Development of molecular analytical methods for in situ detection of marine organisms using microfluidic biological sensor technologies

Abstract

Healthy seas are crucial for the protection of public health and to maximise the economic benefit from the use of the ocean for food production (including from aquaculture), resources, pharmaceuticals, and tourism. Sporadic occurrences of marine harmful algal blooms (HABs), however, can bring calamity to both the health and economic well-being of communities and businesses. In particular, toxigenic blooms can lead to significant harm to marine species and ecosystems, as well as human health and maritime industries. Pseudo-nitzschia is a genus of marine microalgae, belonging to the diatom group, which have been identified as a significant risk to human health and a contributor to ecological degradation from HABs. This is due to the production of domoic acid, a potent neurotoxin that is toxic to marine organisms and mammals. Early detection of domoic acid-producing blooms is necessary to minimise exposure. However, existing methods for discerning Pseudo-nitzschia abundance and the associated risks are long and protracted, labour-intensive and expensive. Pseudo-nitzschia blooms. Subsequently, a suite of novel isothermal assays targeting the dabD gene of Pseudo-nitzschia spp., which is linked to toxin biosynthesis, were designed utilising the LAMP and RPA chemistries. The LAMP assay outperformed the RPA assay in sensitivity and specificity, and its potential use for statutory algal surveillance was demonstrated by measuring Pseudo-nitzschia DNA in seawater samples collected over six months from a known HAB hotspot. To support the potential integration of the novel assays with fieldable instrumentation, a novel ‘Vitrification’ technique was developed for the simple and fast preservation and dry storage of complete reaction mixtures. The ‘shelf-life’ of the preserved reactions was at least six months at room temperature and represents a host of improvements upon existing methods. There were no significant differences in quantification performance between the dry-preserved reagents and freshly prepared reactions that relied on cold- chain-dependent, wet reagents. Finally, a new LOC system referred to as "LAMPTRON" was designed and fabricated from scratch, and developed for real-time detection and quantification of P. multistriata cells in a semi-autonomous fashion. LAMPTRON demonstrates a proof-of-concept for integrating automated DNA extraction with fully preserved DNA analysis, enabling the sensitive detection of toxigenic P. multistriata in a comparable timeframe to the leading commercial systems. These advances offer a faster, more sensitive, and simplified molecular analysis compared to existing statutory surveillance methods, which rely on costly reagents, sophisticated equipment, highly skilled personnel, and centralised laboratories. The ability to detect and quantify Pseudo-nitzschia cells using LOC technology could be modified towards a plethora of microbiological and eDNA targets for the surveillance and early warning of biohazards in aquatic environments. Implementing an early warning system using the state of the art in analytical methods will improve response times, improve accuracy and reduce cost; ultimately this will mitigate risk. Molecular analytical techniques, particularly those based on nucleic acid (DNA or RNA) sequence amplification, are widely adopted in food and water quality assessment, public health protection, and environmental monitoring. These methods can be coupled with portable or deployable instrumentation to provide high integrity, laboratory-quality or better metrology in resource-limited settings. In particular, microfluidic 'lab-on-a-chip' (LOC) technology is at the forefront of de-centralised nucleic acid testing and offers advantages such as reduced reagent and energy consumption, ease of use, rapidity, and stability, all while maintaining high levels of specificity, sensitivity, and precision. Automation of these systems makes analysis possible for non-specialist end-users, increasing their scope of application. This study was undertaken to explore new methods and technology for nucleic acid-based detection of Pseudo-nitzschia towards the provision of an integrated early warning system. It began with the design and testing of novel qPCR-based assay for Pseudo-nitzschia spp. detection and quantification. Then, the assay was combined with a reverse transcription step to investigate RNA-based (gene expression) responses in nutrient-depleted P. multistriata cells to the addition of essential nutrients phosphate, nitrate, and silicate. Nutrient availability was found to significantly influence domoic acid production, highlighting the relationship between nutrient availability and the toxic threat posed by Pseudo-nitzschia blooms. Subsequently, a suite of novel isothermal assays targeting the dabD gene of Pseudo-nitzschia spp., which is linked to toxin biosynthesis, were designed utilising the LAMP and RPA chemistries. The LAMP assay outperformed the RPA assay in sensitivity and specificity, and its potential use for statutory algal surveillance was demonstrated by measuring Pseudo-nitzschia DNA in seawater samples collected over six months from a known HAB hotspot. To support the potential integration of the novel assays with fieldable instrumentation, a novel ‘Vitrification’ technique was developed for the simple and fast preservation and dry storage of complete reaction mixtures. The ‘shelf-life’ of the preserved reactions was at least six months at room temperature and represents a host of improvements upon existing methods. There were no significant differences in quantification performance between the dry-preserved reagents and freshly prepared reactions that relied on cold- chain-dependent, wet reagents. Finally, a new LOC system referred to as "LAMPTRON" was designed and fabricated from scratch, and developed for real-time detection and quantification of P. multistriata cells in a semi-autonomous fashion. LAMPTRON demonstrates a proof-of-concept for integrating automated DNA extraction with fully preserved DNA analysis, enabling the sensitive detection of toxigenic P. multistriata in a comparable timeframe to the leading commercial systems. These advances offer a faster, more sensitive, and simplified molecular analysis compared to existing statutory surveillance methods, which rely on costly reagents, sophisticated equipment, highly skilled personnel, and centralised laboratories. The ability to detect and quantify Pseudo-nitzschia cells using LOC technology could be modified towards a plethora of microbiological and eDNA targets for the surveillance and early warning of biohazards in aquatic environments.

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