The development of immunosensing assays for in vitro diagnostics has attracted great attention in recent years. Various substrate materials and immobilization methods of biomolecules were exploited for immunosensors, but their bioactivity and longevity have been facing serious challenges. To address this limitation, we investigated a natural silk cocoon membrane as immunosensing substrate material. By using its intrinsic properties, the target biomolecules were immobilized on the membrane through directional immunoaffinity recognition. The silk cocoon membrane-based immunosensor showed great potential for both qualitative and quantitative immunoassays, through naked-eye observation or analyzing the change in red color intensity, respectively. The immunosensor exhibited significant detection capability for anti-D (titer 1:1024) sensitized red blood cells. The colorimetric responses of concentrations ranged from 1 μg/mL to 1 ng/mL, and the detection limit for anti-D was 3.4 ng/mL. The immunosensor also showed excellent stability for the immobilized antibodies when stored at 4 and 25 °C; the bioactivity remained unchanged or slightly declined within 40 weeks. Even at 37 °C, the bioactivity began to decline after 12 weeks. This current work highlights the potential of using the natural silk cocoon membrane as a substrate for a versatile and thermally stable immunosensing platform for application in immunoassays.

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. IMPORTANCE: Syphilis 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.

A novel microcarrier based on red blood cells was prepared and characterized. Proof of principle was demonstrated by using sheep red blood cells that were treated with glutaraldehyde to render the cells generally resistant to hemolysis. Magnetic beads of approximately 10 nm in diameter were prepared and attached to the surface of glutaraldehyde-treated sheep red blood cells by electrostatic interaction at various concentrations. The composites were characterized by scanning electron microscopy. Dilute coverage of magnetic beads allowed the red blood cells to be separated from solution in the presence of a magnetic field, eliminating the need for centrifugation. The magnetized glutaraldehyde-treated red blood cells were then functionalized with human immunoglobulin and used in flow cytometry for the determination of antihuman immunoglobulin. The results indicated that such magnetized glutaraldehyde-treated red blood cell may be used as carriers in flow cytometry. The approach may allow the preparation of carriers using other red blood cells.