Miloslav Pravda

Recent applications of electrogenerated chemiluminescence in chemical analysis

Analytical applications of electrogenerated chemiluminescence (ECL) are reviewed with emphasis on the years 1997-2000. Recent developments are described for the ECL of organics, metal complexes and clusters, cathodic ECL on oxide covered electrodes, ECL based immunosensors, DNA-probe assays and enzymatic biosensors. Mechanisms are given for polyaromatic hydrocarbons, luminol/hydrogen peroxide, some cathodic ECL reactions and ruthenium complexes with and without co-reactants. New developments and improvements of techniques and instrumentation and their application to analytes are described. The application of ECL for visualisation of electrochemical processes and imaging of surfaces is mentioned.

Prussian Blue bulk modified screen-printed electrodes for H2O2 detection and for biosensors

A sensor for H(2)O(2) amperometric detection based on a Prussian Blue (PB) bulk modified carbon screen-printed electrode was developed. It has been optimised with respect to the lowest limit of detection achieved. PB was made chemically by the reaction of FeCl(3) with K(4)[Fe(CN)(6)]. The resulting powder, obtained by forced crystallisation induced by acetone, was dried and activated at 150 degrees C for 10 h. PB microparticles (<38 mum) were prepared and mixed with carbon ink. The limit of detection achieved was 0.4 muM with the linear range up to 100 muM of H(2)O(2) with the sensitivity of 137 muA mM(-1) cm(-2), that was comparable with sensors based on electrodeposited PB film. The transducer was applied for a glucose biosensor, that exhibited LOD of 0.22 mM, linear range up to 3 mM, K(M)(app) of 4.6 mM, and the sensitivity of 3.21+/-0.16 muA mM(-1) cm(-2). The peroxide sensor, as well as the glucose biosensor, were totally insensitive to oxygen, ascorbate, urate, and paracetamol.

Disposable amperometric immunosensor for the detection of polycyclic aromatic hydrocarbons (PAHs) using screen-printed electrodes

An amperometric immunosensor for polycyclic aromatic hydrocarbons (PAHs) was developed. The immunosensor was based on disposable screen-printed carbon electrodes. The coating antigen used was phenanthrene-9-carboxaldehyde coupled to bovine serum albumin (BSA) via adipic acid dihydrazide. Antibodies were monoclonal mouse anti-phenanthrene. The enzyme alkaline phosphatase (AP) was used in combination with the substrate p-aminophenyl phosphate (pAPP) for detection at +300 mV (vs. Ag/AgCl). Various assay types were compared. Good results were achieved with an indirect co-exposure competition assay with a LOD of 0.8 ng/ml (800 ppt) and an IC(50) of 7.1 ng/ml (7.1 ppb) for phenanthrene. An indirect competition assay could detect phenanthrene with a LOD of 2 ng/ml (IC(50): 15 ng/ml) and an indirect displacement assay with a LOD of 2 ng/ml (IC(50): 11 ng/ml) at a 5 microl surface coating of 8.8 microg/ml phenanthrene-BSA conjugate. A coating concentration of 2.2 microg/ml allowed detection with a LOD of 0.25 ng/ml (250 ppt) with the indirect competition assay. The influence of the coating concentration on the sensor performance was investigated. Cross-reactivities were tested for 16 important PAHs. Anthracene and chrysene showed strong cross-reactivity, whereas benzo[g,h,i]perylene and dibenzo[a,h]anthracene showed no cross-reactivity.

Novel electrochemical immunosensors for seafood toxin analysis

The current work describes the optimisation of a screen-printed electrode (SPE) system for measurement of a variety of seafood toxins, such as okadaic acid, brevetoxin, domoic acid and tetrodotoxin. A disposable screen-printed carbon electrode coupled with amperometric detection of p-aminophenol at +300 mV vs. Ag/AgCl, produced by the label, alkaline phosphatase, was used for signal measurement. ELISA was primarily used to develop all toxin systems, prior to transferring to SPE. The sensors incorporate a relevant range for toxin detection, by which humans become ill, with detection limits achieved at SPE to the order of ng ml (-1) (ppb) or lower in some cases. The SPE system is simple and cost-effective due to their disposable nature, and analysis time is complete in 30 min. In addition, analyses can be achieved outside of a laboratory environment allowing for in-field measurements. Recovery experiments on selected toxins using the relevant working ranges highlighted the functionality of these systems yielding a +/-10% deviation for the true value.

IMMUNOCHEMICAL DETECTION OF POLYCYCLIC AROMATIC HYDROCARBONS (PAHs)

ABSTRACT This review gives a short introduction in the origin, occurrence and detection of polycyclic aromatic hydrocarbons (PAHs). Emphasis lies on immunochemical detection methods for PAH compounds, such as ELISA, RIA, fluoroimmunosensors, QCM immunosensors and electrochemical immunosensors, but also on immunosorbents and commercial immunoassay kits for PAHs. Possibilities and limitations of these methods compared to standard methods, such as HPLC and GC, are discussed.

New antioxidant mixture for long term stability of serotonin, dopamine and their metabolites in automated microbore liquid chromatography with dual electrochemical detection

An automated microbore liquid chromatographic assay with dual electrochemical detection is described for the determination of serotonin, dopamine and their metabolites, 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylacetic acid and homovanillic acid. Due to the chemical instability of the compounds, the addition of an antioxidant is required for automated analysis over a long period of time (e.g., 20 h). Therefore, the time stability of these substances was tested with different antioxidants. The stability for serotonin and 5-hydroxyindoleacetic acid was poor in acidic medium containing Na2EDTA but could greatly be improved by the addition of L-cysteine and ascorbic acid. Using this assay, the neurotransmitters and their metabolites could easily be determined in microdialysates obtained from different rat brain areas.

ROLE OF CHEMOMETRICS FOR ELECTROCHEMICAL SENSORS

ABSTRACT A review on multivariate chemometric methods used in electrochemistry is presented. Some perspectives and new proposals for applications of different chemometric tools in electrochemistry are given. The main goal is to show advantages of particular chemometric techniques for analysis of some data obtained in electrochemistry and electrochemical sensors. This work focuses on multivariate calibration, classification, pattern recognition, and signal processing.

Development of a disposable immunosensor for the detection of human heart fatty-acid binding protein in human whole blood using screen-printed carbon electrodes

An amperometric immunosensor for the rapid detection of human heart fatty-acid binding protein (H-FABP) in whole blood was developed. Due to its rapid kinetics in plasma, H-FABP is a useful biochemical marker for the early assessment of acute myocardial infarction (AMI). A one-step direct sandwich assay in which analyte and alkaline phosphatase (AP) labelled antibody were simultaneously added to the immobilised primary antibody, was employed using two distinct monoclonal mouse anti-human H-FABP antibodies. p-Aminophenyl phosphate was converted to p-aminophenol by AP and the current generated by its subsequent oxidation at +300 mV vs. Ag/AgCl was measured. Total assay time was 50 min and the standard curve was linear between 4 and 250 ngml(-1). The intra- and inter-assay coefficients of variation were below 9%. No cross-reactivity of the antibodies was found with other early cardiac markers and endogenous substances in whole blood did not have an interfering effect. The overall performance of the sensor, wide working range, good precision and specificity demonstrate its potential usefulness for early assessment of AMI.

Biosensors—42 Years and Counting

Abstract In this presentation, the entire field of biosensor research and commercial applications will be covered, starting with the beginnings of developments in the 1960s, through 42 years to the present. Especially stressed are the fields of immunosensors, a technique dating back to 1964 and one that forms the most active field if research today. Finally, some of the newer biological sensors are mentioned‐antibody fragments and non‐immunoglobulin ligands.

Immunosensor for the determination of okadaic acid based on screen-printed electrode

A disposable immunosensor for okadaic acid (OA), using a screen-printed electrode (SPE), was developed and characterised. Detection of the product, p-aminophenol, resulting from the reaction catalysed by alkaline phosphatase (AP), was carried out using an amperometric three-electrode system poised at a voltage of + 300 mV versus Ag/AgCl. Alkaline phosphatase was used as a label for the antigen, OA, and two kinds of alkaline phosphatase preparation were studied for the conjugation of okadaic acid. The calibration curve for okadaic acid obtained from the conjugate created from low-activity AP, 969 units/mg, was unsatisfactory in terms of sensitivity, but a high-activity conjugate delivered the required sensitivity and limit of detection. Studies on the stability of the sensor with α-OA antibody and OA-AP conjugate showed that the current response decreased drastically after one day. Stabilisation strategies have been formulated to overcome this problem. The calibration curve obtained with the high activity conjugate was linear up to 40 ng/ml of okadaic acid with a minimum concentration of analyte detected of 5 ng/ml and a detection limit of 2 ng/ml.