Claudio Botrè

Acid phosphatase/glucose oxidase-based biosensors for the determination of pesticides

Abstract This work presents new amperometric bienzymatic bioelectrodes for the determination of organophosphorus and carbamic acid type pesticides. Two different kinds of bienzymatic bioelectrodes are presented: a classical bienzymatic electrode, obtained by physicochemical immobilization of purified acid phosphatase (AP) and glucose oxidase (GOD) on the tip of an amperometric H2O2 electrode; and a hybrid biosensor, in which AP has been employed in the form of a thin layer of potato (Solanum tuberosum) tissue, endowed with a high content of enzyme activity. Both the biosensors can selectively detect glucose-6-phosphate (G6P), in the 5.0 × 10−5 −1.2 × 10−3M concentration range. Pesticides are detected, thanks to their high inhibition power towards AP, evaluated by adding the sample stepwise to a buffered solution of G6P, and recording the corresponding current change. The detection limit is therefore a function of the type of pesticide, but it can be as low as 1 μg 1−1 in the case of organophosphorus compounds. The detection limit is generally higher for carbamates, as a consequence of their weaker inhibition power towards acid phosphatase. Both bioelectrodes presented comparable values of the main physicochemical and analytical parameters evaluated for assessing their overall performance; nonetheless the plant tissue based bioelectrode exhibited a longer shelf life and a better reliability of the amperometric results.

Plant tissue electrode for the determination of atrazine

Abstract This work presents a new method for the simple and inexpensive determination of atrazine. The method is based on the use of a novel, partially disposable, plant tissue bioelectrode, which is sensitive to a variety of mono- and polyphenols. The biosensor is obtained by coupling a thin slice of potato ( Solanum tuberosum ) tissue, which contains high levels of the enzyme polyphenoloxidase (PPO), to a commercial O 2 -selective Clark electrode. The concentration of atrazine in aqueous samples can be determined thanks to its inhibitory power toward the catalytic activity of PPO. The low cost of this device and its good analytical performance suggest its application in the field of environmental analysis, especially in the continuous monitoring of atrazine in risk areas.

Plant-tissue electrode for the determination of catechol

Abstract A device based on the presence of polyphenol oxidase in potato ( Solanum tuberosum ) tissue was set up to determine catechol. The sensor is realized by coupling a thin slice of potato tissue with a Clark electrode capable of measuring oxygen evolution from a catechol solution. In the concentration range 2.5 × 10 −5 –2.3 × 10 −4 M there is perfect linearity between the electrode signal and catechol concentration in solution. The high stability and the reliability and reproducibility of the results, tested over a period longer than 3 months, and the sensitivity of the device towards mono- and polyphenol derivatives, allow the application of such a system in physiological and pharmacological quality control.

Cholinesterase based bioreactor for determination of pesticides

Abstract A novel analytical system for the in-flow quantitative determination of acetylcholine (ACh) and acetylcholinesterase (AChE) inhibitors is presented. Reliable and reproducible values in the monitoring of ACh, of carbamic acid derivatives and of organophosphorous compounds have been obtained thanks to a device realized by coupling an AChE-based whole plant tissue column bioreactor with a traditional choline oxidase (ChO) biosensor. AChE was extracted from the inner part of a grapefuit shell (Albedum pomi citreum), coupled to chitin by glutaraldehyde and immobilized into a glass column bioreactor, whose outlet was connected to a thermostated flow-through cell incorporating the ChO biosensor. A sharp improvement in the sensitivity limits was obtained by recycling the sample solution into the bioreactor. The efficacy of the analytical system here presented was checked on standard solutions of ACh and on three of the most commonly used inhibitors of AChE (aldicarb, malathion and paraoxon), widely employed mainly as the active components of pesticides and insecticides.

Effect of Anti-Carbonic Anhydrase Antibodies on Carbonic Anhydrases I and II

Carbonic anhydrase (CA; EC 4.2.1.1) is a zinc enzyme that is widely distributed in the living world and is involved in many biochemical processes that depend on the hydration/dehydration of carbon dioxide/bicarbonate [reviewed in Refs. (1)(2)(3)(4)(5)(6)]. Anti-CA antibodies have been identified, isolated, and purified from patients with a wide range of diseases, for some of which their presence can be a reliable diagnostic indicator (7). Anti-CA I and anti-CA II antibodies (aCAIab and aCAIIab) have recently been isolated from patients with systemic lupus erythematosus (8)(9), polymyositis and systemic sclerosis (9), endometriosis(10)(11), Sjogren syndrome (8)(9)(12)(13), idiopathic chronic pancreatitis (13)(14), primary biliary cirrhosis (PBC) (12)(13)(15)(16), and autoimmune cholangitis (15). It has recently been hypothesized that all of these diseases (many of which can occur concomitantly) may have a common pathogenetic mechanism based on autoimmune reactions against a common antigen. According to this hypothesis, it seems that highly active CA isoenzymes (cytosolic CA II and membrane-bound CA IV) are particularly involved, because CA plays an important role in such biochemical processes as tissue hydration and secretory activities. In some cases, the preincubation of CA with specific inhibitors has blocked its antibody interactions, suggesting that the site of the immunologic reaction may involve the active site of the enzyme (9). In patients with PBC, anti-CA antibodies are often associated with the presence of anti-mitochondrial antibodies, particularly anti-pyruvate dehydrogenase (17)(18)(19), which is the main diagnostic marker of the disease (20)(21). However, anti-CA antibodies have also been detected in the absence of anti-mitochondrial antibodies in patients with PBC and ascites (16). The aim of this study was …

Plant metabolism as an analytical tool: some applications of plant tissue electrodes for the selective determination of catecholamines

The present work deals with the quantitative determination of catechol and other biologically active polyphenols (catecholamines) by means of plant tissue electrodes built up by a whole tissue containing an oxidizing enzyme (polyphenol oxidase, PPO) coupled with an O2 gas-selective electrode. Several tissues have been employed to prepare the biocatalytic layer of the biosensors. The analytical characteristics of the plant tissue electrodes here proposed, tested in catechol standard solutions, are comparable to those of traditional PPO-based enzyme electrodes. Moreover, the whole-tissue biocatalytic membranes are more stable than those prepared by using purified enzymes, thus ensuring longer life times of application. The biosensors presented here are also suitable for the quantitative determination of other catecholamines (epinephrine, norepinephrine, dopamine, l-DOPA).

Automotive catalytic converters and environmental pollution: role of the platinum group elements in the redox reactions and free radicals production

The concentrations of Platinum Group Elements (PGE) (Pt, Rh, Pd and Ir) in the atmosphere of urban areas have been progressively increased as a consequence of their release from the automotive catalytic converters and their adsorption in the particulate material. The PGEs catalytic activity is therefore extended also outside the vehicle converters. In the environment, PGEs come in contact with inorganic and organic structures, in particular with living organisms. Beside the photochemical smog (mainly ozone production/degradation), several redox reactions, involving free radicals and promoted by PGEs, can become responsible for interactions at the metabolic pathways level. The environmental monitoring confirms the increasing concentrations of ozone and of PGEs in urban air. To approach such a complex problem, in vitro experiments have been performed on enzyme kinetics and on antioxidants in living systems. Preliminary data on enzymatic systems (NADH) and on antioxidants (ascorbic acid) in the presence of some PGEs are reported.

A multi-enzyme bioelectrode for the rapid determination of total lactate concentration in tomatoes, tomato juice and tomato paste

This work presents a new multi-enzyme bioelectrode for the combined determination of d(−)-l(+)-lactic acid in samples of whole tomatoes, chopped tomatoes, tomato paste and tomato juice. The principle of the biosensor is based on the catalytic activity of the enzymes l(+)-lactic acid oxidase (LOD), d(−)-lactic acid dehydrogenase (D-LDH) and horseradish peroxidase (HPO). The three enzymes are immobilized on the tip of an amperometric oxygen selective electrode. The total concentration of d(−)-l(+)-lactic acid is proportional to the amount of O2 consumed. The biosensor has been tested for standard solutions of d(−)-l(+)-lactic acid, as well as for real samples. The latter set of experiments has been carried out in three different laboratories, indicating a good repeatability and reproducibility of the obtained results. The main characteristics of the lactate biosensor on real samples (whole tomatoes, chopped tomatoes, tomato paste and tomato juice) have been compared, in terms of both analytical and practical features, with those of a traditionally employed enzymatic-spectrometric technique, based on the oxidation of d-l-lactic acid, catalysed by d-lactate dehydrogenase and l-lactate dehydrogenase, with production of pyruvate and NADH, and on the subsequent spectrometric determination, at λ= 340 nm, of the NADH formed.

Carbonic anhydrase and urease: an investigation in vitro on the possibility of a synergic action.

The indirect interactions between the carbonic anhydrase (CA) and urease (UR) are investigated in the present work using rate determinations detected by combined potentiometric measurements. It is shown that, in accord with the mass-action law for the two enzyme catalyzed reactions, the two enzymes assume a synergic pattern: the increase in the rate of removal of CO2 from the solution facilitated by CA increases the rate of production of NH3 consequent from urea dissociation. The experimental system which has been set up to monitor these interactions consists of a potentiometric apparatus to follow the gaseous exchanges of CO2 and NH3 which take place from a buffered solution containing both CA and UR. The results of the present work are consistent with, and add a further support to the finding of Dodgson and Forster, who first demonstrated in vivo the existence of an indirect linkage between urea production and CA catalytic activity.

Determination of glutamic acid decarboxylase activity and inhibition by an H2O2-sensing glutamic acid oxidase biosensor.

The catalytic activity of the enzyme L-glutamic acid decarboxylase (GAD) is determined by an amperometric method based on a recently developed glutamate-selective biosensor. The biosensor is composed of an amperometric H2O2 electrode and a biocatalytic membrane containing the enzyme glutamic acid oxidase (GAO). The biosensor allows the direct and continuous measurement of GA levels by monitoring the H2O2 produced at the electrode interface as a coproduct of the GAO-catalyzed GA oxidation to alpha-ketoglutaric acid. Since GA is transformed to gamma-aminobutyric acid and CO2 under the catalytic activity of GAD, the rate of GA consumption in solution, monitored by the GAO biosensor, represents a reliable measure of GAD catalytic activity. Additional experiments performed in the presence of different concentrations of the GAD inhibitor valproic acid have shown the suitability of the proposed approach for the study of GAD inhibitors also. Discussion of the main experimental characteristics of this new analytical method is given in terms of sensitivity, reproducibility, and reliability of the experimental results and ease, time, and cost of operation.