A protease-free colorimetric strategy based on an aptamer and DCHA-mediated cyclic amplification for the ultrasensitive biosensing of Escherichia coli

Herein, a novel protease-free colorimetric strategy was proposed for the ultrasensitive biosensing of Escherichia coli ( E. coli , strain CMCC 44102). This strategy employed an E. coli whole-cell aptamer for specific recognition and dual-catalyst hairpin assembly (DCHA)-mediated cyclic amplification. Specifically, four nucleic acid strands (Apt, H1, H2, and S) mediated DCHA cyclic amplification, while the G-quadruplex/hemin complex acted as a DNAzyme to catalyze the oxidation of ABTS 2− for signal generation. The formation of H2-S complexes and dissociated G strands depended on the quantity of Apt-induced DCHA. The relationship between Apt and E. coli concentration was established by measuring the activity of the G-quadruplex/hemin DNAzyme. Under optimal conditions, the proposed method exhibited a sensitivity of 69 CFU·mL −1 with a linear range of 1 × 10 2 to 1 × 10 7  CFU·mL −1 . Furthermore, this method demonstrated good selectivity, with recovery rates ranging from 92.80% to 99.20% and relative standard deviations (RSD) below 3.65% in tap water and milk samples. In summary, this strategy is label-free, protease-free, cost-effective, and rapid. Given its modular design, substitution with aptamers specific to other targets would extend this approach to a wide range of applications.