Estévez M Carmen, Huang Yu-Fen, Kang Huaizhi, O'Donoghue Meghan B, Bamrungsap Suwussa, Yan Jilin, Chen Xiaolan, Tan Weihong
Department of Chemistry, Functional Genomics Shands Cancer Center, Center for Research at the Bio/Nano Interface, University of Florida, Gainesville, FL, USA.
Methods Mol Biol. 2010;624:235-48. doi: 10.1007/978-1-60761-609-2_16.
Aptamers are DNA or RNA oligonucleotide sequences that selectively bind to their target with high affinity and specificity. They are obtained using an iterative selection protocol called SELEX. Several small molecules and proteins have been used as targets. Recently, a variant of this methodology, known as cell-SELEX, has been developed for a new generation of aptamers, which are capable of recognizing whole living cells. We have used this methodology for the selection of aptamers, which show high affinity and specificity for several cancer cells. In this chapter, we describe (1) the process followed for the generation of aptamers capable of recognizing acute leukemia cells (CCRF-CEM cells) and (2) the method of enhancing the selectivity and sensitivity of these aptamers by conjugation with a dual-nanoparticle system, which combines magnetic nanoparticles (MNP) and fluorescent silica nanoparticles (FNP). Specifically, the selected aptamers, which showed dissociation constants in the nanomolar range, have been coupled to MNPs in order to selectively collect and enrich cells from complex matrices, including blood samples. The additional coupling of the aptamer to FNPs offers an excellent and highly sensitive method for detecting cancer cells. In order to prove the potential of this rapid and low-cost method for diagnostic purposes, confocal microscopy was used to confirm the specific collection and detection of target cells in concentrations as low as 250 cells. The final fluorescence of the cells labeled with the nanoparticles was quantified using a fluorescence microplate reader.
适配体是能够以高亲和力和特异性选择性结合其靶标的DNA或RNA寡核苷酸序列。它们是通过一种称为指数富集的配体系统进化技术（SELEX）的迭代筛选方案获得的。几种小分子和蛋白质已被用作靶标。最近，这种方法的一种变体，即细胞SELEX，已被开发用于新一代能够识别完整活细胞的适配体。我们已使用这种方法来筛选对几种癌细胞具有高亲和力和特异性的适配体。在本章中，我们描述了（1）生成能够识别急性白血病细胞（CCRF-CEM细胞）的适配体所遵循的过程，以及（2）通过与结合了磁性纳米颗粒（MNP）和荧光二氧化硅纳米颗粒（FNP）的双纳米颗粒系统偶联来提高这些适配体的选择性和灵敏度的方法。具体而言，所选择的在纳摩尔范围内显示解离常数的适配体已与MNP偶联，以便从包括血液样本在内的复杂基质中选择性收集和富集细胞。适配体与FNP的额外偶联为检测癌细胞提供了一种出色且高度灵敏的方法。为了证明这种快速且低成本的方法用于诊断目的的潜力，使用共聚焦显微镜来确认在低至250个细胞的浓度下对靶细胞的特异性收集和检测。使用荧光酶标仪对用纳米颗粒标记的细胞的最终荧光进行定量。
