Integrating Particle Motion Tracking into Thermal Gel Electrophoresis for Label-Free Sugar Sensing

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 10⁶-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.