Miniaturised analytical systems with chemiluminescence detection for environmental applications

Marle, Leanne

March 2006

Thesis or dissertation

© 2006 Leanne Marle. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.

This thesis details the use of microfluidic devices and chemiluminescence detection in order to develop a portable method of analysis for measuring chemical species in the environment.

Chapter 1 outlines microfluidic technology, including fabrication techniques, fluid manipulation and mixing within the devices. Their advantages for analytical environmental purposes are demonstrated along with a review of their uses in environmental applications. Chemiluminescence detection provides a sensitive method of analysis for measuring chemical species in the environment and chemiluminescence theory and reagents are addressed.

Chapter 2 details the development of a portable battery operated chemiluminescence detection system, which can be used in conjunction with microfluidic devices. The fabrication of the micro fluidic devices used in this work is documented. Different micro fluidic channel manifolds were investigated for chemiluminescence reactions and a serpentine design (200 μm width, 65 μm depth) with a channel length of 206 mm was selected as the most suitable design. Methods of fabrication for incorporating immobilised reagents on solid supports within a microfluidic device were also designed.

Chapter 3 documents the investigation of the luminol-cobalt(II) chemiluminescence reaction within a microfluidic device using the portable chemiluminescence detection system to produce a miniaturised analytical system for the determination of hydrogen peroxide in rainwater. Enhancement of the chemiluminescence signal by 132 %was achieved by means of using the mirror reaction to apply a reflective surface directly VI to the top of the micro fluidic device. Immobilisation techniques for immobilising luminol using adsorption and covalent attachment onto a solid support were investigated as a means of producing a reagentless system, however poor sensitivity was observed and this was not progressed for the analytical system.

Using the luminol-cobalt(II) chemiluminescence reaction within a microfluidic device a method of measuring hydrogen peroxide in the low micromolar concentrations was achieved, producing a limit of detection of 4.7 nmol L⁻¹ with a small sample volume (10 μL min⁻¹). A small reagent consumption size (1.2 mL per hour) and a low waste production size (2.4 mL per hour) were also achieved. This system was then used for the determination of hydrogen peroxide in rainwater samples during rainfall events showing the hydrogen peroxide concentration varied from 0.1 to 3.2 μmol L⁻¹. The method was also applied to the analysis of hydrogen peroxide in snow demonstrating the hydrogen peroxide concentration varied from 0.2 to 0.5 μmol L⁻¹ in samples taken at ground level.

Chapter 4 details the development of a heterogeneous (two site) sandwich immunoassay within a microfluidic device to produce a miniaturised analytical system for the determination of E. coli bacteria in seawater. There is a need for rapid sensitive methods of analysis to measure E. coli in seawater as an indicator of faecal contamination. A review of traditional methods and current research on the area is presented. Immunological techniques based on using antibodies to specifically bind to their respective antigens were found to be the most amenable method of analysis for E. coli and an outline of how they work is shown. HRP was selected as the sensitive enzyme label for the antibody in the sandwich immunoassay. The chemiluminescence detection of HRP using the luminol-hydrogen peroxide VB chemiluminescence reaction was investigated within a microfluidic device, the detection was optimised and p-iodophenol was selected as an enhancer for the reaction. The investigation into the immobilisation of E. coli specific antibodies using covalent attachment onto controlled pore glass is presented, an optimal loading of 1.5 μg g⁻¹ was achieved. The development of an ELISA method for the purpose of screening the antibody for their specificity towards different isolates of E. coli and their non-specificity towards other bacteria is detailed. Finally, a microfluidic immunoassay was developed. Regeneration of the immobilised antibodies was achieved using 0.5 mol L⁻¹ sodium hydroxide, allowing the immobilised antibodies to be reused. The microfluidic immunoassay provided a rapid method for the determination of E. coli with an analysis time of 13 min for each sample. The assay also used a low reagent consumption and waste production. This would enable the rapid testing of a number of small samples to provide high temporal and spatial resolution data. Sensitivity provided a problem with the immunoassay and ways to overcome this were addressed.

Department of Chemistry, The University of Hull
Greenway, Gillian M.
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