An SPR based sensor for allergens detection.

A simple, sensitive and label-free optical sensor method was developed for allergens analysis using α-casein as the biomarker for cow's milk detection, to be used directly in final rinse samples of cleaning in place systems (CIP) of food manufacturers. A Surface Plasmon Resonance (SPR) sensor chip consisting of four sensing arrays enabling the measurement of samples and control binding events simultaneously on the sensor surface was employed in this work. SPR offers several advantages in terms of label free detection, real time measurements and superior sensitivity when compared to ELISA based techniques. The gold sensor chip was used to immobilise α-casein-polyclonal antibody using EDC/NHS coupling procedure. The performance of the assay and the sensor was first optimised and characterised in pure buffer conditions giving a detection limit of 58ngmL-1 as a direct binding assay. The assay sensitivity can be further improved by using sandwich assay format and amplified with nanoparticles. However, at this stage this is not required as the detection limit achieved exceeded the required allergens detection levels of 2µgmL-1 for α-S1-casein. The sensor demonstrated good selectivity towards the α-casein as the target analyte and adequate recoveries from CIP final rinse wash samples. The sensor would be useful tool for monitoring allergen levels after cleaning procedures, providing additional data that may better inform upon wider food allergen risk management decision(s) that are made by food manufacturer. In particular, this sensor could potentially help validate or optimise cleaning practices for a given food manufacturing process.

In silico designed nanoMIP based optical sensor for endotoxins monitoring.

Molecular modelling was used to select specific monomers suitable for the design of molecularly imprinted polymers (MIPs) with high affinity towards endotoxins. MIPs were synthesised using solid-phase photopolymerisation with endotoxins from Escherichia coli 0111:B4 as the template. This technique also allowed the endotoxin template to be reused successfully. Particle size of ~190-220 nm was achieved with low polydispersity index, which confirms the quality of the produced MIPs. For the development of the optical sensor, SPR-2 biosensor system was used by functionalising the gold sensor chip with the MIP nanoparticles using EDC/NHS coupling procedure. The affinity based-endotoxin assay can detect endotoxins in the concentration range of 15.6-500 ng mL(-1). MIP surfaces were regenerated showing stability of the method for subsequent analysis and dissociation constants were calculated as 3.24-5.24×10(-8) M. The developed SPR sensor with the novel endotoxins nanoMIP showed the potential of the technology for endotoxins capture, detection and risk management and also the importance of computational modelling to design the artificial affinity ligands.

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.

Detection of ß-amyloid peptide (1-16) and amyloid precursor protein (APP770) using spectroscopic ellipsometry and QCM techniques: a step forward towards Alzheimers disease diagnostics.

A highly sensitive method of spectroscopic ellipsometry in total internal reflection mode (TIRE) was exploited for detecting ß-amyloid peptide (Aß(1-16)) in the direct immune reaction with monoclonal DE2 antibodies (raised against Aß(1-16)) electrostatically immobilised on the surface of gold. A rapid detection of Aß(1-16) in a wide range of concentrations from 5 µg/ml down to 0.05 ng/ml was achieved using a cost-effective and label-free direct immunoassay format. TIRE dynamic spectral measurements proved that the immune reaction between DE2 monoclonal antibodies and Aß(1-16) is highly specific with the affinity constant K(D)=1.46×10(-8) mol/l. The same DE2 antibodies were utilised for detection of amyloid precursor protein APP(770), a larger protein containing Aß(1-16) domain, using the quartz crystal microbalance (QCM) measurements in liquid. A combination of QCM and TIRE kinetics results allowed the evaluation of the originally unknown concentration of APP(770) in complete medium solution containing other proteins, salts, and amino acids.

DBT degradation enhancement by decorating Rhodococcus erythropolis IGST8 with magnetic Fe3O4 nanoparticles.

Biodesulfurization (BDS) of dibenzothiophene (DBT) was carried out by Rhodococcus erythropolis IGST8 decorated with magnetic Fe3O4 nanoparticles, synthesized in-house by a chemical method, with an average size of 45-50 nm, in order to facilitate the post-reaction separation of the bacteria from the reaction mixture. Scanning electron microscopy (SEM) showed that the magnetic nanoparticles substantially coated the surfaces of the bacteria. It was found that the decorated cells had a 56% higher DBT desulfurization activity in basic salt medium (BSM) compared to the nondecorated cells. We propose that this is due to permeabilization of the bacterial membrane, facilitating the entry and exit of reactant and product, respectively. Model experiments with black lipid membranes (BLM) demonstrated that the nanoparticles indeed enhance membrane permeability.

Evaluation of the potential of applying composting/bioremediation techniques to wastes generated within the construction industry.

The objective of the present study was to evaluate the viability of reducing landfill requirements to satisfy EC Landfill Directive requirements by applying composting/bioremediation techniques to the construction and demolition (C&D) industry waste stream at laboratory scale. The experimental study was carried out in nine test rigs to examine different wood mixtures; untreated timber, creosote treated timber and chromated copper arsenate (CCA) treated timber. Several experimental variables affecting the process were characterised and optimised. These include the best nitrogen additive and optimum moisture content required for composting. Poultry manure was found to be the best nitrogen additive. The optimum moisture content was decreased after the addition of poultry manure. The composting/bioremediation process was evaluated through monitoring the microbial activity, carbon dioxide emissions and toxicity examination of the composted product. A typical temperature profile suggested that untreated and CCA treated mix could be classified as hot composting whereas creosote treated mix could be classified as cold composting. The paper reports on the results obtained during this investigation.

Composting and bioremediation process evaluation of wood waste materials generated from the construction and demolition industry.

The suitability of using bioremediation and composting techniques for diverting construction and demolition (C&D) waste from landfill has been validated in this study. Different timber products from C&D waste have been composted using various composting approaches. The present work demonstrates the quality of compost produced as a result of composting of mixed board product wood waste, which is frequently obtained from the construction and demolition industry. Three compost mixes were prepared by mixing shredded chip board, medium density fibre, hardboard and melamine. Poultry manure, Eco-Bio mixture and green waste were used as nutrient supplements. The results revealed that compost produced from mixtures of poultry manure and green waste used as nutrient supplements improved the performance in plant growth trials (phytotoxicity tests). Results obtained from the experimental study clearly indicate that the composts produced comply with the criterion suggested in BSI PAS 100 (A specification for compost materials) for use in different applications. Composting can also be demonstrated to be a very practical approach to material management including transport reduction to and from the site. The economic suitability of the process will be improved with the increase in landfill tax. In the current regulatory scenario, it is recommended that these materials should be composted at a centralised facility.

DNA arrays, electronic noses and tongues, biosensors and receptors for rapid detection of toxigenic fungi and mycotoxins: a review.

This paper presents an overview of how microsystem technology tools can be applied to the development of rapid, out-of-laboratory measurement capabilities for the determinations of toxigenic fungi and mycotoxins in foodstuffs. Most of the topics discussed are all under investigation within the European Commission-sponsored project Good-Food (FP6-IST). These are DNA arrays, electronic noses and electronic tongues for the detection of fungal contaminants in feed, and biosensors and chemical sensors based on microfabricated electrode systems, antibodies and novel synthetic receptors for the detection of specific mycotoxins. The approach to resolution of these difficult measurement problems in real matrices requires a multidisciplinary approach. The technology tools discussed can provide a route to the rapid, on-site generation of data that can aid the safe production of high-quality foodstuffs.

Surface plasmon resonance sensor for domoic acid based on grafted imprinted polymer.

A molecularly imprinted polymer (MIP) film for domoic acid (DA) was synthesised by direct photo-grafting onto a gold chip suitable for a surface plasmon resonance (SPR) based bioanalytical instrument system, the BIAcore 3000. The gold surface was first functionalised with a self-assembled monolayer of 2-mercaptoethylamine and subsequent carbodiimide chemistry was performed for covalent attachment of the photoinitiator, 4,4'-azobis(cyanovaleric acid). This ensured that the formation of the MIP thin film, comprising 2-(diethylamino) ethyl methacrylate as functional monomer and ethylene glycol dimethacrylate as cross-linker, occurred only at the surface level. Optimisation and control over the grafting procedure were achieved using contact angle measurements and atomic force microscope (AFM) imaging. The surface grafting resulted in the formation of thin and homogeneous MIP film with thickness of 40 nm. A competitive binding assay was performed with free DA and its conjugate with horseradish peroxidase, which was used as a refractive label. The sensor was evaluated for its sensitivity, cross-reactivity, and robustness by using a BIAcore 3000. Likewise, monoclonal antibodies acting as natural receptors for the toxin were studied with the same BIAcore system. Results of a comparison between the artificial and natural receptors are reported. In contrast to monoclonal antibodies, the regeneration of MIP chip did not affect its recognition properties and continuous measurement was possible over a period of at least 2 months.

MIP-based solid phase extraction cartridges combined with MIP-based sensors for the detection of microcystin-LR.

Microsystin-LR is one of the most widespread and dangerous toxins produced by the freshwater Cyanobacteria. The contamination of water supplies with microcystin-LR has been reported in several areas around the world and the development of an easy-to-use, rapid, robust and inexpensive sensor for this toxin is urgently required. In this work an artificial receptor for microcystin-LR was synthesised using the technique of molecular imprinting. The composition of the molecularly imprinted polymer (MIP) was optimised using computer modelling. The synthesised polymer was used both as a material for solid-phase extraction (SPE) and as a sensing element in a piezoelectric sensor. Using the combination of SPE followed by detection with a piezoelectric sensor the minimum detectable amount of toxin was 0.35 nM. The use of MIP-SPE provided up to 1000 fold pre-concentration, which was more than sufficient for achieving the required detection limit for microcystin-LR in drinking water (1 nM). This work is the first example where the same MIP receptor has been used successfully for both SPE and the corresponding sensor.

Screen-printed amperometric biosensors for the rapid measurement of L- and D-amino acids.

Screen-printed three-electrode amperometric sensors incorporating L- and/or D-amino acid oxidase for the general purpose measurement of L- or D-amino acids is described. The working electrode incorporates rhodinized carbon, to facilitate hydrogen peroxide oxidation at a decreased operating potential, and immobilized enzyme. The devices responded to all 20 common L-amino acids and all of the D-amino acids examined, the exceptions being L- and D-proline. Linear response profiles were observed for L-leucine, L-glycine and L-phenylalanine with limits of detection of 0.47, 0.15 and 0.20 mM respectively. The devices were reproducible and exhibited stability over a 56 d test period. The biosensor compares favourably with a standard photometric amino acid test and was used to monitor milk ageing effects. The assay is cheap, simple to perform and rapid, requiring only buffer-electrolyte and a small sample volume.

Immunomagnetic separation with mediated flow injection analysis amperometric detection of viable Escherichia coli O157.

The coupling of an immunological separation (using immunomagnetic beads) with amperometric flow injection analysis detection of viable bacteria is presented. Using a solution containing Escherichia coli O157, the electrochemical response with two different mediators [potassium hexacyanoferrate(III) and 2,6-dichlorophenolindophenol] was evaluated in the FIA system. Antibody-derivatized Dynabeads were used to selectively separate E. coli O157 from a matrix. The kinetics and the capacity parameters regarding the attachment of bacteria to the immunobeads were studied. The immunomagnetic separation was then used in conjunction with electrochemical detection to measure the concentration of viable bacteria. A calibration curve of colony-forming units (cfu) against electrochemical response was obtained. The detection limit for this rapid microbiological method was 10(5) cfu mL-1, and the complete assay was performed in 2 h. Some advantages over ELISA methods are the direct detection of viable cells (and not total bacterial load) and the need for only one antibody (not enzyme-labeled), thus making the assay faster (only one washing step is necessary) and less expensive.

Microbial detection.

There is a widespread need for commercial instrumentation for the rapid and inexpensive detection of microbial contamination of food, industrial waste water and clinical samples. A large number of detection methods have been developed utilizing the optical, electrochemical, biochemical and physical properties of microorganisms. The need for a device which can produce a rapid, accurate, sensitive, real-time analysis for clinical, industrial and environmental applications has led to considerable progress being achieved in recent years in the development of biosensors for microbial detection. This intense research has resulted in the commercialization of several instruments. Techniques used for the quantification of microorganisms are reviewed under the general categories of non-bioelectrochemical and bioelectrochemical methods.