由于i-motif与配体作用效率远不及G-四链体，i-motif DNA不像G-四链体那样与其配体一起被广泛用作DNA纳米分析的信号读出器。针对此问题，我们在双分子i-motif的5’端引入含GA碱基片段，将i-motif核心结构中一条平行双链延长，为荧光配体硫磺素(ThT)提供高效结合位点。基于理论计算，对ThT分子结构进行裁剪，再合成分离，以改善对i-motif的选择性，适合DNA纳米分析。以上设计的i-motif双分子结构和ThT衍生物以圆二色谱、核磁共振、质谱进行表征。然后，以该i-motif来引导DNA纳米脚手架组装，结合ThT读出荧光信号，利用凝胶电泳、原子力显微镜、荧光光谱法对DNA纳米组装过程进行监控。在此基础上，我们将i-motif引导并指示的DNA纳米组装体系应用于酸性生理条件下的无标记适配体传感分析中，为癌细胞表面酸性微环境的pH荧光显微成像提供新思路。

Since the interactions of i-motifs with ligands are far weaker than those of G-quadruplexes, i-motif DNAs have not widely been used together with their ligands as signal reporters for DNA nanoanalysis, unlike G-quadruplexes. To resolve this problem, we introduce a GA-containing nucleotide segment into the 5’-terminal of a bimolecular i-motif and extend one parallel duplex of the i-motif core to provide an efficient binding site for a fluorescent ligand thioflavin T (ThT). Based on theoretical calculation, we tailor the molecular structrue of ThT and then synthesize and separate the target ligand, to improve its selectivity for i-motif structrues and make it applicable to DNA nanoanalysis. The above designed i-motif bimolecular structure and ThT derivate are characterized by circular dichroism, nuclear magnetic resonance, and mass spectrum. Then, we utilize the i-motif to guide the assembly of DNA nanoscaffolds, combined with ThT to report the fluorescent signal. The DNA nanoassembly process is monitored by gel electrophoresis, atomic force microscopy, and fluorescence spectroscopy. With this basis, we further apply the i-motif–directed and –reported DNA nanoassembly system to label-free aptasensor-based analysis under acidic physiological conditions, and provide a new strategy to fluorescent microscopic pH imaging of the acidic microenvironments on cancer cell surfaces.