Exploiting the Potential of Spherical PAM Antenna for Enhanced CRISPR-Cas12a: A Paradigm Shift toward a Universal Amplification-Free Nucleic Acid Test Platform

IF 6.7 1区化学 Q1 CHEMISTRY, ANALYTICAL Analytical Chemistry Pub Date : 2025-01-09 DOI:10.1021/acs.analchem.4c04871

Jiahui Dai, Beibei Wu, Fengxiang Ai, Zhugen Yang, Yanyan Lu, Cai Zinian, Kun Zeng, Zhen Zhang

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Abstract

The CRISPR-Cas12a system has shown tremendous potential for developing efficient biosensors. Albeit important, current CRISPR-Cas system-based diagnostic technologies (CRISPR-DX) highly rely on an additional preamplification procedure to obtain high sensitivity, inevitably leading to issues such as complicated assay workflow, cross-contamination, etc. Herein, a spherical protospacer-adjacent motif (PAM)-antenna-enhanced CRISPR-Cas12a system is fabricated for universal amplification-free nucleic acid detection with a detection limit of subfemtomolar. Meanwhile, the clinical detection capability of this sensor was further verified using gold-standard real-time quantitative polymerase chain reaction through Mycobacterium tuberculosis measurement, which demonstrated its good reliability for practical applications. Importantly, its excellent sensitivity is mainly ascribed to high efficiency of target search induced by a localized PAM-enriched microenvironment and improved catalytic activity of Cas12a (up to 4 folds). Our strategy provides some new insights for rapid and sensitive detection of nucleic acids in an amplification-free fashion.

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利用球形PAM天线增强CRISPR-Cas12a的潜力：向通用无扩增核酸检测平台的范式转变

CRISPR-Cas12a系统已经显示出开发高效生物传感器的巨大潜力。虽然重要，但目前基于CRISPR-Cas系统的诊断技术（CRISPR-DX）高度依赖额外的扩增前程序来获得高灵敏度，不可避免地导致复杂的分析工作流程、交叉污染等问题。本文构建了一个球形原间隔邻近基序（PAM）天线增强的CRISPR-Cas12a系统，用于通用无扩增核酸检测，检测限为亚飞摩尔。同时，通过金标准实时定量聚合酶链反应对结核分枝杆菌进行检测，进一步验证了该传感器的临床检测能力，证明了其具有良好的实际应用可靠性。重要的是，其优异的灵敏度主要归功于局部富集pam的微环境诱导的高效靶标搜索和Cas12a催化活性的提高（高达4倍）。我们的策略提供了一些新的见解，快速和灵敏的检测核酸扩增无方式。

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来源期刊

Analytical Chemistry 化学-分析化学

CiteScore

12.10

自引率

12.20%

发文量

1949

审稿时长

1.4 months

期刊介绍： Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.