Functionalised Rare Earth Up - Conversion Nanoparticles; reagentless fluorimetric nanobiosensors for biological analytes

Quantifying specific proteins and DNA and RNA is vital within biological research, in medicine and in food and environmental monitoring. For example in hospitals, heart attack is confirmed when specific proteins called troponins are released from damaged heart tissue. At present, for proteins the 'Gold Standard' measurement is the Enzyme Linked ImmunoSorbent Assay (ELISA) and related techniques These techniques are complex and time consuming, need dedicated laboratories, highly trained staff and expensive equipment. This means delays in finding out the results, which is often important for medical applications and it is difficult to do the analysis at the patient's bedside, or in the field for environmental or agricultural applications.

We will develop a completely new and very simple analytical system based on rare earth (RE) nanoparticles. RE ions are non-toxic and some are already used for medical imaging like MRI. Ions of RE elements such as Yttrium are very fluorescent and can be attached to binding proteins such as antibodies, or 'artificial antibodies' (Adhirons), termed the "bioreceptor" that recognise a wide range of biochemical targets. When the RE tagged antibody binds to its target, the fluorescence is enhanced. With RE nanoparticles (up-conversion nanoparticles -"UCNP), which are Sodium Yttrium Fluoride nanocrystals, doped with Yterbium and Erbium, the UCNP are not only brighter than the RE ions alone, but also show a phenomenon called up-conversion where infra-red illumination gives visible light fluorescence This produces no background fluorescence, unlike conventional fluorescent molecules and so gives very low background signals. The bioreceptors (antibodies or Adhirons) are then attached for target recognition and target binding again boosts fluorescence.

The physics of fluorescence enhancement upon target binding to RE nanoparticles is not known and measurements will be carried out to discover this. If target binding prevents energy loss from RE nanoparticles to solution we should observe an extended fluorescence lifetime. This will allow us to better design the bare UCNPs. Also, the design on the nanoscale for RE nanoparticles with attached bioreceptors is important for best performance.The positioning and orientation of the bioreceptor onto the UCNP has a strong effect on their performance and will be examined.

Finally, we shall develop RE-tagged bioreceptors and bioreceptor-functionalised against 'model' target proteins. Here, we have chosen two proteins initially. Myoglobin is a well understood protein within our laboratories; it is a biomarker of heart attack and muscle damage generally and there are conventional assay systems like ELISA available for comparison. The second protein is neurophil associated gelatinase lipocalin (NGAL), and is a marker of acute kidney injury (AKI) which often accompanied major trauma and leads ultimately to multi-organ failure and death. We have shown proved that the proposed system works in principle with both myoglobin and NGAL in test solutions. The RE based analysis system will be developed to work in real world fluids such as serum and urine and also for monitoring cell damage from "virtual organ systems" that are models of heart attack and stroke; these require no animal use - they are essentially 'organs on a chip'.

Quantifying specific proteins at low nM to pM levels is vital within biological research.
The current 'Gold Standard' technique is the ELISA, which is complex/ time consuming, and ideally requires automated equipment. Moreover, none of the current assays are capable of real time (or quasi real time) monitoring of proteins released as a result of physiological stress or injury.

The rare earth (RE) elements of the lanthanide series are gaining prominence as inorganic fluors, more recently formulated as up-conversion fluorescent nanoparticles (UCNP) which tyupically comprise NaYF4 nanocrystals doped with a sensitizer (e.g. Yb) and an emitter (e.g. Er). This allows near IKR excitation, (e.g. 980 nm) and produces visible light emission. The benefits are low auto-fluorescence, absorption and deep penetration into biological samples. The UCNP are much less prone to photo-bleaching and their lack of toxicity means that they can be used in cellular assays and in vivo.

Recently, we have shown that UCNP can be functionalised with natural and artificial bioreceptors such as antibodies and Adhirons, and that specific binding of the target myoglobin leads to enhanced fluorescence emission, proportional to its concentration. The physical mechanism for this enhancement is most likely due to reduction of energy loss at the UCNP surface by the bound protein.

Our overall aim is to establish functionalised RE nanoparticles as tools for both research, medical diagnostics and environmental monitoring and our objectives are:
1.Synthesize NaYF4 nanoparticles, doped with Yb3+, Er3+ and other RE ions and characterise excitation and emission characteristics of these UCNP.
2. Functionalise the UCNP with antibodies and Adhiron bioreceptors (= 'fUCNPs') against cardiac, stroke colorectal disease biomarkers.
3. Implement the fUCNP assay in a plate based format.
4. Monitor biomarkers release in real time from a quasi-vivo cell system.

Impact on scientific knowledge: The assay platform developed will greatly facilitate most areas of molecular bioscience and medicine and by speeding up and simplifying measurement of specific proteins. Also, the technology will be much cheaper, as the components for RE particle synthesis and adaption of existing plate readers for near IR illumination are fairly inexpensive. At the moment, although commercial ELISAs and analogous systems are available for many proteins, their use represents a considerable cost both in terms of reagents and the processing equipment needed. The system is also flexible as to the recognition agent used, e.g. antibody or other binding protein.
Knowledge and technology translation: We have already discussed IP protection with the Enterprise and Innovation service at Leeds and proper protection will be in place before dissemination by the usual routes (publication , conferences) but also via the appropriate KTN network events. Technology translation is likely to be via our relationships with commercial antibody suppliers, such as ABCAM (Millner has just graduated a BBSRC CASE PhD student with ABCAM), suppliers of synthetic binding proteins such as the Affimers (Avacta Ltd) or with SMEs such as ELISHA Systems Ltd (of which Millner is a founding director).
Industrial impact: As well as new product lines for immunoassay companies and antibody suppliers, the producers of in vitro tissue models will benefit from the ability to more easily monitor protein release, most likely in real time, from cell cultures and co-cultures following physiological or toxic stress, or administration of drugs. Kirkstall Ltd, manufacturers of "Quasi Vivo" systems and who have a collaboration with the Saha laboratory, have provided a letter of support. At present we are focusing on the BBB system within the Saha laboratory. However, the methods developed for culturing heterogeneous mixtures of closely related cells with supporting matrices and analysis of biomolecules in real-time will be widely applicable to other systems, including lung, kidney, liver and cardiovascular systems .
The ability to follow protein release following drug administration will facilitate pharmaceutical research by enabling an easier cellular assessment of drug action during the discovery phase.

Impact on animal and human welfare: The up-conversion fluorescence system, by facilitating adoption of in vitro models using human primary cells will impact both on animal and human welfare, not only by replacing animals utilised for in vivo and ex vivo screening for novel drugs, but also by reducing the number of animals and volunteers used in pre-clinical and clinical studies to confirm the dose effectiveness of molecules. Thus, the project supports the UK agenda for replacement, refinement and reduction (3Rs) for animal welfare by providing an alternative method of study in an ethically-contentious area of research. Adoption of the alternative model by the pharmaceutical industry as part of the lead-to-hit selection process could also substantially reduce animal use.
Impact on society: There will be an overall society benefit from economic gains as well as from advances in human and veterinary medicine both via research into the basic biology of diseases but also for diagnostics which target molecular biomarkers. There will also be benefit from new and accelerated drug development. Society will also gain from workforce capacity building for the training of highly qualified, interdisciplinary, specialised professionals. Public perception of science and technology will also be enhanced by tangible medical benefits to society.