Atsar Felix S, Bourger Hillary D, Baker Christopher A
Department of Chemistry & Biochemistry, New Mexico State University, Las Cruces, NM, 88003-001, USA.
Department of Chemical and Materials Engineering, New Mexico State University, Las Cruces, NM, 88003-001, USA.
Analyst. 2025 Feb 10;150(4):620-629. doi: 10.1039/d4an01208a.
Hydrodynamic radius () is a descriptive metric of protein structure with the potential to impact drug development, disease diagnosis, and other important research areas of molecular biology. Common instrumental methods for molecular size characterization are disadvantageous due to high sample consumption, measurements made in non-physiological conditions, and/or inaccurate size determinations. Capillary Taylor dispersion analysis (TDA) is a molecular sizing method that utilizes nL sample volumes and achieves absolute size determination without calibration or comparison to standards. One key drawback of TDA is that it reports the concentration-weighted average , which may be limiting in the analysis of complex sample mixtures. Here, we describe the development of a 3D printed instrument to integrate capillary electrophoresis (CE) separations online with TDA size characterization. Dual laser-induced fluorescence detectors were developed to enable two-channel detection using a single PMT and fluorescence filter set, achieving detection limits for AlexaFluor 532 of 0.6 ± 0.4 nM and 1.1 ± 0.2 nM for detectors 1 and 2, respectively. Joule heating during CE separations was observed to introduce bias in subsequent TDA measurements. The effects of Joule heating were mitigated by integrating a water circulating sheath flow on the portion of the capillary used for CE. The utility of CE-TDA in bioanalysis was demonstrated by standard-free peak identification in the ficin digestion of IgG1. CE-TDA was further applied to characterizing denaturation dynamics of the Group II heat resistant protein apolipoprotein A-1 (ApoA), in which was observed to increase from 2.3 ± 0.2 nm at 20 °C to 5.2 ± 0.5 nm while heated at at 90 °C, then returned to a quasi-native state with = 2.9 ± 0.5 nm after cooling to 20 °C. CE-TDA is a powerful analysis mode with potential to impact various domains of bioanalysis. The instrument developed in this work offers a low barrier to entry for researchers interested in adopting CE-TDA.
流体动力学半径（）是蛋白质结构的一种描述性度量，有可能影响药物开发、疾病诊断和分子生物学的其他重要研究领域。由于样品消耗量大、在非生理条件下进行测量和/或尺寸测定不准确，常用的分子大小表征仪器方法存在缺点。毛细管泰勒分散分析（TDA）是一种分子大小测定方法，它使用纳升级的样品体积，无需校准或与标准品比较即可实现绝对尺寸测定。TDA的一个关键缺点是它报告的是浓度加权平均值，这在复杂样品混合物的分析中可能会受到限制。在此，我们描述了一种3D打印仪器的开发，该仪器可将毛细管电泳（CE）分离与TDA尺寸表征在线集成。开发了双激光诱导荧光检测器，以使用单个光电倍增管（PMT）和荧光滤光片组实现双通道检测，检测器1和检测器2对AlexaFluor 532的检测限分别为0.6±0.4 nM和1.1±0.2 nM。观察到CE分离过程中的焦耳热会在随后的TDA测量中引入偏差。通过在用于CE的毛细管部分集成水循环鞘流，减轻了焦耳热的影响。通过在IgG1的无花果蛋白酶消化中进行无标准峰识别，证明了CE-TDA在生物分析中的实用性。CE-TDA进一步应用于表征II组耐热蛋白载脂蛋白A-1（ApoA）的变性动力学，其中在90°C加热时，流体动力学半径从20°C时的2.3±0.2 nm增加到5.2±0.5 nm，然后冷却至20°C后恢复到准天然状态，流体动力学半径为2.9±0.5 nm。CE-TDA是一种强大的分析模式，有可能影响生物分析的各个领域。这项工作中开发的仪器为有兴趣采用CE-TDA的研究人员提供了较低的入门门槛。
