Radiation & Photochemistry Division, Bhabha Atomic Research Centre, Mumbai 400085, India.
Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai 400094, India.
Methods Appl Fluoresc. 2022 Jul 20;10(4). doi: 10.1088/2050-6120/ac804b.
Molecular interactions are fundamental to any chemical or biological processes, and their rates define the operational sequence and control for any desirable product. Here, we deliberate on a recently developed novel fluorescence spectroscopic method, which combines fluorescence photon anti-bunching, photon bunching, time-correlated single-photon counting (TCSPC), and steady-state fluorescence spectroscopy, to study composite chemical reactions with single molecule sensitivity. The proposed method captures the full picture of the multifaceted quenching kinetics, which involves static quenching by ground state complexation and collisional quenching in the excited state under dynamic exchange of fluorophore in a heterogeneous media, and which cannot be seen by steady-state or lifetime measurements alone. Photon correlation in fluorescence correlation spectroscopy (FCS) provides access to interrogate interaction dynamics from picosecond to seconds, stitching all possible stages of dye-quencher interaction in a micellar media. This is not possible with the limited time window available to conventional ensemble techniques like TCSPC, flash photolysis, transient absorption, stop-flow, etc. The basic premises of such unified global analysis and sanctity of extracted parameters critically depends on the minimum but precise description of reaction scheme, for which careful inspection of ensemble spectroscopy data for photo-physical behaviour is very important. Though in this contribution we discussed and demonstrated the merits of photon antibunching and bunching spectroscopy for dye-quencher interaction in cationic cetyltrimethylammonium bromide (CTAB) micellar solution by photo-induced electron transfer mechanism and the influence of micellar charge and microenvironment on the interaction kinetics, but in principal similar arguments are equally applicable to any other interaction mechanisms which alter fluorescence photon correlations, like Förster resonance energy transfer (FRET), proton transfer, isomerisation, etc.
分子相互作用是任何化学或生物过程的基础，它们的速率决定了任何期望产物的操作顺序和控制。在这里，我们讨论了一种最近开发的新型荧光光谱方法，该方法结合了荧光光子反聚束、光子聚束、时间相关单光子计数（TCSPC）和稳态荧光光谱，以研究具有单分子灵敏度的复合化学反应。所提出的方法捕捉到了多方面猝灭动力学的全貌，其中涉及静态猝灭通过基态络合和在动态交换荧光团时的激发态中的碰撞猝灭在非均相介质中，这是单独使用稳态或寿命测量无法看到的。荧光相关光谱（FCS）中的光子相关提供了从皮秒到秒的相互作用动力学的询问，拼接了胶束介质中染料猝灭剂相互作用的所有可能阶段。这是不可能的，因为传统的集合技术（如 TCSPC、闪光光解、瞬态吸收、停流等）可用的时间窗口有限。这种统一的全局分析的基本前提和提取参数的神圣性严重依赖于反应方案的最小但精确描述，对于这种情况，仔细检查集合光谱数据对于光物理行为非常重要。尽管在本贡献中，我们讨论并演示了通过光诱导电子转移机制在阳离子十六烷基三甲基溴化铵（CTAB）胶束溶液中对染料猝灭剂相互作用的光子反聚束和聚束光谱的优点，以及胶束电荷和微环境对相互作用动力学的影响，但原则上类似的论点同样适用于任何其他改变荧光光子相关的相互作用机制，如Förster 共振能量转移（FRET）、质子转移、异构化等。
