细胞器靶向的多参数协同精准分析光学传感器研究

Multi-parameter combination analysis has a great application in life science and medical applications. In this project, we are aiming to solve the vital problems in the field of multi-parameter sensing and imaging, which includes signal fast-decay, poor measurement stability, low precision, usage of large number and many kinds of sensors, uncontrollable distribution of sensors, and high imaging background. Simultaneously sensing and imaging intracellular key parameters (including oxygen, pH and temperature) was selected as an example to show our strategy in designing the targeted multiple nanosensor. We will start our research from screening and synthesizing high-performance fluorescent probes and sensing materials. The multiple nanosensor was constructed based on a new strategy called "space division". Sensing material will be divided into different functional regions according to their functionalities. Their surface will be modified with protecting polymer and targeting groups to form core/shell nanostructure and specifically direct the multiple sensor at an intracellular site of interest. This external surface will also act as a barrier to protect the internal optical probes and prevent their cross-talking to ions and other small molecules. This specially designed structure will allow all sensors have their optimized performances. The multiple optical signals will be analyzed using fluorescence lifetime based sensing and imaging technology, which can significantly improve sensor stability and measurement precision, greatly reduce imaging background, make signal separation easier, and prevent signal cross-talking. Through multi-parameter joint, compensation and reference analysis, we can use less sensors to gain more intracellular key parameters, which enables high-precision sensing at specific sites of interest, and makes real-time monitoring intracellular activities become possible. The application of these targeted multiple sensors will be extended to life science and disease diagnosis.

多参数联合分析在探究生命过程和疾病诊疗领域具有极其重要的生物学和医学价值，本项目针对多参数荧光成像分析中信号衰减快、测量稳定性差、准确度低、传感器使用种类多、数量大、细胞内分布不可控、无法定位分析、荧光成像背景过高等关键难题，以氧气、pH和温度多参数检测传感器为例，筛选、合成高性能荧光探针和传感材料，并以“空间分割”新策略为主线，对同一纳米材料根据功用进行三维空间划区和修饰，构建具有靶向细胞器/受体能力的多参数协同检测纳米传感器，使各传感器能发挥其最佳性能。并建立以荧光寿命测量为核心的多通道协同精准成像分析新技术，通过多参数联合、补偿和参比分析，解决多参数分析中信号串扰和分离难题，提高传感器的稳定性和测量精度，大幅度降低成像背景干扰，实现使用更少传感器得到细胞内更多参数的高空间分辨、精确定位的精准成像分析，使实时跟踪细胞内动态变化成为可能，并拓展多参数传感器在生命科学和疾病综合诊疗中的应用

本项目针对多参数荧光成像分析中测量稳定性差、准确度低、传感器使用种类多、数量大、细胞内分布不可控等关键难题，提出以“空间分割”新策略为基础，设计和合成集多探针于一身的多参数纳米传感器，用于细胞内关键参数的同时检测，从而大幅度减少纳米传感器的用量，降低对细胞正常生命活动过程的影响。研究结果表明项目提出的"空间分割"策略是实现在纳米尺度高度集成多种荧光染料，并实现细胞尺度多参数测量的关键。研究工作应用该策略成功设计合成了3类多参数纳米传感器。这种新策略不仅可以有效地提高染料在纳米多参数传感器中的浓度，有利于增强其亮度；而且能有效地避免染料之间发生浓度淬灭和荧光共振能量转移，从而简化信号分离和分析过程，有利于精准实现细胞内多参数的同时测量。项目执行过程中建立了高效细胞器靶向基团修饰的新方法和荧光染料固定化新技术，制备出发光亮度高、靶向定位效果好、稳定性高的多参数纳米传感器。为了解决多参数分析中的信号分离问题，研究工作发展了基于门控技术的快速磷光寿命测量技术，并与显微成像技术相结合，搭建出了快速磷光寿命成像新装置；利用该装置实现了细胞内溶解氧浓度、Cl-浓度的高稳定、长时间追踪测量；项目执行中还利用快速磷光寿命成像系统测量精准这一技术特点，与氧传感技术相结合，拓展该系统的应用领域，开发出一种基于组合化学策略、不可复制的新型高安全荧光防伪加密技术。项目执行过程中所产生的新装置、新算法、新技术和新材料不仅为研究细胞动态变化工程提供了必需的工具，而且可用于细胞培养过程的监控、疾病机理的研究，高安全防伪加密技术可用于身份核验等一些高级别的安全应用场合。