本课题组前期采用定量蛋白质组技术从非小细胞肺癌骨转移患者血清外泌体中筛到骨转移新标志物αv、β3、β1和CD109，western和流式验证了该结果，但此两种技术灵敏度低、操作繁琐。目标序列激活的Cas12a具有非特异的DNase活性，可高灵敏检测到单分子核酸。HCR简便、低成本地放大核酸信号，其适配体部分可识别外泌体上的靶蛋白。我们近期联合HCR, CRISPR技术，用通用的crRNA识别HCR的重复序列，激活Cas12a的DNase活性，剪切荧光报告分子产生荧光强度(FI)变化，与外泌体靶蛋白含量呈正比，此技术通用、简便、价廉、高灵敏（102/ml）。本项目拟采用价廉的CD109适配体代替昂贵的抗体捕获血清中转移特异外泌体， HCR-CRISPR技术高灵敏一次检测CD109+、αvβ3+和αvβ1+外泌体，并临床验证其诊断骨转移的性能，此技术将提高NSCLC患者无创性骨转移诊断的准确性

We have selected out 3 up-regulated integrin αv, β3, β1 and CD109 from the exosomes of non-small cell lung cancer patients with bone metastases using the quantitative proteomic technique, iTRAQ. The results were confirmed by western blotting and flow cytometry, suggesting that they were potential markers for NSCLC bone metastasis. However, exosomes have to be enriched and purified in advance. Herein, we developed a fluorescence aptasensor for the efficient and direct detection of EV proteins by integrating hybridization chain reaction (HCR) and CRISPR/Cas12a, named the apt-HCR-CRISPR assay. Inspired by the structural versatility of HCR, the rationally designed H0 both acts as the initiator for the HCR for signal amplification and recognizes target proteins on immunization-captured EVs. HCR is also used to generate long repeated DNA barcodes as universal crRNA-targetable units for CRISPR/Cas12a bind-and-cleavage events without the need for time-consuming guiding crRNA redesign each time. The detection is based on the fluorescence intensity (FI) change due to a “collateral effect” of Cas12a/ crRNA/Target complex activated Cas12a promiscuous DNase activity. This project intends to use the cheap CD109-aptamer to capture the metastasis-related exosomes and quantity the protein using Cas12-dependent single-molecule nucleic acid detection system. We aimed to establish the apt-HCR-CRISPR technique to detect CD109, integrin αvβ3 and αvβ1 simultaneously, and explore its application in diagnosing bone metastasis. This technique will improve the accuracy of non-invasive diagnosis in NSCLC patients with bone metastasis