A simple and sensitive fluorescence biosensor based on TdT/Cas12a cascade amplification for assessing sperm DNA integrity

The integrity of sperm DNA is essential for proper fertilization and embryonic development. Various methods exist to evaluate sperm DNA fragmentation. However, they are frequently hindered by high costs, complex procedures, and low precision. Herein, a simple and sensitive fluorescence biosensor was developed using the terminal deoxynucleotidyl transferase (TdT) and CRISPR/Cas12a system for analyzing sperm DNA fragmentation. This biosensor efficiently measures the number of 3′-OH, which correspond to the mean number of sperm DNA breakpoints (MDB). In the presence of TdT, the dATP nucleotide polymerized at the 3′-OH end of the primer, generating numerous polyA-tails, which were detected by CRISPR-guided RNA (crRNA) at the same time. Ultimately, the trans -cleavage activity of CRISPR/Cas12a was activated, which then cleaved the spherical nucleic acids (SNA) reporter, generating amplified fluorescence signal. The proposed method exhibited a broad linear detection for the quantity of 3′-OH ends in single-stranded DNA, from 1 fM to 10 pM, with a limit of detection of 2.5 fM. Moreover, the platform was successfully validated with clinical sperm samples and demonstrated a stronger correlation with sperm motility compared to traditional DNA fragmentation index (DFI) test. Therefore, the developed method offers advantages such as ease of use, elevated sensitivity, and mimimal instrumentation requirements, making it suitable for clinical application.