新型双光子荧光探针用于高灵敏检测硝基还原酶及细胞成像

A Novel Two-photon Fluorescent Probe for Sensitive Determination of Nitroreductase and Live Cell Imaging

以2-乙酰基-6-甲氨基萘为荧光团,对硝基氯甲酸苄酯为识别域,设计合成一种新型双光子荧光探针NNTR。基于单光子和双光子模式考察了NNTR探针的光学特性及其对硝基还原酶(NTR)的荧光响应,发现在NADH催化下,NNTR可与NTR(NTR)反应,5 min后,在单光子激发模式下,510 nm处的发射光强度增加了约350倍,而在双光子激发模式下,810 nm处的发射光强度增加了约500倍,活性截面积可达66 GM(1 GM=1×10^(-50)cm^4·s/photon)。将NNTR探针用于NTR检测,检出限低至22 ng/mL,且具有反应速度快、选择性高、光学稳定性好等特点。考察了探针对HeLa的细胞毒性,并以盖玻片诱导缺氧法使HeLa细胞缺氧,促使NTR过表达,实现了NNTR探针对HeLa活细胞中NTR的成像分析。

A novel two-photon fluorescent probe,NNTR,was synthesized by using 6-methylamino-2-acetyl-naphthalen as a two-photon fluorophore and 4-nitrobenzyl chloridocarbonate as a recognition domain for nitroreductase(NTR).The spectroscopic characteristics and fluorescent response towards NTR were investigated using one-and two-photon modes,respectively.In the presence of NADH,NNTR could react with nitroreductase completely within 5 min.NNTR showed a 350-fold fluorescence enhancement at 510 nm in one-photo model and a 500-fold fluorescence enhancement at 810 nm in two-photo model.The maximal two-photon action cross-section value was calculated to be 66 GM.NNTR presented quick response,high sensitivity and good photostability towards NTR with a detection limit of 22 ng/mL.Cell survival rate of HeLa cells incubating with NNTR probes with different concentrations was investigated.Two-photon microscopic imaging nitroreductase in HeLa Cells treated with 5μmol/L NNTR was successfully performed.