Enzyme-free, Colorimetric DNA Detection System Powered by Autocatalytic Assembly Circuits and G-quadruplex DNAzymes

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. Conclusion: This detection method is simple, cost-effective, and highly sensitive, with potential for point-of-care diagnostics, food safety testing, and environmental monitoring. Its enzyme-free nature, label-free operation, and modular design make it adaptable to other nucleic acid targets.