Abstract
The global rise in syphilis incidence and the challenges of early diagnosis highlight the urgent need for convenient diagnostic tools. In this study, we present a colorimetric immunosensor for the detection of Treponema pallidum antibodies (TP-Ab) using a natural silk cocoon membrane (SCM) as the substrate, based on the principle of enzyme-linked immunosorbent assay (ELISA). The TP monoclonal antibody 3H12 and the chimera antigen TP99, comprising epitopes TP15, TP17, and TP47, were sequentially coupled to functionalized SCM to form the SCM-based TP-Ab immunosensor. The detection was quantified by color extraction, and the Corrected I(red) values were used as a judgment parameter. The TP-Ab immunosensor demonstrated a detection limit of 0.48 mIU/mL, lower than the 0.68 mIU/mL limit of commercial microplate ELISA kits. Furthermore, it exhibited no cross-reactivity with antibodies against other pathogens. Testing with a reference serum panel yielded 100% concordance with the reference results. Comprehensive statistical analysis of the Corrected I(red) values from the reference serum panel and banked samples indicated an optimal cutoff value of 0.0921, achieving 100% sensitivity, 97.96% specificity, and high overall accuracy, with an area under the curve of 0.9995 and a 95% confidence interval of 0.9979-1.00. Moreover, a statistically significant correlation with microplate ELISA was observed (r = 0.9217, P < 0.0001). This reliable ELISA-integrated colorimetric immunosensor, featuring a three-dimensional porous structure and inherent bioactivity, contributes to improving the detection performance and provides a rapid and convenient diagnostic solution for syphilis. It is particularly well-suited for resource-limited settings where on-site testing is critical.IMPORTANCESyphilis remains a major global public health concern, particularly in resource-limited settings where early and accurate diagnosis is often hindered by technical and economic barriers. Here, we report a novel treponemal antibody (TP-Ab) immunosensor based on natural silk cocoon membrane (SCM), a biocompatible and porous material with intrinsic bioactivity. By integrating enzyme-linked immunosorbent assay principles and a recombinant TP15-17-47 chimera antigen, the SCM-based immunosensor enables colorimetric detection of TP-Ab with high specificity and visual readout. Unlike traditional methods that require complex instrumentation or expensive reagents, this platform offers a low-cost, portable, and easy-to-use alternative suitable for on-site or point-of-care testing. The findings highlight the potential of silk-derived biosensors in a promising direction for syphilis screening technologies.