Determination of horseradish peroxidase and a peroxidase-like iron porphyrin at a Nafion-modified electrode.

A catalytic coupling reaction between 4-amino antipyrine and a N,N-disubstituted aniline derivative has been exploited in the indirect electrochemical detection of horseradish peroxidase (HRP) and of a biomimetic catalyst, the iron(III) sulfonated tetraphenyl porphyrin. In the presence of hydrogen peroxide and one of the two catalysts a cationic electroactive quinone-iminium dye P+ was formed and detected by linear scan voltammetry using a screen-printed electrode coated with a Nafion film. Detection limits of 10(-12) M for HRP and 4 x 10(-10) M for the iron porphyrin have been achieved. In conclusion the iron porphyrin is considered to be a promising alternative to the HRP label in enzyme immunoassays with electrochemical detection.

An electrochemical metalloimmunoassay based on a colloidal gold label.

A novel, sensitive electrochemical immunoassay has been developed using a colloidal gold label that, after oxidative gold metal dissolution in an acidic solution, was indirectly determined by anodic stripping voltammetry (ASV) at a single-use carbon-based screen-printed electrode (SPE). The use of disposable electrodes allows for simplified measurement in 35 microL of solution. The method was evaluated for a noncompetitive heterogeneous immunoassay of an immunoglobulin G (IgG) and a concentration as low as 3 x 10(-12) M was determined, which is competitive with colorimetric ELISA or with immunoassays based on fluorescent europium chelate labels. The high performance of the method is related to the sensitive ASV determination of gold(III) at a SPE (detection limit of 5 x 10(-9) M) and to the release of a large number of gold(III) ions from each gold particle anchored on the immunocomplex (e.g., 1.7 x 10(5) gold atoms are theoretically contained in a 18-nm spherical gold particle).

An immunomagnetic electrochemical sensor based on a perfluorosulfonate-coated screen-printed electrode for the determination of 2,4-dichlorophenoxyacetic acid.

A disposable immunomagnetic electrochemical sensor involving a magnetic particle-based solid phase and a Nafion film-coated screen-printed electrode (Nafion-SPE) stuck at the bottom of a polystyrene cylinder (microwell of 300 microL) was developed and evaluated in a competitive immunoassay of the widely used herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The competitive binding of 2,4-D and 2,4-D labled with alkaline phosphatase (AP) for a limited amount of polyclonal anti-2,4-D antibody-coated magnetic beads was monitored electrochemically by measuring the AP labled++ activity bound to the beads. The phosphoric acid ester of [[(4-hydroxyphenyl)amino]-carbonyl]cobaltocenium hexafluorophosphate was used as the AP substrate. This anionic substrate (S-) is enzymatically transformed at pH 9.0 into a cationic phenol derivative (P+) which can be easily accumulated in the polyanionic Nafion coating and determined by cyclic voltammetry. During the enzyme reaction, the AP-associated beads were localized on the surface of the Nafion-SPE with the aid of a magnet, thus effectively increasing the concentration of P+ in the Nafion-modified electrode vicinity. The enzyme generation of P+ close to the electrode surface, and thereby to the Nafion film, resulted in a high amplification of the response. A detection limit of 0.01 microgram L-1 2,4-D was thus achieved. The performance of the sensor was successfully evaluated on river water samples spiked with 2,4-D, indicating that this convenient and sensitive technique offers great promise for decentralized environmental applications.

Simultaneous detection of three drugs labeled by cationic metal complexes at a nafion-loaded carbon paste electrode.

The synthesis of the pentaammine ruthenium(II) complex of N-isonicotinoyl-nortriptyline (NORPy-Ru(2+)) was performed and its electrochemical properties at a nafion-loaded carbon paste electrode were examined. The anodic oxidation of the positively charged labeled antidepressant proceeded at -0.06 V (versus Ag/AgCl, 0.05 M Cl(-)). A detection limit of 0.075 muM (S/N=3) was achieved at physiological pH by square-wave voltammetry after a 5-min preconcentration step, with a linear response over the range 0.075-5.0 muM. With a view to a future triple-analyte immunoassay, the detection of NORPy-Ru(2+) was also examined in the presence of two labeled antiepileptics previously synthesised, i.e. phenytoin labeled by a ferroceneammonium salt (oxidation potential at 0.26 V) and phenobarbital labeled by a cobaltocenium salt (reduction potential at -1.05 V). The simultaneous detection of the three labeled drugs proceeded with analytical performances similar to those corresponding to the separate accumulation of each tracer. However it was observed that the pentaammine ruthenium(II) complexes of pyridine and its derivatives were not stable in the presence of serum, which does not allow for their use as redox cationic labels in a multi-analyte immunoassay to be envisaged.

Ion-exchange voltammetry as a solid-phase microextraction analytical method: factors influencing the mass transfer to perfluorosulfonated ionomer film-coated electrodes and some of their consequences on the current responses.

A simple theoretical model of mass transfer kinetics at an electrode coated with an ion-exchange film polymer is proposed. The model takes into account the analyte depletion in solution and gives the relationship between the ion-exchange voltammetric (IEV) peak current and the initial analyte concentration in the sample matrix. The verification of the model is investigated at disposable Nafion film-coated screen-printed electrodes, using the redox cationic (ferrocenylmethyl)trimethylammonium salt. It is shown that the theoretical model and the experimental data fit satisfactorily insofar as the variation of the extraction and apparent diffusion coefficients of the salt with the film thickness are taken into account. Indeed, the film thickness plays a crucial role for the optimization of the IEV sensitivity, because the physicochemical properties of the recast Nafion polymer are dependent on the amount per unit area of Nafion deposited on the electrode surface.

Renewable perfluorosulfonated ionomer carbon paste electrode for competitive homogeneous electrochemical immunoassays using a redox cationic labeled hapten.

A Nafion-loaded carbon paste electrode (CPE) was prepared and its square wave voltammetric response at a fresh surface was optimized using (ferrocenylmethyl)-trimethylammonium iodide (1) as a model of a cation that accumulates irreversibly. Low current responses could be obtained reproducibly without preconditioning at a CPE loaded with 1-octanol and neutralized Nafion. Use of the former made the method more sensitive, and use of the latter gave lower background currents and stable analytical response over a long period of Nafion-loaded CPE use. Under optimized conditions and using a 5 min accumulation period, the modified electrode gave a linear current response over the range of 5 nM to 1.5 microM 1, a detection limit of 5 nM 1 and a relative standard deviation of 5% for 0.5 microM 1. Using these optimized conditions, the competitive homogeneous immunoassay of phenytoin as a model hapten was carried out with cobaltocenium-labeled phenytoin as tracer. Clinical serum samples (5 microL) of phenytoin at therapeutic concentrations (20-80 microM) could be analyzed using this method.

Ferrocenylethyl phosphate: an improved substrate for the detection of alkaline phosphatase by cathodic stripping ion-exchange voltammetry. Application to the electrochemical enzyme affinity assay of avidin.

The ferrocenylethyl phosphate disodium salt was synthesized and used as a new substrate for alkaline phosphatase (AP). The enzyme-generated ferroceneethanol was selectively and sensitively detected at a Nafion film-coated electrode by anodic preconcentration of the ferricinium salt, followed by cyclic voltammetry. The accumulated ferricinium units could be expelled from the polymer film in their neutral form by cathodic stripping, and so the Nafion-modified electrode could be reused for more than 10 measurements with a standard deviation less than 3%. Values of 0.75 mM for the Michaelis constant and 1.42 µmol s(-)(1) (mg of protein)(-)(1) for the maximal velocity were found. The regenerable Nafion-coated electrode was employed for the indirect detection of AP down to 2 × 10(-)(12) M and for the noncompetitive heterogeneous enzyme assay of avidin, whose detection limit was 5 × 10(-)(12) M.