Utilising novel microfluidic arrays to performing DNA reactions, manipulations, and purification for genomic applications

Project Sumary

There is an urgent and growing need to develop more sophisticated tools and technologies in the global fight against infectious diseases. DNA and RNA sequencing approaches offer promise due to their ability to provide species identification of pathogens, as well as information on antimicrobial resistance and relatednessⁱ. Sequencing has become more accessible since the launch of Oxford Nanopore’s portable MinION sequencer in 2014ⁱⁱ, but there has been less innovation in the extraction and preparation of DNA/RNA for these applications. We aim to close this gap and develop a simple sample-to-sequence solution providing comprehensive genomic information to infectious disease professionals.

The purification of DNA in many molecular biology applications is performed by binding DNA to silica-coated magnetic beads, followed by repeated cycles of suspension, pelleting, and solution exchange. These steps are time-consuming, require a skilled user, and often result in inefficient removal of reactants and contaminants. Deterministic lateral displacement (DLD) arrays are geometric arrays of pillars or posts used in microfluidics to separate particles of different sizes. DLDs were first published in 2004ⁱⁱⁱ and have been utilised in biomedical applications for separating cells of different sizes, such as red and white blood cells. Although the predominant use for DLDs has been size separation^iv, they also offer great potential for controlling reactions and purification for small sample volumes^v.

This project will combine a DLD array, DNA-bound to beads, and laminar flow lanes of various solutions within a microfluidic chamber. As the beads move through the DLD, they are guided by the pillars to follow a diagonal trajectory. This trajectory causes the beads to traverse the laminar flow lanes in series, resulting in the DNA bound to the beads experiencing the different solutions sequentially. The approach creates opportunities to control reactions and perform complex chemistries in small volumes. It is of particular interest to the Pathogen Program project as a tool for automated DNA/RNA library preparation.

We will commence with basic designs, device fabrication, bead recovery, and characterisation methods, with an emphasis on DNA binding and clean-up. This will be followed by performing enzymatic reactions and clean-ups within multiple laminar flow lanes in a single device. Later phases will be open-ended, exploring the potential of the approach for novel chemistry and complex workflows.

The position offers experience of a multi-disciplinary work environment bringing together a diverse range of scientific areas such as: MEMs fabrication, microfluidics, physical analytical chemistry, complex biological samples, DNA manipulation, enzyme kinetics, surface chemistry, and DNA sequencing. The successful candidate will benefit from working across the University of Oxford and the Ellison Institute of Technology combing the expertise from world leading academics and applied scientists with deep experience of biotechnology, using cutting edge science to solve real-world problems.

Potential Supervisors

• Dr James Clarke (Senior Director of Product Science – Pathogen Program, EIT)
• Prof Hagan Bayley (Professor - Department of Chemistry, University of Oxford)
• Prof Molly Stevens FREng FRS (?) (John Black Professor of Bionanoscience - Department of Physiology, Anatomy & Genetics, University of Oxford)

Skills Required

• Physics, physical or analytical chemistry
• Fluidics and fluid modelling

Skills to be Developed

• Experimental design and planning
• Device design and fabrication
• Analytical methods and data analysis
• Critical thinking
• Researching scientific literature
• Discussion and presentation of key findings
• Working in a multi-disciplinary environment
  

University DPhil Courses

• DPhil in Chemistry, Department of Chemistry, https://www.ox.ac.uk/admissions/graduate/courses/dphil-chemistry

Relevant Background Reading

Charalampous T, Kay GL, Richardson H, Aydin A, Baldan R, Jeanes C, Rae D, Grundy S, Turner DJ, Wain J, Leggett RM, Livermore DM, O'Grady J. Nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection. Nat Biotechnol. 2019 Jul;37(7):783-792. doi: 10.1038/s41587-019-0156-5. Epub 2019 Jun 24. PMID: 31235920.

https://nanoporetech.com/blog/a-decade-of-discovery-with-minion-insights-from-two-pioneer-researchers

Huang LR, Cox EC, Austin RH, Sturm JC. Continuous particle separation through deterministic lateral displacement. Science. 2004 May 14;304(5673):987-90. doi: 10.1126/science.1094567. PMID: 15143275.

Hochstetter A, Vernekar R, Austin RH, Becker H, Beech JP, Fedosov DA, Gompper G, Kim SC, Smith JT, Stolovitzky G, Tegenfeldt JO, Wunsch BH, Zeming KK, Krüger T, Inglis DW. Deterministic Lateral Displacement: Challenges and Perspectives. ACS Nano. 2020 Sep 22;14(9):10784-10795. doi: 10.1021/acsnano.0c05186. Epub 2020 Aug 26. PMID: 32844655.

US20240192205A1

(https://patentimages.storage.googleapis.com/a5/fd/fd/64860f5f0d4769/US20240192205A1.pdf)