Development towards a point-of-care system to monitor pregnancy and fertility biomarkers

Wright, Kevin John

August 2015

Thesis or dissertation

© 2015 Kevin John Wright. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.

The aim of this thesis was to develop a point of care (POC) device that could monitor progesterone and oestriol in saliva. These hormones have a key role to play in both female fertility and pregnancy. Understanding the concentration of progesterone in the body is key to understanding a patient’s fertility and pregnancy status. Combining progesterone and oestriol detection can give valuable insight into when labour may commence during pregnancy. This can be achieved by measuring the progesterone to oestriol ratio in saliva samples, throughout the pregnancy progesterone is the more dominant hormone but a few weeks before labour oestriol becomes the more dominant hormone. Saliva was chosen as the biological sample due to the ease of collection as well as it providing a better chemical understanding of the active hormonal concentrations compared to blood. Typical levels in saliva are <31 pg mL⁻¹ rising to >100 ng mL⁻¹ for progesterone and 0 - 5 ng mL⁻¹ for oestriol.

The work presented began with the design of a chemiluminescence immunoassay which could be translated onto a microfluidic device, this approach would provide both good sensitivity and selectivity. Chemiluminescence was chosen as a detection system due to the high sensitivities that can be achieved with simple instrumentation where a CCD camera could be used to also obtain spatial information. The CL assay involved the chemical immobilisation of the antibodies onto glass slides. Silanisation with (3-aminopropyl)triethoxysilane (APTES) in combination with a N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide/N-hydroxysulfosuccinimide (EDC/sulfo-NHS) linker proved the most successful immobilisation method, with a LOD of 33 ±3 pg mL⁻¹ being achieved for progesterone in 10 mM phosphate buffered saline (PBS). This method however lacked reproducibility and did not transfer well on to polymer substrates or the microfluidic devices due to problems with the antibody immobilisation procedure. Immobilisation of anti-progesterone was then investigated on a range of electrode surfaces (Au, glassy carbon and ITO). This immobilisation procedure involved electrochemically depositing nitrobenzene onto the electrode surface followed by an electrochemical reduction of the nitro groups to the corresponding amine. To allow electrochemical detection ferrocene was tagged to the anti-progesterone antibodies to give a redox tag. The antibodies were then immobilised through an EDC/sulfo-NHS linkage. This method proved to be successful and very reproducible. By tagging ferrocene onto the antibody a rigid structure was achieved during the immobilisation procedure allowing the ideal antibody orientation, this process also allowed quantification of the concentration of antibodies on the surface (4.46 x10⁻⁷ mol m⁻²). Electrochemical based immunoassays were successfully carried with a 15 min incubation time for progesterone giving LODs of 1 pg mL⁻¹ for the gold and glassy carbon and 0.1 pg mL⁻¹ for the ITO. The ITO performed better than the other materials due to the electrode being uniformly flat enabling more efficient surface modifications. The methodology was also translated for use with artificial saliva with a LOD of 1.7 pg mL⁻¹ for progesterone.

Once the electrochemical immobilisation platform had been shown to be successful this was taken forward as a potential route to carry out a CL immunoassay. This novel approach utilised the oxidised ferrocene tag on the antibody as the catalyst for the luminol CL reaction. A static system was devised in which the antibodies had been immobilised on to ITO using the electrochemical approach and the CL reagents added by pipette, LODs of 2.35 and 2.54 pg mL⁻¹ were obtained for progesterone and oestriol respectively in saliva after a 30 min incubation time. The static system could therefore be used as a POC device for these hormones meeting the aims of this thesis. The next step was then to start developing a more automated method within a flow cell. Initially the ITO electrode with the pre-prepared immobilised antibody and the oxidised ferrocene tag was incorporated into a macrofluidic device. The CL immunoassay was successfully carried out in the macrofluidic device although the LODs were an order of magnitude higher than those seen from the static system for both progesterone and oestriol in saliva due to the large volume of the flow cell. Finally the ITO electrode with pre-prepared immobilised antibody with the oxidised ferrocene tag was slotted into the microfluidic device that had been designed and measurement were made. There was however problems with the design and possible new designs are discussed.

Department of Chemistry, The University of Hull
Greenway, Gillian M.; Wadhawan, Jay
Sponsor (Organisation)
University of Hull
Qualification level
Qualification name
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