Multiplexed Protein Mapping Using Nanopores and DNA Technology for Cancer Risk Stratification

Cancer is still one of the most challenging diseases which can be inherited to its intra-tumour heterogeneity. Intra-tumour heterogeneity can be defined as the changes within cancer in individual patient. Understanding this heterogeneity can be tackled by detecting different proteins in individual tumour cells to provide a tumour protein profile, which is also known as protein mapping. A promising approach to provide a specific and sensitive mapping of multiple proteins is nanopore sensing.
Nanopore sensing is based on resistive pulse sensing, where a nanopore is used to connect two chambers that have electrolyte solution. When a charge is applied, electrolytes move through the nanopore producing a base line current. This force can be used to drive molecules from one chamber to another, which would result in a partial blocking that induces a drop in current. The drop in current over time corresponds to the size and the charge of the molecule. This can be used to map and quantify structures on the DNA. DNA molecule with protein specific binding sites across its length can be used to identify the presence of certain proteins as each of the protein-protein-binding-site complex would result in a drop in current at a specific DNA location. These DNA constructs can be refereed as DNA carriers.
Here we intend to design DNA carriers with at least 40 different protein binding sites on each carrier to improve upon current technologies that can identify up to 40 proteins; such as single-cell barcode chips and time-of-flight mass cytometry. To differentiate between each DNA carrier, different DNA structures embedded on one end of each DNA carrier can be used as barcoded to identify the different DNA carriers and their corresponding proteins.
The aim of this is to enable mapping of the cancer-specific proteins. This comes with its own challenges, especially the stability of the binding in high salt concentration between the protein binding cites during the nanopore measurements. Stable protein binding will be ensured by the use of DNA-protein crosslinking, aptamer binding, DNA-antibody conjugates, or DNA sequence modification.
To ensure the clinical reliability of the data, well-defined lung cancer cells and oesophagus cells from a potential collaborator will be obtained. Established single-cell isolation and recovery techniques in combination with magnetic separation are to be done to recover DNA carrier from individual cells. The analysis of the protein profiles via the DNA carrier will allow for the quantification of cell-specific proteins landscape.
The outcome of this project will help in providing a clinically reliable risk stratification biosensing method that can aid in optimizing diagnostics and deciding the best course of treatment or intervention for different cancer types.

The primary outputs from the CDT will be cohorts of highly qualified, interdisciplinary postgraduates who are experts in a wide range of sensing activities. They will benefit from a world leading training experience that recognises sensor research as an academic discipline in its own right. The students will be taught in all aspects of Sensor Technologies, ranging from the physical and chemical principles of sensing, to sensor design, data capture and processing, all the way to applications and opportunities for commercialisation, with a strong focus in entrepreneurship, technology translation and responsible leadership. Students will learn in extensive team and cohort engaging activities, and have access to cutting-edge expertise and infrastructure. 90 academics from 15 different departments participate in the programme and more than 40 industrial partners are actively involved in delivering research and business leadership training, offering perspectives for impact and translation and opportunities for internships and secondments. End users associated with the CDT will benefit from the availability of outstanding, highly qualified and motivated PhD students, access to shared infrastructure, and a huge range of academic and industrial contacts.

Immediate beneficiaries of our CDT will be our core industrial consortium partners (MedImmune, Alphasense, Fluidic Analytics, ioLight, NokiaBell, Cambridge Display Technologies, Teraview, Zimmer and Peacock, Panaxium, Silicon Microgravity, etc., see various LoS) who incorporate our cross-leverage funding model into their corporate research strategies. Small companies and start-ups particularly benefit from the flexibility of the partnerships we can offer. We will engage through weekly industry seminars and monthly Sensor Cafés, where SME employees can interact directly with the CDT students and PIs, provide training in topical areas, and, in turn, gain themselves access to CDT infrastructure and training. Ideas can be rapidly tested through industrially focused miniprojects and promising leads developed into funded PhD programmes, for which leveraged funding is available through the CDT.

Government departments and large research initiatives are formally connected to the CDT, including the Department for the Environment, Food and Rural Affairs (DEFRA); the Cambridge Centre for Smart Infrastructure and Construction (CSIC); the Centre for Global Equality (CGE); the National Physics Laboratory (NPL); the British Antarctic Survey (BAS), who all push our CDT to generate impacts that are in the public interest and relevant for a healthy and sustainable future society. With their input, we will tackle projects on assisted living technologies for the ageing population, diagnostics of environmental toxins in the developing world, and sensor technologies that help replace the use of animals in research. Developing countries will benefit through our emphasis on open technologies / open innovation and our exploration of responsible, ethical, and transparent business models. In the UK, our CDT will engage directly with the public sector and national policy makers and regulators (DEFRA, and the National Health Service - NHS) and, with their input, students are trained on impact and technology translation, ethics, and regulatory frameworks.