Sensitivity-enhanced competitive lateral flow immunoassays by polycaprolactone electrospun stacking pad: Estrous determination in whole blood

Lateral flow assays (LFA) are widely adopted in point-of-care diagnostics across a spectrum of applications, due to their simplicity of use and cost-effectiveness. However, in complex biological matrices (e.g., whole blood), LFA sensitivity and analytical performance may be lower than those of laboratory-based techniques. Here, we introduce a polycaprolactone electrospun stacking pad designed to enhance the sensitivity of competitive LFAs. The stacking pad works as an automated pre-incubation step, promoting the analyte interaction with antibody conjugated gold nanoparticles, without affecting the test strip's flow dynamics. We assessed that the stacking pad allows accurate tuning of the flow rate, resulting in a significant increase in sensitivity in whole bovine blood, thereby achieving the required performance for the naked-eye detection of progesterone at the estrous threshold level (2 ng mL⁻¹). The proposed method shows promising potential for broad adaptation to other immunoassays that demand enhanced sensitivity for on-site diagnostics.