Development of a europium-thiosemicarbazide-based nano-optical biosensor for early detection of HE4 in ovarian cancer.

Abstract

Early diagnosis of ovarian cancer remains a major clinical challenge. Human epididymis protein 4 (HE4) has emerged as a promising biomarker for early OC detection. To address limitations of existing HE4 quantification methods, we developed a nano-optical biosensor based on a luminescent europium-thiosemicarbazide (Eu-TSC) complex immobilized within a modified epoxy-cellulose thin-film matrix. The Eu-TSC thin film exhibited characteristic europium excitation and emission bands at 395 nm and 617 nm. Following functionalization with a monoclonal anti-HE4 antibody, the biosensor displayed a concentration-dependent fluorescence quenching response upon HE4 binding. A linear analytical response was obtained over 0.05-0.37 ng mL-1, with LOD of 0.108 ng mL-1. However, the LOQ (0.327 ng mL-1) lies close to the upper limit of the linear range, the practical validated quantification interval is extremely narrow (approximately 0.327-0.37 ng mL-1). This interval is far too narrow to cover the clinical HE4 range, and the present sensor should therefore be regarded as a preliminary proof-of-concept platform rather than a clinically suitable quantitative assay. Time-resolved luminescence measurements are consistent with a quenching-based sensing response, but the present experiments do not distinguish between FRET, PET, or other quenching pathways. Molecular docking and 50 ns molecular dynamics simulations provide a computational rationale for ligand selection and suggest possible surface binding of thiosemicarbazide-based ligands to HE4. Selectivity studies against twelve potential interferents revealed relative responses below 5% for all tested analytes when tested individually at clinically relevant concentrations (n = 3 per interferent; one-way ANOVA with Dunnett's test). However, because interferents were tested individually rather than as pooled mixtures, and because cross-reactivity with structurally related proteins cannot be fully excluded, these selectivity results are preliminary and do not establish excellent selectivity in complex serum. The biosensor was further evaluated using 21 human serum samples (11 OC patients, 10 healthy controls), demonstrating agreement with a commercial HE4 ELISA assay (R2 > 0.98). Recovery values were re-verified for all samples; several values fall outside the commonly accepted 85-115% range and are flagged in Tables 1A and B. Therefore, the analytical accuracy of the present method is preliminary and not satisfactory for clinical quantification. ROC analysis yielded an AUC of 1.000, with 90.9% sensitivity and 100% specificity. These findings are based on a limited case-control cohort (n = 21) and must be interpreted as a preliminary proof-of-concept analytical and clinical evaluation, not as a clinically validated diagnostic platform. Larger prospective, multi-center studies including patients with benign gynecological conditions are required before clinical implementation can be considered.