Juan C. Vidal

An electrochemical competitive biosensor for ochratoxin A based on a DNA biotinylated aptamer

Ochratoxin A (OTA) is one of the most important mycotoxin contaminants of foods, particularly cereals and cereal products, with strict low regulatory levels (of ppb) in many countries worldwide. An electrochemical competitive aptamer-based biosensor for OTA is described. Paramagnetic microparticle beads (MBs) were functionalized with an aptamer specific to OTA, and were allowed to compete with a solution of the mycotoxin conjugated to the enzyme horseradish peroxidase (OTA-HRP) and free OTA. After separation and washing steps helped with magnetic separations, the modified MBs were localized on disposable screen-printed carbon electrodes (SPCEs) under a magnetic field, and the product of the enzymatic reaction with the substrate was detected with differential-pulse voltammetry. In addition to magnetic separation assays, other competitive schemes (direct/indirect aptasensors performed on the SPCEs surface or using gold nanoparticles functionalized with the aptamer) were preliminary tested, optimized and compared. The magnetic aptasensor showed a linear response to OTA in the range 0.78-8.74 ng mL(-1) and a limit of detection of 0.07±0.01 ng mL(-1), and was accurately applied to extracts of certified and spiked wheat samples with an RSD lower than about 8%.

In situ preparation of a cholesterol biosensor: entrapment of cholesterol oxidase in an overoxidized polypyrrole film electrodeposited in a flow system: Determination of total cholesterol in serum

Abstract A straightforward method for constructing cholesterol biosensors by entrapment of cholesterol oxidase within a polypyrrole (PPy) film electropolymerized in a flow system is proposed. This method enables adjustment of the biosensor characteristics and features low reagent consumption. Prior to use, the PPy film is overoxidized to provide it with anion-exclusion properties in order to minimize the interference of electroactive species such as ascorbic acid and uric acid. A comprehensive preliminary study of the influence of 2-propanol and Triton X-100 added to the cholesterol solution on the flow-injection determination of this substrate, and the effects of different experimental variables on the biosensor response and its selectivity against interfering electroactive species was carried out. The proposed cholesterol oxidase based biosensor, named Pt/PPy–ChOx, was applied to the determination of cholesterol in reference serum samples, and the results were consistent with certified values.

Electrochemical affinity biosensors for detection of mycotoxins: A review

This review discusses the current state of electrochemical biosensors in the determination of mycotoxins in foods. Mycotoxins are highly toxic secondary metabolites produced by molds. The acute toxicity of these results in serious human and animal health problems, although it has been only since early 1960s when the first studied aflatoxins were found to be carcinogenic. Mycotoxins affect a broad range of agricultural products, most important cereals and cereal-based foods. A majority of countries, mentioning especially the European Union, have established preventive programs to control contamination and strict laws of the permitted levels in foods. Official methods of analysis of mycotoxins normally requires sophisticated instrumentation, e.g. liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. For about sixteen years, the use of simpler and faster analytical procedures based on affinity biosensors has emerged in scientific literature as a very promising alternative, particularly electrochemical (i.e., amperometric, impedance, potentiometric or conductimetric) affinity biosensors due to their simplicity and sensitivity. Typically, electrochemical biosensors for mycotoxins use specific antibodies or aptamers as affinity ligands, although recombinant antibodies, artificial receptors and molecular imprinted polymers show potential utility. This article deals with recent advances in electrochemical affinity biosensors for mycotoxins and covers complete literature from the first reports about sixteen years ago.

A comparative study of immobilization methods of a tyrosinase enzyme on electrodes and their application to the detection of dichlorvos organophosphorus insecticide

The use of several designs of amperometric enzymatic biosensors based on the immobilized tyrosinase enzyme (Tyr) for determining dichlorvos organophosphate pesticide are described. The biosensors are based on the reversible inhibition of the enzyme and the chronocoulometric measurement of the charge due to the charge-transfer mediator 1,2-naphthoquinone-4-sulfonate (NQS). Tyr becomes active when reducing the quinone form of the mediator molecule (NQS) to the reactive o-diol form substrate of Tyr (H(2)NQS) at the working electrode, thus permitting modulation of the catalytic activity of the enzyme and measurement of the inhibition produced by the pesticide. The full activity of the enzyme reversibly recovers after removal of the pesticide and re-oxidation of H(2)NQS. Tyr was immobilized onto electrodes using different procedures: (i) entrapment within electropolymerized conducting and non-conducting polymers, (ii) covalent attachment to self-assembled monolayers (SAM), (iii) cross-linking with glutaraldehyde (and nafion covering) and (iv) dispersion within carbon-paste electrodes. The mediator was co-immobilized onto the working electrode next to the enzyme and reagentless biosensors were subsequently constructed. In the SAM design (ii) NQS was added to the solution. The analytical properties of the different biosensors based on the competitive inhibition produced by dichlorvos were then compared. A detection limit of about 0.06 microM was obtained for dichlorvos with entrapment of NQS and Tyr within electropolymerized poly(o-phenylenediamine) polymer (oPPD), which was the design that proved to have the best analytical performance.

Ochratoxin A nanostructured electrochemical immunosensors based on polyclonal antibodies and gold nanoparticles coupled to the antigen

Competitive electrochemical immunosensors, based on disposable screen-printed electrodes (SPCEs), have been developed for the determination of the mycotoxin ochratoxin A (OTA). Two indirect immunoassays schemes were assessed using polyclonal antibodies and through the physical adsorption of OTA conjugated to albumin from bovine serum (OTA-BSA) and newly synthesized OTA-BSA bound to gold nanoparticles (OTA-BSA-AuNPs) onto the working electrode surface. After the competition step, detection was facilitated by a secondary aIgG antibody labelled with alkaline phosphatase and differential-pulse voltammetry using α-naphtyl-phosphate as substrate. The performance of the optimized immunosensors in terms of sensitivity, reproducibility and selectivity was studied. The linear working range of the described biosensors ranged between 0.9 and 9.0 ng mL−1 for the OTA-BSA based immunosensor and between 0.3 and 8.5 ng mL−1 for the gold nanostructured immunosensor, with a limit of detection (LOD) equal to 0.86 ng mL−1 (RSD = 10.6%) and 0.20 ng mL−1 (RSD = 8.0%) of OTA, respectively. The nanostructured immunosensor was applied to a certified wheat standard and a non-contaminated wheat material spiked with OTA, obtaining recoveries from about 104 ± 0.07% to 107 ± 0.08%. The influence of the wheat matrix on the analytical performance of the immunoassay was also studied.

Strategies for the improvement of an amperometric cholesterol biosensor based on electropolymerization in flow systems: use of charge-transfer mediators and platinization of the electrode

Different configurations based on an amperometric biosensor with cholesterol oxidase entrapped in a polypyrrole film have been developed with a view to improving the analytical properties of this biosensor. The alternatives considered involve the simultaneous entrapment of the enzyme and a charge-transfer mediator as well as previous platinization of the surface of the Pt electrode. Both artificial (a ferrocene derivative) and natural (flavin nucleotides) mediators were studied as constituents of the charge-transfer process between the enzyme and the electrode. The comparative study of these biosensors, which were prepared in situ in a continuous flow system, made it possible to determine the advantages and disadvantages of each configuration when applied to flow-injection determination of cholesterol.

Extraction-atomic-absorption spectrophotometric determination of lead by hydride generation in non-aqueous media

A method for lead hydride generation in a non-aqueous extraction phase is proposed. The lead hydride generation is carried out in an aliquot of lead pyrrolidine-1-carbodithioate extract in chloroform, by the addition of sodium tetrahydroborate(III) solution in dimethylformamide. Lead is determined by atomic-absorption spectrophotometry at 217.0 nm. The proposed method gives improved sensitivity and eliminates interferences, which is necessary because lead hydride generation in aqueous solution occurs in the presence of oxidants such as potassium dichromate or ammonium peroxydisulphate. The method was applied to the determination of lead in BCS standard steels and air particulates. Good accuracy and precision were obtained.

Improved electrochemical competitive immunosensor for ochratoxin A with a biotinylated monoclonal antibody capture probe and colloidal gold nanostructuring

We report a sensitive electrochemical immunosensor for Ochratoxin A (OTA), which is a frequent mycotoxin contaminant in cereals and other kinds of agricultural commodities, based on a biotinylated monoclonal antibody against OTA (mAbOTA–bi) and the avidin–biotin coupling of the tracer extravidin–horseradish peroxidase (ea–HRP). The analytical performance has been improved with respect to a previously developed immunosensor based on a polyclonal antibody against OTA and a secondary aIgG antibody labeled with alkaline phosphatase as tracer. The immunosensor relies on indirect competitive assay format after the passive physical adsorption of the antigen conjugated to bovine serum albumin (OTA–BSA) or bound to gold nanoparticles (OTA–BSA–AuNPs), and the screen-printed technology for voltammetric (DPV) measurements. The new design is simpler (only one capture probe), requires less time (only one incubation time with antibody), and shows an increased slope at the linear range of the calibration plot due to higher affinity of the monoclonal antibody compared to the polyclonal one. The newly designed immunosensor has a linear dynamic range of 0.15 to 9.94 ng mL−1 of OTA (R ≥ 0.9900), lower detection limit (0.10 ng mL−1 OTA), and a variability between assays of about 10%. The immunosensor was validated with certified wheat samples after extraction of OTA in acetonitrile : water (6/4) (v/v), and allows the determination of OTA in concentration levels well below those permitted in cereals under European Union recommendations (3 ng g−1).

Potentiometric determination of metoclopramide using a double-membrane based ion-selective electrode

Abstract A metoclopramide double-membrane ion-selective electrode, based on an internal conducting membrane made of tetrabutylammonium bromide (BrTBA) and an external membrane used on the ion-pair complex of metoclopramide (MCP) with sodium tetraphenylborate (NaTPB) has been prepared, employing dibutylphthalate (DBP) as plasticiser and poly(vinyl chloride) (PVC) as the inert matrix. The electrode exhibits a linear response within the concentration range 10−1-10−1Mof MCP, with a Nemstian slope (57.9 mV/decade). The reproducibility, stability, response time and selectivity were also investigated. The electrode exhibits very good selectivity for MCP with respect to the investigated substances of biological interest. The effects of ionic strength and pH of the solution were also investigated and the electrode was applied with satisfactory results to the determination of MCP in pure solutions and in a pharmaceutical product using the calibration graphy and standard addition method.

Extraction-atomic-absorption spectrophotometric determination of antimony by generation of its hydride in non-aqueous media

A method for covalent hydride generation in a non-aqueous extraction phase is proposed. The hydride generation is carried out in an aliquot of metal-complex extraction solution by sodium tetrahydroborate(III) in N,N-dimethylformamide solution and anhydrous acetic acid. The proposed method gives improved sensitivity, eleminates interferences and enables hydride generation of difficult elements to be carried out. Antimony is extracted by ammonium pyrrolidine-1-carbodithioate into chloroform and after hydride generation by the proposed method, it is determined by flame AAS. The method is applied to the determination of antimony in BCS standard steels with good accuracy and precision.