School of Chemistry, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.
Henry Wellcome Laboratory for Cell Imaging, Faculty of Science, University of East Anglia, Norwich Research Park, Norwich, NR4 7T, UK.
J Mater Chem B. 2023 Apr 12;11(15):3387-3396. doi: 10.1039/d3tb00103b.
Nitric oxide (NO) plays an important role in the regulation of the immune, cardiovascular and nervous systems. Consequently, being able to monitor and quantify intracellular NO levels would provide a greater understanding of the implications of this molecule in the different biological processes, including, for example, in cancer. Here, we report a broadly applicable two-photon excitable fluorescent nanoprobe able to detect and potentially quantify NO levels in an extensive range of cellular environments. The nanoprobe consists of a thiolated photoinduced electron transfer-based two=photon fluorescent probe attached onto the surface of 2.4 ± 0.7 nm gold nanoparticles (DANPY-NO@AuNPs). The nanoprobe, which can be synthesised in a reproducible manner and exhibits great stability when stored at room temperature, is able to selectively detect NO in solution, with a dynamic range up to 150 μM, and at pH values of biological relevance. DANPY-NO@AuNPs were able to selectively detect endogenous NO in RAW264.7γ NO macrophages and THP-1 human leukemic cells; and endogenous and exogenous NO in endothelial cells. The nanoprobe accumulated in the acidic organelles of the tested cell lines showing negligible toxicity. Importantly, DANPY-NO@AuNPs showed potential to quantify intracellular NO concentrations in MDA-MB-231 breast cancer cells. The biological evaluation of the nanoprobe was undertaken using confocal laser scanning (images and intracellular emission spectra) and multiphoton microscopies, and flow cytometry. Based on their excellent sensitivity and stability, and outstanding versatility, DANPY-NO@AuNPs can be applied for the spatiotemporal monitoring of and NO levels.
一氧化氮（NO）在免疫系统、心血管系统和神经系统的调节中起着重要作用。因此，能够监测和量化细胞内的 NO 水平将有助于更好地理解该分子在不同生物过程中的影响，包括例如癌症。在这里，我们报告了一种广泛适用的双光子激发荧光纳米探针，能够在广泛的细胞环境中检测和潜在地定量 NO 水平。该纳米探针由巯基化的光诱导电子转移型双光子荧光探针组成，附着在 2.4±0.7nm 金纳米颗粒（DANPY-NO@AuNPs）的表面上。该纳米探针可以以可重复的方式合成，并在室温下储存时表现出极好的稳定性，能够在溶液中选择性地检测 NO，动态范围可达 150μM，且在具有生物学相关性的 pH 值范围内也是如此。DANPY-NO@AuNPs 能够选择性地检测 RAW264.7γNO 巨噬细胞和 THP-1 人白血病细胞中的内源性 NO；以及内皮细胞中的内源性和外源性 NO。该纳米探针能够在测试的细胞系的酸性细胞器中选择性地积累，表现出可忽略的毒性。重要的是，DANPY-NO@AuNPs 显示出在 MDA-MB-231 乳腺癌细胞中定量细胞内 NO 浓度的潜力。该纳米探针的生物学评估采用共聚焦激光扫描（图像和细胞内发射光谱）和多光子显微镜以及流式细胞术进行。基于其出色的灵敏度和稳定性以及卓越的多功能性，DANPY-NO@AuNPs 可用于时空监测和NO 水平。
