Photonic Sensing and Dual-mode Bio-Imaging with Rare Earth Upconversion Nanoparticles

Cardiovascular diseases (CVD) which include acute myocardial infarction (AMI) and stroke are the main cause of premature deaths in the world. The nature of CVD demands efficient and simultaneous detection of biomarkers earlier than current approaches that are incapable or time consuming. Atherosclerosis is an inflammatory disease, and inflammatory marker high-sensitivity C-reactive protein (CRP), has been shown to identify additional individuals who are at risk. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is also a marker of vascular inflammation. Thus screening with for example both Lp-PLA2 and hs-CRP may provide a better risk assessment for CVD and stroke than using either test alone.
In this project we have chosen four biomarkers NT-proBNP, LpPLA2, GFAP and IL6 for implementing a multiple biomarker detection and measurement system. Traditional diagnostic tools currently used for detection and assessing atherosclerotic plaques have their limitations regarding the less contrast images received even from modern techniques such as CT and MRI. Although recent advances in imaging techniques together with proteomics and metabolic profiling have enhanced the feasibility of patient screening for the diagnosis, they are not suitable for the "point-of-care" where the early diagnosis may benefit primary prevention and treatment of these diseases. The "gold standard" cardiac angiography is an invasive method with risks associated with the catheterization procedure and there is no reliable non-invasive method to identify and quantify the severity of atherosclerotic plaque formation. Ultrasound scan is currently used for detecting plaque burden non-invasively, particularly in the carotids, but there is no currently available interpretation of this that allows differentiation of unstable vs stable plaques.
In this interdisciplinary project, we will develop lanthanide doped up-conversion nanoparticles (UCNPs) and biomolecular conjugate based assays for the rapid and simultaneous detection and imaging of key biomarkers - for CVD and atherosclerosis. UCNPs have specific excitation and emission wavelengths, and the individual fluorescent lanthanide ions are very sensitive to environmental alterations around them. The project aims to create a scalable manufacturing process for rare earth (RE) upconversion nanoparticles (UCNP) suitable for photoluminescence based rapid bio-sensing and bio-imaging that also preclude signal noise due to autofluorescence (fluorescence from tissues). The crystallinity and RE doping concentration of the UCNPs need improvement for high output visible light emission under infrared laser excitation while being biologically safe. We propose to scale up the manufacturability of the UCNPs suitable for commercial exploitation. These nanoparticles with intense visible light emission under near infrared excitation will be surface functionalised with -Affimers to be suitable for biologically safe and targeted laser based as well as magnetic (MRI) imaging of tissues and organs. Affimers are synthetic binding proteins, developed at the University of Leeds and marketed by Avacta Lifesciences. As a further development, these particles suitably functionalised will be used to create lab-on-a-chip device for simultaneous and rapid sensing of CVD biomarkers. By bringing together imaging and sensing modalities, this project proposes to develop methodologies for identifying, monitoring and finally detecting atherosclerotic tissues. The methodology developed in the project might also be applicable to various forms of cancer biomarker detection and tissue imaging.

At present there are a number of discrete devices in the market for detection of biomarkers of cardiovascular diseases(CVD) separately, primarily based on ELISA requiring different assay procedures and equipment to measure. Most of these measurements are time consuming and would not provide simultaneous detection of multiple biomarkers which is essential for more accurate and early diagnostics of cardiovascular disease. This project aims at using a class of nanoparticles known as rare earth doped upconversion nanoparticles (UCNPs) for making assays and sensor devices which provide the simultaneous detection capability which could be cost effective with high specificity and sensitivity. The incorporation of rare earth ions such as Er3+ in these nanoparticles is the key to their specific narrow emissions spectral features in the visible emission wavelength range under near infrared laser excitation which allows multiplexed sensing. We proposed to use a smaller protein molecule developed by Avacta known as Affimers with these fluorescent nanoparticles to provide targeted detection of individual biomarkers. The use of Affimers is the key to our multiplexed sensing strategy owing to their size and cost advantages over antibodies conventionally used in most sensor assays. Previous studies of UCNPs were mostly focused on their synthesis at laboratory scale for demonstration of certain applications. In order to achieve commercial success, more standard manufacturing process with repeatable performance characteristics are essential, and we are collaborating CPI Ltd, Durham in this respect. Eluceda, another industrial partner in this project will be an early beneficiary of our nanoparticle manufacturing platform as they are intending to use these particles for their anticounterfeiting and DNA based pathogen sensing business. Their expertise in biosensing will also be advantageous to the project in developing the multiple biomarker sensing platform. The nanoparticles and their interface with other molecules/surface will be studied in depth using advanced X-ray techniques in collaboration with EPSRC facilities-SuperSTEM and Diamond Light Sources, to optimise their optical properties, considering the structural aspects of UCNPs. The UCNPs are also attractive as optical as well as magnetic imaging agents and we propose to exploit this aspect for the imaging of atherosclerotic tissues to enable the early detection of CVD. The multidisciplinary approach will deliver the following specific impacts:
i) Expanding the scope of sensing and imaging technology beyond the state-of-the-art providing multiple sensing for early diagnostics of CVD-impact will be based on the new knowhow and IP generated during the project which will be suitable for licensing and commercial development
ii) UCNP production technology achieved in collaboration with CPI which will be available for commercial exploitation-Eluceda could be an early beneficiary with their partnership in the project
iii) Establishing Affimer as key component of the sensor assay-impact on the business of Avacta
iv) Novel dual mode imaging technology for atherosclerosis, early stage research results available which are available for knowledge transfer and medical device development.
Overall, the UK competitiveness in biosensing and bioimaging research will be enhanced by the novel sensing-imaging concepts investigated in the project which will lead to invaluable technology of device development and bioimaging prospects in healthcare through the industrial collaborations. It will also cement out position in the global market that is expected to reach over $25b. The CVD sensors/imaging platforms proposed will provide point of care diagnostics and will be suitable for ambulances and A&Es that can help saving time and cost to NHS in the longer term.