بيان و خالص‌سازي Super Renilla Luciferase8-(Linker-B72)2 متصل‌شونده به گانگليوزيد GM1 و ارزيابي الحاق آن به يك رنگ فلورسنت

According to the World Health Organization report, approximately 17.5% of adults worldwide were affected by infertility issues in 2023. Infertility is the primary factor in about 20% of cases and contributes to 30-40% of cases of male infertility. Several factors can lead to infertility in men, with sperm functional disorders being a significant cause. One such disorder is impaired capacitation. This critical process occurs in the female reproductive system after ejaculation. During this process, non-fertilizing sperm are transformed into sperm capable of fertilizing an egg. During capacitation, GM1 gangliosides in the sperm membrane rearrange and accumulate in the apical-middle region, as a reliable marker for detecting capacitation Given the importance of sperm capacitation and its disorders, it is essential to develop accurate and efficient diagnostic methods in this field. In this research, an attempt was made to create a biosensor based on the Renilla luciferase enzyme called Super Renilla Luciferase8-(Linker-B72)2 with the aim of binding to ganglioside GM1. The results of the eva‎luation of the designed biosensor showed that the binding of peptide B72 did not affect the performance of the Super Renilla Luciferase8 enzyme. The results obtained from the fluorescent microscopy studies showed that the binding of the Super Renilla Luciferase8 enzyme to peptide B72 reduced its binding affinity to ganglioside GM1. Cell detection studies showed that at a concentration of 10 nM of the biosensor, there was a significant difference between the luminescent signal of sperm cells in the capacitated state compared to the non-capacitated state. Despite the positive characteristics that this biosensor has shown so far, presenting this assay as a diagnostic method in the clinical field requires further studies and optimization for future applications. In general, this study attempted to design a biosensor that can detect capacitated sperm and is clinically efficient.

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