摘要/Abstract

次硝酸（Nitroxyl，HNO）由一氧化氮（NO）经单电子还原和质子化作用产生，在生理和病理过程中都有着重要作用.本工作构建了一种基于发光共振能量转移（Luminescence resonance energy transfer，LRET）的比率型上转换纳米探针用于检测生物体系中HNO含量.该探针以上转换纳米颗粒（Upconversion nanoparticles，UCNPs）为能量供体，有机染料Fl-TP为能量受体构建.Fl-TP能特异性识别HNO并与之反应生成Fl-HNO，Fl-HNO在400～500 nm处具有明显的吸收峰，与UCNPs的蓝色发射光谱重叠，从而发生LRET过程.随着HNO浓度增加，Fl-HNO的荧光强度（F_{525 nm}）逐渐增强，UCNPs的荧光强度（F_{480 nm}）逐渐下降，比率信号（F_{525 nm}/F_{480 nm}）逐渐增加，并与HNO浓度对数呈良好的线性关系，该探针的线性检测范围为3～100 μmol·L^－1，检出限23.4 nmol·L^－1.实验结果表明该比率探针特异性强，灵敏度高，并可成功实现在活细胞和生物组织中HNO的检测.
关键词: 上转换荧光纳米颗粒, 发光共振能量转移, 比率信号, 次硝酸, 荧光成像
Nitroxyl (HNO), produced by nitric oxide (NO) with one-electron reduction and protonation, has recently received substantial interest due to its important roles in various biological functions and pharmacological activities. Research indicates that HNO also has many potential pharmacological applications for different diseases. Therefore, the development of a reliable method for HNO assay in biosystems is highly desired. Ratiometric fluorescent probes show significant advantages over traditional “turn-on” ones, because simultaneous measurement of two emission signals can provide a built-in correction and thus minimize the inaccurate fluorescence signal readouts. As far as we know, there is no ratiometric fluorescent probe for HNO detection based on upconversion nanoparticles (UCNPs). Herein, a ratiometric nanoprobe for HNO assay was constructed based on the luminescence resonance energy transfer (LRET) principle by using UCNPs with a core-shell structure (NaYbF₄:30%Gd@NaYF₄:2%Yb:1%Tm) as the energy donor and an organic dye Fl-TP as the potential energy acceptor. The oleate-coated UCNPs (OA-UCNPs) and Fl-TP were assembled through hydrophobic interaction to construct the upconversion nanoprobe (termed as Fl-TP-UCNPs). Because of the ring-closed form, Fl-TP displayed weak absorption and was non-fluorescent, which blocked the LRET process. After reaction with HNO, the triphenylphosphine moiety left and released Fl-HNO with the fluorescent ring-open form. Fl-HNO showed strong absorption in the range of 400～500 nm, which completely overlapped with the blue luminescence of UCNPs and triggered the LRET process between UCNPs and Fl-HNO. Thus, the luminescence from UCNPs around 480 nm decreased and the emission from Fl-HNO around 525 nm increased with a [HNO]-dependent manner. The ratiometric luminescence intensity F_{525 nm}/F_{480 nm} showed a good linear relationship (R²＝0.9914) to the logarithm of AS (Angeli’s salt, a generally used HNO donor) concentration in the range of 3～100 μmol·L^－1 and the limit of detection was 23.4 nmol·L^－1. The excellent sensitivity, stability, selectivity and low cytotoxicity endow Fl-TP-UCNPs with the superior capability for HNO assay in vitro and in vivo. We found that Fl-TP-UCNPs probe is appropriate for monitoring HNO in living cells as well as imaging HNO in liver tissues. This probe may be a powerful tool for HNO assay in various physiological processes.
Key words: upconversion nanoparticles, luminescence resonance energy transfer, ratiometric signal, nitroxyl, fluorescence bioimaging


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