背景:快速,灵敏的比色检测方法对于疾病诊断至关重要,特别是那些涉及如弗林蛋白酶的蛋白酶,牵涉到各种条件,包括癌症.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,推动新型检测策略的发展。因此,开发一个简单的,无酶,高灵敏度的快速比色分析方法对弗林蛋白酶的检测势在必行。
结果:这里,我们在这项工作中首次提出了一种比色法来检测弗林蛋白酶,利用具有增强催化活性的G-四链体/血红素DNA酶的组装。具体来说,设计包含弗林蛋白酶识别肽和用于信号扩增的侧翼DNA序列的肽-DNA缀合物(PDC)以促进DNA酶组装。经过弗林治疗,PDC裂解引发环状催化发夹组装反应,通过发夹1(HP1)和发夹2(HP2)形成互补双链结构,使HP1中的G-四链体序列更接近HP2上的血红素。此外,所得的G-四链体/血红素DNA酶表现出强烈的过氧化物酶样活性,能够催化ABTS2-的比色反应以检测弗林蛋白酶。我们的方法显示出高灵敏度,快速反应,以及与复杂样本矩阵的兼容性,实现低至1.1pM的检测极限。
结论:这项工作中报道的DNA酶表现出强大的催化活性,使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实现视觉弗林蛋白酶检测,在生物医学和临床诊断应用中具有重要的应用前景。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。
BACKGROUND: 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.
RESULTS: 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.
CONCLUSIONS: 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.