The design, development and application of a novel electrochemical biosensor/sensor system for the real-time monitoring of in-vitro cell toxicity

• Main Author: Rawson, Frankie James
• Published: University of the West of England, Bristol 2009
• Subjects: 571.966
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.557140

In recent years there has been a reported requirement by the pharmaceutical and agrochemical industry that there is a need to monitor In vitro cell toxicity in real time, and also a necessity for using cell models that mimic the In vivo situation more closely. The aim of this project was to provide a solution for these key issues set out above. This was achieved by developing, characterising and implementing the use of electrochemical micro-biosensors and micro-sensors manufactured from screen printed electrodes. These were then used for monitoring cytotoxicity in HepG2 spheroids which mimic more closely their corresponding organ in vivo. In chapter two a new method of using screen-printed carbon electrodes (SPCEs) incorporating the electrocatalyst cobalt phthalocyanine (CoPC) for the manufacture of microband electrodes for hydrogen peroxide detection is described. Tubular microband electrode (TMBEs) fabricated using an organic based ink displayed superior properties over plain microbands. TMBEs were demonstrated to display steady state currents which, is indicative of microelectrode behaviour. The current density values obtained from the voItammogram was compared to that obtained for a conventional sized CoPC-SPCE, and the values were 5618 and 35.65 μA cm -2, respectively. Cyclic voltammetry was carried out for the same electrodes; using 7 mM H202 prepared in phosphate buffer at scan rates between 1 and 50 mV s-I and no significant increase in current response was observed. The application of these tubular microband CoPC-SPCEs, to the measurement of H202 using chronoamperometry was investigated. A calibration study was performed and the plot showed a sensitivity value of 252 μA mM-1 cm-2 and a lower detection limit of70 μM. Although this data was promising for later biosensor construction it is known that the organic based ink used to screen-print the sensors would not allow for direct incorporation of the enzyme which would be later incorporated to allow for monitoring of biomarkers important in cell toxicity. Therefore, in chapter three microband electrodes screen-printed using a water based ink were investigated which would allow for enzyme incorporation in to the inks subsequent to screen-printing and thus allowing for the formation of biosensors. Plain microband electrodes fabricated from this water based carbon ink displayed superior characteristics over the TMBEs fabricated from the same ink for hydrogen peroxide monitoring, this included higher current density values, stir independence and these were more reproducible. Therefore the plain microband was the design choice for future sensor and biosensor construction. In chapter three and four screen-printed sensors and biosensors were constructed using a water based ink with the incorporation of lactate oxidase or glucose oxidase. In chapter 5, organic based carbon ink incorporating Meldola's blue was added prior to printing allowing for a one step print process. These were then cut forming microband biosensors and sensors, allowing for the monitoring of the analytes lactate, glucose and NADH respectively. Lactate microband biosensors could monitor lactate and was measured over a dynamic range of 1-10 mM which was linear up to 6mM; a calculated lower limit of detection of 289 μM was ascertained. Microband sensors for glucose determination were used and could measure glucose over the concentration range 1-14 mM. A linear range was obtained up to 6 mM glucose (y = 3.05x) giving a R2 value of 0.99 and an intra-electrode coefficient of variation over this range of 10%. A sensitivity value over this range of 3.1 nA mM-I was obtained with a theoretical lower detection limit of 258 μM. Microband sensors incorporating the electrocatalyst were used to measure NADH over a dynamic range 0.2-3.5 mM. A linear range was obtained up to 400 IlM NADH (y = 0.0076x + 0.7665) giving a R2 value of 0.999 and a typical inter-electrode coefficient of variation over this range of 18% (n=5). A sensitivity value over this range of 7.6 nA mM-I was obtained with a theoretical lower detection limit of 63 IlM. The microband screen-printed sensors for lactate, glucose and NADH were then used to monitor cytotoxicity by monitoring the release of their corresponding analyte. This was performed over a 24 hour period in which HepG2 spheroids were exposed to the model hepatotoxin galactosamine. The results provide proof of principle that the developed electrochemical sensors could follow the above biomarkers which were good indicators of cell induced toxicity caused by the galactosamine.