Gareth Hughes firstname.lastname@example.org
The design, development and application of novel, screen-printed amperometric glutamate biosensors
The aim of the studies presented in this thesis was to develop a screen-printed electrochemical biosensor for the measurement of glutamate and to apply this device to the determination of the analyte in food, serum and toxicity studies.
Chapter 1 serves as an introduction to both the physiological significance of glutamate and the fundamental principles underpinning the electrochemical techniques used throughout this thesis.
Chapter 2 is a review chapter, separated into two main sections. The first section details glutamate biosensors fabricated with glutamate oxidase (GluOx), the second section details biosensors fabricated with glutamate dehydrogenase (GLDH). The immobilization techniques, ease of fabrication and sample preparation techniques employed are compared. Biosensor characteristics such as sensitivity, limit of detection and linear range are summarised within a table.
The studies described in Chapter 3 focus on the development of a non-reagentless glutamate biosensor. A Meldola’s Blue screen-printed carbon electrode (MB-SPCE) was employed as the base transducer. The biosensor was constructed by drop coating the biopolymer chitosan (CHIT) and GLDH onto the surface of the MB-SPCE. For this study, NAD+ was present in free solution. Meldola’s Blue served as the electrocatalyst, whereby NADH produced by the GLDH/NAD+ reaction, was electrocatalytically oxidised at a low operating potential (+0.1V (vs. Ag/AgCl)). The applied potential, temperature, pH and concentration of the co-factors required for the biosensor operation were optimised in this study. The assay exhibited a linear range of 12.5 µM to 150 µM, limit of detection of 1.5 µM, response time of 2s and a sensitivity of 0.44 nA/ µM. The optimised biosensor was subsequently applied to the determination of endogenous and fortified concentrations of glutamate in both serum and food samples (OXO cubes). The serum was fortified with and the resulting mean recovery was 96% with a CV of 3.3% (n = 6). For the food sample, an unfiltered beef OXO cube was analysed for monosodium glutamate (MSG) content. The endogenous content of MSG was 125.43 mg/g, with a CV of 8.98% (n = 6). The solution was fortified with 100mM of glutamate and a resulting mean recovery of 91% with a CV of 6.39% (n = 6) was determined.
In Chapter 4, the glutamate biosensor was further developed in order to produce a reagentless device whereby the cofactor NAD+ and GLDH were immobilized on to the surface of the electrode utilising CHIT. The reagentless device was developed in order to monitor glutamate release from human liver carcinoma cells (HepG2) as a result of cell toxicity from exposure to paracetamol. The biosensor was miniaturised in the form of a microband biosensor, whereby one dimension of the electrode is of micrometre size and the other millimetre size. Micro bands exhibit unique diffusion properties in comparison to conventional sized electrodes. Calibration studies were carried out with an applied potential of +0.1V (vs. Ag/AgCl) using both phosphate buffer and cell media. In phosphate buffer the following microband biosensor characteristics were determined: linear range; 25 - 125µM, sensitivity; 0.0636 nA/µM and a theoretical limit of detection of 1.20µM. In cell media; linear range; 25 – 150 µM, sensitivity; 0.128 nA/µM and a theoretical limit of detection of 4.2µM. As the HepG2 cells were grown in an incubator at a fixed temperature and pH, studies were carried out at pH 7, 37ºC, in a 5% CO2 atmosphere. The miniaturised biosensor was applied to the determination of glutamate and the quantification was done by standard addition in cell media after 24 hours exposure to various concentrations of paracetamol. The average endogenous concentrations for glutamate released from the HepG2 cells was 52.07µM (CoV: 13.74%, n = 3), 93.30µM (CoV: 18.41%, n = 3) and 177.14µM (CoV: 14.54% n = 3) for 1mM, 5mM, 10mM doses of paracetamol respectively. The microband biosensor was also applied to the real time monitoring of glutamate over 8 hours. The standard deviations for the final current generated after eight hours are as follows; 1mM (coefficient of variation (CoV): 3.3%), 5mM (CoV: 9.056%) and 10mM (CoV: 13.18%). The study showed that the magnitudes of the steady state currents increased in proportion to the concentration of added paracetamol. The study also demonstrated the possibility of applying microband biosensors, over extended time periods, for toxicity studies; there is no significant removal of analyte owning to the small biosensor dimensions.
Chapter 5 describes the development of a reagentless conventional sized glutamate biosensor whereby the cofactor NAD+ and GLDH were immobilized using a combination of multi-walled carbon nanotubes (MWCNT), CHIT and additional water based MB in a layer-by-layer fashion. The MWCNT/CHIT/MB combination facilitates electron transfer to the surface of working electrode. The MWCNT/CHIT also entraps GLDH and the NAD+ on the surface of the electrode. The pH, temperature, optimum applied potential, concentrations of NAD+, CHIT and the addition of water-based MB were optimised. The electrocatalyst MB allowed a operating potential of +0.1V (vs. Ag/AgCl) to be utilised. The biosensor was examined with standard glutamate solutions and the following biosensor characteristics were determined; linear range; 7 - 105µM, LOD; 3 µM, sensitivity; 0.39 nA/µM, response time 20-30s. A food sample was analysed for MSG and found to contain 90.56 mg/g with a CV of 7.52% (n = 5). The reagentless biosensor was also applied to the determination of glutamate in serum. The endogenous concentration was found to be 1.44mM (n = 5), CV: 8.54%. The recovery of glutamate in fortified serum was 104% (n = 5), CV of 2.91%. The results indicate that the new biosensor holds promise for food and biomedical studies.
|Institution Citation||Hughes, G. The design, development and application of novel, screen-printed amperometric glutamate biosensors. (Thesis). University of the West of England|
|Keywords||screen printing, amperometric glutamate biosensors|
THESIS AMENDED GHUGHES FINAL.pdf