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Globally, the most prevalent cyanobacterial toxins, in blooms from fresh and brackish waters, are the cyclic peptide toxins of the microcystin family. The need for on-site algal-toxin monitoring has become increasingly urgent due to the amplified demand for fresh-water and for safe, ‘toxinfree’ shellfish and fish stocks. Along with routine testing of shellfish stocks for biotoxins including microcystin, the EU also require routine monitoring for the presence of the causative algal species. Herein, we describe a novel, Lab-On-A-Disc (LOAD) platform which has been developed to assess microcystin toxin levels in-situ. Using recombinant antibody technology, the LOAD platform combines immunofluorescence with centrifugally driven microfluidic liquid handling to achieve a next-generation disposable device for high throughput sampling. A low-complexity, ‘LED-Photodiode’ based optical sensing system was tailor-made for the system, which allows the fluorescence signal of the microcystin-specific reaction to be quantified. This system can rapidly and accurately detect the presence of microcystin in less than 10 minutes with minimum user interaction and maximum reproducibility. This method provides a low cost diagnostic alternative to the current laborious and costly methods used for toxin monitoring.

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 technology development presented here is a fully integrated and portable sample-to-answer centrifugal microfluidics-based system for the detection of toxic cyanobacteria – Microcystin-LR in fresh water. Our unique system employs highly-specific recombinant chicken anti-microcystin antibodies, prepared in-house, with a 3D-printed ‘LASER-photo¬diode’ fluorescent detection technique, also developed in-house. The system has high analytical specificity and sensitivity for detection of toxins below the regulatory limit with intra/inter day coefficient of variation of less than 20%. Dissolvable-film based valving technique was used for flow actuation and integration of multiple assays on the centrifugal cartridge. This new approach forms the basis of a cost efficient, USB-controlled water quality monitoring system. Technically, this integrated system consists of two components; a microfluidic disc (figure 1.A), the disc-holder fabricated and assembled from a 3D-printed casing, with electronic components housed in device. The 5-layered microfluidic disc consists of five reservoirs (figure 1.B), each with a separate venti-lation, aligned radially with inter-connected microchannels. A competitive immunoassay format is utilised to detect free toxin (figure 1.C). Sensitivity, reproducibility and ease-of-use are key features of this monitoring device. The ‘top-down’ optical detection system has been modified for improved detection sensitivity, as well as the elimination of external noise.

The characterization of a polyclonal antibody produced via immunization with an [Os(bpy)2dcbpy] hapten is described. Bpy is 2,2′-bipyridine and dcbpy is 2,2′-bipyridine-4,4′-dicarboxylic acid. The cross-reactivity of the antibody for the Ru(II) analogue of the hapten was also investigated. Large increases in the emission and luminescent lifetime of a series of Os and Ru complexes were observed on binding of the antibody. Association equilibrium constants were derived from luminescence titration data and were found to be 5.6 × 108 and 5.0 × 108 M−1 for [Os(bpy)2dcbpy] and [Ru(bpy)2dcbpy], respectively. Spectroscopic changes were likely due to the exclusion of H2O from the complex/antibody binding cleft and blocking of vibrational relaxation pathways of the Os/Ru excited state. D2O/H2O experiments confirmed that the antibody protected approx. 82% of [Os(bpy)2dcbpy] and 80% of [Ru(bpy)2dcbpy] from excited state deactivation by the aqueous solvent.

A polyclonal antibody raised against morphine-3-glucuronide (M3G, the main metabolite of heroin and morphine) was used in the development of a novel assay format using a surface plasmon resonance (SPR)-based biosensor. Previously developed assays have generated calibration curves based on differences in the quantity of response units binding to the surface of a chip coated with the analyte. The novel assay described here was based on the development of a standard curve using the slope of a series of consecutive binding interactions. Using this format, regeneration between each assay cycle was no longer required. This increased the useable life span of the chip surface and, as a result, decreased the cost associated with the assay. Thus, at least 15 binding interactions could be carried out before the saturation of antibody on the surface of the chip caused the response to deviate significantly from linearity. After 15 nonregenerated binding interactions, the slope still remained within 1.5% of the slope after a single binding event. Analysis time, and the sample volumes required were also markedly decreased while sensitivity was enhanced. The inhibition assay developed had a detection range of 270 to 17,500 pg ml 1 .

A recombinant single-chain variable fragment (scFv) antibody to morphine-3-glucuronide (M3G) was produced using genetic material obtained from the spleen cells of mice immunised with a morphine-3-glucuronide-bovine serum albumin (M3G-BSA) conjugate. Immunoglobulin light (VL) and heavy (VH) chain genes were amplified and cloned into pAK vectors for generation of recombinant antibody fragments in Escherichia coli. A competition ELISA assay was developed in PBS to characterise the ability of the antibody fragments to recognise free drug and the detection limits were found to be as low as 3 ng ml−1. Surface plasmon resonance-based inhibition immunoassays were developed. The recombinant antibody was pre-incubated with various concentrations of free drug followed by injection over a morphine-3-glucuronide-thyroglobulin (M3G-THY) immobilised surface. The response of antibody binding to the surface of the chip was inversely proportional to the amount of free drug in solution. Regeneration conditions for antibody binding to the surface were optimised resulting in a binding-regeneration capacity of at least 30 cycles. The inhibition assay for M3G was tested with assay ranges between 3 and 195 ng ml−1 and 3 and 97 ng ml−1 in PBS and urine, respectively.

The production and characterization of recombinant antibodies to aflatoxin B[SUB1] (AFB[SUB1]), a potent mycotoxin and carcinogen is described. The antibody fragments produced were then applied for use in a surface plasmon resonance-based biosensor (BIAcore), which measures biomolecular interactions in 'real-time'. Single chain Fv (scFv) antibodies were generated to aflatoxin B1 from an established phage display system, which incorporated a range of different plasmids for efficient scFv expression. The scFv's were used in the development of a competitive ELISA, and also for the development of surface plasmon resonance (SPR)-based inhibition immunoassays. They were found to be suitable for the detection of AFB[SUB1], in this format, with the assays being sensitive and reproducible.

An assay utilizing CE, has been developed for studying the in vitro metabolism of 7-hydroxycoumarin to 7-hydroxycoumarin-glucuronide. A reaction mixture containing a crude preparation of bovine uridine diphosphate (UDP) glucuronyl transferase (UDPGT) and the substrates uridine diphosphate glucuronic acid (UDPGA) and 7-hydroxycoumarin was prepared. Aliquots were removed from the reaction mixture at specific time intervals and analyzed on a 57-cm untreated silica capillary for the presence of 7-hydroxycoumarin-glucuronide. Samples were electrophoresed in a 100 mM phosphate/11 mM deoxycholic acid (sodium salt)/acetonitrile electrolyte buffer, pH 7.0, at 30 kV, with detection at 320 nm. The method allowed the determination of 7-hydroxycoumarin-glucuronide without sample cleanup, with a limit of detection of 2 micrograms/mL, and a linear detection range of 0 microgram/mL to 100 micrograms/mL. The 7-hydroxycoumarin-glucuronide, concentrations were calculated from calibration curves of standards prepared in enzyme solution. The initial rate of production of 7-hydroxycoumarin-glucuronide, in the first 70 min, was 3.1 +/- 0.13 nmol/mL/min/mg protein. The method was fast and reliable with percentage relative standard deviations for concentration of 7-hydroxycoumarin-glucuronide, over time, of less than 10%.

Fatty-add binding proteins (FABP) and myeloperoxidases (MPO) are associated with many chronic conditions in humans and considered to be important biomarkers for diagnosis of cardiac diseases. Here we assemble a new electrical biosensor platform based on graphene-coated interdigitated electrode arrays (IDE-arrays) towards ultrafast, label-free screening of heart type-FABP and MPO. Arrays of nanoscale (nanoIDE) and microscale (microIDE) electrode-arrays were fabricated on wafer-scale by combining nanoimprint and photolithography processes. Chemical vapor deposition grown multilayer graphene was transferred onto nano/microIDE-arrays and used as a high surface-to-volume ratio electrical transducer. Novel biofunctional layers of specially engineered anti-h-FABP and anti-MPO single-chain fragment variables (scFv) were immobilized onto graphene-coated IDE-array sensor platform for electrical detection of h-FABP and MPO in physiological saline. scFv fragments show increased sensitivity in comparison to the state-of-the-art competitive ELISA for their higher affinity towards target analytes. Deploying FABP and MPO specific scFvs as receptor molecules onto our high sensitivity graphene-coated IDE-arrays with identical sensor characteristics and assays covering clinically relevant concentrations in physiological saline, we demonstrate realization of a simple and versatile biosensor platform capable of high performance cardiac-bioassays for point-of-care applications. Stiftung Rheinland-Pfalz fur Innovation [1082]; Euroimmun AG; European Commission FP7 Programme through the Marie Curie Initial Training Network PROSENSE [317420]; DCU INVENT; Imperial sources

The synthesis and characterisation of ruthenium (II) bis-(1 ,10-phenanthroline) and bis-(2 ,2 1-bipyridyl) complexes containing amino and isothiocyanate phenanthroline and pyridine ligands are described. These complexes were characterised using HPLC, UV/vis spectroscopy, electrochemistry, NMR, IR, and emission spectroscopy. HPLC was used to determine the purity of the various complexes. Electronic spectra and electrochemical measurements indicate that most likely, the 2 ,2 '-bipyridyl and 1 ,10-phenanthroline ligands are the emitting ligands and the amino and isothiocyanate phenanthroline and pyridine ligands act as the spectator ligands. These complexes were subsequently conjugated to biological materials, including albumins, immunoglobulins and poly-L-lysine. The complexes were bound at different sites on the biomolecules, via the lysine residues, the carbohydrate moieties and via the tyrosine residues. The effect of conjugation on the spectroscopic properties of the complexes was examined. The absorption spectra were found to exhibit changes in the MLCT band with the absorption wavelength maximum experiencing slight red shifts together with general band broadening effects. The emission decay behaviour was also investigated. The unbound complexes were found to exhibit single exponential decay behaviour and the bound complexes exhibited essentially double exponential behaviour. From the double exponential decay fits, the first short-lived species was suggested to be a quenched bound species whilst the second species probably arises from a bound species which is considerably protected from the effects of quenching molecules. The synthesis and characterisation of a number of bis-(1 ,10-phenanthroline) (phen), and bis-(4,4'-dimethyl- 2, 2'-bipyridyl) (dmbpy), ruthenium (II) complexes containing a series of pyridyltriazole ligands are described. The ruthenium ion may bind via the Nl'/ N 2 ' or n4' of the triazole ring. The most favourable coordination mode being dependent on the position of a substituent on the triazole ring. Coordination isomers were obtained and separated by semi-preparative HPLC methods. The electrochemical results suggest that the phen and dmbpy ligands are the emitting ligands and that the pyridyltriazole ligands are the spectator ligands. The acid-base chemistry of those pyridyltriazole complexes which can undergo protonation / deprotonation reactions was investigated and the results substantiated the coordination modes proposed using NMR data for the complexes. The ground state pKa values show that there is a strong a-donating effect from the triazole ring to the ruthenium ion. Also, the excited state pKa* values are lower than the ground state pKa values which indicates that the pyridyltriazole ligands do not participate in the emission process. This study completes investigations which were initially conducted on the 2 ,2 '-bipyridyl analogues.