensitivity of 0.37 μA l μmol⁻¹ and detection limit of 0.04 μmol l⁻¹ were obtained with this sensor. The repeatability of the proposed sensor, evaluated in term of relative standard deviation, was verified to be 1.4% for 10 measurements of 0.2 μmol l⁻¹ nitrite solution. Interference caused by common ions has been investigated in simulated mixtures containing high concentration level of interfering ions and the sensor was found to be tolerant against these ions. The developed sensor was applied for the nitrite determination in water samples and the results were in agreement with those obtained by a comparative method described in the literature. The average recovery for these samples was 100.1 (±0.7)%.     

Synthesis of Functionalized MWCNTs Decorated with Copper Nanoparticles and Its Application as a Sensitive Sensor for Amperometric Detection of H2O2

Functionalized‐multiwall carbon nanotubes decorated with redox active copper nanoparticles have been fabricated for sensitive enzyme‐less H₂O₂ detection. The new nanocomposite was characterized by Transmission electron microscopy, energy dispersive X‐ray analysis and cyclic voltammetry. The response of the modified electrode to H₂O₂ was examined using amperometry at ?0.45 V vs. Ag/AgCl in a buffer solution at pH 10.0. The developed sensor displayed linear concentration ranges of 0.5–10.0 and 10.0–10000.0 µmol L^?1 with a detection limit of 0.3 µmol L^?1. The proposed sensor displayed good selectivity for H₂O₂ detection in the presence of common interferences such as ascorbic acid.     

A New Approach how to Define the Coefficient of Electroactivity of Adenine and Its Twelve Derivatives Using Flow Injection Analysis with Amperometric Detection

We studied the electrochemical behaviour of adenine derivates (adenosine, 2‐aminopurine, 2,6‐diaminopurine, 6‐benzyl‐aminopurine, adenosine monophosphate, cyclic adenosine monophosphate, nicotinamide adenine dinucleotide, adenosine triphosphate, S‐adenosyl‐L ‐methionine, and synthetic derivatives AD‐3, AD‐6 and AD‐9) using flow injection analysis with electrochemical detection using a glassy carbon electrode. The influences of pH, flow rate and potential on the signal height of the studied derivates were tested. The optimal pH was 3, the flow rate of the mobile phase 0.75 mL min^?1 and the potential 1100 mV. Further, we attempted to characterize each of the studied derivatives by mathematical equations and classic analytical parameters. The lowest detection limit was estimated for adenine as 0.9 nM and 2‐aminopurine as 0.5 nM.     

Determination of three Tumor Biomarkers (Homovanillic Acid,Vanillylmandelic Acid,and 5‐Hydroxyindole‐3‐Acetic Acid) Using Flow Injection Analysis with Amperometric Detection

Flow injection analysis with amperometric detection (FIA‐AD) at screen‐printed carbon electrodes (SPCEs) in optimum medium of Britton‐Robinson buffer (0.04 mol ? L^?1, pH 2.0) was used for the determination of three tumor biomarkers (homovanillic acid (HVA), vanillylmandelic acid (VMA), and 5‐hydroxyindole‐3‐acetic acid (5‐HIAA)). Dependences of the peak current on the concentration of biomarkers were linear in the whole tested concentration range from 0.05 to 100 μmol ? L^?1, with limits of detection (LODs) of 0.065 μmol ? L^?1 for HVA, 0.053 μmol ? L^?1 for VMA, and 0.033 μmol ? L^?1 for 5‐HIAA (calculated from peak heights), and 0.024 μmol ? L^?1 for HVA, 0.020 μmol ? L^?1 for VMA, and 0.012 μmol ? L^?1 for 5‐HIAA (calculated from peak areas), respectively.     

Multi‐Channel Piezoelectric Quartz Crystal Sensor with Principal Component Analysis and Back‐Propagation Neural Network for Organic Pollutants from Petrochemical Plants

A multi‐channel piezoelectric quartz crystal gas sensor comprising arrays coated with various organic materials and a home‐made computer interface for data processing were prepared and employed to detect six kinds of common organic pollutants from petrochemical plants including benzene, styrene, chloroform, octane, hexene and hexyne. The principal component analysis (PCA) method was employed to select six kinds of appropriate coating materials for these organic pollutants from 22 adsorbents onto piezoelectric crystals. After performing a PCA assay, six representative coating materials, namely Polyisobutylene, Poly(dimethylsiloxane) (SE30), 4‐tert‐Butylcalix[6]arene, Cholesteryl chloroformate, C60‐Polyphenyl acetylene (C60‐PPA) and Ag(I)/cryptand‐2,2/Ethylene diamine/NH₃/Polyvinyl chloride were selected. Moreover, effects of coating load of adsorbents and concentration of pollutants were also investigated. Three kinds of recognition techniques including 2D PCA score map, radar plot and back‐propagation neural network (BPN) were employed for qualitative analysis of these organic pollutants, and a quantitative analysis method could be established by creating calibration curves for each organic pollutant. This homemade multi‐channel piezoelectric quartz crystal gas sensor showed a good detection limit of 0.068‐1.127 mg/L for these organic pollutants. The multi‐channel piezoelectric gas sensor exhibited good reproducibility with a relative standard deviation (RSD) of 1.1‐9.6%. Furthermore, this multi‐channel piezoelectric crystal detection system with BPN recognition technique was also utilized to successfully distinguish and identify each component of the mixture of organic gas samples. Multivariate linear regression (MLR) analysis was employed to quantitatively compute the concentration of species in the organic mixtures.