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Cyclic peptide cyanobacterial toxins, in particular Microcystis aeruginosa, pose a serious health risk to humans and animals alike [1], [2]. Occurring mostly in fresh and brackish water, they have been identified to cause cancer promotion and liver damage [3]. Herein, we describe a portable, microfluidic-based system for in-situ detection of algal toxins in fresh water. The Atalanta system is a novel, portable and sample-to-answer platform for the detection of toxic cyanobacteria – Microcystin-LR in fresh water. Atalanta utilises the partnership of highly-specific recombinant chicken anti-microcystin antibodies, prepared in-house, with a 3D-printed ‘LASER-photo¬diode’ fluorescent detection method, also developed in-house. A competitive immunoassay format is utilised to detect free toxin. Furthermore, dissolvable-film (DF) based flow-actuation facilitates full assay inte¬gration. This new approach will form the basis of a cost efficient, USB-controlled water quality monitoring system. The Atalanta detection system consists of two components; the microfluidic Atalanta disc and the disc-holder. The Atalanta disc-holder was fabricated and assembled from a 3D-printed casing, with electronic components housed in device. The 5-layered microfluidic disc consists of five reservoirs, each with a separate venti¬lation, aligned radially with inter-connected microchannels. Each reservoir represents a functional assay step. First, microcystin conjugate is coated to the functionalised surface of the reservoir 3 prior to assembling the disc. A freshwater sample in reservoir 1 is pre-incubated with recombinant antibodies labelled with fluorophore (Alexa 647) in reservoir 2. This is then spun into reservoir 3 for detection through a competitive immunoassay using Microcystin-LR. Low fluorescence signal indicates high Microcystin-LR concentration in the sample.

STRA6 is a plasma membrane protein that mediates the transport of vitamin A, or retinol, from plasma retinol binding protein (RBP) into the cell. Mutations in human STRA6 are associated with Matthew-Wood syndrome, which is characterized by severe developmental defects. Despite the obvious importance of this protein to human health, little is known about its structure and mechanism of action. To overcome the difficulties frequently encountered with the production of membrane proteins for structural determination, STRA6 has been expressed in Pichia pastoris as a fusion to green fluorescent protein (GFP), a strategy which has been a critical first step in solving the crystal structures of several membrane proteins. STRA6-GFP was correctly targeted to the cell surface where it bound RBP. Here we report the large-scale expression, purification and characterisation of STRA6GFP. One litre of culture, corresponding to 175 g cells, yielded about 1.5 mg of pure protein. The interaction between purified STRA6 and its ligand RBP was studied by surface plasmon resonance-based binding analysis. The interaction between STRA6 and RBP was not retinol-dependent and the binding data were consistent with a transient interaction of 1 mole RBP/mole STRA6.

Episodes of toxin-producing phytoplankton occur worldwide, causing both animal and human fatalities. Toxicity occurs through consumption of phycotoxins, including azaspiracid, which accumulate in filter-feeding shellfish. Microcystins are hepatotoxins, produced mainly by freshwater cyanobacteria. Aflatoxins are potent, fungal hepatocarcinogens, which occur mainly in food and feed products. The purpose of this research was to examine the cytotoxic and immunosuppressive effects of aflatoxins (B1, B2 and G1), azaspiracid-1 and microcystin-LR in vitro, using the murine macrophage cell line, J774A.1. The results clearly demonstrated that azaspiracid and microcystin had a significant effect on host defence functions, through deregulation of IL-6, IL-10, IL12p40 and TNF-α cytokine expression. Microcystin exposure significantly decreased IL-1β expression. ‘Toll-like’ receptor (TLR2 and CD14) expression was altered following aflatoxin exposure, while apoptotic marker (caspase-1) expression was affected following microcystin exposure. This knowledge should be taken into consideration in the implementation of detection limits, aimed at minimising risks to human health through toxin exposure. Increased awareness of the hazards presented by toxins led to the requirement for recombinant antibodies for these targets, for incorporation into sensitive detection immunoassays. This thesis describes the production of leprine and avian immune libraries for azaspiracid and microcystin, respectively. Attempts were made to isolate azaspiracid-specific antibodies with little success. Phage display was utilised to successfully isolate two single chain antibody fragments (scFvs) to microcystin from the avian library. Error-prone PCR resulted in the isolation of a mutant clone which displayed a 2.3-fold improvement in sensitivity by ELISA, with an LOD of 1.4 ng/mL. The mutant scFv displayed an altered cross-reactivity profile to the microcystin variants tested using Biacore™ inhibition analysi

In this article we introduce a novel technology that utilizes specialized water dissolvable thin films for valving in centrifugal microfluidic systems. In previous work (William Meathrel and Cathy Moritz, IVD Technologies, 2007), dissolvable films (DFs) have been assembled in laminar flow devices to form efficient sacrificial valves where DFs simply open by direct contact with liquid. Here, we build on the original DF valving scheme to leverage sophisticated, merely rotationally actuated vapour barriers and flow control for enabling comprehensive assay integration with low-complexity instrumentation on "lab-on-a-disc" platforms. The advanced sacrificial valving function is achieved by creating an inverted gas-liquid stack upstream of the DF during priming of the system. At low rotational speeds, a pocket of trapped air prevents a surface-tension stabilized liquid plug from wetting the DF membrane. However, high-speed rotation disrupts the metastable gas/liquid interface to wet the DF and thus opens the valve. By judicious choice of the radial position and geometry of the valve, the burst frequency can be tuned over a wide range of rotational speeds nearly 10 times greater than those attained by common capillary burst valves based on hydrophobic constrictions. The broad range of reproducible burst frequencies of the DF valves bears the potential for full integration and automation of comprehensive, multi-step biochemical assay protocols. In this report we demonstrate DF valving, discuss the biocompatibility of using the films, and show a potential sequential valving system including the on-demand release of on-board stored liquid reagents, fast centrifugal sedimentation and vigorous mixing; thus providing a viable basis for use in lab-on-a-disc platforms for point-of-care diagnostics and other life science applications.

In this article we introduce a novel technology that utilizes specialized water dissolvable thin films for valving in centrifugal microfluidic systems. In previous work (William Meathrel and Cathy Moritz, IVD Technologies, 2007), dissolvable films (DFs) have been assembled in laminar flow devices to form efficient sacrificial valves where DFs simply open by direct contact with liquid. Here, we build on the original DF valving scheme to leverage sophisticated, merely rotationally actuated vapour barriers and flow control for enabling comprehensive assay integration with low-complexity instrumentation on ‘‘lab-on-a-disc’’ platforms. The advanced sacrificial valving function is achieved by creating an inverted gas-liquid stack upstream of the DF during priming of the system. At low rotational speeds, a pocket of trapped air prevents a surface-tension stabilized liquid plug from wetting the DF membrane. However, high-speed rotation disrupts the metastable gas/liquid interface to wet the DF and thus opens the valve. By judicious choice of the radial position and geometry of the valve, the burst frequency can be tuned over a wide range of rotational speeds nearly 10 times greater than those attained by common capillary burst valves based on hydrophobic constrictions. The broad range of reproducible burst frequencies of the DF valves bears the potential for full integration and automation of comprehensive, multi-step biochemical assay protocols. In this report we demonstrate DF valving, discuss the biocompatibility of using the films, and show a potential sequential valving system including the on-demand release of on-board stored liquid reagents, fast centrifugal sedimentation and vigorous mixing; thus providing a viable basis for use in lab-on-a-disc platforms for point-of-care diagnostics and other life science applications.

Several surface plasmon resonance (SPR) biosensor assays were developed and validated for the detection of anthelmintic veterinary drugs in liver tissue and milk using a QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction procedure. The first screening assay was developed to detect 11 benzimidazole carbamates in milk and liver. In bovine milk the assay showed a limit of detection (LOD) of 2.7 μg kg-1 and a detection capability (CCβ) of 5 μg kg-1. Analyte recovery was in the range 81 to 116% and the assay was found to be fit for purpose when its performance was compared to UPLC-MS/MS analyses of milk from cows treated with benzimidazole products. In bovine liver the LOD (32 μg kg-1) and the CCβ (50 μg kg-1) were determined and the analyte recovery was in the range 77-132%. All non-compliant samples were identified when the assay performance was tested by analysing liver from animals treated with benzimidazole drugs and comparing the results with a UPLC-MS/MS confirmatory method. A screening assay was developed for four amino-benzimidazoles in liver. The LOD (41 μg kg-1) and the CCβ (75 μg kg-1) were determined and the analyte recovery was in the range 103-116%. A screening assay for thiabendazole and 5-hydroxy-thiabendazole in ovine liver tissue using a novel recombinant antibody fragment (Fab) was developed. The LOD (12.3 μg kg-1), the CCβ (20 μg kg-1) and analyte recovery (86-107%) satisfied the criteria required for thiabendazole screening in liver tissue. A biosensor to detect triclabendazole residues in liver tissue was developed through the immobilization of amino-triclabendazole via a glutaraldehyde homo-bifunctional crosslinker. Several experiments were required to reduce non-specific binding in this assay. An LOD of 105 μg kg-1 was determined which was close to the maximum residue limit (MRL) in liver matrix (100 μg kg-1).A biochip array was developed and validated to screen orange juice for fungicide and pesticide residues. The LOD for carb

In this article, we report on poly(amidoamine) dendrimers (PAMAM) as coupling agents for recombinant single-chain (ScFv) antibodies to nanoparticle (NP) labels, for use in immunoassay. We present a simple theory for the kinetics of particle capture onto a surface by means of an antibody–antigen reaction, in which the important parameter is the fraction of the particle surface that is active for reaction. We describe how increasing the generation number of the linking dendrimers significantly increased the fraction of the NP surface that is active for antigen binding and consequently also increased the assay kinetic rates. Use of dendrimers for conjugation of the NP to the antibody resulted in a significantly higher surface coverage of active antibody, in comparison with mono-valent linker chemistry. As a direct consequence, the increase in effective avidity significantly out-weighed any effect of a decreased diffusion coefficient due to the NP, when compared to that of a molecular dye-labelled antibody. The signal to noise ratio of the G4.5 dendrimer-sensitised nanoparticles out-performed the dye-labelled antibody by approximately four-fold. Particle aggregation experiments with the multi-valent antigen CRP demonstrated reaction-limited aggregation whose rate increased significantly with increasing generation number of the dendrimer linker.

Porphyromonas gingivalis is one of the major periodontitis-causing pathogens. P. gingivalis secretes a group of proteases termed gingipains, and in this study we have used the RgpB gingipain as a biomarker for P. gingivalis. We constructed a naive camel nanobody library and used phage display to select one nanobody toward RgpB with picomolar affinity. The nanobody was used in an inhibition assay for detection of RgpB in buffer as well as in saliva. The nanobody was highly specific for RgpB given that it did not bind to the homologous gingipain HRgpA. This indicated the presence of a binding epitope within the immunoglobulin-like domain of RgpB. A subtractive inhibition assay was used to demonstrate that the nanobody could bind native RgpB in the context of intact cells. The nanobody bound exclusively to the P. gingivalis membrane-bound RgpB isoform (mt-RgpB) and to secreted soluble RgpB. Further cross-reactivity studies with P. gingivalis gingipain deletion mutants showed that the nanobody could discriminate between native RgpB and native Kgp and RgpA in complex bacterial samples. This study demonstrates that RgpB can be used as a specific biomarker for P. gingivalis detection and that the presented nanobody-based assay could supplement existing methods for P. gingivalis detection.

The attachment of mouse immunoglobulin G (IgG) and anti-mouse IgG antibodies onto carbon nanotubes (CNTs), using either non-covalent or covalent means was investigated. The resultant CNTs were characterised using a variety of techniques including enzyme-linked and fluorescence-linked immunoassays, UV–visible-NIR and Raman spectroscopy, transmission electron microscopy and cyclic voltammetry. TEM images of the adsorbed antibody on the CNTs show that the covalent modification approach was successful, whereas the non-covalent approach resulted in no electrochemically detectable labelled antibody. Direct electrical communication between CNTs covalently linked to peroxidase-labelled antibodies was observed during cyclic voltammetry, which suggests applications in developing carbon-nanotube-based immunosensors.

The fungal pathogen Pst causes yellow rust disease in wheat plants leading to crop losses. The organism spreads by releasing wind-dispersed urediniospores from infected plants. In this study a library of novel monoclonal antibodies (mAbs) was developed against Pst urediniospores. Nine mAb-producing cell lines were cloned and their cross-reactivities characterised against a panel of airborne fungal spores representing genera commonly found in the same environment as Pst. Two specific mAbs were used to develop a competitive ELISA (Pst mAb4) and a subtractive inhibition ELISA (Pst mAb8). Standard curves for both assays had good intra- and interday reproducibility. The subtractive inhibition ELISA had greater sensitivity with a detection limit of 1.5 105 spores ml1. Cross-reactivity studies of Pst mAb8 in the subtractive inhibition ELISA, showed reaction with other Puccinia spores only, suggesting that common epitopes exist within this genus. The biosensor-compatible Pst mAb8 assay principle developed in this study has the potential to be implemented in future ‘label-free’ in-the-field systems for Pst detection.

This paper describes a biosensor-based method for detection of fungal spores using Surface Plasmon Resonance (SPR). The approach involves the use of a mouse monoclonal antibody (Pst mAb8) and a SPR sensor for label-free detection of urediniospores from the model organism Puccinia striiformis f.sp. tritici (Pst). In the subtractive inhibition assay, urediniospores and Pst mAb8 were mixed, urediniospore-bound Pst mAb8 removed by centrifugation and the remaining Pst mAb8 quantified using the SPR sensor. Assay conditions were optimised and a detection limit of 3.1 x 105 urediniospores/ml was achieved. Spiked Pst samples were further examined in a background of a related spore and it was found that Pst quantification was possible in this mixture. This study represent the first use of SPR technology for fungal spore detection as well as the first report of a successful biosensor-based detection strategy for Pst.

This paper describes a biosensor-based method for detection of fungal spores using Surface Plasmon Resonance (SPR). The approach involves the use of a mouse monoclonal antibody (Pst mAb8) and a SPR sensor for label-free detection of urediniospores from the model organism Puccinia striiformis f.sp. tritici (Pst). In the subtractive inhibition assay, urediniospores and Pst mAb8 were mixed, urediniospore-bound Pst mAb8 removed by centrifugation and the remaining Pst mAb8 quantified using the SPR sensor. Assay conditions were optimised and a detection limit of 3.1 x 105 urediniospores/ml was achieved. Spiked Pst samples were further examined in a background of a related spore and it was found that Pst quantification was possible in this mixture. This study represent the first use of SPR technology for fungal spore detection as well as the first report of a successful biosensor-based detection strategy for Pst.

The fungal pathogen Pst causes yellow rust disease in wheat plants leading to crop losses. The organism spreads by releasing wind-dispersed urediniospores from infected plants. In this study a library of novel monoclonal antibodies (mAbs) was developed against Pst urediniospores. Nine mAb-producing cell lines were cloned and their cross-reactivities characterised against a panel of airborne fungal spores representing genera commonly found in the same environment as Pst. Two specific mAbs were used to develop a competitive ELISA (Pst mAb4) and a subtractive inhibition ELISA (Pst mAb8). Standard curves for both assays had good intra- and interday reproducibility. The subtractive inhibition ELISA had greater sensitivity with a detection limit of 1.5 105 spores ml1. Cross-reactivity studies of Pst mAb8 in the subtractive inhibition ELISA, showed reaction with other Puccinia spores only, suggesting that common epitopes exist within this genus. The biosensor-compatible Pst mAb8 assay principle developed in this study has the potential to be implemented in future â¿¿label-freeâ¿¿ in-the-field systems for Pst detection.