量子点-刚性芽胞组合的可编码微球研究

At the leading edge of analytical chemistry, barcode fluorescence microspheres with advantages such as low cost, eco-friendly, high code capacity, and high stability have become a crucial technology in many analytical methods (e.g., multiplexed suspension arrays). Based on the properties of Bacillus spores (e.g., different morphology, and mechanically rigid and protective shell) as well as the quantum dots (e.g., minimal spectral width, and remarkable photostability), for the first time, we present a simple and high-throughput biological method to prepare multicolor-coded microspheres by embedding different emitting QDs onto the surface of spore-based microspheres. .(1).The quantum dots loaded onto the surface of different Bacillus spores (e.g., Bacillus subtilis, Bacillus megaterium, and Bacillus sphaericus) were respectively investigated. The effect factors (e.g., the properties of the spore surface and quantum dots, and the reaction conditions) for the characterizations of color-coded microspheres were analyzed in detail. Accordingly, the mechanism of the quantum dots loaded onto the surface of spores was studied in detail. Based on these researches, we try to find out the rules and methods to regulate and improve the characterizations of the color microspheres, which can provide theoretical basis and technical guidance for the preparation spore-based color microspheres. .(2).Pseudorabies, PRRS, ring, and Japanese encephalitis virus can cause a mixed infection polyinfection event, which lead to porcine reproductive disturbance. Using this mixed infection polyinfection event as an example, the developed suspension microarrays were used for multiplex immunoassays of animal diseases. .The success of this project will lay the foundation of theory and technology of the developed suspension array method for rapid multiplex targets analysis in the research fields such as differential diagnosis of mixed infection polyinfection events, proteomic, genomic, metabonomics and drug screening.

成本低、环境友好、编码容量高、荧光性能稳定的编码微球是分析化学研究的前沿，也是多种分析技术推广应用的核心和关键。我们提出利用具有刚性外壳的细菌芽胞、结合荧光量子点发展新颖的、高容量编码微球。本课题拟：（1）以枯草芽胞杆菌、巨大芽胞杆菌和球形芽胞杆菌为例，分析芽胞表面分子结构和组成、量子点的化学组成和表面分子组成等因素对荧光微球性能的影响，研究量子点-芽胞微球相互作用分子机制，发展调控并提高编码微球性能的规律和方法，为建立基于芽胞的编码微球新方法提供理论依据和技术指导。（2）以引起猪繁殖障碍的伪狂犬、蓝耳病、圆环和乙型脑炎病毒混合感染为例，构建新型悬浮芯片技术实现同时快速检测上述病原的混合感染事件。本项目的成功将为这一新技术用于多病原混合感染事件、蛋白质组学、基因组学、代谢组学及药物筛选等基础和应用研究中探测多目标分子奠定理论和技术基础。

基于微球的悬浮/液相生物芯片具有多功能性、高通量、高敏感及高灵活性等特点，在实现高通量、高灵敏度、多目标同时测定领域有无可比拟的优点和优势，特别适用于微纳、复杂样品中多靶标的同时快速测定；其应用领域已从基础研究扩展到临床诊断、高通量药物筛选、蛋白质组学、基因组学、环境监测等应用研究。针对当前化学法制备编码微球过程中的问题，本课题提出一种采用单细胞微生物（如：芽胞、酵母、大肠杆菌以及蓝细菌）为模板制备功能微球，以无机量子点CdTe及CdTe/CdS/ZnS、碳量子点及荧光染料制作彩色编码微球的新方法。研究结果表明：1）采用生物法制备功能微球具备绿色环保、可以大量生产、简单、快速、粒径均一、易于控制以及价格低廉等优势；2）基于微生物细胞的功能微球表面有大量的、天然的功能基团（如;氨基、羧基以及巯基等），非常有利于荧光材料在其表面的可控组装；3）可以实现荧光-形貌差异的复合编码方式提高编码容量；4）MOFs材料的刚性、多孔结构，非常有利于进一步提高细胞微球的机械强度和比表面积，并能有效提高生物识别分子（捕获探针）的结合密度，进而提高检测灵敏度；5）无机量子点会在微球表面发生团聚，这种团聚会造成采用流式细胞仪对浓度梯度解码失效。这种团聚效应可以采用粒径小的碳量子点及有机荧光染料进行解决。采用戊二醛偶联的方法可以将碳量子点及有机荧光染料锁在微球内并防止泄漏，从而使采用流式细胞仪解码效率得到大大提高。6）荧光编码微球被成功用于引起猪繁殖综合征的三种病毒（猪蓝耳、猪圆环、猪伪狂犬病）的同时快速检测。在本项目的支持下，本课题组较为出色的完成了预期目标，共发表SCI论文13篇、获得批准发明专利两项、申请发明专利1项。本项目的成功对于发展基于微球的悬浮生物芯片分析的新理论、提高对复杂样品的分析通量、降低使用成本、扩大应用范围以及促进国民经济发展有十分重要的科学研究意义和价值。