Cornejo Mario A, Linz Thomas H
Department of Chemistry, Wayne State University, 5101 Cass Ave, Detroit, Michigan 48202, United States.
ACS Sens. 2025 Jan 24;10(1):204-212. doi: 10.1021/acssensors.4c02042. Epub 2025 Jan 3.
Bioanalytical sensors are adept at quantifying target analytes from complex sample matrices with high sensitivity, but their multiplexing capacity is limited. Conversely, analytical separations afford great multiplexing capacity but typically require analyte labeling to increase sensitivity. Here, we report the development of a separation-based sensor to sensitively quantify unlabeled polysaccharides using particle motion tracking within a microfluidic electrophoresis platform. Carboxymethyl dextran (20 kDa) was spiked into Pluronic thermal gel along with fluorescent nanoparticles (200 nm diameter) and loaded into single-channel microfluidic devices. Upon voltage application, the soluble sugar enriched into a concentrated band that induced motion of the insoluble particles as it passed. Bead displacement was tracked over time to produce electropherograms where peak areas were proportional to analyte concentrations. Key studies herein established the range of acceptable operating conditions (e.g., gel concentration, temperature) to characterize how the temperature-dependent rigidity of thermal gel influenced the analysis. Data processing strategies were then evaluated to identify conditions (e.g., exposure intervals, particle averaging, motion directionality) to maximize sensitivity. The quantitative response of the method was evaluated over a broad concentration range (0.5-5000 nM) where detection limits were found to be 520 pM for the 20 kDa sugar, providing a 10-fold superior mass LOD than a gold standard UV-vis absorbance method. Studies into the detection mechanism found that sensitivity was dependent on the molecular weight of the sugar as larger sugars produced greater responses. Collectively, these studies established best practices for integrating particle sensing into thermal gel separations for label-free polysaccharide quantitation.
生物分析传感器擅长从复杂样本基质中高灵敏度地定量目标分析物，但其多重分析能力有限。相反，分析分离具有很强的多重分析能力，但通常需要对分析物进行标记以提高灵敏度。在此，我们报告了一种基于分离的传感器的开发，该传感器利用微流控电泳平台内的粒子运动跟踪来灵敏地定量未标记的多糖。将羧甲基葡聚糖（20 kDa）与荧光纳米颗粒（直径200 nm）一起加入普朗尼克热凝胶中，并加载到单通道微流控装置中。施加电压后，可溶性糖富集形成一个浓缩带，当它通过时会引起不溶性颗粒的运动。随着时间的推移跟踪珠子的位移以产生电泳图，其中峰面积与分析物浓度成正比。本文的关键研究确定了可接受的操作条件范围（例如凝胶浓度、温度），以表征热凝胶的温度依赖性刚性如何影响分析。然后评估数据处理策略以确定条件（例如曝光间隔、粒子平均、运动方向性）以最大化灵敏度。在较宽的浓度范围（0.5 - 5000 nM）内评估了该方法的定量响应，发现对于20 kDa的糖，检测限为520 pM，比金标准紫外 - 可见吸光光度法的质量检测限高10倍。对检测机制的研究发现，灵敏度取决于糖的分子量，因为较大的糖产生更大的响应。总的来说，这些研究确立了将粒子传感集成到热凝胶分离中进行无标记多糖定量的最佳实践。
