College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, 250014, China.
Center for Disease Control and Prevention of Weihai City, Weihai, 264200, China.
Talanta. 2024 Jan 1;266(Pt 2):125089. doi: 10.1016/j.talanta.2023.125089. Epub 2023 Aug 18.
Human alkyladenine DNA glycosylase (hAAG) is essential for repairing alkylated and deaminated bases, and it has become a prospective diagnosis biomarker and a therapeutic target for disease treatment. However, most of hAAG assays suffer from complicated reaction scheme, poor specificity, long assay time, and limited sensitivity. Herein, we report a novel single probe-based catalytic quantum dot (QD) Förster resonance energy transfer (FRET) nanosensor for simple and sensitive detection of hAAG activity. In this assay, hAAG induces the generation of 3' OH terminus via the excision of I base and the cleavage of AP site by APE1, subsequently initiating strand displacement reaction to produce numerous ssDNA signal probes. These probes can self-assemble on the QD surface to induce efficient FRET between QD and Cy5. This assay is very simple with the involvement of only a single probe for the achievement of both specific sensing and efficient signal amplification. Moreover, each signal probe contains multiple Cy5 moieties, and multiple signal probes can assemble on a single QD to greatly enhance the FRET efficiency. This nanosensor exhibits a detection limit of 3.60 × 10 U/μL and it is suitable for measuring enzymatic kinetics, screening inhibitor, and quantifying cellular hAAG activity with single-cell sensitivity.
人类烷基腺嘌呤 DNA 糖基化酶 (hAAG) 对于修复烷基化和脱氨碱基至关重要,它已成为疾病治疗中具有前景的诊断生物标志物和治疗靶标。然而,大多数 hAAG 测定方法存在反应方案复杂、特异性差、测定时间长、灵敏度有限等问题。在此,我们报告了一种新颖的基于单探针的催化量子点(QD)荧光共振能量转移(FRET)纳米传感器,用于简单、灵敏地检测 hAAG 活性。在该测定中,hAAG 通过 APE1 切除 I 碱基和 AP 位点的切割,诱导产生 3' OH 末端,随后启动链置换反应,产生大量的单链 DNA 信号探针。这些探针可以在 QD 表面自组装,从而在 QD 和 Cy5 之间诱导有效的 FRET。该测定非常简单,仅涉及单个探针,即可实现特异性传感和高效信号放大。此外,每个信号探针都包含多个 Cy5 部分,并且多个信号探针可以组装在单个 QD 上,从而大大增强 FRET 效率。该纳米传感器的检测限为 3.60×10 U/μL,适用于测量酶动力学、筛选抑制剂以及以单细胞灵敏度定量细胞内 hAAG 活性。