Tehran University of Medical Sciences Acta Biochimica Iranica 0001-5261 3 1 2025 03 21 36 45 EN Rajabian Davood Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran Hadi Ravan Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran Salari Shahram student Master's Degree Human Genetics School of Medicine , Mashhad University of Medical Sciences, Mashhad, Iran. 2025 04 27 2025 06 05 Objectives: To develop an enzyme-free colorimetric DNA detection system combining autocatalytic assembly circuits with G-quadruplex DNAzymes for highly sensitive and visual DNA identification. Methods: The platform utilized toehold-mediated strand displacement (TMSD) integrated with autocatalytic hairpin circuits and a G-quadruplex DNAzyme. Buffer conditions, hemin concentration, temperature, and reaction time were optimized. Spectrophotometric detection at 416 nm was employed, and NUPACK software was used for oligonucleotide design validation. Results: The optimized system exhibited a detection limit as low as 31 pM. Clear electrophoretic band patterns confirmed correct structure formation. The system showed high reproducibility, sensitivity, and a visible signal without enzymatic components. Conclusions: This detection method is simple, cost-effective, and highly sensitive, with potential for point-of-care diagnostics, food safety testing, and environmental monitoring. The enzyme-free nature, label-free operation, and modular design make it adaptable to other nucleic acid targets.    https://abi.tums.ac.ir/index.php/abi/article/view/108 https://abi.tums.ac.ir/index.php/abi/article/download/108/100