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

Hart, J. P.; Pemberton, R. M.; Hughes, G.; Hughes, Gareth; Pemberton, Roy; Fielden, P. R.; Hart, John P.

Abstract

© 2016 Elsevier B.V. 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+0.1V (vs. Ag/AgCl). All experiments were carried out at the following conditions: pH 7, temperature 37°C, atmosphere 5% CO2. The linear range of the device was found to be 25–125μM in phosphate buffer (75mM, containing 0.05M NaCl) and 25–150μM in cell culture medium. The limits of detection (LOD) were found to be 1.2μM and 4.2μM based on three times signal to noise, using PBS and culture medium respectively. The sensitivity was calculated to be 106nAμM−1cm−2 and 210nAμM−1cm−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 (1mM, 5mM and 10mM) 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 24h 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 8h. 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.

• Journal Article Type: Article
• Acceptance Date: Jun 2, 2016
• Online Publication Date: Jun 9, 2016
• Publication Date: Aug 24, 2016
• Deposit Date: Jul 18, 2016
• Publicly Available Date: Jun 9, 2017
• Journal: Analytica Chimica Acta
• Print ISSN: 0003-2670
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 933
• Pages: 82-88
• DOI: https://doi.org/10.1016/j.aca.2016.06.005
• Keywords: glutamate, microband, biosensor, cell toxicity, amperometric, HepG2
• Public URL: https://uwe-repository.worktribe.com/output/919888
• Publisher URL: http://dx.doi.org/10.1016/j.aca.2016.06.005
• Contract Date: Jul 18, 2016