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

Davood  Rajabian; Hadi  Ravan; Shahram  Salari

doi:10.18502/abi.v3i1.19349

Davood Rajabian 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
Shahram Salari School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

DOI: https://doi.org/10.18502/abi.v3i1.19349

Keywords: DNA Nanotechnology, DNAzyme, TMSD, Colorimetric Technique

Abstract

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.