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Pemberton R.M.,University of the West of England | Xu J.,University of the West of England | Pittson R.,Gwent Electronic Materials Ltd | Drago G.A.,Applied Enzyme Technology Ltd | And 3 more authors.
Biosensors and Bioelectronics | Year: 2011

Microband biosensors, screen-printed from a water-based carbon ink containing cobalt phthalocyanine redox mediator and glucose oxidase (GOD) enzyme, were used to monitor glucose levels continuously in buffer and culture medium. Five biosensors were operated amperometrically (Eapp of +0.4V), in a 12-well tissue culture plate system at 37°C, using a multipotentiostat. After 24h, a linear calibration plot was obtained from steady-state current responses for glucose concentrations up to 10mM (dynamic range 30mM). Within the linear region, a correlation coefficient (R2) of 0.981 was obtained between biosensor and spectrophotometric assays. Over 24h, an estimated 0.15% (89nmol) of the starting glucose concentration (24mM) was consumed by the microbiosensor. The sensitivity of the biosensor response in full culture medium was stable between pHs 7.3 and 8.4.Amperometric responses for HepG2 monolayer cultures decreased with time in inverse proportionality to cell number (for 0 to 106cell/ml), as glucose was being metabolised. HepG2 3D cultures (spheroids) were also shown to metabolise glucose, at a rate which was independent of spheroid age (between 6 and 15 days). Spheroids were used to assay the effect of a typical hepatotoxin, paracetamol. At 1mM paracetamol, glucose uptake was inhibited by 95% after 6h in culture; at 500μM, around 15% inhibition was observed after 16h. This microband biosensor culture system could form the basis for an in vitro toxicity testing system. © 2010 Elsevier B.V.

Pemberton R.M.,University of the West of England | Cox T.,QinetiQ | Cox T.,University of the West of England | Tuffin R.,QinetiQ | And 12 more authors.
Sensors (Switzerland) | Year: 2014

This report describes the design and development of an integrated electrochemical cell culture monitoring system, based on enzyme-biosensors and chemical sensors, for monitoring indicators of mammalian cell metabolic status. MEMS technology was used to fabricate a microwell-format silicon platform including a thermometer, onto which chemical sensors (pH, O2) and screen-printed biosensors (glucose, lactate), were grafted/deposited. Microwells were formed over the fabricated sensors to give 5-well sensor strips which were interfaced with a multipotentiostat via a bespoke connector box interface. The operation of each sensor/biosensor type was examined individually, and examples of operating devices in five microwells in parallel, in either potentiometric (pH sensing) or amperometric (glucose biosensing) mode are shown. The performance characteristics of the sensors/biosensors indicate that the system could readily be applied to cell culture/toxicity studies. © 2014 by the authors; licensee MDPI, Basel, Switzerland.

Pemberton R.M.,University of the West of England | Rawson F.J.,University of the West of England | Xu J.,University of the West of England | Pittson R.,Gwent Electronic Materials Ltd | And 4 more authors.
Microchimica Acta | Year: 2010

Microband biosensors were fabricated from a screen-printed water-based carbon ink containing cobalt phthalocyanine redox mediator and glucose oxidase or lactate oxidase enzyme. The microbiosensors were characterised for their ability to monitor ferrocyanide and H2O2 in phosphate buffer solution: sigmoidal cyclic voltammograms, high current density values and steady-state amperometric responses confirmed the existence of radial-diffusion-limiting microelectrode behaviour. The lactate microband biosensors were then used, in conjunction with a screen-printed Ag/AgCl reference and platinum counter electrode, to monitor lactate levels in culture medium, with a linear range of 0.5-5 mM, sensitivity of 20 nA.mM-1, and dynamic range up to >9 mM. The lactate microband biosensors could operate continuously in culture medium over extended times (up to 24 h) at 37 °C. These biosensors were then applied to detect changes in lactate release from cultured cells in response to toxic challenge: m-dinitrobenzene (500 μM) caused a reduction in lactate production by high-passage number HepG2 single cells; D-galactosamine (20 mM) induced release of lactate by HepG2 spheroid cultures. This novel use of microband biosensors in cell culture has the potential for further application in toxicity monitoring, in both environmental and pharmaceutical areas. © 2010 Springer-Verlag.

Pemberton R.M.,University of the West of England | Cox T.,QinetiQ | Tuffin R.,QinetiQ | Sage I.,QinetiQ | And 11 more authors.
Biosensors and Bioelectronics | Year: 2013

A water-based carbon screen-printing ink formulation, containing the redox mediator cobalt phthalocyanine (CoPC) and the enzyme glucose oxidase (GOx), was investigated for its suitability to fabricate glucose microbiosensors in a 96-well microplate format: (1)the biosensor ink was dip-coated onto a platinum (Pt) wire electrode, leading to satisfactory amperometric performance; (2)the ink was deposited onto the surface of a series of Pt microelectrodes (10-500 μm diameter) fabricated on a silicon substrate using MEMS (microelectromechanical systems) microfabrication techniques: capillary deposition proved to be successful; a Pt microdisc electrode of ≥100 μm was required for optimum biosensor performance; (3)MEMS processing was used to fabricate suitably sized metal (Pt) tracks and pads onto a silicon 96 well format base chip, and the glucose biosensor ink was screen-printed onto these pads to create glucose microbiosensors. When formed into microwells, using a 340 μl volume of buffer, the microbiosensors produced steady-state amperometric responses which showed linearity up to 5. mM glucose (CV=6% for n=5 biosensors). When coated, using an optimised protocol, with collagen in order to aid cell adhesion, the biosensors continued to show satisfactory performance in culture medium (linear range to 2. mM, dynamic range to 7. mM, CV=5.7% for n=4 biosensors). Finally, the operation of these collagen-coated microbiosensors, in 5-well 96-well format microwells, was tested using a 5-channel multipotentiostat. A relationship between amperometric response due to glucose, and cell number in the microwells, was observed. These results indicate that microphotolithography and screen-printing techniques can be combined successfully to produce microbiosensors capable of monitoring glucose metabolism in 96 well format cell cultures. The potential application areas for these microbiosensors are discussed. © 2012 Elsevier B.V.

Zikos C.,Greek National Center For Scientific Research | Evangelou A.,Greek National Center For Scientific Research | Karachaliou C.-E.,Greek National Center For Scientific Research | Gourma G.,Greek National Center For Scientific Research | And 9 more authors.
Chemosphere | Year: 2015

Carbendazim is a fungicide widely used for controlling fungi affecting fruits, vegetables, field crops etc. Determination of carbendazim in water, soil and various crops is frequently required to assure compliance with national/European regulations. A polyclonal antibody recognizing carbendazim was developed by using commercially available 2-(2-aminoethyl) benzimidazole, 2-benzimidazole propionic acid and 2-mercaptobenzimidazole as immunizing haptens; each of the above derivatives was directly conjugated to the carrier protein keyhole limpet hemocyanin and a mixture of the conjugates was administered to New Zealand white rabbits. Immunochemical functionality of the antisera and the corresponding isolated antibody (whole IgG fraction) was evaluated through titer and displacement curves in an in-house developed ELISA, which employed a 2-mercaptobenzimidazole - functionalized lysine-dendrimer as the immobilized hapten. As shown with ELISA-displacement curves, the above antibody could recognize carbendazim as well as other benzimidazole-type fungicides, i.e. benomyl and thiabendazole, and also intact benzimidazole, while it did not cross-react with the structurally different pesticides carbaryl and imazalil. Considering the rather simple approach which has led to its development and its highly promising immunochemical profile, the new antibody may be exploited in immunoanalytical systems for detecting benzimidazole-type pesticides e.g. in samples of environmental interest. The above antibody is being currently tested as a biorecognition element in the novel FOODSCAN cell biosensor platform for pesticide residue detection based on the Bioelectric Recognition Assay technology. © 2014 Elsevier Ltd.

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