Lab-on-a-chip technology for in situ molecular analysis of marine microorganisms

Abstract

In situ monitoring of ocean biology is typically done in the form of sample collection during ship based cruises or other field expeditions, and sample analysis either on-board the ship or in a laboratory at a later time. However such expeditions can be expensive, labour intensive, and only follow pre-defined courses and locations. Alternative methods are used and in development in an effort to provide flexibility and reduced costs. Molecular methods are valuable tools in ecological studies, offering high specificity, reliability and excellent limits of detection. Techniques including polymerase chain reaction (PCR) and nucleic acid sequence based amplification (NASBA) are now routinely used for the monitoring of microorganisms in water (e.g. pathogens, biotoxins and viruses). The use of these techniques, however, is time consuming, costly, and requires substantial training and specially adapted laboratories, making them impractical in many field applications or where infrastructure is limited.

Recent advances in microfluidic research have demonstrated that molecular techniques (e.g. qPCR, NASBA) can be applied on lab-on-a-chip (LOC) systems for the in situ detection of target organisms or biomarkers in natural waters. Microfluidic devices (i.e. LOC systems) can automatically perform multiple laboratory functions on a single integrated and compact chip. NASBA is particularly attractive for LOC applications targeting RNA, because it is an isothermal amplification assay and does not require complex microfluidic systems. A NASBA assay, targeting toxic microalgae Karenia brevis, has been successfully automated using a LOC-based, bench-top sensor “LabCardReader” here at the National Oceanography Centre of Southampton (NOCS).

The purpose of this work has been to develop and optimise a fully automated molecular assay on a novel lab-on-a-chip system, which performs RNA-based amplification, for the detection and quantification of marine microorganisms. This PhD provides significant developments in the field of lab-on-chip technology, as well as in situ sample collection and processing for harmful algal bloom (HAB) monitoring. This thesis reports on (1) the optimisation of an internal control (IC) NASBA assay for the detection and quantification of microorganisms while examining the validity of the technique under varying physiological states and growth conditions; (2) the long-term preservation and storage of NASBA reaction components; (3) the development of a fully automated, fully preserved, lab-on-a-chip-based IC-NASBA protocol for real-time detection and quantification of microorganisms; (4) the development of a simple, rapid, and reliable sample collection and concentration method, aimed for in situ application and compatibility with nucleic acid analysis techniques.

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Identifiers

ORCID for Dr Matt Mowlem: orcid.org/0000-0001-7613-6121

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