State Key Laboratory for Organic Electronics and Information Displays, Jiangsu Key Laboratory for Biosensors, Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, Nanjing, 210023, China.
State Key Laboratory for Organic Electronics and Information Displays, Jiangsu Key Laboratory for Biosensors, Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, Nanjing, 210023, China; State Key Laboratory of Applied Optics, Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences, Changchun, 130033, China.
Biosens Bioelectron. 2023 Jul 1;231:115273. doi: 10.1016/j.bios.2023.115273. Epub 2023 Mar 28.
Nondestructive separation/enrichment and reliable detection of extremely rare circulating tumor cells (CTCs) in peripheral blood are of considerable importance in tumor precision diagnosis and treatment, yet this remains a big challenge. Herein, a novel strategy for nondestructive separation/enrichment and ultra-sensitive surface-enhanced Raman scattering (SERS)-based enumeration of CTCs is proposed via aptamer recognition and rolling circle amplification (RCA). In this work the magnetic beads modified with "Aptamer (Apt)-Primer" (AP) probes were utilized to specifically capture CTCs, and then after magnetic separation/enrichment, the RCA-powered SERS counting and benzonase nuclease cleavage-assisted nondestructive release of CTCs were realized, respectively. The AP was assembled by hybridizing the EpCAM-specific aptamer with a primer, and the optimal AP contains 4 mismatched bases. The RCA enhanced SERS signal nearly 4.5-fold, and the SERS strategy has good specificity, uniformity and reproducibility. The proposed SERS detection possesses a good linear relationship with the concentration of MCF-7 cells spiked in PBS with the limit of detection (LOD) of 2 cells/mL, which shows good potential practicality for detecting CTCs in blood with recoveries ranging from 100.56% to 116.78%. Besides, the released CTCs remained good cellular activity with the normal proliferation after re-culture for 48 h and normal growth for at least three generations. The proposed strategy of nondestructive separation/enrichment and SERS-based sensitive enumeration is promising for reliable analysis of EpCAM-positive CTCs in blood, which is expected to provide a powerful tool for analysis of extremely rare circulating tumor cells in complex peripheral blood for liquid biopsy.
非破坏性分离/富集和可靠检测外周血中极其罕见的循环肿瘤细胞（CTC）在肿瘤精准诊断和治疗中具有重要意义，但这仍然是一个巨大的挑战。在此，提出了一种通过适体识别和滚环扩增（RCA）实现非破坏性分离/富集和基于表面增强拉曼散射（SERS）的超敏 CTC 计数的新策略。在这项工作中，用“适体（Apt）-引物（Primer）（AP）探针修饰的磁性珠被用于特异性捕获 CTC，然后在磁分离/富集后，分别实现了 RCA 增强的 SERS 计数和苯甲脒核酸酶切割辅助的 CTC 的非破坏性释放。AP 通过将 EpCAM 特异性适体与引物杂交组装而成，最佳的 AP 含有 4 个错配碱基。RCA 增强了 SERS 信号近 4.5 倍，并且 SERS 策略具有良好的特异性、均匀性和重现性。所提出的 SERS 检测与 PBS 中掺入 MCF-7 细胞的浓度呈良好的线性关系，检出限（LOD）为 2 个细胞/mL，显示出在血液中检测 CTC 具有良好的实际应用潜力，回收率范围为 100.56%至 116.78%。此外，释放的 CTC 经再培养 48 小时后具有良好的细胞活性，并能正常增殖至少三代。这种非破坏性分离/富集和基于 SERS 的敏感计数策略有望用于可靠分析血液中的 EpCAM 阳性 CTC，有望为复杂外周血中极其罕见的循环肿瘤细胞的分析提供强大的工具，用于液体活检。
