3D Printed Instrument for Taylor Dispersion Analysis with Two-Point Laser-Induced Fluorescence Detection
3D Printed Instrument for Taylor Dispersion Analysis with Two-Point Laser-Induced Fluorescence Detection
复制标题
用于泰勒色散分析和两点激光诱导荧光检测的 3D 打印仪器
DOI：
10.1021/acs.analchem.1c04566
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发表时间：
2022
影响因子：
7.4
通讯作者：
Baker, Christopher A.
中科院分区：
文献类型：
--
作者：
Moser, Meagan R.;Smith, Claire M.;Gutierrez, Genoveve G.;Baker, Christopher A.
精确控制工程生物分子和药物化合物的大小通常对其功能至关重要。用于尺寸表征的标准方法，例如动态光散射或尺寸排阻色谱法，可能是样品密集型的，并且可能无法提供质量或浓度受限的生物系统所需的灵敏度。泰勒色散分析（TDA）是一种经过验证的分析方法，用于直接、无需校准的粒度测定，仅使用nL-pL样品体积。在TDA中，扩散系数在数学上转化为流体动力学半径，通过在良好控制的层流条件下表征分析物的谱带展宽来确定。在这里，我们描述了用于TDA的3D打印仪器的设计和开发，这是第一个提供双点激光诱导荧光（LIF）检测的仪器。该仪器利用完全3D打印的喷射器作为真空源，在毛细管内实现精确和稳定的压力驱动流动，与标准机械泵（1.53%）相比，产生的静压对施加的气体压力的线性响应（R2= 0.997）和静压稳定性（0.05% RSD）提高了30倍。LIF检测系统的设计方面进行了优化，以最大限度地提高激发和发射光轴的S/N，并实现了高灵敏度，如80 pM的检测限的蛋白R-藻红蛋白和低nM的检测限的三个额外的荧光团证明。该仪器的实用性通过在pM浓度下测定R-藻红蛋白的大小来证明。
Precisely controlling the size of engineered biomolecules and pharmaceutical compounds is often critical to their function. Standard methods for size characterization, such as dynamic light scattering or size exclusion chromatography, can be sample intensive and may not provide the sensitivity needed for mass- or concentration-limited biological systems. Taylor dispersion analysis (TDA) is a proven analytical method for direct, calibration-free size determination which utilizes only nL-pL sample volumes. In TDA, diffusion coefficients, which are mathematically transformed to hydrodynamic radii, are determined by characterizing band broadening of an analyte under well-controlled laminar flow conditions. Here, we describe the design and development of a 3D printed instrument for TDA, which is the first such instrument to offer dual-point laser-induced fluorescence (LIF) detection. The instrument utilized a fully 3D printed eductor as a vacuum source for precise and stable pressure-driven flow within a capillary, evidenced by a linear response in generated static pressure to applied gas pressure (R2= 0.997) and a 30-fold improvement in stability of static pressure (0.05% RSD) as compared to a standard mechanical pump (1.53%). Design aspects of the LIF detection system were optimized to maximize S/N for excitation and emission optical axes, and high sensitivity was achieved as evidenced by an 80 pM limit of detection for the protein R-Phycoerythrin and low nM limits of detection for three additional fluorophores. The utility of the instrument was demonstrated via sizing of R-Phycoerythrin at pM concentrations.