Use of Tris-NH4Cl in modified G4/hemin DNAzyme assay with duplexed probes extends life of colorized radical product

The split G-quadruplex/hemin DNAzyme exhibits peroxidase-like activity and catalyzes the oxidation of colorless 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS2−) to the green-colored radical ABTS∙− in the presence of H2O2. The two major challenges to overcome are the relatively low catalytic activity and the short color retention after catalytic action with the substrate ABTS2−. We improved the sensitivity of the G-quadruplex DNAzyme-based DNA assay by utilizing split (duplex) DNAzyme hybridization and adjusting reagent concentrations under limited temperature and pH conditions. Significant improvements in sensitivity and stability were achieved using the activator Triton X-100 and NH4+ as compared to traditional K+. In addition, we demonstrate that the rapid color bleaching of the catalytic substrate ABTS2− is due to the disproportionation of the green-colored reaction product ABTS∙− by H2O2 rather than the degradation of hemin by H2O2. Since residual H2O2 remains after the primary catalytic reaction, the Tris-NH4Cl buffer preserves the radical by removing H2O2 that remains after completion of the primary reaction before it scavenges the radical product. This improved split G-quadruplex DNAzyme formulation can be used to detect various targets conveniently and rapidly, and the test results are stable for more than three days.