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'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 37 °C, 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 ∼60 s 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 h incubation. The concentrations of glutamate were found to be 52 μM, 93 μM and 177 μM, released on exposure to 1 mM, 5 mM and 10 mM paracetamol respectively. The second method involved the continuous monitoring of glutamate released from HepG2 cells upon exposure to paracetamol over 8 h. The concentrations of glutamate released in the presence of 1 mM, 5 mM and 10 mM paracetamol, increased in proportion to the drug concentration, ie: 16 μM, 28 μM and 62 μM respectively. This result demonstrates the feasibility of using this approach to monitor early metabolic changes after exposure to a model toxic compound.
authors: Centre for Research in Biosciences, Faculty of Health and Applied Sciences, University of the West of England, Bristol, Coldharbour Lane, Bristol, BS16 1QY, United Kingdom.
authors: G
authors: G
authors: Hughes
authors: Centre for Research in Biosciences, Faculty of Health and Applied Sciences, University of the West of England, Bristol, Coldharbour Lane, Bristol, BS16 1QY, United Kingdom.
authors: R M
authors: RM
authors: Pemberton
authors: Department of Chemistry, Lancaster University, Bailrigg, Lancaster, LA1 4YB, United Kingdom.
authors: P R
authors: PR
authors: Fielden
authors: Centre for Research in Biosciences, Faculty of Health and Applied Sciences, University of the West of England, Bristol, Coldharbour Lane, Bristol, BS16 1QY, United Kingdom. Electronic address: john.hart@uwe.ac.uk.
authors: J P
authors: JP
authors: Hart
mesh_terms: D000082:Acetaminophen / Q000633:toxicity*; D015374:Biosensing Techniques; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000032:analysis* / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: D000082:Acetaminophen; D018698:Glutamic Acid
keywords: Amperometric; Biosensor; Cell toxicity; Glutamate; HepG2; Microband