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When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given e.g. AUTHOR (year of submission) "Full thesis title", University of Southampton, name of the University School or Department, PhD Thesis, pagination http://eprints.soton.ac.uk UNIVERSITY OF SOUTHAMPTON SCHOOL OF OCEANOGRAPHIC SCIENCE Lab-on-a-chip Systems for the Analysis of Phytoplankton RNA By Mahadji Bahi Thesis for degree of doctor of Engineering / Bioelectronics January 2013 1 Abstract Monitoring microorganisms in natural water is central to understanding and managing risks to human health and ecosystems. Some phytoplankton can produce toxic blooms which are harmful to aquatic ecosystems and human health. Kariena brevis is responsible for Harmful Algal Blooms and produces brevetoxin which can lead to gastrointestinal and neurological problems in mammals. Traditional methods for Harmful Algal Bloom monitoring require sample collection and preservation for later study in laboratories where they are generally processed using microscopy which can take many hours or days. Laboratory equipment for this application has been adapted for ship-board use. Portable instrument systems that incorporate sample preparation and detection have been also developed for environmental applications. However, very few are suitable for deployment in the environment (either as a hand-held or in situ system) and often require laboratory infrastructure or personnel to facilitate sample collection and processing. Current in situ systems are large, expensive, and require expert users to operate them. Thus these existing systems do not provide marine science with the high spatial resolution data required to enable a better understanding of the diversity, function and community structure of marine microorganisms. Ideal in situ sensors should provide sample analysis over wide areas and at many depths for long periods of time. This remains a significant challenge. One possible solution is to develop numerous cheap sensors which could be incorporated into autonomous underwater vehicles or an argofloats network. Micro systems are excellent candidates as when mature, they could be mass produced to enable them to meet this particular spatial mapping requirement. The use of fully automatic and accurate micro total analysis systems, also known as lab-on-a-chip, can overcome the challenges of highly integrated in situ systems for incorporation into environmental monitoring vehicles and stations. Lab-on-a-chip technology appears well suited for environmental monitoring with its main advantages being the possibility of miniaturization, portability, reduced reagent consumption and automation. Molecular biology tools combined with microfluidic technology have been seen as a potential technical solution for in situ environmental applications. The purpose of this work has been to develop key functions in independent microchips that perform elements of a complete biological assay for ribonucleic acid phytoplankton metrology from the sample preparation to the detection step. Specifically the system is being developed to analyse the large subunit of the ribulose-bisphosphate carboxylase (rbcL) gene of phytoplankton Kariena brevis, a species responsible for Harmful Algal Blooms. This thesis reports the development of three lab-on-a-chip devices which perform microalga cell lysis, nucleic acid purification and real-time ribonucleic acid detection. The aim was to demonstrate proof-of concept for each device separately in order to obviate the need to tackle the complications of system integration (which remains a challenge), while understanding performance needed and comparing that achieved to the most likely scenarios for real-world applications. Future research should integrate these separate chips into an integrated single chip design to achieve fully automated chips with “sample-in” to “answer-out” capability. 2 Acknowledgements I would like to express my great thanks to my supervisors Doctor Matthew Mowlem and Professor Peter Statham for their guidance, encouragement and support during the course of my study. 3 Declaration of Authorship I, Mahadji Majid Bahi, declare that the thesis entitled ‘Lab-on-a-chip Systems for the Analysis of Phytoplankton RNA’ and the work presented in the thesis is both my own and have been generated by me as the result of my own original research. I confirm that: this work was done wholly or mainly while in candidature for a research degree at the University of Southampton ; where any part of this thesis has previously been submitted for a degree or any other qualification at this University or any other institution, this has been clearly stated; where I have consulted the published work of others, this is always clearly attributed; Where I have quoted from the work of others, the source is always given. With the exception of such quotations, this thesis is entirely my own work; I have acknowledged all main sources of help; Where the thesis is based on work done by myself jointly with others, I have made clear exactly what was done by others and what I have contributed myself; part of this work have been published as: o “Electroporation and lysis of marine microalga Karenia brevis for RNA extraction and amplification” by M. M. Bahi, M.-N. Tsaloglou, M. Mowlem and H. Morgan. (published) (Bahi, Tsaloglou et al. 2010) o “On-chip real-time nucleic acid sequence-based amplification for RNA detection and amplification” by M.-N. Tsaloglou, M. M. Bahi, E.M Waugh, H. Morgan and M. Mowlem. (published)(Tsaloglou, Bahi et al. 2011) 4 Contents Abstract ................................................................................................................................................................... 2 Acknowledgements ................................................................................................................................................. 3 Declaration of Authorship ....................................................................................................................................... 4 Contents .................................................................................................................................................................. 5 List of Figures ......................................................................................................................................................... 9 List of Tables ........................................................................................................................................................ 14 Nomenclature ........................................................................................................................................................ 15 Chapter 1 Introduction .......................................................................................................................................... 17 1.1. Research motivations and objectives ......................................................................................................... 19 1.1.1. Main criteria and solutions for nucleic acid in situ sensors ................................................................ 23 1.1.1.a Low cell number detection ........................................................................................................... 23 1.1.1.b Viable cells detection ................................................................................................................... 24 1.1.1.c Transportable system and assay cost ............................................................................................ 24 1.1.1.d Ease of use ................................................................................................................................... 25 1.1.2. Scope and outline of this thesis .......................................................................................................... 26 1.1.3. Statement of novelty ........................................................................................................................... 27 1.1.4. Contribution of the author and co-workers ......................................................................................... 28 1.1.5. Additional activities during my PhD .................................................................................................. 29 1.2. Nucleic acid analysis overview .................................................................................................................. 31 1.2.1. Introduction to PCR and NASBA ...................................................................................................... 32 1.3. Current nucleic acid amplification-based lab-on-a-chip devices with “sample-in” to “answer-out” capability .........................................................................................................................................................