Browsing by Author "Psoma, Sotiria D."

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    Fluorescence-based optical biosensors for clinical and environmental applications
    (Cranfield University, 1996-12) Psoma, Sotiria D.; Turner, Anthony P. F.
    The aim of this thesis was to investigate the feasibility of simultaneous utilisation of pH and oxygen-dependent fluorescent indicators for the development of a novel fibre-optical fluorescence-based bio sensor. This approach would be used to measure simultaneously changes in the two indicator species generated by a single enzyme-catalysed reaction in response to one analyte where both the indicators and the enzyme are immobilised in the same sol-gel matrix, and to offer more accurate and reliable results using this portable optical biosensor in the clinical and environmental fields. HPTS (1-hydroxypyrene-3,6,8-trisulfonic acid) and tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate, respectively, were used as the target fluorescent indicators; these two indicators had no cross sensitivity separate or in the same solution and well-separated emission bands at 510 nm and 610 mn, respectively. The catalytic oxidation of glucose by the enzyme glucose oxidase was initially investigated using the two indicators, and subsequently the same principle was applied in other biocatalysed oxidations such as of lactate, xanthine and phenol. Substrate concentration was assessed by simultaneously measuring two parameters: oxygen consumption, through the reduction of the fluorescence intensity of tris(2,2'-bipyridyl) ruthenium(II) chloride hexahydrate; and the production of acid, through pH changes affecting the fluorescence intensity of HPTS.A thorough spectroscopic study of the enzymatic oxidation of glucose was performed using glucose oxidase in solution in a cuvette, in the presence of both indicators. A number of combinations of wavelengths of the indicators for excitation and fluorescence were utilised in order to establish calibration curves with the optimum performance for glucose detection in the diabetic range. Similarly results were taken from the kinetic studies of lactate oxidase, xanthine oxidase and polyphenol oxidase for the detection of lactate and xanthine in blood and phenol in water at ppb-levels, using the above principle. The application and characterisation of immobilisation techniques for the fluorescence-based blood-glucose b iosenor were carried out. The advantages of the microcapsulation sol-gel method over conventional immobilisation techniques for application in an optical biosensor, were elucidated and this immobilisation technique was implemented for glucose and phenol detection. Finally, additional solution studies were conducted and used to evaluate the implementation and performance of the above method when used for the detection and measurement of glucose concentration in biological samples such as human serum.
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    A novel enzyme entrapment in SU-8 microfabricated films for glucose micro-biosensors
    (Elsevier, 2010-12-15) Psoma, Sotiria D.; van der Wal, Peter D.; Frey, Olivier; de Rooij, Nicolaas F.; Turner, Anthony P. F.
    The present work investigates the utilisation of the widely used SU-8 photoresist as an immobilisation matrix for glucose oxidase (GOx) for the development of glucose micro-biosensors. The strong advantage of the proposed approach is the simultaneous enzyme entrapment during the microfabrication process within a single step, which is of high importance for the simplification of the BioMEMS procedures. Successful encapsulation of the enzyme GOx in “customised” SU-8 microfabricated structures was achieved through optimisation of the one-step microfabrication process. Although the process involved contact with organic solvents, UV-light exposure, heating for pre- and post-bake and enzyme entrapment in a hard and rigid epoxy resin matrix, the enzyme retained its activity after encapsulation in SU-8. Measurements of the immobilised enzyme's activity inside the SU-8 matrix were carried out using amperometric detection of hydrogen peroxide in a 3-electrode setup. Films without enzyme showed negligible variation in current upon the addition of glucose, as opposed to films with encapsulated enzyme which showed a very clear increase in current. Experiments using films of increased thickness or enzyme concentration, showed a higher response, thus proving that the enzyme remained active not only on the film's surface, but also inside the matrix as well. The proposed enzyme immobilisation in SU-8 films opens up new possibilities for combining BioMEMS with biosensors and organic electronics.
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    A proof of concept of a BioMEMS glucose biosensor using microfabricated SU-8 films
    (Cranfield University, 2009-12) Psoma, Sotiria D.; Turner, Anthony P. F.
    The present project investigated and proved the concept of developing a novel BioMEMS glucose micro-biosensor using a simple one-step microfabrication process of the widely used SU-8 polymer. More specifically, the study focused on the investigation of the suitability of the SU-8 polymer as a matrix for enzyme immobilisation that is carried out during the microfabrication process. A comparative study between commercially available SU-8 and “customised” SU-8 solutions showed that the optimum concentration of photo-initiator for stress reduction can be achieved easier with “customised” SU-8 solutions. The most appropriate type of microstructure for the SU-8 matrix and the corresponding required microfabrication process were defined and encapsulation of the enzyme GOx in the SU-8 solution was accomplished. A detailed experimental investigation of the immobilised enzyme’s activity inside the SU-8 matrix, was carried out using amperometric detection of hydrogen peroxide in a 3-electrode setup. SU-8 films were immersed in a buffer solution and the platinum working electrode was brought in close contact with the film. Films without enzyme showed negligible variation in current upon the addition of glucose, as opposed to films with encapsulated enzyme which showed a very clear increase in current. Experiments using films of increased thickness or enzyme concentration, showed a higher response, thus proving that the enzyme remained active not only on the film’s surface, but inside the matrix as well. In the fluorescence spectroscopy experiments, the utilisation of the tris (4,7- diphenyl-1,10-phenanthroline) ruthenium(II) dichloride oxygen indicator, which was also captured in the polymer matrix during the microfabrication process, was proven to be very sensitive to glucose concentration changes during the glucose oxidation and there was no photo-bleaching. The experimental investigations proved that the proposed concept of using SU-8 matrices for the immobilisation of biomolecules, is a valid proposal for the construction of a BioMEMS glucose biosensor. An important outcome was the successful immobilisation of glucose oxidase in SU-8 microfabricated structures. The enzyme still showed activity despite the “hostile” conditions during microfabrication The proof of principle of enzyme immobilisation in SU-8 films opens up new possibilities for combining BioMEMS with biosensors and organic electronics.