Department of Chemistry, College of Sciences, Northeastern University, Shenyang 110819, China.
Analyst. 2022 Sep 12;147(18):4008-4013. doi: 10.1039/d2an01180h.
Acetylcholinesterase (AChE) is an essential serine hydrolase associated with neurodegenerative diseases and serves as a biomarker. In this work, a ratiometric fluorescence strategy for the sensitive assay of AChE activity is developed based on the decomposition of MnO by the enzymatic hydrolysis product and DNAzyme-mediated strand cleavage. The sensing system is termed as MnO/DNAzyme. DNAzyme locked by H2 strands is inactive in the absence of AChE. The fluorescence emission of fluorescein amidite (FAM)-labeled DNA (DNA-F) at 518 nm is quenched by neutral red (NR) and the fluorescence of NR at 632 nm is simultaneously enhanced due to fluorescence resonance energy transfer (FRET). The presence of AChE triggers the hydrolysis of the substrate acetylcholine (ATCh) to enzymatic thiocholine (TCh), which reduces MnO nanowires to Mn and releases the attached H1 strands into the solution. The H1 strands hybridize with H2 strands through the strand displacement reaction. Meanwhile, the activated DNAzyme cleaves the RNA nucleotide of the DNA-F signal probe to release FAM. The fluorescence of FAM at 518 nm is thus recovered, corresponding to a decrement of NR emission at 632 nm owing to the blocking of FRET. The fluorescence ratio of / serves as a signal readout for the AChE assay within 5 × 10-10 U mL, with a limit of detection (LOD) of 2.7 × 10 U mL. The feasibility of this method was demonstrated by the measurement of AChE activity in human blood, which reveals its promising potential in clinical assays.
乙酰胆碱酯酶（AChE）是一种与神经退行性疾病相关的重要丝氨酸水解酶，可用作生物标志物。在这项工作中，基于酶解产物分解 MnO 和 DNA 酶介导的链切割，开发了一种用于灵敏测定 AChE 活性的比率荧光策略。该传感系统被称为 MnO/DNA 酶。在没有 AChE 的情况下，被 H2 链锁定的 DNA 酶没有活性。荧光素胺（FAM）标记的 DNA（DNA-F）的荧光发射在 518nm 处被中性红（NR）猝灭，并且由于荧光共振能量转移（FRET），NR 在 632nm 处的荧光同时增强。存在 AChE 会触发底物乙酰胆碱（ATCh）的水解生成酶促硫代胆碱（TCh），将 MnO 纳米线还原为 Mn 并将附着的 H1 链释放到溶液中。H1 链通过链置换反应与 H2 链杂交。同时，激活的 DNA 酶切割 DNA-F 信号探针的 RNA 核苷酸以释放 FAM。因此，FAM 在 518nm 处的荧光恢复，对应于由于 FRET 阻断而导致 NR 在 632nm 处发射的减少。/的荧光比值用作 AChE 测定的信号读出，其在 5×10-10 U mL 内的检测限（LOD）为 2.7×10 U mL。通过测量人血液中的 AChE 活性验证了该方法的可行性，这表明它在临床测定中具有广阔的应用前景。
