Demonstration of labeless detection of food pathogens using electrochemical redox probe and screen printed gold electrodes.

The demonstration of a labeless immunosensor for the detection of pathogenic bacteria using screen printed gold electrodes (SPGEs) and a potassium hexacyanoferrate(II) redox probe is reported. Gold electrodes were produced using screen printing and the gold surfaces were modified by a thiol based self assembled monolayer (SAM) to facilitate antibody immobilisation. SAMs based on the use of thioctic acid (TA), mercaptopropionic acid (MPA) and mercaptoundecanoic acid (MUA) were evaluated. Following antibody immobilisation via the optimum SAM, the redox behaviour and diffusion co-efficient (D) of the potassium hexacyanoferrate(II) probe was monitored in the absence and presence of analyte. In the presence of analyte, a change in the apparent diffusion co-efficient of the redox probe was observed, attributable to impedance of the diffusion of redox electrons to the electrode surface due to the formation of the antibody-bacteria immunocomplex. No change in the diffusion co-efficient was observed when a non-specific antibody (mouse IgG) was immobilised and antigen added. The system has been demonstrated with Listeria monocytogenes and Bacillus cereus.

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.

Use of oxygen sensors to non-destructively measure the oxygen content in modified atmosphere and vacuum packed beef: impact of oxygen content on lipid oxidation.

The ability of optical oxygen sensors to monitor the levels of oxygen in raw and cooked beef was investigated. Raw and cooked beef slices were vacuum packaged and cooked beef slices were modified atmosphere packaged MAP, (60% N(2): 40% CO(2)) and held under refrigerated display (4 °C) for 15 or 35 days for MAP and vacuum packed samples, respectively. Oxygen sensors attached to the inside of the lidding material in modified atmosphere packages, or inserted into vacuum packages, were capable of monitoring changes in oxygen levels in all packaged samples. Lipid oxidation of samples was measured at regular intervals. Oxygen contents detected, ranged from 1.15 to 1.26% and 0.07-0.55% in MAP and vacuum packed samples, respectively. Samples containing greatest levels of oxygen were most oxidised and cooked samples were significantly (P<0.05) more oxidised than raw samples.

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 H(2)O(2) 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.

A rapid interferent-free assay for the determination of LD-1 in biological samples.

A highly specific enzyme linked immunosorbent assay (ELISA) for the detection of lactate dehydrogenase-1 (LD-1) has been developed. A competition assay is described with polyclonal antibody to LD-1 passively adsorbed on an ELISA 96-well plate, with non-labelled and labelled LD-1 antigen competing. The LD-1 antigen is conjugated to alkaline phosphatase (ALP) using the one step glutaraldehyde method. A linear range of 10-4000 U/L was obtained, and cross-reactivity was only observed with LD-2. It was possible to eliminate this cross-reactivity by carrying out the final incubation step at 65 degrees C. The developed assay was applied to the analysis of spiked serum and plasma samples and the recoveries obtained were acceptable.

The use of electrochemically grown polymers on metallized electrodes to reduce electrode fouling in biological matrices.

The use of electrochemically grown polymers has expanded dramatically in the last couple of years, and they are now well established as membranes for immobilizing components. The evidence here for their anti-fouling properties is good. The poly(1,3-diaminobenzene)-covered electrodes performed well in the buffer, urine, plasma and serum samples, but not so well in the blood. The Ru/Rh/Pt, Rh/Rh and the Pt-on-glassy carbon electrodes covered with poly(1,3-diaminobenzene) were the best electrodes in the blood. The Pt disc seemed to exhibit the largest irrepeatability in most of the biological matrices.

Increasing the sensitivity of Listeria monocytogenes assays: evaluation using ELISA and amperometric detection.

An immunosensor for the detection of Listeria monocytogenes was developed. ELISA and amperometric studies were run in parallel to develop a more sensitive and rapid assay for the bacterium. Conditions for the immunosensor were primarily characterised using ELISA. A direct sandwich assay was employed and the affinities of two polyclonal (goat and rabbit) and one monoclonal (mouse) anti-L. monocytogenes antibodies were compared using this format. Owing to low sensitivity being obtained with all antibodies, biotin-avidin amplification and an indirect sandwich assay were employed. The system was then transferred to screen-printed electrodes (SPEs), the primary antibody being immobilised by cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and the mode of detection being amperometric. Various parameters (limit of detection, working range, incubation time, cross-reactivity) of the systems were characterised. The effect of direct incubation in milk is also discussed. The final immunosensor had a working range of 1 x 10(6)-1 x 10(3) cells ml-1 and a detection limit of 9 x 10(2) cells ml-1. The assay took about 3.5 h to complete.

Development of an ultrasensitive immunoassay for rapid measurement of okadaic acid and its isomers.

This report highlights the characteristics of an okadaic acid immunoassay with limits of detection in the subfemtomole range. Two different immunoassay formats were investigated and their characteristics compared in relation to linear ranges, limits of detection, and cross-reactivity with other seafood toxins present in water and/or mussel samples. The developed ELISA system can be manipulated to quantitatively measure total diarrhetic shellfish poisoning (DSP) content or for okadaic acid and dinophysistoxin-1 individual concentrations by variation of the format of the immunoassay. Real mussel samples were validated in percentage recovery test. Calibration curves were established, and aliquots of real samples were tested. Very good recoveries were attained, highlighting the validity of the ELISA system to accurately determine the DSP concentration in mussel samples.

An immunosensor based on the glucose oxidase label and optical oxygen detection.

A novel optical immunosensor setup is described which uses glucose oxidase enzyme as a label in conjunction with a luminescence lifetime-based oxygen sensor and phase measurements. The oxygen sensor membranes prepared on microporous filters were used as a solid phase on which the immunoassay was carried out. These sensing materials in combination with a new measurement setup provided high sensitivity for the detection of oxidase enzymes, being at nanogram per milliliter level, i.e., 10(-11)-10(-12) M, with respect to glucose oxidase and its conjugates. Experimental data on the sensitivity were validated using theoretical equations and calculations. Using the new measurement setup and IgG-anti-IgG as a model, a number of different sensing materials were studied aimed to optimize the immunosensor and evaluate its performance. This approach was then applied to a practical system for the detection of human lactate dehydrogenase isoenzymes. It provided similar sensitivity of approximately 1 ng/mL, which is comparable to that of standard ELISA. The attributes of the new immunosensor approach are discussed with respect to performance and versitility.

The use of polymers coupled with metallised electrodes to allow H(2)O(2) detection in the presence of electrochemical interferences.

One of the most important parameters to be considered when developing a biosensor based on the use of oxidase enzymes is the selective determination of H(2)O(2) in the presence of easily oxidisable interferents. In this work one approach was taken to overcome this problem-the use of metallised carbon electrodes in conjunction with polymers. Polymers, both conducting and nonconducting, have recently become very interesting materials for the suppression of interferences. They are easy to grow on any electrode surface and the extensive range of polymers available provides a huge scope for the large variety of sensors that exist today. They can be grown in organic or aqueous media. In this work, three polymers (polypyrrole, polyaniline and 1,3-diaminobenzene) were examined for their interferent-preventing potential on several types of electrodes. Previous work carried out at the Laboratory of Sensor Development has shown the co-deposition of ruthenium and rhodium on carbon to provide an electrode surface which is highly catalytic and selective towards H(2)O(2)[1]. The co-deposition of Ru, Rh and Pt, as well as Pt on Ru-Rh electrodes was investigated and all these transducers were coupled with the use of the polymers for enhanced elimination of interferences with highly promising results obtained. The best system was seen to be a Ru-Rh metallised electrode polymerised with poly(1,3-diaminobenzene). At an applied potential of +100 mV the response to H(2)O(2) was approximately 200 times greater than the response of any of the potential interferences.

A new interference-free lysine biosensor using a non-conducting polymer film.

An electrochemical biosensor for the determination of lysine to be used for rapid evaluation of food quality has been developed. Platinum electrodes have been coated by electropolymerisation with 1,2-diaminobenzene (1.2-DAB) using cyclic voltammetry. The reduction in the oxidation of interferents compared with the bare platinum electrode was 100% for ascorbic acid, 99% for acetaminophen and 99% for cysteine. The enzyme L-lysine-alpha-oxidase was then immobilised onto the polymer layer by passive adsorption and a calibration curve for lysine constructed. This gave a linear range of 1 x 10(-5) mol/l to 1 x 10(-3) mol/l and a limit of detection of 2 x 10(-7) mol/l.

Amperometric bienzymic sensor for aspartame.

An amperometric enzyme electrode for the determination of aspartame was developed by covalent immobilization of alcohol oxidase and alpha-chymotrypsin. A platinum based hydrogen peroxide electrode was used as the detector. Excellent sensitivity was obtained using batch, flow-through and flow injection methods with detection limits of 2 x 10(-7), 4 x 10(-7) and 10(-6) mol l-1, respectively. Different strategies for eliminating interfering compounds, including the introduction of an additional alcohol oxidase-catalase membrane and signal subtraction using an alcohol electrode, were employed. A recovery study on seven food samples was carried out and the results were satisfactory.

Quartz crystal microbalance detection of Vibrio cholerae O139 serotype.

A piezoelectric (PZ) quartz crystal microbalance (QCM) biosensor for the rapid detection of Vibrio cholerae serotype O139 has been developed. The antibody to this serotype was immobilized on the gold transducer surface of a 10 MHz AT cut PZ crystal. Solutions containing known antigen concentrations were then incubated for 1 h on the antibody-bound transducer. The biosensor was able to detect 10(5) cells per ml of O139 versus a background of O1 (Ogawa) serotype.

Non-invasive biosensors in clinical analysis.

Several amperometric biosensors have been developed and applied for the non invasive determination of metabolites in body fluids. Advantages of saliva or sweat analysis are the ease of sample collection and that samples can be collected more frequently with much less stress on the patient. An alcohol biosensor has been developed with a hydrogen peroxide based electrode utilizing immobilized alcohol oxidase. Immobilization parameters have been optimized to increase the stability of the enzyme. An auter hydrophobic gas membrane was used to improve the selectivity of the probe. A hydrogen peroxide based amperometric biosensor has also been developed that utilized the enzyme glucose oxidase. The biosensor was applied to the determination of sera and saliva glucose content. Two hydrogen peroxide based amperometric biosensors that utilized lactate oxidase were also developed for determination of lactate in saliva and sweat. To discriminate against electroactive substances, the biosensor for assay of lactate in saliva utilized a dual electrode with one side active and one inactive, while the biosensor for assay of sweat lactate content utilized a hydrophobic hydrogen peroxide membrane to improve selectivity. Lactate content of saliva and sweat samples were measured after an intense physical exercise. A new procedure to measure glucose via transbuccal mucosa was developed using a dual glucose probe similar to that used for lactate. Correlation between glucose in blood and in transbuccal mucosa has been evaluated.

Recent developments in piezoelectric immunosensors. A review.

The development of piezoelectric (PZ) devices as immunosensors is reviewed. The recent advances in sensor design have stimulated great interest in PZ technology and facilitated diverse applications in a variety of matrices. Methods of antibody immobilization on crystals and several applications are reported including microgravimetric immunoassays, microbial toxins and other contaminants.

Amperometric determination of urea using an NADH-dependent coupled enzyme.

Enzyme electrodes for the amperometric measurement of urea were prepared by co-immobilizing l-glutamate dehydrogenase and urease onto an Immobilon-AV affinity membrane with attachment to a glassy carbon electrode. Reduced nicotinamide adenine dinucleotide (NADH) was used as the electroactive species. The electrochemical oxidation of NADH was monitored at +1.0 V vs. Ag/AgCl. The enzyme immobilized electrode was linear over the range of 2.0 x 10(-5) to 2 x 10(-4)M. The response time of the electrode was 3 min and the optimum pH of enzyme immobilized membrane was pH 7.4-7.6 (Dulbecco's buffer solution). It was stable for at least two weeks and 50 assays. There were no interferences from other physiological material, except for high levels of ascorbic acid.

A fiber optic biosensor for sulfite analysis in food.

The sulfite fiber optic biosensor developed herein is based on the enzymatic oxidation reaction of sulfite catalyzed by sulfite oxidase (SOD). The consumption of O(2) is measured with an O(2) fiber optic sensor which monitors the fluorescence quenching of the indicator, perylene, by molecular oxygen. Perylene is immobilized into a polymer matrix and attached to the end of a fiber bundle forming an O(2) sensor. The enzyme, sulfite oxidase is immobilized on a pre-activated membrane and mounted onto the O(2) sensor. Several analytical characteristics of this sulfite biosensor were investigated including dynamic range, reversibility, reproducibility, stability and selectivity. The sulfite contents of various food samples, e.g. dried fruits, potato flakes, lemon juice were determined and the results obtained were in good agreement with the standard AOAC method.