专一识别和超灵敏检测Aβ早期聚集体的金纳米增强型超分子荧光传感新方法

The supramolecular fluorescent probe enhanced by gold nanoparticles(AuNPs) will be built in the project, and the probe will be specific recognition and ultrasensitive detection the earliest β-amyloid peptide (Aβ) oligomerss which is one of the important diagnostic biomarkers of the Alzheimer's disease (AD). The fluorescence probe will be assembled according to the properties of strong fluorescence of pyrene, specific recognition of Aβ by Aβ16-20 united flexible chains with different lengths and amplification the fluorescence signal by AuNPs enriching the fluorophores. The specific recognition of the free earliest Aβ oligomers by Aβ16-20 will be achieved according to supramolecular self-assembly. The ultra-low concentration determination of Aβ in the cerebrospinal fluid and plasma will be obtained based on AuNPs enhance significantly the fluorescence signal of pyrene. The rapid screening of Aβ inhibitors will be realized accordding to the fluorescence switching function of the probes. Atomic force microscopy and scanning electrochemical microscopy will assist to verify the results of the fluorescence method. The biosensing principles of identification the earliest Aβ oligomers will be developed based on the supramolecular principle.The supramolecular fluorescence sensing new method enhanced by AuNPs for identification and determination of the earliest Aβ oligomers with high selectivity, high sensitivity, low cost and rapid speed will be built. The method will be conducive to the early diagnosis of AD.

该项目构筑金纳米增强型超分子荧光探针，用来专一识别和超灵敏检测阿尔茨海默病（AD）的重要生物标志物-β-淀粉样蛋白( Aβ)早期积聚体。项目基于芘的强荧光性能、不同长度柔性链连接的Aβ16-20多肽片段对Aβ的专一识别性能和金纳米粒子富集荧光基团扩增荧光信号的性能组装荧光探针。依据探针的Aβ16-20多肽片段与自由Aβ间的超分子自组装实现对Aβ早期聚集体的专一性识别；依据金纳米粒子大幅度增强芘的荧光信号 实现脑脊髓液和血浆中超低浓度Aβ的测定；依据所合成的探针具有荧光开关功能进行Aβ抑制剂的快速筛选；以原子力显微镜和扫描电化学显微镜辅助验证荧光方法研究结果；发展基于超分子识别Aβ早期聚集体的生物传感原理，建立高选择性、高灵敏度、低成本、快速地识别、测定Aβ早期聚集体的金纳米增强型超分子荧光传感新方法，为AD的早期确诊提供依据。

阿尔茨海默病(Alzhemer’s disease，AD)是一种中枢神经系统退行性疾病，研究证明大脑中不溶的β-淀粉样蛋白(amyloid β-peptide，Aβ)是组成老年斑的主要成分，也是AD的神经细胞病变的特征。因此，阻止Aβ纤维的形成是治疗AD的最有效方法之一。同时血液中Aβ浓度的检测对AD的早期确诊及治疗有重要意义。本项目的研究包含两方面内容：⑴Aβ解聚药物的筛选；⑵Aβ浓度的检测。.⑴Aβ解聚药物的筛选：利用原子力显微镜、紫外-可见吸收光谱和荧光光谱研究了药物小分子贝加因、人参皂苷Rb1、香兰素、天麻素与Aβ1-42的相互作用，结果表明，贝加因、人参皂苷Rb1、香兰素对Aβ具有明显的解聚作用，荧光光谱法没有检测到天麻素与Aβ相互作用的明显信号。此研究对开发新型AD候选药物有参考意义。.⑵Aβ浓度的检测：①以Aβ16-20多肽片段为识别基团，制备了Pyrene-GFFVL-KK-LVFFG-Pyrene和Pyrene-GFFVL -Ahx-K-Ahx-LVFFG-Pyrene两种荧光探针，用以检测Aβ1-42和Aβ1-40，没有很好的响应信号，根据实验现象分析，原因可能是识别的多肽片段的专一识别能力不够。②以核酸适配体为识别基团合成了两种Aβ适配体荧光探针：T-SO 508(5'to3'：GCCTGTGGTGTTGGGGCGGGTGCG，5'quasar 670，3'BHQ3)和，T-SO 509(5'to3'：GCCTGTGGTGTTGGGGCGGGTGCG，3'TAMRA5'JOE，3'TAMRA），并研究其对Aβ的识别行为，结果发现两种探针可以对Aβ1-40有响应，但响应信号不稳定，原因可能是所配制Aβ的状态不稳定。③利用马来酰亚胺与半胱氨酸上巯基的高亲和性，将β-淀粉样蛋白特异性地结合在聚集诱导荧光探针上，基于聚集诱导发光增强现象监测β-淀粉样蛋白的聚集过程。该方法具有高灵敏、高选择性、稳定性好，易控制等优点。可监测微摩尔至纳摩尔的Aβ1-42和Aβ1-40含量，且检测速度比利用ANS（8-苯胺-1-萘磺酸）等探针的监测方法提高15倍，可定性或定量地监测β淀粉样蛋白聚集过程。