An ultrasensitive hybridization chain reaction-amplified CRISPR-Cas12a aptasensor for extracellular vesicle surface protein quantification
An ultrasensitive hybridization chain reaction-amplified CRISPR-Cas12a aptasensor for extracellular vesicle surface protein quantification
复制标题
用于细胞外囊泡表面蛋白定量的超灵敏杂交链式反应扩增 CRISPR-Cas12a 适体传感器
DOI：
10.7150/thno.49047
复制
发表时间：
2020-01-01
期刊：
影响因子：
12.4
通讯作者：
Liu, Wanli
中科院分区：
文献类型：
--
作者：
Xing, Shan;Lu, Zedong;Liu, Wanli
肿瘤衍生的细胞外囊泡（TEV）蛋白生物标志物有助于癌症诊断和预后评估。然而，缺乏可靠和方便的定量方法来评估TEV蛋白，阻碍了它们的临床应用。研究方法：在此，基于杂交链反应（HCR）和CRISPR-Cas 12 a的双重扩增，我们开发了apta-HCR-CRISPR检测方法，用于直接高灵敏度检测TEV蛋白。通过HCR扩增TEV蛋白靶向适体以产生包含多个CRISPR RNA（crRNA）可靶向条形码的长重复序列，并且通过CRISPR-Cas 12 a附带切割活性进一步扩增信号，从而产生荧光信号。结果：通过检测TEV蛋白标志物核仁素和程序性死亡配体1（PD-L1），验证了所建立的策略。两种方法的检测限（LOD）值均低至102个微粒/微升，灵敏度比适体-ELISA至少高104倍，比apta-HCR-ELISA高102倍。我们直接将我们的检测方法应用于来自50 µL血清的循环TEV的临床分析，揭示了核仁素+TEV用于鼻咽癌（NPC）诊断和PD-L1+ TEV用于治疗监测的潜在应用。结论：该方法简单易行，适用于临床样品中TEV蛋白的高灵敏度和多功能定量分析。
Tumor-derived extracellular vesicle (TEV) protein biomarkers facilitate cancer diagnosis and prognostic evaluations. However, the lack of reliable and convenient quantitative methods for evaluating TEV proteins prevents their clinical application. Methods: Here, based on dual amplification of hybridization chain reaction (HCR) and CRISPR-Cas12a, we developed the apta-HCR-CRISPR assay for direct high-sensitivity detection of TEV proteins. The TEV protein-targeted aptamer was amplified by HCR to produce a long-repeated sequence comprising multiple CRISPR RNA (crRNA) targetable barcodes, and the signals were further amplified by CRISPR-Cas12a collateral cleavage activities, resulting in a fluorescence signal. Results: The established strategy was verified by detecting the TEV protein markers nucleolin and programmed death ligand 1 (PD-L1). Both achieved limit of detection (LOD) values as low as 102 particles/µL, which is at least 104-fold more sensitive than aptamer-ELISA and 102-fold more sensitive than apta-HCR-ELISA. We directly applied our assay to a clinical analysis of circulating TEVs from 50 µL of serum, revealing potential applications of nucleolin+ TEVs for nasopharyngeal carcinoma cancer (NPC) diagnosis and PD-L1+ TEVs for therapeutic monitoring. Conclusion: The platform was simple and easy to operate, and this approach should be useful for the highly sensitive and versatile quantification of TEV proteins in clinical samples.