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

对特定蛋白质、DNA和RNA进行定量在生物学研究、医学、食品和环境监测中至关重要。例如，在医院里，当被称为肌钙蛋白的特定蛋白质从受损的心脏组织中释放出来时，心脏病发作就被证实了。目前，对于蛋白质的“黄金标准”测量是酶联免疫吸附测定（ELISA）和相关技术。这些技术复杂且耗时，需要专门的实验室、训练有素的工作人员和昂贵的设备。这就意味着要延迟获得结果，而这对于医学应用来说非常重要，在患者床边或者在环境或农业应用领域很难进行分析。我们将开发一种全新的、非常简单的基于稀土（RE）纳米颗粒的分析系统。稀土离子是无毒的，有些已经用于医学成像，如MRI。稀土元素的离子如钇是非常荧光的，可以附着在结合蛋白上，如抗体或“人工抗体”（Adhirons），称为“生物受体”，可以识别广泛的生化靶标。当RE标记的抗体与其靶标结合时，荧光增强。使用RE纳米颗粒（上转换纳米颗粒-“UCNP”），其是掺杂有镱和铒的氟化钇钠纳米晶体，UCNP不仅比单独的RE离子更亮，而且还显示出称为上转换的现象，其中红外照明给出可见光荧光。生物受体（抗体或Adhiron）然后被附着用于靶识别，并且靶结合再次增强荧光。靶与RE纳米颗粒结合后荧光增强的物理学尚不清楚，将进行测量以发现这一点。如果目标结合防止能量从RE纳米颗粒到溶液的损失，我们应该观察到延长的荧光寿命。这将使我们能够更好地设计裸UCNP。此外，纳米级设计的RE纳米粒子与连接的生物受体是重要的最佳性能。的定位和方向的生物受体上的UCNP有一个强大的影响他们的性能，并将进行检查。最后，我们将开发RE标记的生物受体和生物受体功能化对“模型”靶蛋白。首先，我们选择了两种蛋白质。肌红蛋白是我们实验室中非常了解的蛋白质;它通常是心脏病发作和肌肉损伤的生物标志物，并且有常规的检测系统如ELISA可用于比较。第二种蛋白质是嗜神经细胞相关明胶酶脂质运载蛋白（NGAL），是急性肾损伤（阿基）的标志物，AKI通常伴随重大创伤并最终导致多器官衰竭和死亡。我们已经证明，所提出的系统的工作原理与肌红蛋白和NGAL在测试溶液中。基于RE的分析系统将被开发为在真实的世界流体中工作，例如血清和尿液，并且还用于监测来自“虚拟器官系统”的细胞损伤，所述虚拟器官系统是心脏病发作和中风的模型;这些不需要动物使用-它们本质上是“芯片上的器官”。

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'.