高等学校化学学报 ›› 2020, Vol. 41 ›› Issue (6): 1252-1261.doi: 10.7503/cjcu20200026

• 分析化学 • 上一篇    下一篇

基于双色荧光传感器的癌细胞成像及microRNA定量检测

白翠婷,岳仁叶,罗列高,马楠*()   

  1. 苏州大学材料与化学化工学部, 苏州 215123
  • 收稿日期:2020-01-13 出版日期:2020-06-10 发布日期:2020-02-26
  • 通讯作者: 马楠 E-mail:nan.ma@suda.edu.cn
  • 基金资助:
    国家自然科学基金(21522506);国家自然科学基金(21475093);国家高技术研究发展计划(2014AA020518);青年千人计划、 江苏高校优势学科建设工程项目和苏州大学启动基金

Quantitative Analysis of MicroRNA Content by Fluorescence Imaging in Cancer Cells Using Dual-color Fluorescence Nanosensor

BAI Cuiting,YUE Renye,LUO Liegao,MA Nan*()   

  1. College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, China
  • Received:2020-01-13 Online:2020-06-10 Published:2020-02-26
  • Contact: Nan MA E-mail:nan.ma@suda.edu.cn
  • Supported by:
    † National Natural Foundation of Chinathe (No.2014AA020518),(21522506);National Natural Foundation of China(21475093);National High-Tech R&D Program of China(2014AA020518);the 1000-Young Talents Plan, the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD), and Startup Funds from Soochow University, China

摘要:

癌细胞中microRNA(miRNA)的灵敏成像对于疾病的诊断治疗具有重要意义, 其中miRNA-21通常在多种癌细胞中异常表达. 本文将DNA功能化的金纳米颗粒与发射波长分离的荧光染料FAM和Cy5.5修饰的DNA通过含有光控基团PC-linker的DNA4作为桥梁进行自组装, 构建了纳米传感器GDC. 将302 nm紫外光作为启动开关, 用其照射该体系时, Cy5.5修饰的DNA3被释放, 其荧光强度可作为内参比信号, 用于标定进入细胞的组装体含量; 细胞中miRNA-21作为催化分子, 与外加燃料Fuel DNA共同作用下可实现催化放大, FAM修饰的DNA2被释放且被猝灭的荧光信号得以恢复, 并作为检测信号. 通过2种荧光信号强度(FL)的检测及FLFAM/FLCy5.5比值的计算, 达到定量分析细胞中miRNA含量的目的. 该体系可扣除因细胞内组装体含量不同造成的背景信号误差, 不仅能显著提高检测准确度, 还因存在催化循环而大大降低了检出限, 比传统方法至少降低了3个数量级. 该传感器的检出限为23.1 pmol/L, 通过定量计算得出HeLa细胞中miRNA的含量为0.0236 nmol/L.

关键词: 金纳米颗粒, 催化放大, MicroRNA, 荧光成像, 定量分析

Abstract:

MicroRNA(miRNA) plays an important role in cellular function and gene expression with abnormal expression level or varieties. Given this, miRNA imaging in cancer cells was regarded as the effective approach for disease diagnosis and treatment. Herein, we built a sensitive nanosensor with gold nanoparticles(GNP) and two type of dye-modified DNA, FAM-DNA2 and Cy5.5-DNA3. DNA4 containing photocleavable linker(PC-linker), was used as the bridge for self-assembly of nanosensor. 302 nm UV light was used as the startup switch. Cy5.5-DNA3 was released from assembly with the irradiation of UV light, the fluorescence intensity was used as internal reference signal to standardize the quantity of nanoparticles into cells. MiRNA-21 that working as catalytic molecule can induce the assembly of nanosensors, resulting to release FAM-DNA2 in the presence of Fuel DNA, at the same time, the recovery fluorescence of FAM was used as detection signal. By combination of the fluorescence intensity of FAM and Cy5.5(FLFAM/FLCy5.5), we can calculate the quantity of miRNA and nanosensors in cancer cells, which avoided the existence of background signal caused by the different contents of intracellular nanoparticles. Importantly, this nanosensor is of great stability, biocompatibility, and can be used for cellular fluorescence imaging. In addition, the limit of detection(LOD) was about three orders of magnitude lower than traditional non-catalytic method and calculated to be 23.1 pmol/L. The quantity of miRNA-21 in Hela cells was calculated to be 0.0236 nmol/L.

Key words: Gold nanoparticles, Catalytic amplification, MicroRNA, Fluorescence imaging, Quantitative analysis

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