Jiangsu Engineering Laboratory of Smart Carbon-Rich Materials and Device, Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China.
Henan Key Laboratory of Polyoxometalate Chemistry, College of Chemistry and Chemical Engineering, Henan University, Kaifeng 475004, China.
Anal Chem. 2021 Apr 27;93(16):6567-6572. doi: 10.1021/acs.analchem.1c00829. Epub 2021 Apr 13.
Flap endonuclease 1 (FEN1) is a structure-specific nuclease that cleaves the 5' single-stranded protrusion (also known as 5' flap) during Okazaki fragment processing. It is overexpressed in various types of human cancer cells and has been considered as an important biomarker for cancer diagnosis. However, conventional methods for FEN1 assay usually suffer from complicated platform and laborious procedures with a limited sensitivity. Here, we developed a dual-signal method for sensitive detection of FEN1 on the basis of duplex-specific nuclease actuated cyclic enzymatic repairing-mediated signal amplification. Once the 5' flap of the double-flap DNA substrate was cleaved by target FEN1, the cleaved 5' flap initiated strand-displacement amplification to produce plenty of G-rich DNA (G) sequences. These G sequences that self-assembled into G-quadruplexes in the presence of hemin revealed horseradish-peroxidase-like catalytic activities as well as fluorescence enhancement of thioflavin T. The UV-vis signal showed a good linear relationship with the logarithm of FEN1 activity ranging from 0.03 to 1.5 U with a detection limit of 0.01 U. The fluorescence signal correlated linearly with the logarithm of FEN1 activity ranging from 0.001 to 1.5 U with a detection limit of 0.75 mU. In addition, FEN1 can be visualized not only by colorimetry but also by fluorescence (under ice-water mixture conditions). This reliable, accurate, and convenient method would be a potential powerful tool in point-of-care testing applications and therapeutic response assessment.
核酸内切酶 1（FEN1）是一种结构特异性核酸内切酶，可在冈崎片段加工过程中切割 5'单链突出物（也称为 5' 发夹）。它在各种类型的人类癌细胞中过度表达，已被认为是癌症诊断的重要生物标志物。然而，传统的 FEN1 测定方法通常存在平台复杂和程序繁琐的问题，灵敏度有限。在这里，我们基于双链特异性核酸酶激活的循环酶修复介导的信号放大，开发了一种用于敏感检测 FEN1 的双信号方法。一旦双发夹 DNA 底物的 5' 发夹被靶 FEN1 切割，切割的 5' 发夹就会启动链置换扩增，产生大量富含 G 的 DNA（G）序列。这些 G 序列在血红素存在下自组装成 G-四链体，显示出辣根过氧化物酶样催化活性以及噻唑橙的荧光增强。紫外-可见信号与 FEN1 活性的对数呈良好的线性关系，其范围从 0.03 到 1.5 U，检测限为 0.01 U。荧光信号与 FEN1 活性的对数呈线性相关，范围从 0.001 到 1.5 U，检测限为 0.75 mU。此外，不仅可以通过比色法而且可以通过荧光法（在冰水混合物条件下）可视化 FEN1。这种可靠、准确和方便的方法将成为即时检测应用和治疗反应评估中的潜在有力工具。
