Polymeric Probe for Sensitive Diagnosis of Serum Endothelial Cell Adhesion Molecules in Rheumatic Heart Disease

Abstract

Rheumatic fever is an immunologically mediated inflammatory disease that develops following group A streptococcal infection and triggers valvular inflammation that can progress to chronic rheumatic heart disease (RHD), a major cause of cardiovascular morbidity and mortality in low-resource countries. Early diagnosis remains critical for preventing irreversible valvular damage. Elevated levels of circulating endothelial cell adhesion molecules (CAM), particularly E-selectin; ICAM-1; and VCAM-1 have been associated with disease severity, highlighting their potential as diagnostic biomarkers. Here, we report on the development of a polymeric fluorescent probe based on N-(2-hydroxypropyl)methacrylamide (HPMA) for the sensitive detection of serum E-selectin and VCAM-1. The probe incorporates a high-affinity CAM-binding peptide (Esbp or Vbp), fluorescent reporters (FITC or IR783), and a hexa-histidine (His₆)-tag to enable plate surface immobilization. Polymers were synthesized with increasing IR783 molar content to induce fluorescence quenching at high dye loadings while preserving activation upon interaction with serum biomarkers. Their sensing performance was evaluated in both buffer and serum using fluorescence-based assays. Surface-immobilized probes demonstrated dose-dependent detection of labeled E-selectin and VCAM-1 in the microgram scale (1–10 µg/ml) in PBS. In serum, the probe exhibited specific and quantitative binding to E-selectin, while VCAM-1 binding was reduced, likely due to interference from abundant serum proteins. Optimization of IR783 content in the polymer revealed that 2.5 mol % dye loading enabled effective fluorescence activation upon interaction with E-selectin, whereas higher dye loadings led to self-quenching and reduced responsiveness, due to IR783-driven self-assembly that hindered signal activation at the tested biomarker concentrations. The optimized probe successfully detected E-selectin in spiked serum samples. This study demonstrates a simple, modular HPMA-based sensing platform for detecting circulating CAM that can be readily adapted to a broad range of serum biomarkers. By utilizing polymer-based probes that are inherently more stable and lower in cost than antibody-based reagents, the platform addresses key limitations of current diagnostics and offers a promising route toward affordable, rapid assays for RHD in low-resource settings. Further optimization may enhance sensitivity and expand biomarker coverage to improve clinical applicability.