Immunoassay development for the detection of sperm-derived miRNAs : Towards a biosensing platform for male fertility assessment

Sammanfattning

Male infertility represents a significant global health challenge, and sperm-derived microRNAs (miRNAs) have emerged as promising biomarkers associated with fertility and embryo development in in vitro fertilization (IVF). This study aimed to develop a fluorophore-linked immunosorbent assay (FLISA) for miRNA detection based on DNA–RNA hybrid recognition using the S9.6 antibody. The assay was implemented as a proof-of-concept system using fluorescent detection of labeled DNA probes and synthetic miRNA targets. Key assay components, including fluorophore response, hybrid formation, antibody immobilization, and S9.6 affinity for DNA–RNA hybrids were experimentally evaluated. The fluorophore standard curve showed a linear concentration-dependent response, and formation of DNA–RNA hybrids was verified by Bioanalyzer RNA Nano analysis. Furthermore, a fluorescence polarization assay suggested preferential binding of the S9.6 antibody to DNA–RNA hybrids over single-stranded DNA. Similarly, the FLISA system showed indications of higher fluorescence signals for DNA–RNA hybrids compared to single-stranded DNA controls; however, the observed signals did not consistently exceed background levels, limiting reliable target detection. In conclusion, although key components of the system were successfully validated, the assay did not achieve the sensitivity required for reliable miRNA detection at biologically relevant concentrations. Further optimization, particularly through quantitative characterization of S9.6 binding affinity and improved signal amplification, is required before the assay format can be translated into point-of-care diagnostic applications for male fertility assessment.