Ramadan A. Abuknesha

Immunosensors for pesticide determination in natural waters

Abstract The development and application of immunosensors for environmental monitoring has grown steadily in recent years. In this review, immunosensors developed in the last few years are highlighted regarding their importance and practical contribution to environmental analysis with particular emphasis on monitoring of pesticide levels. Different transduction elements and mechanisms used for the detection of the physicochemical change(s) produced by the biological interaction are shown, as well as their application to the development of real-time measurement devices or systems. In addition, examples concerning the analysis of pesticides in natural water samples obtained using a recently developed optical immunosensor are given. Validation of immunosensor measurements with conventional chromatographic techniques is also reported. The immunosensor system detection mechanism is based on a solid-phase fluoroimmunoassay combined with an optical transducer chip chemically modified with an analyte derivative. Applications to the analysis of atrazine, simazine, paraquat, alachlor, 2,4-D and isoproturon in water are also reviewed.

Integrated optical fluorescence multisensor for water pollution

An integrated optical multisensor for organic pollutants has been realised, and characterised for a single analyte. The sensor exploits fluorescence immunoassay in the evanescent field of channel waveguides to enable rapid, simultaneous and high-sensitivity fluorescence detection of up to 32 pollutants in water. The chemical modification used to render the surface specific to analytes allows automatic regeneration for immediate reuse. The system has been demonstrated for the key pollutant estrone and a detection limit below 1 ng/L has been achieved.

A membrane-based ELISA assay and electrochemical immunosensor for microcystin-LR in water samples.

We describe within this paper the development of an affinity sensor for the detection of the cyanobacterial toxin microcystin-LR. The first stage of the work included acquiring and testing of the antibodies to this target. Following the investigation, a heterogeneous direct competitive enzyme-linked immunosorbent assay (ELISA) format for microcystin-LR detection was developed, achieving a detection limit, LLD(80) = 0.022 μg L(-1). The system was then transferred to an affinity membrane sorbent-based ELISA. This was an amenable format for immunoassay incorporation into a disposable amperometric immunosensor device. This membrane-based ELISA achieved a detection limit, LLD(80) = 0.06 μg L(-1). A three-electrode immunosensor system was fabricated using thick-film screen-printing technology. Amperometric horseradish peroxidase transduction of hydrogen peroxide catalysis, at low reducing potentials, versus Ag/AgCl reference and carbon counter electrodes, was facilitated by hydroquinone-mediated electron transfer. A detection limit of 0.5 μg L(-1) for microcystin-LR was achieved. Similar levels of detection could be obtained using direct electrochemical sensing of the dye produced using the membrane-based ELISA. These techniques proved to be simple, cost-effective, and suitable for the detection of microcystin-LR in buffer and spiked tap and river water samples.

A sensitive method of detecting proteins on dot and Western blots using a monoclonal antibody to FITC

Monoclonal antibodies to FITC were produced and shown to be specific for the fluorochrome. Molecular weight marker proteins labelled with FITC could be detected after SDS-PAGE and transfer onto nitrocellulose using anti-FITC followed by an anti-mouse IgG-alkaline phosphatase conjugate. The molecular weight of an antigen common to Legionella pneumophila and recognised by a monoclonal antibody could be determined accurately on a Western blot when FITC labelled markers were used as internal standards. The FITC-anti-FITC system was shown to be extremely sensitive, detecting 23.7 amol of BSA-FITC conjugate (equivalent to 1.42 x 10(7) molecules of FITC) in a dot blot assay.

A model to assess the infection potential of jet injectors used in mass immunisation

Jet injectors are needleless injectors that penetrate skin with high-pressure fluid. They have potential advantages over needles and syringes in mass immunisation programs, but concerns over their capacity to transfer blood-borne viruses have been a barrier to acceptance. Hepatitis B infection can transmit in 10 pl of blood; detection of such low volumes presents severe difficulties to such assessments. A model to assess jet injector safety was developed using injection of an inert buffer into calves and assaying the next injector discharge, representing the next dose of vaccine, for blood using a highly sensitive ELISA. Four injectors were tested: two with reusable heads and direct skin contact, one with single-use injector heads and one where the injector head discharged at a distance from the skin. All injectors tested transmitted significant (over 10 pl) volumes of blood; the volumes and frequency of contamination varied with injector. The source of the contamination was consistent with contamination by efflux of injected fluid and blood from the pressurised pocket in tissue that is formed during injection. This insight should inform the design of safe jet injectors.

Asexual sporulation signalling regulates autolysis of Aspergillus nidulans via modulating the chitinase ChiB production.

Aims:  Elucidation of the regulation of ChiB production in Aspergillus nidulans.

Rapid detection of Escherichia coli in water using a hand-held fluorescence detector

The quantification of pathogenic bacteria in an environmental or clinical sample commonly involves laboratory-based techniques and results are not obtained for 24-72 h after sampling. Enzymatic analysis of microbial activity in water and other environmental samples using fluorescent synthetic substrates are well-established and highly sensitive methods in addition to providing a measure of specificity towards indicative bacteria. The enzyme beta-d-glucuronidase (GUD) is a specific marker for Escherichia coli and 4-methylumbelliferone-beta-D-glucuronide (MUG) a sensitive substrate for determining the presence of E. coli in a sample. However, currently used procedures are laboratory-based and require bench-top fluorimeters for the measurement of fluorescence resulting from the enzyme-substrate reaction. Recent developments in electronic engineering have led to the miniaturisation of fluorescence detectors. We describe the use of a novel hand-held fluorimeter to directly analyse samples obtained from the River Thames for the presence of E. coli. The results obtained by the hand-held detector were compared with those obtained with an established fluorescent substrate assay and by quantifying microbial growth on a chromogenic medium. Both reference methods utilised filtration of water samples. The miniaturised fluorescence detector was used and incubation times reduced to 30 min making the detection system portable and rapid. The developed hand-held system reliably detected E. coli as low as 7 cfu/mL river water sample. Our study demonstrates that new hand-held fluorescence measurement technology can be applied to the rapid and convenient detection of bacteria in environmental samples. This enables rapid monitoring to be carried out on-site. The technique described is generic and it may, therefore, be used in conjunction with different fluorescent substrates which allows the assessment of various target microorganisms in biological samples.

Enzyme-immunoassay for the determination of metallothionein in human urine: application to environmental monitoring

The objectives of this study were to develop an enzyme immunoassay for metallothioneins in human urine using a polyclonal antiserum and to demonstrate a possible relationship between the level of this biomarker and heavy metal exposure. The antiserum was raised in sheep against horse metallothionein conjugated to carboxylated bovine serum albumin. The antibody was used to construct a two-step competitive ELISA procedure. Human urine was treated with activated charcoal powder to remove traces of metallothioneins and known amounts of pure metallothioneins were added to provide standards for a standard curve. Metallothionein levels were measured in two groups of children living in areas of mild and high environmental pollution due mainly to heavy metals. A comparison was made between the biomarker levels and the levels of cadmium and lead in urine samples in the two groups. A group of children from a non-polluted area acted as controls. The results show that the detected levels of metallothioneins appear to correspond to levels of the two heavy metals studied and that there was an apparent relationship to the environmental exposure. Thus according to results of this study the increase in the metallothionein excretion seems to provide an indication of previous of exposure to metals. The ELISA procedure is sensitive and robust and can be used to screen large numbers of samples and is more rapid than the physical procedures currently used for analysis of these proteins. The assay can therefore be used as an additional tool for screening at-risk populations where either environmental or occupational exposure to divalent heavy metals is suspected.

Labeling of biotin antibodies with horseradish peroxidase using cyanuric chloride

In this report, we describe a two-step protocol for labeling of an affinity-purified antibody to biotin with horseradish peroxidase (HRP) using cyanuric chloride (CC) as a bridge. The enzyme was first modified with CC, and following chromatography on a PD-10 column, the activated HRP was incubated with the antibody to effect coupling of the two proteins. Assessment of the conjugate product was carried out using ELISA and SDS-PAGE electrophoresis where evidence for high antibody activity, high specific activity of the conjugate preparation, coupling of nearly all the antibody and over 90% of the enzyme was shown. The titer of the conjugate exceeded 1/100,000. High molecular weight complexes were observed in the SDS-PAGE results, indicating an efficient conjugation procedure. The presence of high molecular weight complexes indicated an efficient conjugation procedure. The protocol is simple, and the conjugation steps can be completed in 27 h once the preparatory phase has been carried out; the method is entirely generic and may be applied to labeling of any antibody.

Removal of detergents from protein extracts using activated charcoal prior to immunological analysis.

The use of dextran-coated activated charcoal (DCC) powder to absorb solubilising detergents from cell lysates is described. Normal embryonic epithelial cells were lysed in the presence of sodium dodecyl sulphate (SDS). The detergent was then absorbed with DCC to facilitate analysis of polycystin-1 with antibody-based methods. Polycystin-1 is a membrane protein that is involved in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD). The adverse effect of SDS on antibody-polycystin-1 binding was studied and the improvement resulting from its removal demonstrated using enzyme-linked immunosorbent assays (ELISAs). The results indicate that DCC can be used in a simple manner to remove highly reactive membrane-solubilising reagents from protein mixtures prior to immunological analysis. This procedure may be relevant to a variety of other techniques that are normally affected by detergents.