Tumor-derived exosomes (TEXs) are extracellular vesicles that are continuously released into the blood by tumor cells and carry specific surface markers of the original tumor cells. Substantial evidence has implicated TEXs as attractive diagnostic markers for cancer. However, the detection of TEXs in blood at an early tumor stage is challenging due to their very low concentration. Here, we established a method called PLA-RPA-TMA assay that allows TEXs to be detected with high sensitivity and specificity. Based on two proximity ligation assay (PLA) probes that recognize a biomarker on a TEX, we generated a unique surrogate DNA signal for the specific biomarker, which was synchronously amplified twice by recombinase polymerase amplification (RPA) coupled with transcription-mediated amplification (TMA), and then the products of the RPA-TMA reaction were quantitatively detected using a gold nanoparticle-based colorimetric assay. We established proof-of-concept evidence for this approach using TEXs from nasopharyngeal carcinoma (NPC) cells, with a detection limit of 10² particles/mL, and reported the measurement of plasma Epstein-Barr virus latent membrane protein 1 (LPM1)-positive (LMP1⁺, accuracy: 0.956) and epidermal growth factor receptor (EGFR)-positive (EGFR⁺, accuracy: 0.906) TEXs as potent early diagnostic biomarkers for NPC.

肿瘤来源的外来体（TEXs）是细胞外囊泡，它们被肿瘤细胞连续释放到血液中，并带有原始肿瘤细胞的特定表面标记。大量证据表明，TEXs是癌症的诱人诊断标记。然而，由于其浓度非常低，因此在肿瘤早期检测血液中的TEX具有挑战性。在这里，我们建立了一种称为PLA-RPA-TMA分析的方法，该方法可以以高灵敏度和特异性检测TEX。基于识别TEX上生物标志物的两个邻近连接测定（PLA）探针，我们生成了特定生物标志物的独特替代DNA信号，该信号通过重组酶聚合酶扩增（RPA）和转录介导扩增（TMA）同步扩增了两次），然后使用基于金纳米粒子的比色测定法定量检测RPA-TMA反应的产物。我们使用来自鼻咽癌（NPC）细胞的TEX建立了这种方法的概念验证证据，检出限为10^2个颗粒/ mL，并报告了血浆爱泼斯坦-巴尔病毒潜伏膜蛋白1（LPM1）阳性（LMP1 ⁺，准确性：0.956）和表皮生长因子受体（EGFR）阳性（EGFR ⁺，准确性：0.906）的测量TEXs作为NPC的有效早期诊断生物标志物。