DNA纳米传感器荧光成像技术

DNA Nanosensor Fluorescence Imaging Microscopy

把基于量子点的DNA纳米传感器模型应用到ICCD荧光显微成像系统中,利用量子点的量子产率高、荧光寿命长、激发谱宽而发射谱窄、发射波长可由材料尺寸调谐等特性,以量子点为供体,Cy5(一种小分子荧光染料)为受体,结合ICCD系统的全内反射荧光成像功能、实时成像功能和双通道成像功能,证实了DNA纳米传感器可以在溶液中检测到含30个碱基的单链目标DNA片段。实时拍摄了溶液中链霉亲和素包被的量子点对两头分别连着Cy5和生物素的单链DNA片段(Cy5-ssDNA-Biotin)的捕获过程。并在活的中国仓鼠卵巢细胞样品中加入链霉亲和素包被的量子点和Cy5-ssDNA-Biotin进行实时荧光成像,拍摄到链霉亲和素包被的量子点和Cy5-ssDNA-Biotin进入细胞中并发生FRET的过程,初步表明了DNA纳米传感器在活细胞内进行DNA(或RNA)片段检测的可行性。

Quantum dots have many excellent optical properties such as high quantum yield, long fluorescence lifetime, wide excitation spectrum and narrow emission spectrum, tunable emission wavelength and so on, thus have become a newpopular type of fluorescence probes in these years. Quantum do,based DNA nanosensor comprising streptavidin-conjugated quantum dots, capture probes with biotin and reporter probes with Cy5 was designed to detect DNA or RNA segments. Capture probes and reporter probes were connected by the target DNA or RNA segments so that quantum dots and Cy5s could be together and FRET （flu orescence resonance energy transfer） could be detected. In the present work, quantum-dot-based DNA nanosensor was combined with ICCD fluorescence microscopy imaging system through the authors＇ experiments. Using the total internal reflection fluores cence （TIRF）, FRET between quantum dots and Cy5s was recorded by ICCD showing that segments of single-stranded target DNA with 30-base length were detected in solution using DNA nanosensor. When Cy5-ssDNA-Biotins were added into streptavidin-conjugated quantum dots in solution, by real time recording, the FRET efficiency was found to increase with time, which indicated the process of streptavidin-conjugated quantum dots capturing Cy5 ssDNA Biotins. It was also observed that streptavidin-conjugated quantum clots and CyS-ssDNA-Biotins could both enter living Chinese hamster ovary cells and have FRET. The process of streptavidin-conjugated quantum dots capturing CyS-ssDNA Biotins was detected in the cells as well and Cy5s were photobleached after a long time of irradiation. It has been proved that detecting DNA or RNA segments in living cells with DNA nanosensor is possible.