面向便携式血液诊断的组合荧光探针芯片的研究

Point-of-care testing (POCT) has advantages of being portable, fast and low-cost. The related medical devices are trending to be smarter, less invasive, and used more by home patients. Coulter principle and flow cytometry, two major techniques involved to develop portable blood counting for POCT, limit the possibility of preparing blood samples on a smart and miniaturized platform, and therefore, are hard to achieve the goal of counting white blood cells with 5-part differential by home-users. This proposal is based on our previous work “large field-of-view image-based blood counting”, and aims for a novel sample preparation method - “combinatorial fluorescent reporters on a chip” for accurately counting red blood cells, platelets and white blood cells with 5-part differential. The idea is to study thermodynamics and kinetics of individual fluorescent dyes staining sub-types of white blood cells, and then identify an optimized dye combination and reaction condition for staining and differentiating among these types, especially highlighting the most challenging components eosinophils and basophils. We plan to compare our method with clinical results. If it is successful as expected, the idea will provide a significant solution for the sample preparation of portable blood counting, and move this field one important step forward.

床旁诊断（又称POCT）具有便携、廉价、快速的突出优点，可以满足未来医学诊断微创、智能化和个性化发展的要求。目前便携式血液诊断中血细胞计数普遍采用的流式细胞术和库尔特原理，在方法上限制了血液样品前处理的微型化和智能化，不能实现白细胞五分类个人自行诊断的最终目标。本申请基于前期工作“血细胞大视场显微成像计数”的新原理，旨在发明一种新的血样制备方法，即组合荧光探针芯片，对微量血液智能化染色，区分并准确计数红细胞、血小板和白细胞的五种分类。其途径是研究不同荧光探针对白细胞染色的化学平衡和动力学机制，以此为依据找到一个反应条件的制衡点，使微量组分嗜酸性粒细胞和嗜碱性粒细胞从计数图中突显出来，从而突破上述便携式血细胞计数的难题。我们将在初步临床试验中实现这种组合荧光探针芯片的理念，使其较快过渡到初步临床应用阶段，为便携式血液诊断仪器的开发提供有效的思路和技术手段。

目前的医疗系统普遍依赖于昂贵的诊断设施，研发便携式的诊断方法与仪器以实现床旁检测势在必行。本项目开发了一套面向便携式血细胞计数的简单、高效的样品制备方法，提出了直接制备不稀释血样和低倍稀释血样的策略，以及相应的两种制作成像样品池的方案，用于更准确的检测白细胞的三分类；并用循环肿瘤细胞模拟血液中微量罕见的细胞类型，开发了特异性多肽修饰的磁珠正向富集目标细胞的样品制备方法；同时改进了“大视场细胞显微成像计数系统”，使装置的物理尺寸、重量、成本都大幅降低。这项研究为“如何在一个小型或便携式的仪器平台上，实现极微量复杂样品中不同类型物质的定量区分”这一化学测量学领域的科学问题，开拓了新的思路。相应的技术手段为满足未来血细胞计数诊断的微创、即时性、智能化、个性化的发展需求，提供了有效的解决方案和仪器平台，并为癌症的早期诊断、化疗疗效和预后复发的评估提供了一种潜在的床旁诊断的方法。

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