Two orders of magnitude improvement in detection limit of lateral flow assays using isotachophoresis

Lateral flow immunoassays (LFA) are one of the most prevalent point-of-care (POC) diagnostics due to their simplicity, low cost, and robust operation. A common criticism of LFA tests is that they have poor detection limits compared to those of analytical techniques, like ELISA, which confines their application as a diagnostic tool. The low detection limit of LFA and associated long equilibration times are due to kinetically limited surface reactions that result from low target concentration. Here, we use isotachophoresis (ITP), a powerful electrokinetic preconcentration and separation technique, to focus target analytes into a thin band and transport them to the LFA capture line, resulting in a dramatic increase in the surface reaction rate and equilibrium binding. We show that ITP is able to improve the limit of detection (LoD) of LFA by 400-fold for 90 s assay time and by 160-fold for a longer 5 min time scale. ITP-enhanced LFA (ITP-LF) also shows up to 30% target extraction from 100 μL of the sample, whereas conventional LFA captures less than 1% of the target. ITP improves the LoD of LFA to the level of some lab-based immunoassays, such as ELISA, and may provide sufficient analytical sensitivity for application to a broader range of analytes and diseases that require higher sensitivity and lower detection limits.