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

Abstract

Disclaimer UWE has obtained warranties from all depositors as to their title in the material deposited and as to their right to deposit such material. UWE makes no representation or warranties of commercial utility, title, or fitness for a particular purpose or any other warranty, express or implied in respect of any material deposited. UWE makes no representation that the use of the materials will not infringe any patent, copyright, trademark or other property or proprietary rights. UWE accepts no liability for any infringement of intellectual property rights in any material deposited but will remove such material from public view pending investigation in the event of an allegation of any such infringement. 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% CO 2 .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 3 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.

Biosensors Integration in Blood–Brain Barrier-on-a-Chip: Emerging Platform for Monitoring Neurodegenerative Diseases

“…Also, in this case electrochemical enzymatic sensors based on glutamate oxidase or glutamate dehydrogenase are excellent candidates for this purpose. 71 , 72 …”

“…For LDH detection, an electrochemical enzymatic sensor functionalized with lactate oxidase and a redox mediator can be used, such as that reported by Park et al Another real-time biomarker for cell cytotoxicity analysis is glutamate that can be affected by neuronal toxicity. Also, in this case electrochemical enzymatic sensors based on glutamate oxidase or glutamate dehydrogenase are excellent candidates for this purpose. , …”

Fabrication of Miniaturised Screen‐printed Glucose Biosensors, Using a Water‐based Ink, and the Evaluation of their Electrochemical Behaviour

“…Another advantage of microband biosensors is that they exhibit steady‐state behaviour in the absence of stirring. Consequently, changes in the concentration of the target biomarker, which occur during cytotoxic challenge to the cells, can be readily measured .…”

Optimization of the Design and Operating Conditions of an Amperometric Biosensor for Glutamate Concentration Measurements in the Blood Plasma

“…Their work was based on the enzymatic reactions of glutamate oxidase [20][21], glutamate dehydrogenase [22][23], and glutamate synthetase and glutamate decarboxylase [24][25][26]. The enzymes were immobilized on NiÀ Pd nanoparticles [27], fiber optic detector [28], solid electrode based on unplasticized chitosan [29], gold electrode modified with polyaniline and nanoparticles of polypropylor [30], and thick-film platinum electrode [31].…”