Alleviating the "Sample to Sequence" Bottleneck Using Novel Microfluidic Lab-on-a-Chip Nucleic Acid Extraction Technologies

This research will develop and evaluate a new system, based on pre-existing technology and expertise within the UK and Japan, which will improve the way in which we can detect, measure and study ocean biology based on species-specific genetic sequences. Current, best methods for the identification, enumeration and analysis of genetic sequences in the ocean rely upon the collection of water samples, which are returned to a centralized and highly resourced laboratory where they are processed and analyzed by highly trained technical staff. This takes time, delaying potentially important results (e.g. the presence and quantity of harmful species), and is expensive, limiting the number of samples that can be processed and ultimately reducing the resolution with which we can monitor ocean biology. This is now more important than ever as the oceans respond to changing climatic and anthropogenic influences. A key limiting step in this endeavor is the process of removing genetic material from the sample, whether it be whole cells, organisms or their remnants, and purifying it to the point at which it can be measured accurately; the 'extraction bottleneck.' Existing, automated sample processing robots are typically bulky, complicated, power hungry, prohibitively expensive, and not widely available.

Microfluidic Lab on a Chip (LOC) technologies reduce the scale of analytical processes traditionally performed on a lab bench. For example, miniature pipes (typically one tenth of a millimetre across) together with miniature pumps, valves and optics are used to take in sample, and manipulate it along with a suite of reagents to undertake a relatively complex laboratory process in a fraction of the time with minimal sample / chemical consumption and robotically, thus obviating the need for a specialist. In this project, we will capitalize upon the advantages of LOC technology to address the extraction bottleneck with a novel device that will interface with 'front-end' samplers and 'back-end' analyzers to form an integrated, genetic sensor platform. This will be tailored for the detection and quantification of a range of target organisms of high importance to human health, ocean ecology and ocean-centric industries. The project will demonstrate proof of concept that the integration of LOC genetic extraction with existing samplers and analytics can significantly improve the resolution and ease with which we can monitor fundamental biological variables.

As a user-friendly, compact, high-throughput, and contamination free DNA and RNA extraction platform, the proposed Extraction and Purification System (EPS) will alleviate the current 'extraction bottleneck' that requires time-consuming manual sample processing by specialist molecular biologists. We anticipate that the EPS will catalyse the uptake of DNA and RNA analytics, saving time and money across industries. This project therefore has the potential to inspire innovative approaches and open new industrial arenas for the application of nucleic acid analytics, thus stimulating economic growth.

An extensive Knowledge Exchange Programme is proposed, to benefit governmental, regulatory, and industrial stakeholders interested in microbiology or environmental DNA (eDNA) technologies. We propose to work directly with the energy industry, fisheries, as well as regulatory and governmental bodies interested in the detection of Harmful Algal Blooms (HABs), Faecal Indicator Bacteria (FIB), conservation (invasive or endangered species), nuisance (gelatinous) organisms and biogeochemically important species detection.

The EPS will expedite the development of broadly applied, robust, miniaturised in situ DNA and RNA sensors. Therefore it has the potential to provide sensor technology to address most of the identified "Essential Ocean Variables" by the Global Ocean Observing System and regional priorities of members of the United Nations Convention of Biological Diversity.