School of Chemistry and Chemical Engineering, Shandong University, 250100, Jinan, PR China.
Department of Oncology, Research Center of Basic Medicine, Jinan Central Hospital, Cheeloo College of Medicine, Shandong University, 250012, Jinan, PR China.
Talanta. 2021 Jul 1;229:122284. doi: 10.1016/j.talanta.2021.122284. Epub 2021 Mar 16.
Tumor progression is a complicated process influenced by multiple factors, in which the acidic tumor microenvironment (TME) and altered tumor-associated membrane proteins (TA-MPs) are closely involved. Monitoring the status of these factors is of significance for tumor progression research. Here, we develop a novel probe for simultaneously imaging the acidic TME and TA-MPs in situ. In this probe, i-motif-forming sequences (strand I) are conjugated to a gold nanoparticle (AuNP) via gold-sulfur bonds for acid-response. Extended aptamers (strand A) for protein recognition are labeled with Cy3 and Cy5 respectively at two ends. The extended part of strand A hybridizes with strand I to quench Cy3 by the proximal AuNP, and the protein recognition part hybridizes with a strand labeled with BHQ2 (strand Q) to quench Cy5. When the integrated probe encounters an acidic TME, the strand I fold into i-motif quadruplexes and release the AQ duplexes from the AuNP, enabling Cy3 to be lit to indicate the acidic TME. The aptamers in AQ duplexes bind to target proteins, removing the hybridization between strand A and Q thus leading to the fluorescence recovery of Cy5 for in-situ imaging of the proteins. Fluorescence measurement and confocal microscopy imaging showed that the probe could sensitively respond to the alteration in acidity from pH 7.4 into pH 6.5, which is coincide with the acidity gap of extracellular microenvironment between normal and tumor cells. Besides, it enabled the in-situ imaging of MUC1 proteins on living cell surface, revealing their expression level and distribution. This probe demonstrates a new approach for simultaneously imaging the acidic TME and TA-MPs, providing a useful tool for multifactor research of tumor progression.
肿瘤进展是一个受多种因素影响的复杂过程，其中酸性肿瘤微环境（TME）和改变的肿瘤相关膜蛋白（TA-MPs）密切相关。监测这些因素的状态对于肿瘤进展研究具有重要意义。在这里，我们开发了一种新的探针，用于原位同时成像酸性 TME 和 TA-MPs。在该探针中，通过金-硫键将发夹结构形成序列（链 I）连接到金纳米颗粒（AuNP）上，以响应酸性。用于蛋白质识别的扩展适体（链 A）分别在两端用 Cy3 和 Cy5 标记。链 A 的扩展部分与链 I 杂交，通过近端 AuNP 猝灭 Cy3，而蛋白质识别部分与标记有 BHQ2（链 Q）的链杂交，以猝灭 Cy5。当集成探针遇到酸性 TME 时，链 I 折叠成 i-motif 四联体，并从 AuNP 上释放 AQ 双链体，使 Cy3 被点亮以指示酸性 TME。AQ 双链体中的适体与靶蛋白结合，从而消除了链 A 和 Q 之间的杂交，导致 Cy5 的荧光恢复，从而实现蛋白质的原位成像。荧光测量和共聚焦显微镜成像表明，该探针可以对从 pH 7.4 到 pH 6.5 的酸度变化敏感响应，这与正常和肿瘤细胞之间细胞外微环境的酸度间隙一致。此外，它还能够对活细胞表面上的 MUC1 蛋白进行原位成像，揭示其表达水平和分布。该探针为同时成像酸性 TME 和 TA-MPs 提供了一种新方法，为肿瘤进展的多因素研究提供了一种有用的工具。
