Universal All-In-One Lateral Flow Immunoassay with Triple Signal Amplification for Ultrasensitive and Simple Self-Testing of <i>Treponema pallidum</i> Antibodies

Lateral flow immunoassay (LFIA) is valued for its simplicity and rapidity for on-site screening, however, it experienced false negatives in real sample analysis due to low sensitivity. Although many signal amplification techniques can improve the sensitivity, they usually require additional complicated steps. To address these issues, taking <i>Treponema pallidum</i> (<i>T. pallidum</i>) antibodies as a model detecting target, herein, we report an all-in-one LFIA (AIO-LFIA) with triple-step signal amplification to significantly improve sensitivity while maintaining simplicity. This LFIA utilizes a biotin-streptavidin system for initial signal amplification, followed by introducing a release controller with a specific imprinted structure for timed multicomponent release, which avoids the extra steps when adding components in traditional LFIA. Particularly, a 3D-printed programmed metal in situ growth (MISG) device is integrated to localize signal enhancement at specific sites, overcoming limitations of traditional MISG and substantially reducing reagent usage and assay time, and the nitrocellulose membrane surface was much cleaner than the conventional approach, which facilitates signal readout. After optimization, the proposed AIO-LFIA is capable of visual detection down to 1 pg/mL<i>T. pallidum</i> antibodies in 15 min, 1000-fold lower than the gold nanoparticle-based LFIA. In clinical testing of 152 samples, the AIO-LFIA can distinguish all positive samples, outperforming commercial LFIA which missed those positive samples with relatively low antibody levels. Thus, this study presents a universal ultrasensitive and reliable AIO-LFIA strategy for infectious diseases self-testing, providing an effective promising prospect to address the challenge over emerging infectious diseases in the future.