State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin, 300071, China.
Nanoscale. 2019 Aug 8;11(31):14793-14801. doi: 10.1039/c9nr01817d.
Acetylcholinesterase (AChE) plays a vital role in nerve conduction through rapidly hydrolyzing the neurotransmitter acetylcholine (ACh) and is correlated with Alzheimer's disease. In this work, a label-free single-particle enumeration (SPE) method for the quantitative detection of acetylcholinesterase (AChE) activity is developed. The design is based on the fluorescence resonance energy transfer (FRET) between fluorescent conjugated polymer nanoparticles (FCPNPs) and MnO2 nanosheets. The fluorescence of FCPNPs can be effectively quenched by MnO2 nanosheets via hydrogen bonding interaction. In the presence of acetylcholinesterase (AChE), acetylthiocholine (ATCh) could be hydrolyzed to thiocholine (TCh), which can reduce MnO2 to Mn2+ and trigger the decomposition of MnO2 nanosheets. As a result, the fluorescence of FCPNPs is restored. Taking advantage of the superior brightness and stable fluorescence emission from individual FCPNPs, the accurate quantification of AChE is achieved by statistically counting the fluorescent particles on the glass slide surface. A linear range from 5 to 1600 μU mL-1 is obtained for AChE assay and the limit-of-detection (LOD) is 1.02 μU mL-1, which is far below the spectroscopic measurements in bulk solution. In the human serum sample, satisfactory recovery efficiencies are determined in a range of 91.0%-103.0%. Furthermore, pesticide carbaryl as an inhibitor of AChE activity was detected. The LOD is 1.12 pg mL-1 with linear responses ranging from 5 to 300 pg mL-1, which demonstrates the feasibility of this approach for AChE inhibitor screening. As a consequence, the label-free SPE-based method affords a promising platform for the sensitive detection of target molecules in the future.
乙酰胆碱酯酶（AChE）在神经传导中起着至关重要的作用，它通过快速水解神经递质乙酰胆碱（ACh），与阿尔茨海默病密切相关。在这项工作中，开发了一种用于定量检测乙酰胆碱酯酶（AChE）活性的无标记单颗粒计数（SPE）方法。该设计基于荧光共轭聚合物纳米粒子（FCPNPs）和 MnO2 纳米片之间的荧光共振能量转移（FRET）。MnO2 纳米片通过氢键相互作用可以有效地猝灭 FCPNPs 的荧光。在存在乙酰胆碱酯酶（AChE）的情况下，乙酰硫代胆碱（ATCh）可以被水解为硫代胆碱（TCh），TCh 可以将 MnO2 还原为 Mn2+并触发 MnO2 纳米片的分解。结果，FCPNPs 的荧光得到恢复。利用单个 FCPNPs 的优异亮度和稳定的荧光发射，通过统计计算在玻片表面的荧光颗粒，可以实现对 AChE 的准确定量。AChE 测定的线性范围为 5 至 1600 μU mL-1，检测限（LOD）为 1.02 μU mL-1，远低于在体相溶液中的光谱测量。在人血清样品中，在 91.0%-103.0%的范围内确定了令人满意的回收率。此外，还检测了作为 AChE 活性抑制剂的农药carbaryl。LOD 为 1.12 pg mL-1，线性响应范围为 5 至 300 pg mL-1，表明该方法用于 AChE 抑制剂筛选具有可行性。因此，无标记 SPE 基方法为未来目标分子的敏感检测提供了有前景的平台。
