Schaub Jeffrey M, Best Quinn A, Zhao Cheng, Haack Richard A, Ruan Qiaoqiao
Applied Research and Technology, Core Diagnostics, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL, 60064-6016, United States.
Biochem Biophys Rep. 2025 Mar 8;42:101971. doi: 10.1016/j.bbrep.2025.101971. eCollection 2025 Jun.
Luminescent dyes are commonly modified to improve their solubility, permeability, or spectral properties. However, changing the chemical structure influences the absorption of light and thus the compound-specific molar absorption coefficient (), which also confounds the compound's concentration in solution. The accurate determination of the molar absorption coefficient of new luminescent molecules is labor intensive and challenging when a limited amount of material is available for testing. To address this problem, we developed three techniques combined with UV-Vis spectrophotometry to closely approximate the molar absorption coefficient of various light-emitting dyes. The first technique uses Electrospray Mass Spectrometry to obtain a high-resolution incorporation ratio of a dye-labeled protein. The second approach utilizes covalent linking of the unknown dye to a dye with a known absorption coefficient. In the third method, we used fluorescence correlation spectroscopy to determine the fluorophore concentration in solution. We test each method with well-characterized fluorescent dyes and an uncharacterized chemilumiphore. Each technique produced calculated absorption coefficients comparable to the published reference values, although each presented unique limitations that reduced accuracy under certain conditions. Nevertheless, the techniques could be incorporated into current compound evaluation workflows and require only a small amount of sample, two significant advantages over traditional methods for characterizing new luminescent compounds.
发光染料通常经过修饰以改善其溶解性、渗透性或光谱特性。然而，改变化学结构会影响光的吸收，进而影响化合物特定的摩尔吸收系数（），这也会混淆化合物在溶液中的浓度。当可用于测试的材料数量有限时，准确测定新发光分子的摩尔吸收系数既费力又具有挑战性。为了解决这个问题，我们开发了三种与紫外可见分光光度法相结合的技术，以紧密逼近各种发光染料的摩尔吸收系数。第一种技术使用电喷雾质谱法获得染料标记蛋白质的高分辨率掺入率。第二种方法利用未知染料与具有已知吸收系数的染料进行共价连接。在第三种方法中，我们使用荧光相关光谱法测定溶液中的荧光团浓度。我们用特征明确的荧光染料和未表征的化学发光体对每种方法进行测试。尽管每种技术在某些条件下都存在降低准确性的独特局限性，但每种技术产生的计算吸收系数都与已发表的参考值相当。然而，这些技术可以纳入当前的化合物评估工作流程，并且只需要少量样品，这是与表征新发光化合物的传统方法相比的两个显著优势。
