Selective Nanobrush Sensors (SNS) for Label-free Diagnosis of Neurodegenerative Disorders

Alzheimer's, and Parkinson's are major neurological diseases, with a large economic, societal and personal burden. To date, there is no laboratory diagnostic test that can identify these neurological disorders ahead of observable clinical manifestations. There are emerging biomarkers and key proteins linked to these diseases, but existing detection methods are cumbersome due to lack of sensitivity and specificity, and low-abundance in clinical samples. There is an enormous need for adaptable technology capable of discriminating and monitoring biomarkers, to generate a fundamental transformation in diagnostics, and enabling a better understanding.

In both diseases, small soluble protein aggregates such as amyloid beta or alpha synuclein have been identified as important targets for the development of drugs and diagnostics. These small aggregates, however, are exceptionally challenging to detect as they can have heterogeneous sizes, ultra-low concentrations (picomolar) and comprise only a small fraction (lower than 1%) of the total concentration in a clinical sample. This project unites physical sciences, life sciences, and nanomedicine with the aim of developing label-free single-molecule sensors with robust, tuneable and selective recognition chemistry that overcome these limitations. This will be achieved by using what we dub a "smart nanobrush sensor" (SNS), based on nanopores functionalised with aptamer nanobrushes (short single-stranded nucleic acids synthesised specifically bind the target analytes) and a novel mode of electronic detection. Our aims are to focus on delivering a technology to discriminate, label-free, amyloid beta or alpha synuclein oligomer subpopulations and their concentrations in complex media, and ultimately, cerebrospinal fluid samples obtained from AD and PD patients.

These novel sensors will close a wide technological gap that is currently holding back the biomarker analysis of small proteins and their aggregates in clinical samples. It will enable accurate discrimination of analyte populations, as well as their size, shape and concentration. The developed technology can provide transformative methods to identify disease markers and can pave the way for a broad range of techniques to study pathogenesis not only in neurodegenerative diseases, but also in diseases caused by protein misfolding and aggregation (e.g. cancers linked to p53 aggregation). We envisage a stepwise progression in sensor design from the chemical laboratory, to in vitro biomarker diagnostics capable of monitoring molecular changes for early stage prognosis and disease progression.

Advances in our understanding of disease processes emphasise the need for biosensors that are much more sensitive and selective for their target molecules. The majority of methods for detecting biomarkers in vitro often require fluorophores, electrochemical probes, magnetic beads, or active enzymatic labelling which results in extended and potentially complex steps between sample extraction and detection.

This project is the result of the synergy of world-class expertise in nanoscale sensing, biomedical/electronic engineering, and neuroscience to develop next generation biosensors, dubbed the smart nanobrush sensors (SNS), capable of discrimination and monitoring of two exemplar molecules (alpha synuclein, amyloid-beta) in cerebrospinal fluid. The proposed technology is based on the development of aptamer modified solid-state nanopores, tailored for use in vitro. Nanopores are a class of single-molecule detectors that have been used successfully for label-free analysis; However, conventional nanopores lack selectivity, making it challenging to discriminate target molecules in complex clinical samples. The novelty of the SNS relies on the nanopore, having its inner surface coated with aptamer brushes selected to recognise individual biomarkers. The target molecules are identified by transient blockades of the ion current during their transport through the nanopore. The target-aptamer bond is then dissociated by the electric field inside the nanopore, leading to full sensor recovery and simultaneously allowing for quantification of the analyte concentration. The implementation of the SNS will provide 1) a novel tool for accurate and early clinical diagnosis, 2) fundamental insight into neurodegenerative diseases, 3) capability to monitor disease progression. The ultimate aim will be to translate the SNS from a lab-based concept to a clinically viable tool to improve quality of life.

The proposed research directly addresses the societal grand challenge of maintaining health across the whole lifecourse. The proposed technologies are ideally aligned to significantly advance BBSRC strategic priorities including 1) develop and apply new tools in areas such as chemical biology, high resolution structural analysis, 'omics, biomarkers and bioimaging, 2) generate new knowledge of the biological mechanisms of development and the maintenance of health across the lifecourse, and ultimately 3) promote new ways of working to accelerate the translation of basic bioscience to benefit the health of the population across all stages of life.

This highly multidisciplinary project will introduce new possibilities and tools to the scientific and medical communities illustrated here by experiments exploring AD and PD. Its success will open a pathway towards a new generation of molecular sensors that have no comparison in world practice and to our knowledge. The development of novel biosensing technologies is a key area of research and aligns to promoting Government policies in healthcare. This technology will create new platforms and industrial applications to exploit the IP and in addition this could also enhance existing nanopore technologies. This research will augment other biosensing developments in the UK to help maintain the strong global position that the UK has in this field. Ultimately, this programme will invest in the BBSRC delivery plan to improve quality of life to support healthy independent living by enabling early stage diagnostics and at the same time reducing costs associated with medical implementation.

We will facilitate data access and sharing through different mechanisms. The primary mechanism for dissemination of the data will be through open access scientific publications and meetings, such as journal papers, conference proceedings, industry visits etc. As the project is very promising in terms of output, it will be possible to share results in high impact journals (e.g. Nature series) for the work associated with WP3-4. More fundamental aspects will be published in journals such Nano Letters, ACS Nano.

Our aim is that this project to lead to the definition of a new analytical platforms and in turn will ultimately generate the basis for novel clinical tools and intellectual property of commercial value. Hence there are likely to be genuine licensing opportunities or alternatively spin-out opportunities. All investigators have a successful patent portfolio and we have extensive experience in working with Imperial Innovations, the technology development arm of Imperial College London, to support the creation, development, protection and commercialisation of pioneering technologies. Meetings with the investigators will be held every 5 months specifically to explore the IP landscape and freedom to operate.

A key objective of this proposal is in the academic and professional development the postdoctoral researchers. They will have the opportunity to further their careers as an academic will be motivated to access training initiatives available at Imperial College such as Creativity & Ideas Generation, Networking, Time Management, Presentation and Communication Skills, Science & the Media, Project Management, Research Ethics, Technical Writing Skills, Statistics, The lead investigators have been in discussions with industrial partners to secure short-term placements within the project. In addition to knowledge transfer and real-life validation of the developed technologies, this will also enable the successful career progression of the PDRA, both in academic or industrial setting, depending on their future career choices. The skills and capabilities achieved by this training programme will increase the availability of highly skilled workers in the UK that will be an advantage in a knowledge-based economy.