Department of Chemistry, Mississippi State University, Mississippi State, Mississippi 39762, USA.
Appl Spectrosc. 2010 Oct;64(10):1078-85. doi: 10.1366/000370210792973497.
Using bovine serum albumin (BSA) as the model protein, normal Raman spectra of fluorescein isothiocyanate (FITC) conjugated protein were systematically studied for the first time using both solution and the drop coating deposition Raman (DCDR) sampling techniques. The FITC-BSA Raman spectra are dominated by the FITC Raman features that are strongly pH dependent. Current DCDR detection sensitivity obtained with a 10:1 FITC-BSA conjugate is 45 fmol in terms of total protein consumption and ∼15 attomol at laser probed volume. Unlike the FITC-BSA solution Raman spectra, where the FITC Raman features are photostable, concurrent FITC fluorescence and Raman photobleaching is observed in the DCDR spectra of FITC-BSA. While the FITC Raman photobleaching follows a single exponential decay function with a time constant independent of the FITC labeling ratio, the fluorescence background photobleaching is much more complicated and it depends strongly on the FITC labeling ratio and sample conditions. Mechanistically, the FITC Raman photobleaching is believed to be due to photochemical reaction of the FITC molecules in the electronically excited state. The FITC fluorescence photobleaching involves both concentration quenching and photochemical quenching, and the latter may involve a photochemical intermediate that is fluorescence inactive but Raman active.
首次采用溶液和滴涂沉积拉曼（DCDR）采样技术，以牛血清白蛋白（BSA）为模型蛋白，系统研究了荧光素异硫氰酸酯（FITC）缀合蛋白的正常拉曼光谱。FITC-BSA 的拉曼光谱主要由 FITC 拉曼特征主导，这些特征强烈依赖于 pH 值。目前，使用 10:1 的 FITC-BSA 缀合物，通过 DCDR 检测获得的总蛋白消耗检测灵敏度为 45 fmol，激光探测体积检测灵敏度约为 15 飞摩尔。与 FITC-BSA 溶液拉曼光谱不同，FITC 拉曼特征在其中具有光稳定性，在 FITC-BSA 的 DCDR 光谱中同时观察到 FITC 荧光和拉曼光漂白。虽然 FITC 拉曼光漂白遵循与 FITC 标记比无关的单指数衰减函数，但荧光背景光漂白要复杂得多，并且强烈依赖于 FITC 标记比和样品条件。从机制上讲，FITC 拉曼光漂白被认为是由于 FITC 分子在电子激发态下的光化学反应。FITC 荧光光漂白涉及浓度猝灭和光化学猝灭，后者可能涉及一种荧光失活但拉曼活性的光化学中间产物。
