Department of Clinical Laboratory, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Sun Yat-sen University Cancer Center, 651 Dongfeng Road East, Guangzhou, 510060, PR China.
Laboratory of Oncology Science and Molecular Biology, ShunDe Hospital of the Guangzhou University of Chinese Medicine, No. 12 Jinsha Avenue, Shunde District, Foshan, 528333, PR China.
Talanta. 2021 Jan 1;221:121670. doi: 10.1016/j.talanta.2020.121670. Epub 2020 Sep 17.
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 (10-10 particles/mL), which was found to be sufficient to effectively detect TEV proteins directly in low-volume (50 μl) 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.
肿瘤衍生的细胞外囊泡 (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，缺乏用于 TEV 中超痕量膜蛋白的高灵敏度分析方法，阻碍了它们在 TEV 中的临床应用。在此，通过结合特异性结合 TEV 上蛋白质靶标的适体、基于 PCR 的指数扩增和 CRISPR/Cas12a 实时 DNA 检测，我们开发了一种新的技术，称为适体-CRISPR/Cas12a 测定法，用于从细胞系和复杂生物体液中检测 CD109 和 EGFR TEV。该平台能够以低至 100 个颗粒/mL 的浓度高度敏感地检测 CD109 和 EGFR TEV，线性范围跨越 6 个数量级（10-10 个颗粒/mL），这足以有效地直接在低体积（50 μl）样品中检测 TEV 蛋白。此外，临床血清样本分析验证了血清 CD109 和 EGFR TEV 水平的组合在区分 NPC 与健康对照方面具有很高的诊断准确性，AUC 为 0.934（95%CI：0.868-1.000），灵敏度为 84.1%，特异性为 85.0%。此外，放射治疗后应答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/Cas12a 测定法可快速方便地直接在血清中检测超痕量的 CD109 和 EGFR TEV，它可能有助于 NPC 的诊断和预后。
