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摘要
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 degrees C, atmosphere 5% CO2. The linear range of the device was found to be 25-125 mu M in phosphate buffer (75 mu M, containing 0.05 M NaCl) and 25-150 mu M in cell culture medium. The limits of detection (LOD) were found to be 1.2 mu M and 4.2 mu M based on three times signal to noise, using PBS and culture medium respectively. The sensitivity was calculated to be 106 nA mu M-1 cm(-2) and 210 nA mu M-1 cm(-2) in PBS and cell medium respectively. The response time was similar to 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 mu M, 93 mu M and 177 mu M, 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 mu M, 28 mu M and 62 mu M respectively. This result demonstrates the feasibility of using this approach to monitor early metabolic changes after exposure to a model toxic compound.
- 出版日期2016-8-24
