17β-Estradiol point-of-care test by commercial pregnancy test strips based on target-triggered CRISPR/Cas12a cleavage activity

17β-Estradiol (E2) serves as both a reproductive accelerator and growth promoter, yet its uncontrolled application induces severe endocrine dysfunction. Point-of-care testing (POCT) has emerged as a promising analytical platform for on-site E2 quantification, due to its inherent advantages in terms of sensitive, specific, cost-effective, and instrument-free. We developed a novel strategy combining the high collateral cleavage activity of CRISPR/Cas12a with the strong amplification power of dual-cycle reaction, translating target presence into a colorimetric signal on pregnancy test strip (PTS). In the presence of E2, the dual-cycle amplification was initiated to produce HP2–HP3 (Hairpin DNA 2–Hairpin DNA 3) duplexes. These duplexes then bound to the Cas12a-crRNA complex, activating its trans-cleavage activity. The activated Cas12a subsequently cleaved the ssDNA linkers in the MBs (magnetic beads)-ssDNA (single-stranded DNA)-hCG (human chorionic gonadotropin) conjugates, releasing hCG for quantitative E2 detection through strip color intensity measurement. By integrating CRISPR/Cas12a's high cleavage efficiency with dual-cycle amplification, a visible biosensor was developed, with a linear range of 1.0 × 10⁻¹–200.0 pM and a detection limit of 0.0403 pM for E2. In conclusion, an E2 detection platform characterized by prominent sensitivity and convenience was established for monitoring spiked E2 in milk and urine, providing a significant reference for the POCT detection of other targets.