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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. Source


Brandao D.,Autonomous University of Barcelona | Liebana S.,Applied Enzyme Technology Ltd | Campoy S.,Autonomous University of Barcelona | Cortes M.P.,Autonomous University of Barcelona | And 2 more authors.
Biosensors and Bioelectronics | Year: 2015

Simultaneous detection of Salmonella enterica, Listeria monocytogenes and Escherichia coli based on triple-tagging multiplex PCR and electrochemical magneto genosensing on silica magnetic particles is reported. A set of tagging primers were selected for the specific amplification of yfiR (375bp), hlyA (234bp) and eaeA (151bp), being one of the primers for each set labelled with fluorescein, biotin and digoxigenin coding for S. enterica, L. monocytogenes and E. coli, respectively. Afterwards, electrochemical magneto genosensing of the bacteria was achieved by using silica magnetic particles as a carrier and three different electrochemical reporters, specific for each pathogen. This method was able to clearly distinguish among the pathogenic bacteria tested within 50min, with detection limits ranging from 12 to 46pgμL-1. © 2015 Elsevier B.V. Source


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. Source


Brandao D.,Autonomous University of Barcelona | Liebana S.,Applied Enzyme Technology Ltd | Campoy S.,Autonomous University of Barcelona | Alegret S.,Autonomous University of Barcelona | Pividori M.I.,Autonomous University of Barcelona
Talanta | Year: 2015

Abstract This paper addresses a comparative study of immunomagnetic separation of Salmonella using micro and nano-sized magnetic carriers. In this approach, nano (300 nm) and micro (2.8 μm) sized magnetic particles were modified with anti-Salmonella antibody to pre-concentrate the bacteria from the samples throughout an immunological reaction. The performance of the immunomagnetic separation on the different magnetic carriers was evaluated using classical culturing, confocal and scanning electron microscopy to study the binding pattern, as well as a magneto-actuated immunosensor with electrochemical read-out for the rapid detection of the bacteria in spiked milk samples. In this approach, a second polyclonal antibody labeled with peroxidase as electrochemical reporter was used. The magneto-actuated electrochemical immunosensor was able to clearly distinguish between food pathogenic bacteria such as Salmonella enterica and Escherichia coli, showing a limit of detection (LOD) as low as 538 CFU mL-1 and 291 CFU mL-1 for magnetic micro and nanocarriers, respectively, in whole milk, although magnetic nanoparticles showed a noticeable higher matrix effect and higher agglomeration effect. These LODs were achieved in a total assay time of 1 h without any previous culturing pre-enrichment step. If the samples were pre-enriched for 8 h, the magneto immunosensor based on the magnetic nanoparticles was able to detect as low as 1 CFU in 25 mL of milk (0.04 CFU mL-1). © 2015 Elsevier B.V. Source


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. Source

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