Department of Chemistry and Center for Nanoscience & Nanotechnology, National Sun Yat-sen University, No. 70 Lienhai Rd., Kaohsiung 80424, Taiwan; Department of Environmental Engineering, Da-Yeh University, No. 168, University Rd., Dacun, Changhua 515006, Taiwan.
Department of Chemistry and Center for Nanoscience & Nanotechnology, National Sun Yat-sen University, No. 70 Lienhai Rd., Kaohsiung 80424, Taiwan.
Spectrochim Acta A Mol Biomol Spectrosc. 2024 Mar 15;309:123781. doi: 10.1016/j.saa.2023.123781. Epub 2023 Dec 22.
Addressing the limitations observed in previous studies, where the quantitative range of nanoprobes for detecting K and adenosine triphosphate (ATP) did not cover concentrations found within living cells, the present study aimed to develop ratiometric nanoprobes that can accurately sense changes in K and ATP levels in living cells and quantify them in human fluids. The proposed nanoprobes consisted of recognition flares modified with 6-carboxyfluorescein (FAM) and 5-carboxytetramethylrhodamine (TAMRA), along with thiolate single-stranded DNA (ssDNA) and molybdenum disulfide nanosheets (MoS NSs). The thiolate ssDNA acts as a linker between the flares and the MoS NSs, directly forming a functional nanostructure at room temperature. The direct conjugation of labeled flares to the MoS NSs simplifies the fabrication process. In the absence of K and ATP, the hybridization of flares and thiolate ssDNA caused FAM to move away from TAMRA, suppressing the fluorescence resonance energy transfer (FRET) process. However, upon the introduction of K and ATP, the flares undergo a structural transformation via the formation of G-quadruplex formation and the generation of hairpin-shaped structures, respectively. This structural change leads to the release of the flares from the ssDNA-conjugated nanosheet surface. The release of the flares brings FAM and TAMRA into close proximity, allowing FRET to occur, leading to FRET and static quenching. By monitoring the ratio between the fluorescence intensities of FAM and TAMRA, the concentration of K (5-100 mM) and ATP (0.3-5 mM) can be accurately determined by the proposed nanoprobes. The advantages of these nanoprobes lie in their ability to provide ratiometric measurements, which enhance the accuracy and reliability of the quantification process. The proposed nanoprobes offer potential applications as ratiometric imaging probes for monitoring K and ATP-related reactions in living cells, providing valuable insights into cellular processes. Additionally, they can be employed for determining the levels of K and ATP in human fluids, offering potential diagnostic applications in various clinical settings.
针对先前研究中观察到的局限性，即用于检测钾和三磷酸腺苷 (ATP) 的纳米探针的定量范围没有涵盖活细胞内存在的浓度，本研究旨在开发比率型纳米探针，能够准确感知活细胞内钾和 ATP 水平的变化，并对其进行定量。所提出的纳米探针由用 6-羧基荧光素 (FAM) 和 5-羧基四甲基罗丹明 (TAMRA) 修饰的识别花饰以及硫醇单链 DNA (ssDNA) 和二硫化钼纳米片 (MoS NSs) 组成。硫醇 ssDNA 作为花饰和 MoS NSs 之间的连接体，在室温下直接形成功能纳米结构。标记花饰直接与 MoS NSs 缀合简化了制造过程。在不存在 K 和 ATP 的情况下，花饰和硫醇 ssDNA 的杂交导致 FAM 远离 TAMRA，抑制荧光共振能量转移 (FRET) 过程。然而，当引入 K 和 ATP 时，花饰分别通过形成 G-四链体形成和发夹状结构发生结构转变。这种结构变化导致花饰从 ssDNA 缀合的纳米片表面释放。花饰的释放使 FAM 和 TAMRA 靠近，允许 FRET 发生，导致 FRET 和静态猝灭。通过监测 FAM 和 TAMRA 的荧光强度比，可以通过所提出的纳米探针准确确定 K（5-100 mM）和 ATP（0.3-5 mM）的浓度。这些纳米探针的优点在于它们能够提供比率测量，从而提高定量过程的准确性和可靠性。所提出的纳米探针作为用于监测活细胞中与 K 和 ATP 相关反应的比率成像探针具有潜在应用，为细胞过程提供有价值的见解。此外，它们可用于测定人液中 K 和 ATP 的水平，在各种临床环境中具有潜在的诊断应用。
