基于量子点-甲酚紫FRET比率探针的活细胞硫化氢成像

A Frster Resonance Energy Transfer Ratiometric Probe Based on Quantum Dot-Cresyl Violet for Imaging Hydrogen Sulfide in Living Cells

硫化氢(H_2S)作为一种重要的气体信号分子,其细胞水平异常与多种疾病的发生发展密切相关。本研究针对现存荧光探针的胞内非特异性响应问题,发展了一种基于量子点-甲酚紫的荧光能量共振转移(F9rster resonance energy transfer,FRET)探针,利用比率荧光分析,实现活细胞内H_2S的灵敏成像。通过一步超声乳化法制备量子点纳米球,再结合叠氮甲酚紫(CV-N_3)制得无机-有机杂化QDS-N3比率探针。此纳米探针尺寸均一,粒径约为120 nm,酸性稳定性高,且无细胞毒性。H2S可将CV-N_3上的N3还原成NH2,生成的氨基甲酚紫(CV-NH_2)作为FRET-受体,吸收量子点纳米球的橙色荧光,产生红光信号。通过两个波长的荧光强度的比率分析可有效消除光源强度波动和细胞内复杂环境等的干扰,最终实现了活细胞内H2S成像。

Hydrogen sulfide(H2S)has been confirmed as a significant endogenous gaseous signaling molecule involved in various physiological processes.To monitor H2S in living cells,a Forster resonance energy transfer(FRET)ratiometric probe based on quantum dot-cresyl violet was developed.In this work,quantum dot nanospheres(QDS)were firstly synthesized via a facile ultrasonication emulsion strategy,and the mixture chloroform solution containing hydrophobic quantum dots and COOH-functionalized amphiphilic polymer were successfully transferred into the oil-in-water micelle.The negatively charged quantum dot nanospheres with quantum dots embedded in the polymer matrixes were successfully fabricated after the evaporation of chloroform.And then,these quantum dot nanospheres were condensed with positively charged cresyl violet-azide(CV-N3)via electrostatic interaction to obtain the QDS-N3complexes.The as-prepared QDS-N3complexes were monodispersed nanospheres with an average diameter of about120nm.These complexes were taken up by the cell through endocytosis,and they were still stable even in wide pH range.In addition,the QDS-N3complexes exhibited no cellular toxicity which was verified by MTT assay.In this ratiometric probe,CV-N3as a FRET acceptor was conjugated to quantum dot nanospheres.The quantum dots emitted at591nm and served as the FRET donor;once the aryl azide on the CV-N3was reduced to aniline by H2S,the probe emitted at620nm.The ratiometric probe allowed the elimination of interference of excitation for preparing nanosensors for monitoring various small molecular in living cells.