用于致病菌代谢产物高选择检测的超高灵敏表面等离子共振传感元件

The accurate detection of pathogens metabolites are extremely important for clinical diagnosis of disease and corresponding medication. The main detection ways of pathogens metabolites are mcroscope to observe the living bacteria cultured, enzyme-linked immunosorbent,etc. All the methods are complicated to operate with the limited sensitivity. In particularly, these methods always need a long time to take with the delayed treatment of the illness. It is urgent to develop new detection methods and means with ultra highly sensitivity, rapidness and high-accuracy to meet the need of clinical diagnosis. Surface plasmon resonance (SPR) has been widely used in biology, chemistry, medicine and other fields with many advantages, such as non-fluorescently labeling, fast response, real-time detection, etc. A concerted amplification of the ultra highly sensitive SPR method based on the conformational change and electromagnetic coupling is proposed in this project. As the subject of the sensitive film, the monomer and conjugated polymer are rationally designed to interact with bacterial endotoxin and 1, 3-?-D glucan with the conformational changing characteration. The SPR signal induced by conformational changes can be amplified with the combination of the electromagnetic field coupling between the inorganic noparticles and the gold film. By virtue of controlling the organic molecular structure and aggregation, urtra highly sensitive detection for some important biological species especially can be carried out by SPR components, such as lipopolysaccharide, 1,3-?-D glucan.The highly sensitive SPR components will be further developed to specifically monitor pathogens metabolic. The study in this project possesses theoretical direction value and practical significance for the development of new highly sensitive SPR detection devices.

致病菌代谢产物的准确检测在临床上对疾病确诊及对应用药极为重要，目前主要采用活菌培养显微镜观察、酶联免疫等方法，存在着操作复杂，灵敏度有限，特别是耗时过长延误病情等问题，亟需发展简便高灵敏、快速、高精准度的检测的方法与手段。表面等离子共振检测技术具有无荧光标记、响应快、能实时检测等优点，已在生物、化学、医药等领域广泛应用，而现有表面等离子共振技术在灵敏度与选择性方面无法满足这些物种准确检测的要求，本项目拟针对大肠杆菌代谢产物细菌内毒素和真菌代谢产物1,3-?-D葡聚糖的结构特点，通过设计具有构象变化的功能化聚合物材料，结合纳米颗粒与金膜电磁场耦合作用，放大敏感膜构象变化引起的表面等离子共振信号，实现细菌内毒素和1,3-?-D葡聚糖的超高灵敏检测。在此基础上，开发出可用于致病菌代谢产物特异性检测的表面等离子共振元件，该研究对发展新型超高灵敏表面等离子共振检测元件具有理论指导意义和实际应用价值。

致病菌代谢产物的检测对临床上确诊疾病类型和用药极为重要，目前检测方法存在耗时，操作复杂，灵敏度有限，且需要消耗大量的生化试剂等问题，因此亟需高灵敏、快速、高精准度检测的方法和手段。表面等离子共振检测技术（SPR）具有无荧光标记、响应快、能实时检测等优点，已在生物、化学、医药等领域广泛应用。我们针对革兰氏阴性菌及其代谢产物脂多糖（LPS）的结构特点，通过三单体共聚产生的构象变化的有机聚合物球作为SPR芯片自组装敏感膜主体，实现多种注射用水（生理盐水和葡萄糖注射液）、0.9%泮托拉唑钠NaCl注射液和0.2%0甲磺酸左氧氟沙星NaCl注射液中33 fM的LPS灵敏检测， LPS浓度的对数与SPR信号在3.3×10-14 到1.0×10-11 M范围能成很好的线性关系，相关系数达到0.99以上。制备可与LPS的作用且有构象变化的短肽，实现了5%血清中10 fM的LPS检测，LPS浓度的对数与SPR信号在3.3×10-14到1.0×10-10 M范围能成很好的线性关系，相关系数达到0.99以上；结合生物大分子和小分子修饰表面等离子共振芯片实现了对活细胞的检测，并获得了细胞与分子的结合速率和解离速率，在此基础上结合小分子与菌膜LPS具有特异性作用，实现了100 CFU/mL革兰氏阳性菌高选择性检测。通过特殊结构的单链DNA与汞离子的反应，验证了SPR芯片对表面敏感膜构象变化的灵敏反应，实现血清中0.01ng/mL汞离子检测。制备了可用于电磁场耦合增强的金纳米颗粒，基于SERS基底实现了对0.26 ug/L农药无氯苯酚的灵敏检测。以上结果在Chemical Sensors and Actuators B: Chemical, RSC Advance，和Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 国际刊物发表文章4篇， SPIE会议论文全文1篇，正在整理文章3篇，发表会议摘要论文6篇。申请专利4项，其中PCT国际专利1项，授权中国专利2项。培养在读硕士3名，毕业硕士3名，其中两名硕士研究生获得国家奖学金，一名研究生参加第二届材料化学亚太国际会议国际会议，获得优秀墙报奖，完成了项目的预期研究目标。