神经氨酸酶检测试剂的合成及其在流感病毒检测中的应用

The flu which is a zoonotic infectious disease caused by influenza viruses seriously jeopardizes public health and the development of animal husbandry. Until now, sequence-dependent nucleic acid amplification technology (i.e., polymerase chain reaction, PCR) is the common method for detecting influenza virus in the laboratory, which requires higher detection conditions and operational skills. Neuraminidase (NA) is a crucial surface glycoprotein of influenza virus, which plays crucial roles during infections. However, only very limited methods for neuraminidase detection were reported. There is an urgent need for the development of a general, convenient method to detect influenza viruses by detecting influenza viral neuraminidases. In view of the poor sensitivity and poor specificity of the existing neuraminidase detection probes, we intend to develop two new types of neuraminidase detection reagents and investigate their detection effects on various neuraminidases. The first is based on the aggregation-induced emission (AIE) effect and the second is related with the glucose released in the neuraminidase-catalyzed hydrolysis reaction. For the first case, we design several modified-sialic acid-functionalized tetraphenylethene luminogens with excellent hydrophilicity, good stability and with high sensitivity and unique selectivity towards influenza viral neuraminidases. Upon influenza viral neuraminidases triggering deglycosylation to uncage the sialic acid moieties, the probes will be rejuvenated to realize neuraminidase detection sensitively. For the second case, we will synthesize several disaccharides composed by modified-sialic acid and glucose. Upon influenza viral neuraminidases triggering the hydrolysis of disaccharides specifically, the liberated glucose could be detected easily by glucometer to realize neuraminidase detection. Since the 4, 7, and 5 positions of sialic acid will be modified based on the structure of influenza viral neuraminidases, the detection specificity towards influenza viral neuraminidases will be improved greatly. This project owns great scientific value and has vast application prospect.

流感是流感病毒引起的人畜共患传染病，序列依赖的核酸扩增技术是实验室检测流感病毒的常用方法，该方法要求的检测条件较高。流感病毒神经氨酸酶在流感传染中起关键酶作用，以神经氨酸酶为检测靶点发展高效的流感病毒检测方法具有重要的现实意义。本项目拟针对现有的神经氨酸酶检测探针灵敏性差、特异性差等问题，发展两类流感病毒神经氨酸酶检测试剂并评价其应用价值。一类是基于聚集诱导发光（AIE）效应，设计特定位点修饰的唾液酸单元“包裹”的四苯乙烯化合物，合成系列水溶性的“糖基AIE”探针，利用酶的水解作用，发展“off-on”型的荧光探针,提高检测的灵敏性；另一类是设计合成特定位点修饰的唾液酸化葡萄糖，通过检测酶水解该二糖后释放出的葡萄糖，间接检测流感病毒神经氨酸酶。由于我们对两类试剂中唾液酸母体的4位、7位和5位进行了特定修饰，其可提高对流感病毒神经氨酸酶检测的特异性。本项目具有重要的科学价值和广阔的应用前景。

在本项目的支持下，开展了糖基聚集诱导发光类检测试剂的合成和在神经氨酸酶检测等方面的应用研究。在合成方面，我们以糖苷化反应为研究重点，尝试将以糖基硫苷为供体，(Tol)2SO/Tf2O为促进剂的糖苷化方法应用于4位含氮修饰的唾液酸糖苷化反应中，初步得到多个4位修饰的唾液酸苷化合物；同时，我们发现该方法可用于高产率高选择性地合成Kdo糖复合物，并高效地合成了系列Kdo糖复合物。在检测应用方面，我们系统综述了基于唾液酸衍生物开展神经氨酸酶和Siglecs检测的研究及糖基聚集诱导发光材料在物质检测领域中的研究进展；合成了系列含糖基的荧光材料应用于神经氨酸酶、人参皂苷、次氯酸、氰根等的检测。例如，我们合成了四唾液酸修饰的四苯乙烯化合物(TPE4S)和含三个唾液酸单元的四苯乙烯化合物(TPE3S)，基于神经氨酸酶的水解作用，TPE4S和TPE3S可普适性用于神经氨酸酶的灵敏检测，TPE4S还可用于原人参二醇类人参皂苷的检测。本项目合成了4位胍基取代的唾液酸化四苯乙烯化合物（TPE-SG）等，初步研究显示，该类化合物对流感病毒神经氨酸酶有检测效果；合成了含乳糖和腙基团的四苯乙烯化合物（THG-1）,基于次氯酸和腙之间的水解反应，实现了对次氯酸的检测；合成了含乳糖和乙烯基丙二腈单元的四苯乙烯化合物（TPELC），基于乙烯基丙二腈单元和CN-间的亲核加成反应，实现了对氰根的检测。研究表明，经过合理设计，糖基聚集诱导发光材料可作为一类性质优良的探针用于多种生物分子的检测。