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

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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'.