高分辨光流控显微成像技术及应用研究

The demand of point-of-care greatly urges optical microsocpy, a vital tool for modern medicine, to be portable. Considered as coming manstream technique in the field of POC, optofluidic microscopy (OFM) is one of the efficient ways to miniaturize a light microscope. So far, miniaturization of microscope is obtained at the cost of resolution, and the limitation of OFM is that we cannot get a high resolution image. It still remains a challenge at both academic and technique to increase the resoltution of OFM to a high or a super high level. The reason is that equipments to produce excitation area smaller than sub-diffraction which are gennerally complicated and giant cannot be intergrated with OFM easily due to the huge difference in their size. With our knowledge and experience in super-resolution imaging and microfluidics, we strongly believe that it is feasible to achieve both high resolution and miniaturization of OFM by nanopore array imaging. In this proposal, we are going to build a lensless OFM based on a nanpore array in order to take high resolution picture at both single-particle and ensemble levels, which will definitely be the fundamental to the high resolution portable microscope.

光学显微成像技术是现代医学不可或缺的检验分析手段，使之微型化至便携或手持程度是现场即时检测(POC)的需求。基于光流控芯片的显微成像技术（OFM）是光学显微镜微型化的有效途径，也被认为是未来用于POC领域的主流方法。现阶段，OFM已经具有了最基础的成像功能，能实现便携甚至手持，但主要问题在于成像品质不理想，分辨率不够高，不能满足准确、精细的POC成像要求。而已有的高分辨乃至超高分辨成像技术所需仪器复杂、体积庞大、价格昂贵，难以直接与OFM相耦合。提高微型显微成像技术的分辨率至实验室适用量级的高分辨率水平，在科学和技术层面都是一个挑战。本项目在课题组已有的超高分辨率显微成像和微流控芯片研究工作的基础上，提出利用纳孔限域成像技术实现高分辨率光流控显微成像的研究思路，拟实现单颗粒和宏观层面上及细胞体系中亚微米级别的高分辨率成像，为进而研制微型化高分辨率光学显微镜，满足POC的高层次需求奠定基础。

重要生物分子的高分辨高灵敏成像传感方法的构建有利于精准诊断和精准治疗。数字传感方法具有检测灵敏度高，使用样品量少，避免使用信号的强度变化带来的分析误差等优点。本项目构建了几种数字分析方法，主要包括（一）以金纳米颗粒为标志物，以彩色CCD检测到单个金纳米粒子与团聚金颗粒的颜色差异，通过计数团聚金颗粒的个数设计了高灵敏汞离子传感方法。（二）以量子点为标记物，通过量子点蓝移不同步性识别并计数量子点团聚体或二聚体的个数，分别构建了血液中肝素和肿瘤标志物的定量分析方法。（三）以金颗粒和量子点分别标记捕获抗体和检测抗体，当形成免疫复合物时，发生等离子共振能量转移，金颗粒的散射强度下降，通过单颗粒水平上计量散射强度下降程度与抗原浓度的关系，实现肿瘤标志物分子的定量。（四）以聚苯乙烯微球为标志物，通过磁珠的浓缩与分离，将全部微球沉降在基底上，实现绝对定量，以CEA为模型，达到4.9aM的检测限。这些方法的构建为进一步实现肿瘤标志物的早期筛查打下基础。