State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering , Nanjing University , Nanjing 210023 , PR China.
Anal Chem. 2019 Mar 5;91(5):3374-3381. doi: 10.1021/acs.analchem.8b04661. Epub 2019 Feb 20.
As an investigative tool, live-cell imaging requires superior probe design to guarantee imaging quality and data validity. The ability to simultaneously address the robustness, sensitivity, and consistency issues in a single-assay system is highly desired, but it remains a largely unsolved challenge. We describe herein a probe-design strategy called a nanoamplicon comparator (NAC) and demonstrate its proof-of-concept utility in intracellular microRNA (miRNA) imaging. This novel designer architecture builds upon spherical nucleic acids (SNAs) for robustness, catalytic hairpin assembly (CHA) for sensitivity, and upconversion nanoparticles (UNPs) for consistency. A catalytic circuit comprising a UNP-hairpin-DNA (UNP-HDNA) conjugate and a hairpin-DNA-organic-fluorophore (HDNA-F) conjugate as probe responds to target miRNA and generates the UNP-HDNA-HDNA-F complex as an NAC for quantitative UNP-to-organic-fluorophore-luminescence-resonance-energy-transfer (LRET) imaging against a native UNP-emission reference channel. An imaging application with miR21 shows the ability to monitor miRNA-expression levels across different cell lines and under an external stimulus.
作为一种研究工具，活细胞成像需要出色的探针设计来保证成像质量和数据有效性。人们高度期望能够在单一分析系统中同时解决稳健性、灵敏度和一致性问题，但这仍然是一个尚未解决的挑战。本文描述了一种称为纳米放大器比较器（NAC）的探针设计策略，并展示了其在细胞内 microRNA（miRNA）成像中的概念验证实用性。这种新型设计结构基于球形核酸（SNA）提高稳健性、催化发夹组装（CHA）提高灵敏度，以及上转换纳米粒子（UNP）提高一致性。由上转换纳米粒子-发夹 DNA（UNP-HDNA）缀合物和发夹 DNA-有机荧光团（HDNA-F）缀合物组成的催化回路作为探针，响应靶 miRNA 并生成 UNP-HDNA-HDNA-F 复合物作为 NAC，用于对天然 UNP 发射参考通道进行定量 UNP-到-有机荧光团-荧光共振能量转移（LRET）成像。miR21 的成像应用表明，能够跨不同细胞系和外部刺激监测 miRNA 表达水平。
