Identification and characterization of circulating tumor cells (CTCs) from blood samples of patients with cancer can help monitor parameters such as disease stage, disease progression and therapeutic efficiency. However, the sensitivity and specificity of current multivalent approaches used for CTC capture is limited by the lack of control over the ligands’ position. In this Protocol Update, we describe DNA-tetrahedral frameworks anchored with aptamers that can be configured with user-defined spatial arrangements and stoichiometries. The modified tetrahedral DNA frameworks, termed ‘n-simplexes’, can be used as probes to specifically target receptor−ligand interactions on the cell membrane. Here, we describe the synthesis and use of n-simplexes that target the epithelial cell adhesion molecule expressed on the surface of CTCs. The characterization of the n-simplexes includes measuring the binding affinity to the membrane receptors as a result of the spatial arrangement and stoichiometry of the aptamers. We further detail the capture of CTCs from patient blood samples. The procedure for the preparation and characterization of n-simplexes requires 11.5 h, CTC capture from clinical samples and data processing requires ~5 h per six samples and the downstream analysis of captured cells typically requires 5.5 h. The protocol is suitable for users with basic expertise in molecular biology and handling of clinical samples.

从癌症患者的血液样本中识别和表征循环肿瘤细胞 (CTC) 有助于监测疾病阶段、疾病进展和治疗效率等参数。然而，目前用于 CTC 捕获的多价方法的灵敏度和特异性由于缺乏对配体位置的控制而受到限制。在此协议更新中，我们描述了以适体锚定的 DNA 四面体框架，可以使用用户定义的空间排列和化学计量进行配置。修饰后的四面体 DNA 框架，称为“n-单纯形”，可用作特异性靶向细胞膜上受体-配体相互作用的探针。在这里，我们描述了针对 CTC 表面表达的上皮细胞粘附分子的 n-单纯体的合成和使用。 n-单纯体的表征包括测量由于适体的空间排列和化学计量而导致的与膜受体的结合亲和力。我们进一步详细介绍了从患者血液样本中捕获 CTC 的情况。 n-单纯形的制备和表征过程需要 11.5 小时，从临床样品中捕获 CTC 和数据处理每六个样品需要约 5 小时，捕获细胞的下游分析通常需要 5.5 小时。该协议适合具有分子生物学和临床样本处理基础知识的用户。