Continuous Monitoring of Tumor Necrosis Factor Alpha Using Online Affinity Micro Free-Flow Electrophoresis

Real-time monitoring of biochemical messengers such as Tumor Necrosis Factor Alpha (TNF-α) is critical for understanding dynamic biological processes. However, current detection methods are limited in their ability to provide real-time data.

Traditional methods for quantifying biochemical messengers, like ELISA, offer only static snapshots, missing the dynamic fluctuations crucial for real-time understanding. Recent approaches have attempted continuous monitoring by coupling immunoassays with detection techniques such as capillary electrophoresis (CE) and fluorescence anisotropy (FA). Our research introduces a simplified, continuous, real-time monitoring platform that integrates micro free-flow electrophoresis (μFFE) with affinity reagents, specifically aptamers.

μFFE provides a powerful approach for continuous, real-time analysis of biomolecular interactions. To achieve selective detection, we employ aptamers, whose small size and high stability offer distinct advantages over antibodies. Upon binding to target molecules, aptamers exhibit a greater shift in electrophoretic mobility, which enhances resolution between bound and unbound species during μFFE. This compact, non-competitive system enables high-resolution, real-time quantification of TNF-α.

Significant outcomes using this approach included clear separation of bound and unbound aptamer peaks, establishment of a linear calibration curve for TNF-α that confirms the feasibility of the assay, and reproducible signals achieved through optimization of buffer composition and aptamer handling. Ongoing work is focused on enhancing temporal response and improving the assay’s limit of detection.

The insights gained from this work could pave the way for advancements in diagnostics and therapeutic monitoring, with broader applications in disease research and clinical diagnostics.