A novel colorimetric immunosensor based on silk cocoon membrane-integrated ELISA for treponemal antibody detection

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.