Signal-on label-free electrochemical lateral flow immunoassay with secondary antibody-mediated mass amplification for...

Chlamydia trachomatis is a prevalent, often asymptomatic sexually transmitted infection, while molecular tests remain constrained by cost and infrastructure. Here, we report a label-free electrochemical lateral flow immunoassay (eLFIA) designed to address these challenges through an intuitive signal-on readout implemented in an indirect competitive format. A secondary antibody-mediated interfacial mass-loading strategy was introduced to amplify steric blocking and improve signal discrimination without requiring reporter labels. Mechanistically, sequential binding of primary and secondary antibodies forms a bulky protein assembly at the electrode-membrane interface, increasing charge-transfer resistance and reducing the effective electroactive area, thereby amplifying signal discrimination between positive and negative samples. Assay configurations were evaluated and fitted using a four-parameter logistic model. Incorporation of a secondary antibody delivered approximately a five-fold sensitivity enhancement relative to the single-antibody format. The optimized platform achieved a limit of detection of 10 ng/mL with a practical quantitative working range of ∼10-500 ng/mL (CV < 15%). Accuracy in simulated urine, artificial saliva, and simulated vaginal fluid yielded recoveries of ∼80-120% with matrix-independent calibration behavior. A portable NFC-enabled reader demonstrated the signal-on response, supporting potential translation to home-based testing. This secondary antibody-mediated signal-on, label-free eLFIA provides a scalable and user-oriented platform for C. trachomatis screening and surveillance, with potential utility in home-based testing to support infection control.