Detection of Sequence. specific DNA Based on Colorimetric Reaction Catalyzed by Mimic Enzyme Combined With Isothermal Exponential Amplification

With the combination of isothermal exponential amplification reaction (IEXPAR) and the chromogenic reaction catalyzed by mimic enzyme formed by hemin and G-quadruplex (G4), we have established a novel colorimetric approach to detect sequence-specific DNA. Through designing amplification template sequence, IEXPAR is initiated by target DNA and produces large amounts of single-strand DNA with G-rich sequences which can fold into G4 structure in the presence of potassium ion. The G4 can bind hemin to form a kind of mimic enzyme (DNAzyme) of peroxidase, which effectively catalyze the H2O2-mediated oxidation of 2-azinobis(3-ethylbenzothiazoline) - 6-sulfonic acid (ABTS), giving rise to a change in solution color. The maximal absorption wavelength of reaction products is at 421 nm. The experimental parameters including the concentration of hemin, ABTS, H2O2 and time of IEXPAR and colorimetric reaction, are optimized systematically. Using the chromogenic reaction, the proposed method can determine target-DNA in the range of 100 fmol/L-10 nmol/L. This method can also clearly discriminate the complete complementary DNA sequence and single-base mismatched DNA, indicating its high specificity.