Construction And Sensing Application Of DNA-based Artificial Enzyme

Compared with natural enzymes,artificial enzymes are advantageous in several aspects,such as low cost,ease of mass production,high stability,long-term storage,and size/composition dependent activity.Until now,lots of artificial enzymes have been studied to mimic various natural enzymes for wide applications.Herein,DNA-based artificial enzymes are facilely constructed and the critical factors of their peroxidase mimetics activities are investigated.Then,those DNA-based artificial enzymes are designed for colorimetric sensings with high sensitivity and selectivity.Firstly,DNA-based peroxidase mimetics are facilely constructed through Cu?II?-coordination with different oligonucleotides involving G₂₀,C₂₀,A₂₀ and T₂₀,respectively,with high peroxidase mimicking activity as well as high stability against proteins.Peroxidase-like activities of DNA-Cu?II?complexes are greatly associated with the sequence composition of DNA templates,which decrease in the following order: G₂₀>C₂₀>A₂₀>T₂₀.G₂₀-Cu?II?complex?[Cu2+]/[base]=0.05?possesses the Km value of 0.257 mM toward 3,3?,5,5?-tetramethylbenzidine and 102.3 mM toward hydrogen peroxide at 25 oC.G₂₀-Cu?II?complexes are employed to develop a colorimetric turn-on assay of alkaline phosphatase with high sensitivity and selectivity,on the basis of pyrophosphate-induced inhibition of their intrinsic peroxidase-like activities.The limit of detection is achieved as 0.84 U/L with the linear response region of ₂₀^<sub>200 U/L.Such colorimetric assay system is probably applicable for the quantitative determination of ALP in biological fluids.Next,a novel G-quadruplex DNAzyme based probe has been designed for the colorimetric sensing of target DNA.This neotype G-quadruplex based DNAzyme with high peroxidase-like activity exhibits the Km value of 0.153 mM toward 3,3',5,5'-tetramethylbenzidine and 64.1 mM toward hydrogen peroxide.Then this G-quadruplex-hemin complex is employed to develop a colorimetric turn-off assay of target DNA with high sensitivity and selectivity,on the basis of target DNA-induced inhibition of its intrinsic peroxidase-like activities.This sensor allows the detection of target DNA at a concentration as low as 0.4 nmol L-1 with the linear response region of 5⁷0 nmol L-1.The proposed sensor is easy to fabricate,which avoids the tedious and expensive labeling procedures,and exhibits high selectivity against single-base mismatched DNA.