Chen Wenwen, Cao Rongkai, Su Wentao, Zhang Xu, Xu Yuhai, Wang Peng, Gan Zhongqiao, Xie Yingying, Li Hongjing, Qin Jianhua
CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
Dalian Polytechnic University, Dalian, China.
Lab Chip. 2021 May 4;21(9):1759-1770. doi: 10.1039/d0lc01311k.
Tumor-derived exosomes have been recognized as promising biomarkers for early-stage cancer diagnosis, tumor prognosis monitoring and individual medical treatment. However, it is a huge challenge to separate exosomes from trace biological samples in clinics for disease diagnosis. Herein, we propose a simple, quick, and label-free method for isolating circulating exosomes from serum of patients. The strategy synergistically integrates chitosan electrostatic-adsorption, micro-patterned substrates, and microfluidic shuttle flow control to enable the capture/release of circulating exosomes in a simple manner. Using this microchip, we can isolate exosomes from trace samples (10 μl) with relative purity over 90% and high RNA recovery ratio over 84% within 15 minutes, which is impossible for traditional ultracentrifugation methods. We then validate the application of the microchip using 24 serum samples from clinical breast cancer and breast fibroma patients. The isolated exosomes are subjected to miRNA sequencing and RT-PCR, followed by pathway prediction analysis. The results showed that exosomes were relevant to the invasion and metastasis of breast cancer cells and hsa-miR-18a-3p might have the potential to become a new biomarker for distinguishing breast cancer from breast fibroma (AUC = 0.83, P value = 0.019). This established method is simple, quick and easy to operate with integration. And it may pave a new way for clinical research on exosomes and tumor relevant diagnosis.
肿瘤来源的外泌体已被公认为是早期癌症诊断、肿瘤预后监测及个体化医疗的有前景的生物标志物。然而，在临床中从微量生物样本中分离外泌体用于疾病诊断是一项巨大挑战。在此，我们提出一种简单、快速且无需标记的从患者血清中分离循环外泌体的方法。该策略协同整合了壳聚糖静电吸附、微图案化底物和微流控穿梭流控制，以简单的方式实现循环外泌体的捕获/释放。使用这种微芯片，我们能够在15分钟内从微量样本（10 μl）中分离出相对纯度超过90%且RNA回收率超过84%的外泌体，这是传统超速离心方法无法做到的。然后，我们使用来自临床乳腺癌和乳腺纤维瘤患者的24份血清样本验证了该微芯片的应用。对分离出的外泌体进行miRNA测序和RT-PCR，随后进行通路预测分析。结果表明，外泌体与乳腺癌细胞的侵袭和转移相关，且hsa-miR-18a-3p可能有潜力成为区分乳腺癌与乳腺纤维瘤的新生物标志物（AUC = 0.83，P值 = 0.019）。这种已建立的方法简单、快速且易于集成操作。并且它可能为外泌体及肿瘤相关诊断的临床研究开辟一条新途径。
