Bogdan Bucur

Affinity Methods to Immobilize Acetylcholinesterases for Manufacturing Biosensors

Abstract Two new affinity methods to immobilize acetylcholinesterase (AChE) onto screen‐printed electrodes (SPE) are described and compared for the fabrication of SPE: one based on metal chelate affinity (MCA) and one based on the Concanavalin A (Con A), a sugar residue binding lectine. The manufacturing and the optimization procedure for each type of biosensor are discussed with respect to no‐specific binding, functionalization of the SPE surface and the amount of enzyme used. A linear response range to acetylthiocholine substrate between 10 and 100 µmol L−1 was obtained for Con A sensors and between 1 and 60 µmol L−1 with the MCA sensors. The possibilities to reuse the sensors fabricated using both affinity immobilization chemistries were also discussed. The optimized sensors were used to study the inhibitory effects of organophosphorus pesticides on immobilized AChE activity. A detection limit of 5 × 10−7 mol L−1 chlorpyrifos methyl‐oxon (ee AChE sensor) and 2 × 10−9 mol L−1 paraoxon (dm AChE sensor) was achieved for the sensors with the enzyme attached via Con A and NTA–Ni, respectively.

Determination of Silver(I) by Differential Pulse Voltammetry Using a Glassy Carbon Electrode Modified with Synthesized N-(2-Aminoethyl)-4,4'-Bipyridine

A new modified glassy carbon electrode (GCE) based on a synthesized N-(2-aminoethyl)-4,4′-bipyridine (ABP) was developed for the determination of Ag(I) by differential pulse voltammetry (DPV). ABP was covalently immobilized on GC electrodes surface using 4-nitrobenzendiazonium (4-NBD) and glutaraldehyde (GA). The Ag(I) ions were preconcentrated by chemical interaction with bipyridine under a negative potential (−0.6 V); then the reduced ions were oxidized by differential pulse voltammetry and a peak was observed at 0.34 V. The calibration curve was linear in the concentration range from 0.05 μM to 1 μM Ag(I) with a detection limit of 0.025 μM and RSD = 3.6%, for 0.4 μM Ag(I). The presence of several common ions in more than 125-fold excess had no effect on the determination of Ag(I). The developed sensor was applied to the determination of Ag(I) in water samples using a standard addition method.

Spectrophotometric determination of organophosphoric insecticides in a FIA system based on AChE inhibition

Abstract This work presents a FIA system for pesticide analysis in water based on irreversible inhibition of acetylcholine esterase by organophosphoric insecticides with spectrometric determination of thiocholine with Ellmans reagent. The enzyme was immobilized on amminated glass pearls kept into a reactor and reactivated with 2-PAM after each inhibition step. The total toxic substances of real sample is expressed as equivalent paraoxon and calculated based on relative inhibition of the enzyme measuring the FIA peaks high before and after inhibition. The calibration graph was linear between 2 and 70 ppb paraoxon at an incubation time of 20 min. The LOD was 1.3 µg/L. A complete analysis lasted 35 min. The method was used for analysis of real water samples.

Electrode-modified with nanoparticles composed of 4,4′-bipyridine-silver coordination polymer for sensitive determination of Hg(II), Cu(II) and Pb(II)

We have modified a glassy carbon electrode (GCE) with nanoparticles composed of a 4,4′-bipyridine-silver coordination polymer (CP) and showed that this CP can be applied to the sensitive differential pulse voltammetric analysis of the ions Hg(II), Cu(II) and Pb(II). The coordination polymer was prepared by mixing a solution of silver nitrate and 4,4′-bipyridine at room temperature. The surface of the GCE was modified with an organic layer of synthesized 1-[(4-nitrophenyl)methyl]-4,4′-bipyridinium and silver ions, which caused the binding of the added Ag-bipy CP. Anodic (oxidative) peaks of the electrode were at +300 mV for Hg(II), −70 mV for Cu(II), and at −540 mV for Pb(II) [versus Ag/AgCl]. Under optimal conditions, calibration graphs were linear in concentration ranges from 0.2 to 10 μg L−1 for Hg(II), from 1.3 to 6.4 μg L−1 for Cu(II), and from 4.1 to 20.7 μg L−1 for Pb(II). The respective detection limits were 0.09 μg L−1 Hg(II), 0.71 μg L−1 Cu(II) and 2.3 μg L−1 Pb(II). Relative standard deviation was 3.2% at a level of 4 μg L of Hg(II) for n = 10. The modified electrode was applied to the analysis of Hg(II) in spiked fish samples, and of Cu(II), Pb(II), and Hg(II) in spiked plant samples, and recoveries ranged from 90 to 108%. This is the first paper that presents the use of 4,4′-bipyridine-silver coordination polymer for heavy metal electrochemical detection.

Biosensor based on inhibition of monoamine oxidases A and B for detection of β-carbolines.

β-Carbolines are inhibitors of monoamine oxidases (MAO-A and MAO-B) and can be found in foods, hallucinogenic plant or various drugs. We have developed a fast analysis method for β-carbolines based on the inhibition of MAO. The enzymes were immobilized on screen-printed electrodes modified with a stabilized film of Prussian blue that contain also copper. We have used benzylamine as substrate for the enzymatic reaction and the hydrogen peroxide was measured amperometrically at -50 mV. The detection limits obtained were 5.0 µM for harmane and 2.5 µM for both harmaline and norharmane. The MAO-A is inhibited by all three tested β-carbolines (harmane, norharmane, and harmaline) while MAO-B is inhibited only by norharmane. The presence of norharmane in mixtures of β-carbolines can be identified based on the difference between the cumulative inhibition of MAO-A by all β-carbolines and MAO-B inhibition. The developed biosensors were used for food analysis.

Bienzymatic biosensor for rapid detection of aspartame by flow injection analysis.

A rapid, simple and stable biosensor for aspartame detection was developed. Alcohol oxidase (AOX), carboxyl esterase (CaE) and bovine serum albumin (BSA) were immobilised with glutaraldehyde (GA) onto screen-printed electrodes modified with cobalt-phthalocyanine (CoPC). The biosensor response was fast. The sample throughput using a flow injection analysis (FIA) system was 40 h−1 with an RSD of 2.7%. The detection limits for both batch and FIA measurements were 0.1 μM for methanol and 0.2 μM for aspartame, respectively. The enzymatic biosensor was successfully applied for aspartame determination in different sample matrices/commercial products (liquid and solid samples) without any pre-treatment step prior to measurement.

Critical Evaluation of Acetylthiocholine Iodide and Acetylthiocholine Chloride as Substrates for Amperometric Biosensors Based on Acetylcholinesterase

Numerous amperometric biosensors have been developed for the fast analysis of neurotoxic insecticides based on inhibition of cholinesterase (AChE). The analytical signal is quantified by the oxidation of the thiocholine that is produced enzymatically by the hydrolysis of the acetylthiocholine pseudosubstrate. The pseudosubstrate is a cation and it is associated with chloride or iodide as corresponding anion to form a salt. The iodide salt is cheaper, but it is electrochemically active and consequently more difficult to use in electrochemical analytical devices. We investigate the possibility of using acetylthiocholine iodide as pseudosubstrate for amperometric detection. Our investigation demonstrates that operational conditions for any amperometric biosensor that use acetylthiocholine iodide must be thoroughly optimized to avoid false analytical signals or a reduced sensitivity. The working overpotential determined for different screen-printed electrodes was: carbon-nanotubes (360 mV), platinum (560 mV), gold (370 mV, based on a catalytic effect of iodide) or cobalt phthalocyanine (110 mV, but with a significant reduced sensitivity in the presence of iodide anions).

L-Cysteine Determination Based on Tyrosinase Amperometric Biosensors without Interferences from Thiolic Compounds

This paper presents an enzymatic analysis method for selective detection of L-cysteine (L-Cys) without interferences from other thiols like cysteamine (CA) and mercaptoacetic acid (MAC). The amperometric biosensors are based on tyrosinase (Tyr) that converts catechol to o-quinone. The L-Cys detection is based on the fact that all thiols (including L-Cys) react with o-quinone producing electroinactive compounds and only interfering thiols (CA and MAC) inhibit Tyr. One sample was analyzed twice: with Tyr immobilized on WE surface to quantify enzyme inhibition by thiolic interferents and with Tyr free in solution to investigate the reactions between quinone and all thiolic compounds.

Electrodeposited Organic Layers Formed from Aryl Diazonium Salts for Inhibition of Copper Corrosion

Copper substrates deposed on a gold screen-printed electrode were covered with different aryl diazonium salts by electrodeposition at 0.25 mA for 30 or 300 s. Seven compounds were investigated: 4-aminophenylacetic acid, 4-aminophenethyl alcohol, 4-fluoroaniline, 4-(heptadecafluorooctyl)aniline, 4-aminoantipyrine, 4-(4-aminophenyl)butyric acid and 3,4,5-trimethoxyaniline. Quantitative monitoring of the electrodeposition process was carried out by electrogravimetry using quartz crystal microbalance (QCM). The electrodeposited mass varies between 26 ng/cm2 for 4-fluoroaniline formed during 30 s to 442 ng/cm2 for 4-phenylbutyric acid formed during 300 s. The corrosion inhibition properties of aryl-modified layers have been studied in buffer citrate with pH = 3 or 3.5% NaCl solutions using electrochemical noise (ECN) and Tafel potentiodynamic polarization measurements. A corrosion inhibiting efficiency up to 90% was found. The highest corrosion inhibition was obtained for 4-(4-aminophenyl)butyric acid and the lowest for 4-fluoroaniline. A relation between the inhibition efficiency and the chemical nature of the substituents in the protective layer was found.

Advances in Enzyme-Based Biosensors for Pesticide Detection

The intensive use of toxic and remanent pesticides in agriculture has prompted research into novel performant, yet cost-effective and fast analytical tools to control the pesticide residue levels in the environment and food. In this context, biosensors based on enzyme inhibition have been proposed as adequate analytical devices with the added advantage of using the toxicity of pesticides for detection purposes, being more “biologically relevant” than standard chromatographic methods. This review proposes an overview of recent advances in the development of biosensors exploiting the inhibition of cholinesterases, photosynthetic system II, alkaline phosphatase, cytochrome P450A1, peroxidase, tyrosinase, laccase, urease, and aldehyde dehydrogenase. While various strategies have been employed to detect pesticides from different classes (organophosphates, carbamates, dithiocarbamates, triazines, phenylureas, diazines, or phenols), the number of practical applications and the variety of environmental and food samples tested remains limited. Recent advances focus on enhancing the sensitivity and selectivity by using nanomaterials in the sensor assembly and novel mutant enzymes in array-type sensor formats in combination with chemometric methods for data analysis. The progress in the development of solar cells enriched the possibilities for efficient wiring of photosynthetic enzymes on different surfaces, opening new avenues for development of biosensors for photosynthesis-inhibiting herbicides.