黑色素瘤是一种高度侵袭性的肿瘤，也是皮肤癌相关死亡的重要原因。及时诊断和治疗需要鉴定黑色素瘤细胞分泌的外泌体中的特异性生物标志物。在本研究中，采用具有尺寸匹配选择性的无标记表面增强拉曼光谱（SERS）方法来检测与黑色素瘤细胞（B16-F10）共培养的成纤维细胞（L929）刺激环境中释放的外泌体中的膜蛋白。 。为了促进外泌体的正常分泌，采用微等离子体处理温和诱导共培养的细胞，并稍微增加细胞周围的应激水平，以便后续使用SERS方法进行检测。首先，活性氧/活性氮的变化（评估细胞微环境中的 ROS/RNS）浓度以及健康细胞的活力和增殖。结果表明，微等离子体处理增加了细胞外 ROS/RNS 水平，同时适度减少细胞增殖，而不会显着影响细胞存活。其次，从L929、B16-F10以及不同微等离子体处理时间的共培养细胞的培养基中分离的分泌性外泌体的粒径在短处理时间的单细胞条件下没有显着增加，但在短处理时间下可能会发生变化。共培养条件或更长的处理时间。第三，对于与膜蛋白生物标志物相关的SERS信号，外泌体标志物CD9、CD63和CD81可以分配给943-1030和1304-1561 cm-1范围内的显着拉曼位移，而L929和B16的特征SERS峰-F10细胞最有可能分别位于1394/1404、1271和1592 cm-1。因此，这种微等离子体诱导的共培养模型提供了一种有前途的临床前方法来了解皮肤黑色素瘤/成纤维细胞分泌的外泌体的诊断潜力。此外，具有尺寸匹配选择性的无标记SERS方法提供了一种筛选黑色素瘤细胞分泌的外泌体中生物标志物的新方法，旨在减少标记试剂的使用和传统上所需的处理时间。版权所有©2023。由Elsevier B.V.出版。

Melanoma is a highly aggressive tumor and a significant cause of skin cancer-related death. Timely diagnosis and treatment require identification of specific biomarkers in exosomes secreted by melanoma cells. In this study, label-free surface-enhanced Raman spectroscopy (SERS) method with size-matched selectivity was used to detect membrane proteins in exosomes released from a stimulated environment of fibroblasts (L929) co-cultured with melanoma cells (B16-F10). To promote normal secretion of exosomes, micro-plasma treatment was used to gently induce the co-cultured cells and slightly increase the stress level around the cells for subsequent detection using the SERS method.Firstly, changes in reactive oxygen species/reactive nitrogen species (ROS/RNS) concentrations in the cellular microenvironment and the viability and proliferation of healthy cells are assessed. Results showed that micro-plasma treatment increased extracellular ROS/RNS levels while modestly reducing cell proliferation without significantly affecting cell survival. Secondly, the particle size of secreted exosomes isolated from the culture medium of L929, B16-F10, and co-cultured cells with different micro-plasma treatment time did not increase significantly under single-cell conditions at short treatment time but might be changed under co-culture condition or longer treatment time. Third, for SERS signals related to membrane protein biomarkers, exosome markers CD9, CD63, and CD81 can be assigned to significant Raman shifts in the range of 943-1030 and 1304-1561 cm-1, while the characteristics SERS peaks of L929 and B16-F10 cells are most likely located at 1394/1404, 1271 and 1592 cm-1 respectively.Therefore, this micro-plasma-induced co-culture model provides a promising preclinical approach to understand the diagnostic potential of exosomes secreted by cutaneous melanoma/fibroblasts. Furthermore, the label-free SERS method with size-matched selectivity provides a novel approach to screen biomarkers in exosomes secreted by melanoma cells, aiming to reduce the use of labeling reagents and the processing time traditionally required.Copyright © 2023. Published by Elsevier B.V.