Ampholyte-free microfluidic free-flow isoelectric focusing via ion-exchange membranes for particle separation

Free-flow isoelectric focusing (FFIEF) is a powerful technique for continuous and high-resolution separation of biomolecules based on their isoelectric points (pIs). However, conventional FFIEF platforms rely heavily on carrier ampholytes (CAs), which are expensive, interfere with detection methods, and often limit reproducibility. Although microfluidic FFIEF (μ-FFIEF) devices offer advantages such as low reagent consumption and integration with lab-on-a-chip systems, the inability to form a stable pH gradient without CAs has hindered practical applications. Therefore, this study addresses the key challenge of establishing a stable, ampholyte-free pH gradient in a microfluidic FFIEF system.

We developed a μ-FFIEF device that generates a continuous and stable pH gradient via proton injection through patterned Nafion ion-exchange membranes, eliminating the need for CAs. Systematic optimization identified 90 V and a 15 μL/min flow rate as optimal conditions for stable pH gradient formation from pH 2 to 9. The electric current remained consistent for over 60 min of operation, confirming the device's electrical and operational stability. Under these optimized conditions, three representative analytes-fluorescent polystyrene particles, Escherichia coli (E. coli), and mRNA-lipid nanoparticles (mRNA-LNPs)-were successfully separated according to their distinct pIs. Zeta potential analysis and fluorescence intensity profiles confirmed spatial resolution and reproducibility of separation. These results confirm that the μ-FFIEF platform enables precise, label-free separation of biologically relevant particles based on their electrochemical properties.

This study presents an ampholyte-free μ-FFIEF platform based on electrochemically driven pH gradient formation using ion-selective membranes. With continuous operation, minimal sample requirements, and compatibility with real biological samples, this system offers strong potential for integration into preparative bioanalysis and lab-on-a-chip analytical workflows. Its ampholyte-free design also facilitates downstream analysis by minimizing interference with spectroscopic or mass spectrometric detection methods.