School of Medical Technology, Guangdong Medical University, Dongguan, 523808, China; Laboratory Medicine Center, Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
Center for Clinical Laboratory Diagnosis and Research, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, Guangxi, 533000, China; Key Laboratory of Research on Clinical Molecular Diagnosis for High Incidence Diseases in Western Guangxi of Guangxi Higher Education Institutions, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise 533000, Guangxi, China.
Talanta. 2024 Dec 1;280:126773. doi: 10.1016/j.talanta.2024.126773. Epub 2024 Aug 25.
APE1, an essential enzyme for DNA repair, is overexpressed in various cancers and has been identified as a potential biomarker for cancer diagnosis. However, detecting APE1 at low expression levels in the early stage of cancer presents a significant obstacle. Here, we introduced a novel localized Cas12a-based cascade amplification (LCas12a-CA) method. This method confined both the terminal deoxynucleotidyl transferase and the crRNA/Cas12a complex onto the surfaces of gold nanoparticles (AuNPs). This confinement not only boosts the stability of the multiple enzymes but also induces a substrate channeling effect. As a result, it significantly accelerates the reaction rate and enhances the sensitivity of APE1 detection. Upon the addition of APE1, the AP sites within the APE1 primer can be recognized and cleaved by APE1, exposing the 3'-OH ends. In the presence of LCas12a-CA, polyA sequences are generated at 3'-OH ends with the help of TdT and dATP. The sequences directly enter the Cas12a system, activating the trans-cleavage activity of Cas12a, thereby cutting the reporters on the surface of AuNPs and releasing fluorescence. Our platform demonstrates a detection limit (LOD) as low as 2.51 × 10 U/mL, which is more than 60 times lower than that of free Cas12a-CA. Furthermore, the LCas12a-CA exhibits enhanced resistance ability in extreme environments and has been proven effective for the detection of APE1 in clinical samples. Overall, this work offers a promising platform for robust biosensing in cancer diagnosis and prognosis.
APE1 是一种参与 DNA 修复的必需酶，在各种癌症中过表达，已被确定为癌症诊断的潜在生物标志物。然而，在癌症的早期阶段检测低表达水平的 APE1 存在很大的障碍。在这里，我们引入了一种新的基于局部 Cas12a 的级联扩增 (LCas12a-CA) 方法。该方法将末端脱氧核苷酸转移酶和 crRNA/Cas12a 复合物同时限定在金纳米粒子 (AuNPs) 的表面。这种限制不仅提高了多种酶的稳定性，还诱导了底物通道效应。因此，它显著加快了反应速度，提高了 APE1 检测的灵敏度。在加入 APE1 后，APE1 引物中的 AP 位点可被 APE1 识别和切割，暴露出 3'-OH 末端。在 LCas12a-CA 的存在下，TdT 和 dATP 帮助在 3'-OH 末端生成 polyA 序列。这些序列直接进入 Cas12a 系统，激活 Cas12a 的转切割活性，从而切割 AuNPs 表面的报告分子并释放荧光。我们的平台检测限 (LOD) 低至 2.51×10 U/mL，比游离 Cas12a-CA 低 60 多倍。此外，LCas12a-CA 在极端环境下表现出增强的抗性能力，并已被证明可有效用于临床样本中 APE1 的检测。总的来说，这项工作为癌症诊断和预后的稳健生物传感提供了一个很有前景的平台。
