Aptamer-based CRISPR/Cas12a assay for the ultrasensitive detection of extracellular vesicle proteins
Aptamer-based CRISPR/Cas12a assay for the ultrasensitive detection of extracellular vesicle proteins
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基于适体的 CRISPR/Cas12a 检测用于细胞外囊泡蛋白的超灵敏检测
DOI：
10.1016/j.talanta.2020.121670
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发表时间：
2021-01-01
期刊：
影响因子：
6.1
通讯作者：
Liu, Wanli
中科院分区：
文献类型：
--
作者：
Li, Huilan;Xing, Shan;Liu, Wanli
肿瘤来源的细胞外囊泡（TEV）已成为鼻咽癌（NPC）诊断和预后生物标志物的有前途的来源。然而，缺乏高灵敏度的分析方法，对超痕量膜蛋白的TEVs阻碍了他们的临床应用的TEVs。本文中，通过结合特异性结合TEV上蛋白质靶点的适体、基于PCR的指数扩增和CRISPR/Cas 12 a实时DNA检测，我们开发了一种新技术，称为适体-CRISPR/Cas 12 a测定，用于检测细胞系和复杂生物流体中的CD 109（+）和EGFR（+）TEV。该平台能够在低至100个粒子/mL的水平上高度灵敏地检测CD 109（+）和EGFR（+）TEV，线性范围跨越6个数量级（10（2）-10（8）个粒子/mL），发现这足以直接有效地检测低体积（50 μ l）样品中的TEV蛋白。临床血清标本分析证实，联合检测CD 109（+）和EGFR（+）TEV对鼻咽癌的诊断准确率较高，AUC为0.934（95%CI：0.868-1.000），敏感性为84.1%，特异性为85.0%。此外，放射治疗后反应者中两种生物标志物的急剧下降表明它们在放射治疗监测中的潜在作用。鉴于适体-CRISPR/Cas 12 a检测快速方便地直接检测血清中超低浓度的CD 109（+）和EGFR（+）TEV，它可能有助于NPC诊断和预后。
Tumor-derived extracellular vesicles (TEVs) have emerged as promising sources of diagnostic and prognostic biomarkers for nasopharyngeal carcinoma (NPC). However, the lack of high-sensitivity analytic methods for ultratrace membrane proteins on TEVs hamper their clinical application of TEVs. Herein, by combining aptamers that specifically bind to protein targets on TEVs, PCR-based exponential amplification and CRISPR/Cas12a real-time DNA detection, we developed a novel technique, termed the aptamer-CRISPR/Cas12a assay, to detect CD109(+) and EGFR(+) TEVs from cell lines and complex biofluids. The platform enables highly sensitive detection of CD109(+) and EGFR(+) TEVs at as low as 100 particles/mL with a linear range spanning 6 orders of magnitude (10(2)-10(8) particles/mL), which was found to be sufficient to effectively detect TEV proteins directly in low-volume (50 mu l) samples. Furthermore, clinical serum sample analysis verified that the combination of serum CD109(+) and EGFR(+) TEV levels yielded high diagnostic accuracy, with an AUC of 0.934 (95% CI: 0.868-1.000), a sensitivity of 84.1% and a specificity of 85.0%, in discriminating NPC from healthy controls. Moreover, the dramatic decrease in both biomarkers in responders after radiotherapy indicated their potential roles in radiotherapy surveillance. Given that the aptamer-CRISPR/Cas12a assay rapidly and conveniently detects ultralow concentrations of CD109(+) and EGFR(+) TEVs directly in serum, it could be useful in NPC diagnosis and prognosis.