One-Step Label-Free Electrochemical Lateral Flow Immunosensor for SARS-CoV-2 Antigen Detection

Rapid and ultrasensitive diagnostic tests for COVID-19 remain crucial, yet conventional lateral flow antigen kits are limited by their reliance on labeled probes and suboptimal sensitivity at low viral loads. Here, we present a label-free electrochemical antigen test kit (free-EATK) that enables one-step detection of the SARS-CoV-2 N protein without the need for conjugate pads, covalently labeled redox probes, or signal normalization schemes. The system integrates a nitrocellulose-coated electrode, a redox pad preloaded with [Ru-(NH₃)₆]³⁺, and a sample inlet. Upon sample application, the immunocomplex forms directly at the sensing zone, followed by diffusion of the redox mediator toward the electrode surface. Signal generation is achieved through direct anodic square wave voltammetry, offering sharp oxidation peaks without additional surface modification or multistep protocols. The method achieves a detection limit of 0.69 pg/mL, with high reproducibility (RSD < 10%, <i>n</i> = 10), sensitivity of 91.7%, and specificity of 100% across clinical samples (<i>n</i> = 24). The free-EATK offers a simple, robust, and reproducible alternative for early stage infectious disease screening, particularly in settings where conventional labels or complex assay formats are impractical.