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Rare Earth based Upconversion Luminescent Nanomaterials as novel bioprobes for the detection of cardiac biomarkers

Periodic Reporting for period 1 - RECORDER (Rare Earth based Upconversion Luminescent Nanomaterials as novel bioprobes for the detection of cardiac biomarkers)

Reporting period: 2016-07-13 to 2018-07-12

The objectives of the project include the synthesis of upconversion nanoparticles (UCNPs) and adopting the application of upconversion fluorescence phenomena for the detection of cardiac biomarkers and fabrication of a multianalyte system. As the part of achieving the final target of biosensing using the phenomena of upconversion, we have developed cubic as well as hexagonal UCNPs with very high fluorescence emission when excited using near infrared (NIR) radiation. UCNPs have been synthesised with variation in host/sensitizer/activator and were optimised with respect to photoluminescence and were correlated with structural features including nano size distribution. The main materials chemistry part including synthesis of upconversion particles (using solution, hydrothermal and solvothermal methods) and optimisation of conjugation of bioreceptors such as reduced antibodies and Affimers to selected UCNPs has successfully been carried out. Silica substrates were also successfully fabricated. The biosensing of selected markers were done using the upconversion phenomena where a limit of detection of 10nM have been achieved. Attempts are going on to reduce the detection limit in the range 0.1-0.01 nM and even pM level. Different attempts are going on in order to use the substrate based detection technology for multimarker detection. Possibilities of using UCNPs for imaging were also attempted as a part of application of UCNPs.
Upconversion nanoparticles have been prepared by solution method, hydrothermal and solvothermal methods. The cubic and hexagonal phases of the upconversion nanoparticles synthesised have been confirmed by XRD and the particles sizes in the nanorange have been established by TEM and DLS. Advanced TEM characterization have been attempted for specific systems. Upconversion nano systems with different hosts BaYF5 and NaYF4 with Yb3+ as sensitizer and Er3+ as activator have been synthesised. Upconversion systems with various sensitisers/activators have also been synthesised. The hydrothermal methods cause a little aggregation with highly fluorescent red emission with cubic phase, while solvothermal method gives particles with hexagonal phase with very intense green fluorescence emission hexagonally shaped particles. The toxicity has been assessed by adopting MTT assay and the samples are found to be non toxic for further advanced applications. Silica substrates have been synthesised using sol-gel method. Initial biosensing trials have been done with cubic nanoparticles. Even if cubic and hexagonal particles have been synthesised, the hexagonal particles synthesised by solvothermal method were found to be much more ideal with respect to fluorescence intensity. Silanisation procedures were adopted as a part of attaching the bioreceptors to UCNPs. Trials have been made to silanize and bioconjugate with reduced antibodies, reduced antibodies and Affimers for biodetection. As initial trials, a competitive assay for ofloxacin using hexagonal antibodies were found to be successful. The detection of biomarkers using upconversion nanoparticles have been done for selected biomarkers and the limit of detection is found to be in the 10nM range. Trials are going on to reduce the detection limit by modifying the particle size and detection procedures. It has also been identified that these upconversion nanoparticles are ideal candidates for fluorescence imaging. Synthesis of NaGdF4/ Yb3+/Er3+ was also carried out expecting its application with respect to its dual property-magnetic as well as optical. Even if the optical properties and nanosize properties were characterised, the magnetic properties for dual mode detection and imaging is still in the preliminary stage. Conjugation of A15 peptides with hexagonal UCNPs were carried out with the aim of targeting thrombus diseased site and this work also in the initial stage. Collaboration with different schools involved in the project has been established. The upconversion nanotechnology and the advanced applications and challenges are established in University of Leeds through this project. The current work leads to a publication in J. Biophotonics (Just accepted, where the researcher is the First and Corresponding Author) and a publication in Scientific reports (Scientific Reports, (2018) 8:1106 | DOI:10.1038/s41598-018-19415-w) where the researcher is a co-author. Researcher presented two plenary talks in two international conferences (5th International Conference on Nanotechnology in Medicine cum 11th International Forum on Innovative Technologies for Medicine (ITMED), 2018, June 26-28th (2018), Manchester, UK.; 23rd International Conference on Nanomaterials and Nanotechnology-Nanomaterials 2018, March 15-16 (2018), London, UK). Discussions are going on with Research and Innovation at University of Leeds regarding IP aspects of UCNP synthesis and modifications, but this is in the very preliminary stage. The industrial links created by the researcher will be supportive for taking this work forward and for effective commercialisation in future.
Recently, we have shown that upconversion nanoparticles (UCNPs) can be functionalised with bioreceptors such as antibodies and Affimers, and the specific binding of the target protein leads to variation in fluorescence emission, proportional to its concentration. We identified the application of the UCNPs in multimodal imaging.
The impact of the work done involves the importance of the particle synthesis for detection technologies. Detection of biomarkers seeks high importance and multiplexing is one of the novel aspects we could consider in future using the rare earth UCNPs, as different sensitizer/activator could be used for different emission profiles with the single 980 nm excitation source. The industrial collaborations developed during this period involve prospects of future collaboration with Centre for Process and Innovation Ltd., UK and Promethean Particles Ltd., UK who are interested in scaling up of the particles and processes and biosensing assay development for multimarker detection of various markers using our technology developed. The Fellow and Supervisor had interactions with the technologists involved and discussed the chance for commercialisation in future. Merck Co. also showed interest in our processs of UCNP based biodetection. The developmental applications including clinical trials using these particles will also be a next stage of this project, in collaboration with Leeds Institute for Cardiovascular and Metabolic Medicine. A competitive assay was developed for the detection of ofloxacin antibiotic as a part of trial before accepting the method for cardiac biomarker detection and was found to be successful. We have identified the importance of the nanoparticles in the area of imaging and certain procedures have been developed in order to conjugate peptides which could specifically target the thrombus site. This work is at a very early stage. But could have huge impact in the medical field. This has been done while the optimisation of different conjugation procedures to the nanoparticles have been carried out. On the basis of the results achieved in the RECORDER project, the researcher applied for BeCurious, an initiative by University of Leeds to demonstrate the challenging technologies developed at University of Leeds.