Li Yan, Li Yichen, Hu Yueli, Liu Rui, Lv Yi
Key Laboratory of Green Chemistry and Technology, Ministry of Education, College of Chemistry, Sichuan University, Chengdu 610064, Sichuan, China.
Analytical and Testing Center, Sichuan University, Chengdu 610064, Sichuan, China.
Anal Chem. 2025 Mar 11;97(9):5049-5056. doi: 10.1021/acs.analchem.4c05961. Epub 2025 Feb 20.
The CRISPR-Cas system, particularly CRISPR-Cas12a and CRISPR-Cas13a, has been widely utilized in constructing various biosensors due to their "-cleavage" ability as a means of signal amplification. However, this universal "-cleavage" characteristic also presents a challenge for realizing CRISPR-Cas multiplexed bioanalysis. Besides, potential signal cascading interference and complicated design are notable obstacles in CRISPR-Cas multiplexed bioanalysis. Herein, we propose a mass spectrometry method that leverages the CRISPR-Cas12a/13a system to achieve simultaneous detection of ctDNA and miRNA. Based on the properties of the CRISPR-Cas12a/13a system, two types of nanoparticle reporter probes have been engineered, using cancer-related biomarkers ctDNA and miR-21 as our model analytes. The nanoparticle tags, which intrinsically incorporated millions of detectable atoms, combined with the CRISPR-Cas12a/Cas13a system's "-cleavage" ability, allow the proposed mass spectrometry strategy to achieve fmol-level detection limits without any nucleic acid amplification procedures. The assay was successfully applied to human serum samples, demonstrating its potential for early disease diagnosis and progression tracking.
CRISPR-Cas系统，尤其是CRISPR-Cas12a和CRISPR-Cas13a，因其作为信号放大手段的“-切割”能力，已被广泛用于构建各种生物传感器。然而，这种普遍的“-切割”特性也给实现CRISPR-Cas多重生物分析带来了挑战。此外，潜在的信号级联干扰和复杂的设计是CRISPR-Cas多重生物分析中的显著障碍。在此，我们提出一种利用CRISPR-Cas12a/13a系统实现同时检测ctDNA和miRNA的质谱方法。基于CRISPR-Cas12a/13a系统的特性，以癌症相关生物标志物ctDNA和miR-21作为模型分析物，设计了两种类型的纳米颗粒报告探针。纳米颗粒标签本身包含数百万个可检测原子，结合CRISPR-Cas12a/Cas13a系统的“-切割”能力，使所提出的质谱策略无需任何核酸扩增程序即可实现fmol级的检测限。该检测方法已成功应用于人类血清样本，证明了其在疾病早期诊断和进展跟踪方面的潜力。
