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Title: Micromachined sensors for single-cell signalling
Author: Cai, Xinxia
ISNI:       0000 0001 3513 8403
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2001
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The fabrication of microelectrodes integrated within microtitre chambers for ultra-low volume amperometric determination of lactate continues to be of interest in the subject of cell screening. Devices were fabricated using photolithography to give highly reproducible sensors integrated within high aspect ratio analytical chambers with volumes of between 160 ~ 400 pL. These chambers were either circular or square with a depth of 20 ?m, defined using the photoepoxy, SU8. In addition to the 3- microelectrode sensor, a 2-microelectrode sensor was also studied, which consists of a working microelectrode and an internal pseudo-microreference (combined with the counter electrode). The electrochemical characterization of a miniaturized sensor using a model redox compound of FMCA was discussed. Meanwhile, the detection of hydrogen peroxide, the redox enzyme linked electrochemical assay of glucose and the regeneration of the micro planar electrodes were investigated. 'Bulk' and pL-scale lactate measurements were carried out using the micromachined sensors. A microfluidic dispensation system was developed to deliver very low titres (6.5 pL) into a low volume micro-electrochemical cell. The determination of lactate was optimised using an enzyme-linked assay based upon lactate oxidase. This involved the amperometric determination of hydrogen peroxide at 640 mV vs. an internal pseudo AglAgCl reference (in the 2 electrode configuration). The system (including the microfluidic device, the microfluidic dispenser) had an observed detection limit of 15-fmol of lactate (as 3sigma above background). The sensor sensitivity was not limited by the ability of the sensor to resolve the amperometric signal, but rather the capability of the fluidic dispenser to deliver analyte in a reproducible and quantitative fashion at volumes below 6.5 pL. A linear calibration curve of the charge transferred and the amount of injected lactate in the range between 65 and 266 fmol was obtained. For the study of lactate measurements from single heart cells, the biocompatability of various materials used in the electrochemical devices was evaluated. The biocompatibility of SU-8 was demonstrated. It was shown that direct contact of the strongly oxidizing AgCl layer with a single myocyte could cause the myocyte to promptly die. Therefore, a two-electrode configuration, with a platinized working electrode and a combined reference and counter platinum electrode, acting as a pseudo-reference electrode, was also used for single-cell measurements. On the basis of the theory of lactate metabolism and the anoxia model, the dynamic electrochemical measurements of lactate from healthy and anoxic single heart cells were obtained. The lactate content after metabolic inhibition was approximately three times that of the unpoisoned cell. The efflux of high level lactate was measured in real time after the injection of FCCP at high concentration at 150 ~ 200 muM. Whereas, the lactate content of cells poisoned by contact with the AglAgCl was similar to that of a healthy cell. The lactate signal produced from cells, inserted using drawn pipettes, was higher for cells with higher metabolic rate than cells that were quiescent and weakly contracting. In addition to standard photolithography techniques, the use of SU-8 and PDMS, as well as "soft lithography" techniques were explored for microsensor arrays and microfluidics applications. An integrated microfluidic biosensor chip and on-line flow injection analysis (FIA) monitoring system was designed. In summary, this thesis describes a generic method of fabricating a single cell sensor employing pL-scale volume and based on oxidase-reductase enzymes and microfluidics in a lab-on-a-chip format. The next technological advancement would be the immobilisation of enzymes on the microelectrodes, leading to improve stability and avoiding expensive enzyme strategies as a means to every response in the future. In the future, integration of individual enzyme sensor arrays and fluorescence sensors needs to be performed for the simultaneous detection of various biomolecules from a single cell.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biosensors; Heart cell signalling