Fernando Porcelli

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.

Characterization of biosensors based on membranes containing a conducting polymer

Abstract A new type of biosensor based on the coupling of an enzyme to an ion-selective membrane containing a conducting polymer is evaluated. The results obtained with the enzyme field- effect transistor (ENFET) and the ion-selective electrode (ISE) for the determination of creatinine and urea are compared. The presence of the conducting polymer significantly lowers the detection limit for creatinine by one decade to 10 −7 and 10 −4 M for the ENFET and ISE, respectively. The determination of urea in urine and serum with the ENFET was carried out, and the results correlated well with those obtained by spectrophotometry.

Immunoenzymatic assay using CHEMFET devices

Abstract An IMMUNOFET device in which an antibody is immobilized onto the gate surface by means of an immunochemical membrane containing Protein A has been realized. Such a membrane is chemically linked to the device surface by using a layer of polyaminosiloxane. The antigen-antibody interaction is detected by an immunoenzymatic assay which allows the response of the immunoreaction to be amplified using as marker an enzyme (e.g., glucose oxidase) that is able to produce, during the catalysed reaction, a pH variation detected by the ISFET device. The possibility of employing this immunosensor in biomedical applications and environmental monitoring is shown using as a model system human IgG (hIgG) and the herbicide atrazine respectively.

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.

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).

Peroxidase based amperometric biosensors for the determination of γ-aminobutyric acid

Abstract This work presents the realization of enzymatic bioelectrodes, suitable for the determination of γ-aminobutyric acid (GABA). The biosensors are based on the catalytic activity of the enzymes γ-aminobutyric glutamic transaminase (GABA-T), succinic semialdehyde dehydrogenase (SSDH) and horseradish peroxidase (HPO). The first two enzymes are usually indicated by the general term “GABASE”. All the biosensors presented in this work are realized by immobilizing the enzyme HPO on the tip of an amperometric oxygen selective electrode: the resulting NADPH-sensitive biosensor is used in combination with GABASE to determine the concentration of GABA in aqueous samples. Since SSDH depends on the NADP+/NADPH equilibrium, it follows that, in the presence of HPO, the NADPH formed is oxidized to NADP, and the decrease in the concentration of dissolved oxygen is proportional to the concentration of NADPH and, in turn, to that of GABA. The experiments were performed either with GABASE free in solution or co-immobilized with HPO on the surface of the oxygen electrode. In the latter case, the immobilization of the three enzymes has been performed either on a single membrane or on two separated membranes. In both cases there is an almost perfect linearity between the electrode signal and GABA concentration in the range 5.0 × 10−5–1.2 × 10−3M, with a lower detection limit of 2.0 × 10−5M; but the single-membrane biosensor showed a better overall performance, especially in terms of repeatability of measurements and lifetime of operation.

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.

Plant tissue biosensors for the determination of biogenic diamines and of their amino acid precursors: effect of carbonic anhydrase

Abstract A new class of plant tissue biosensors has been set up and applied to the determination of biogenic diamines. All the biosensors presented in this work are based on the amperometric determination of H2O2 produced by the enzymatic oxidation of diamines (putrescine and cadaverine) by the diamine oxidase (DAO) contained in the cotyledon of legumes (pea and lentil). Putrescine and cadaverine were quantitatively determined by means of these plant tissue electrodes in a range of concentrations between 0.5 and 320 μM and between 0.5 and 200 μM, respectively. The same lentil tissue-based sensor was then used, in combination with a lysine decarboxylase (LDC) membrane, for the realization of a hybrid enzyme electrode for the determination of lysine. The performances of the sensor here proposed, tested in lysine standard solutions, were comparable to those of traditional lysine bienzyme electrodes. A similar enzyme electrode was assembled for the determination of ornithine: in this case the lentil tissue diamine oxidase sensor was used in combination with an adequate amount of ornithine decarboxylase (ODC), added directly into the measuring cell. The effect of carbonic anhydrase (CA) on the speed of the LDC- and ODC-catalysed reactions was also evaluated. The synergistic interaction observed between the decarboxylation reaction, operated by either LDC or ODC, and the faciliated transport of CO2, operated by CA, is discussed both in terms of its analytical relevance and of its broader physiopathologic implications.

Determination of l-glutamate and l-glutamine in pharmaceutical formulations by amperometric l-glutamate oxidase based enzyme sensors

An amperometric biosensor for the direct determination of L-glutamate was developed by chemical bonding of L-glutamate oxidase (GAO) on a carboxylic Nylon membrane with polyazetidine prepolymer (PAP), and using a hydrogen peroxide electrode as indicating sensor. The biosensor is specific for L-glutamate and the peculiar analytical properties (linearity range, reproducibility, accuracy) were experimentally determined. Furthermore, the same basic biosensor was also modified to be used and characterized for the direct determination of L-glutamine. This L-glutamine biosensor was obtained by coimmobilizing, on two separate membranes, glutamic acid oxidase and glutaminase (GMN) on the same biosensor. The two sensors were then used for the determination of glutamate and L-glutamine contained in pharmaceutical formulations and the results were compared with those obtained by other analytical methods.

Fabrication and characterization of ENFET devices for biomedical applications and environmental monitoring

Abstract We have realized an enzyme-modified FET device (ENFET) in which the enzyme has been chemically linked to the FET surface by means of a layer of polymerized [3-(2-aminoethyl)aminopropyl]trimethoxysilane (AEAPS). The enzyme has been bound to the amino functional groups of the polysiloxane surface together with bovine serum albumin by using cross-linking agents, such as glutaraldehyde, and the obtained enzymatic membrane has been characterized by FT-IR directly onto the gate surface. The polysiloxane layer has proved to be a good interface between the enzyme and the device surface allowing a biosensor with the characteristics of good stability and reproducibility to be obtained. In this paper two examples of the fabrication and the performances of this kind of biosensor for urea determination in biomedical applications and pesticide detection in environmental control are shown, employing immobilized urease or acetylcholinesterase ENFET devices, respectively.