CRISPR/Cas13a Signal Amplification Linked Immunosorbent Assay for Femtomolar Protein Detection
CRISPR/Cas13a Signal Amplification Linked Immunosorbent Assay for Femtomolar Protein Detection
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用于飞摩尔蛋白检测的 CRISPR/Cas13a 信号放大连锁免疫吸附测定
DOI：
10.1021/acs.analchem.9b04403
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发表时间：
2020-01-07
影响因子：
7.4
通讯作者：
Zhou, Xiaoming
中科院分区：
文献类型：
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作者：
Chen, Qian;Tian, Tian;Zhou, Xiaoming
酶联免疫吸附测定 (ELISA) 是分析和临床研究中使用的基本技术。然而，传统的 ELISA 仍然不够灵敏，无法检测超低浓度的生物标志物，以早期诊断癌症、心血管风险、神经系统疾病和传染病。在此，我们展示了一种利用基于 CRISPR/Cas13a 的信号输出放大策略的机制，该策略通过 T7 RNA 聚合酶转录和 CRISPR/Cas13a 附带裂解活性双重放大输出信号。该过程称为 CRISPR/Cas13a 信号放大连锁免疫吸附测定 (CLISA)。该方法通过检测炎症因子人白细胞介素 6 (人 IL-6) 和肿瘤标志物人血管内皮生长因子 (人 VEGF) 进行了验证，检测限 (LOD) 值分别为 45.81 fg/mL (2.29 fM) 和 32.27 fg/mL (0.81 fM)，表明 CLISA 的灵敏度比传统方法至少高 10(2) 倍酶联免疫吸附试验。
The enzyme-linked immunosorbent assay (ELISA) is a basic technique used in analytical and clinical investigations. However, conventional ELISA is still not sensitive enough to detect ultralow concentrations of biomarkers for the early diagnosis of cancer, cardiovascular risk, neurological disorders, and infectious diseases. Herein we show a mechanism utilizing the CRISPR/Cas13a-based signal export amplification strategy, which double-amplifies the output signal by T7 RNA polymerase transcription and CRISPR/Cas13a collateral cleavage activity. This process is termed the CRISPR/Cas13a signal amplification linked immunosorbent assay (CLISA). The proposed method was validated by detecting an inflammatory factor, human interleukin-6 (human IL-6), and a tumor marker, human vascular endothelial growth factor (human VEGF), which achieved limit of detection (LOD) values of 45.81 fg/mL (2.29 fM) and 32.27 fg/mL (0.81 fM), respectively, demonstrating that CLISA is at least 10(2)-fold more sensitive than conventional ELISA.