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A novel reagentless glutamate microband biosensor for real-time cell toxicity monitoring

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A novel reagentless glutamate microband biosensor for real-time cell toxicity monitoring. / Hughes, G.; Pemberton, R. M.; Fielden, Peter Robert et al.
In: Analytica Chimica Acta, Vol. 933, 24.08.2016, p. 82-88.

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Hughes G, Pemberton RM, Fielden PR, Hart JP. A novel reagentless glutamate microband biosensor for real-time cell toxicity monitoring. Analytica Chimica Acta. 2016 Aug 24;933:82-88. Epub 2016 Jun 9. doi: 10.1016/j.aca.2016.06.005

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Hughes, G. ; Pemberton, R. M. ; Fielden, Peter Robert et al. / A novel reagentless glutamate microband biosensor for real-time cell toxicity monitoring. In: Analytica Chimica Acta. 2016 ; Vol. 933. pp. 82-88.

Bibtex

@article{faaa93711d7d4cb4a5eaae37343191f5,
title = "A novel reagentless glutamate microband biosensor for real-time cell toxicity monitoring",
abstract = "A reagentless glutamate biosensor was applied to the determination of glutamate released from liver hepatocellular carcinoma cells (HepG2) in response to toxic challenge from various concentrations of paracetamol. A screen printed carbon electrode (SPCE) containing the electrocatalyst Meldola{\textquoteright}s Blue (MB-SPCE) served as the electron mediator for the oxidation of NADH.A mixture of the enzyme glutamate dehydrogenase (GLDH), cofactor nicotinamide adenine dinucleotide (NAD+) and the biopolymer chitosan (CHIT) were drop-coated onto the surface of the transducer (MB-SPCE) in a simple one step fabrication process.The reagentless biosensor was used with amperometry in stirred solution at an applied potential of +0.1 V (vs. Ag/AgCl). All experiments were carried out at the following conditions: pH 7, temperature 37ºC, atmosphere 5% CO2.The linear range of the device was found to be 25 – 125 μM in phosphate buffer (75 mM, containing 0.05 M NaCl) and 25 – 150 μM in cell culture medium. The limits of detection (LOD) were found to be 1.2 μM and 4.2 μM based on three times signal to noise, using PBS and culture medium respectively. The sensitivity was calculated to be 106 nA μM-1 cm-2 and 210 nA μM-1 cm-2 in PBS and cell medium respectively. The response time was ∼60s in an agitated solution.HepG2 cells were exposed to various concentrations of paracetamol (1 mM, 5 mM and 10 mM) in order to investigate the drug-induced release of glutamate into the culture medium in real time. Two toxicity studies were investigated using different methods of exposure and analysis.The first method consisted of a single measurement of the glutamate concentration, using the method of standard addition, after 24 hours incubation. The concentrations of glutamate were found to be 52μM, 93μM and 177μM, released on exposure to 1mM, 5mM and 10mM paracetamol respectively.The second method involved the continuous monitoring of glutamate released from HepG2 cells upon exposure to paracetamol over 8 hours. The concentrations of glutamate released in the presence of 1mM, 5mM and 10mM paracetamol, increased in proportion to the drug concentration, ie: 16μM, 28μM and 62μM respectively. This result demonstrates the feasibility of using this approach to monitor early metabolic changes after exposure to a model toxic compound.",
keywords = "Glutamate, Microband, Biosensor, Cell toxicity, Amperometric, HepG2",
author = "G. Hughes and Pemberton, {R. M.} and Fielden, {Peter Robert} and Hart, {J. P.}",
year = "2016",
month = aug,
day = "24",
doi = "10.1016/j.aca.2016.06.005",
language = "English",
volume = "933",
pages = "82--88",
journal = "Analytica Chimica Acta",
issn = "0003-2670",
publisher = "Elsevier Science B.V.",

}

RIS

TY - JOUR

T1 - A novel reagentless glutamate microband biosensor for real-time cell toxicity monitoring

AU - Hughes, G.

AU - Pemberton, R. M.

AU - Fielden, Peter Robert

AU - Hart, J. P.

PY - 2016/8/24

Y1 - 2016/8/24

N2 - A reagentless glutamate biosensor was applied to the determination of glutamate released from liver hepatocellular carcinoma cells (HepG2) in response to toxic challenge from various concentrations of paracetamol. A screen printed carbon electrode (SPCE) containing the electrocatalyst Meldola’s Blue (MB-SPCE) served as the electron mediator for the oxidation of NADH.A mixture of the enzyme glutamate dehydrogenase (GLDH), cofactor nicotinamide adenine dinucleotide (NAD+) and the biopolymer chitosan (CHIT) were drop-coated onto the surface of the transducer (MB-SPCE) in a simple one step fabrication process.The reagentless biosensor was used with amperometry in stirred solution at an applied potential of +0.1 V (vs. Ag/AgCl). All experiments were carried out at the following conditions: pH 7, temperature 37ºC, atmosphere 5% CO2.The linear range of the device was found to be 25 – 125 μM in phosphate buffer (75 mM, containing 0.05 M NaCl) and 25 – 150 μM in cell culture medium. The limits of detection (LOD) were found to be 1.2 μM and 4.2 μM based on three times signal to noise, using PBS and culture medium respectively. The sensitivity was calculated to be 106 nA μM-1 cm-2 and 210 nA μM-1 cm-2 in PBS and cell medium respectively. The response time was ∼60s in an agitated solution.HepG2 cells were exposed to various concentrations of paracetamol (1 mM, 5 mM and 10 mM) in order to investigate the drug-induced release of glutamate into the culture medium in real time. Two toxicity studies were investigated using different methods of exposure and analysis.The first method consisted of a single measurement of the glutamate concentration, using the method of standard addition, after 24 hours incubation. The concentrations of glutamate were found to be 52μM, 93μM and 177μM, released on exposure to 1mM, 5mM and 10mM paracetamol respectively.The second method involved the continuous monitoring of glutamate released from HepG2 cells upon exposure to paracetamol over 8 hours. The concentrations of glutamate released in the presence of 1mM, 5mM and 10mM paracetamol, increased in proportion to the drug concentration, ie: 16μM, 28μM and 62μM respectively. This result demonstrates the feasibility of using this approach to monitor early metabolic changes after exposure to a model toxic compound.

AB - A reagentless glutamate biosensor was applied to the determination of glutamate released from liver hepatocellular carcinoma cells (HepG2) in response to toxic challenge from various concentrations of paracetamol. A screen printed carbon electrode (SPCE) containing the electrocatalyst Meldola’s Blue (MB-SPCE) served as the electron mediator for the oxidation of NADH.A mixture of the enzyme glutamate dehydrogenase (GLDH), cofactor nicotinamide adenine dinucleotide (NAD+) and the biopolymer chitosan (CHIT) were drop-coated onto the surface of the transducer (MB-SPCE) in a simple one step fabrication process.The reagentless biosensor was used with amperometry in stirred solution at an applied potential of +0.1 V (vs. Ag/AgCl). All experiments were carried out at the following conditions: pH 7, temperature 37ºC, atmosphere 5% CO2.The linear range of the device was found to be 25 – 125 μM in phosphate buffer (75 mM, containing 0.05 M NaCl) and 25 – 150 μM in cell culture medium. The limits of detection (LOD) were found to be 1.2 μM and 4.2 μM based on three times signal to noise, using PBS and culture medium respectively. The sensitivity was calculated to be 106 nA μM-1 cm-2 and 210 nA μM-1 cm-2 in PBS and cell medium respectively. The response time was ∼60s in an agitated solution.HepG2 cells were exposed to various concentrations of paracetamol (1 mM, 5 mM and 10 mM) in order to investigate the drug-induced release of glutamate into the culture medium in real time. Two toxicity studies were investigated using different methods of exposure and analysis.The first method consisted of a single measurement of the glutamate concentration, using the method of standard addition, after 24 hours incubation. The concentrations of glutamate were found to be 52μM, 93μM and 177μM, released on exposure to 1mM, 5mM and 10mM paracetamol respectively.The second method involved the continuous monitoring of glutamate released from HepG2 cells upon exposure to paracetamol over 8 hours. The concentrations of glutamate released in the presence of 1mM, 5mM and 10mM paracetamol, increased in proportion to the drug concentration, ie: 16μM, 28μM and 62μM respectively. This result demonstrates the feasibility of using this approach to monitor early metabolic changes after exposure to a model toxic compound.

KW - Glutamate

KW - Microband

KW - Biosensor

KW - Cell toxicity

KW - Amperometric

KW - HepG2

U2 - 10.1016/j.aca.2016.06.005

DO - 10.1016/j.aca.2016.06.005

M3 - Journal article

VL - 933

SP - 82

EP - 88

JO - Analytica Chimica Acta

JF - Analytica Chimica Acta

SN - 0003-2670

ER -