快速、灵敏的比色检测方法对于疾病诊断至关重要，特别是那些涉及弗林蛋白酶等蛋白酶的疾病，这些蛋白酶与包括癌症在内的多种疾病有关。传统的弗林蛋白酶检测方法在现场检测的灵敏度和实用性方面存在局限性，这促使了新型检测策略的开发。因此，开发一种简单、无酶、快速、高灵敏度的弗林蛋白酶检测比色分析方法势在必行。本文中，我们首次提出了一种利用G-四联体组装来检测弗林蛋白酶的比色方法。 /氯化血红素DNAzyme 具有增强的催化活性。具体而言，设计包含弗林蛋白酶识别肽和用于信号放大的侧翼DNA序列的肽-DNA缀合物(PDC)以促进DNA酶组装。弗林蛋白酶处理后，PDC 裂解触发循环催化发夹组装反应，通过发夹 1 (HP1) 和发夹 2 (HP2) 形成互补的双链结构，使 HP1 中的 G-四链体序列更接近 HP2 上的氯高铁血红素。此外，所得的 G-四链体/氯化血红素 DNAzyme 表现出强大的过氧化物酶样活性，能够催化用于弗林蛋白酶检测的 ABTS2- 比色反应。我们的方法表现出高灵敏度、快速响应以及与复杂样品基质的兼容性，实现了低至1.1 pM的检测限。这项工作中报道的DNAzyme表现出强大的催化活性，实现了高灵敏度和良好的检测效率。通过消除对外源酶的需求，我们的方法无需昂贵的仪器和试剂即可进行视觉弗林蛋白酶检测，有望在生物医学和临床诊断应用中发挥重要作用。鉴于肽序列的各种设计和 DNA 的可编程性，它可以很容易地应用于分析其他有用的肿瘤生物标志物。版权所有 © 2024 Elsevier B.V. 保留所有权利。

Rapid and sensitive colorimetric detection methods are crucial for diseases diagnosis, particularly those involving proteases like furin, which are implicated in various conditions, including cancer. Traditional detection methods for furin suffer from limitations in sensitivity and practicality for on-site detection, motivating the development of novel detection strategies. Therefore, developing a simple, enzyme-free, and rapid colorimetric analysis method with high sensitivity for furin detection is imperative.Herein, we have proposed a colorimetric method in this work for the first time to detect furin, leveraging the assembly of G-quadruplex/hemin DNAzyme with enhanced catalytic activity. Specifically, a peptide-DNA conjugate (PDC) comprising a furin-recognition peptide and flanking DNA sequences for signal amplification is designed to facilitate the DNAzyme assembly. Upon furin treatment, PDC cleavage triggers a cyclic catalytic hairpin assembly reaction to form the complementary double-stranded structures by hairpin 1 (HP1) and hairpin 2 (HP2), bringing the G-quadruplex sequence in HP1 closer to hemin on HP2. Moreover, the resulting G-quadruplex/hemin DNAzymes exhibit robust peroxidase-like activity, enabling the catalysis of the colorimetric reaction of ABTS2- for furin detection. Our method demonstrates high sensitivity, rapid response, and compatibility with complex sample matrices, achieving a detection limit as low as 1.1 pM.The DNAzyme reported in this work exhibits robust catalytic activity, enabling high sensitivity and good efficiency for the detection. By eliminating the requirement for exogenous enzymes, our approach enables visual furin detection without expensive instrumentation and reagents, promising significant utility in biomedical and clinical diagnostic applications. Given the various design of peptide sequence and the programmability of DNA, it can be readily applied to analyzing other useful tumor biomarkers.Copyright © 2024 Elsevier B.V. All rights reserved.