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

量化特定的蛋白质、DNA和RNA在生物研究、医学、食品和环境监测中至关重要。例如，在医院，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，心脏病发作就被确认了。目前，对蛋白质的金标准测量是酶联免疫吸附分析(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。这意味着延迟发现结果，这通常对医疗应用很重要，而且很难在患者的床边或现场进行环境或农业应用的分析。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，一些已经被用于医学成像，如核磁共振。稀土元素的离子，如Y，是非常荧光的，可以连接到结合蛋白上，如抗体或被称为“生物受体”的“人造抗体”(Adhirons)，它识别一系列的生化目标。当RE标记的抗体与其靶标结合时，荧光增强。掺Yb和Erb的稀土纳米粒子(上转换纳米粒子--UCNP)是氟化钇钠纳米晶体，UCNP不仅比单独的稀土离子更亮，而且还显示出一种被称为上转换的现象，其中红外光发出可见光荧光，这不会产生背景荧光，不像传统的荧光分子，因此提供非常低的背景信号。然后，生物受体(抗体或粘附素)被连接起来进行靶标识别，靶标结合再次增强荧光。目标与稀土纳米颗粒结合后的荧光增强的物理机制尚不清楚，将进行测量以发现这一点。如果靶结合阻止了从稀土纳米粒子到溶液的能量损失，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露的UCNP。此外，具有生物受体的稀土纳米粒子的纳米级设计对于最佳性能是重要的。生物受体在UCNP上的定位和取向对其性能有很大影响，将被检测。最后，我们将开发RE标记的生物受体和针对目标蛋白的生物受体功能化。在这里，我们最初选择了两种蛋白质。肌红蛋白在我们的实验室中是一种众所周知的蛋白质；它通常是心脏病发作和肌肉损伤的生物标记物，而且有传统的检测系统，如ELISA法可供比较。第二种蛋白是神经细胞相关明胶酶脂蛋白(NGAL)，是急性肾损伤(AKI)的标志，AKI常伴有严重创伤，最终导致多器官衰竭和死亡。我们已经证明，所提出的系统在原则上与肌红蛋白和NGAL一起在测试溶液中工作。基于RE的分析系统将被开发成在真实世界的体液中工作，如血清和尿液，还用于监测作为心脏病发作和中风模型的“虚拟器官系统”的细胞损伤；这些系统不需要动物使用--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'.