双光子荧光光镊多通道定量分析新方法及其在多种甲型流感病毒同时实时检测中的应用

In this project, based on the principle of optical trapping of microparticle using optical tweezers, we aim to develop a novel two-photon fluorescence multichannel approach for the detection of influenza A virus with high-speed and exceptionally high sensitivity. The polystyrene microspheres and highly fluorescent quantum dots (QDs) are used to capture the viruses through an immuno sandwich mode by forming microsphere-virus-QDs complexes. By automatic 3D scanning the focused near-infrared pulse laser beam to trapp the single polystyrene microsphere though monitoring the backscattering of the microsphere to probe the microsphere-virus-QDs complexes, the target viruses can be quantified by two-photon fluorescence detection. All the coexists outside the optical trap do not interfere with the measurements because the two-photon excitation fluorescence signal occurs only within the focal volume of the focused laser light, leading to a detection volume on the order of 1 fL, thus low amount of viruses in very tiny sample volume can be probed with high sensitivity. In this method a high-sensitivity linear CCD is used to construct a polychromator to detect the signal of QDs with different fluorescence peaks that can be excited with the same laser line, therefore simultaneously different kinds of viruses may be captured and quantitatively measured. This method has the advantages such as high sensitivity, good selectivity, high speed,tiny sample volume with no preparation. Most importantly, it is potential for simultaneous and real-time detection of multiple influenza A viruses in the same sample.

本项目基于光镊捕获微米粒子的基本原理，构建一种快速、超高灵敏实时检测甲型流感病毒的双光子荧光检测新方法。首先利用聚苯乙烯微球和荧光纳米量子点材料，通过双抗夹心免疫反应，识别和捕获目标病毒，并形成微球-病毒-量子点复合物。再通过聚焦近红外脉冲激光的三维自动扫描并定量监测微球表面的散射光以捕获上述微球，同时对该复合物进行双光子荧光探测以实现对病毒的定量分析。由于本方法只探测位于光阱处的微球-病毒-量子点复合物所产生双光子荧光，因而溶液中共存物质不产生信号干扰，且探测的体积仅为1fL左右，使得该方法可以用于超高灵敏的极少量病毒的检测。同时，由于本方法以高灵敏度线阵CCD为检测器构成多色仪，且利用不同荧光发射波长的量子点可被同一波长激发光所激发的特点实现不同量子点标记多种不同病毒的同时检测。该方法具有灵敏度高、选择性好、速度快、样品用量少且无需预处理，并有望实现多种甲型流感病毒的同时实时检测。

本项目基于光镊捕获微米粒子的基本原理，构建了一种快速、超高灵敏实时检测甲型流感病毒DNA和其它多种靶分子的双光子荧光检测新方法。首先利用聚苯乙烯微球和荧光纳米量子点材料，通过双抗夹心免疫反应或核酸杂交反应，识别和捕获目标分子，并形成微球-靶分子-量子点复合物。再通过聚焦近红外脉冲激光形成光镊以捕获上述微球，同时采用EMCCD对该复合物进行双光子荧光探测以实现对靶分子的定量分析。由于本方法只探测位于光阱中心微球的双光子荧光信号，因而溶液中共存物质不产生信号干扰，且探测的体积仅为1fL左右，使得该方法可以用于超高灵敏的极少量病毒DNA等靶分子的检测。本方法具有灵敏度高、选择性好、速度快、样品用量少且无需预处理，实现了同一复杂样品（如全血清）中多种甲型流感病毒DNA或其它靶分子的同时定量检测。