Bioinspired Construction Of DNA-based Catalysts And Their Application On Visible Detection

Since native enzymes are the catalysts for regulating biological processes,they have several advantages involving high efficiency,high substrate specificity and mild reaction condition.However,there also exist some limitations in their practical applications,such as low stability,high price and poor storage stability.To date,diverse artificial catalysts have emerged to mimic the functions of natural enzymes,which have been widely used in the fields of chemical engineering,medicine,food,agriculture and environment.Herein,DNA-based biocatalysts have been designed and constructed by regulating the sequence composition and the secondary structure of oligonucleotides,and successfully applied in the visible detection of exonuclease and alkaline phosphatase in human blood with high sensitivity and selectivity.Firstly,a series of short oligonucleotides with different number of guanines?T_xG_y,x+y=5,y=0³?,as well as?T₃G₂?_n and?T₂G₃?_n?n=1¹0?with various repeat units were designed to construct DNA-based peroxidase mimetics through directCu???-coordination.Contiguousguaninesarecrucialtointrinsic peroxidase-like activities of DNA-Cu???complexes.The catalytic activity of T₂G₃-Cu???is 36-times higher than that of Cu²⁺alone.With the increase of repeat units,the catalytic performance of?T₃G₂?_n-Cu???and?T₂G₃?_n-Cu????n=1¹0?undergo three distinct stages,namely rapid increase,slow increase and slight decrease.The optimal repeat units in these two systems was ere determined to be 8 and 6,respectively.In the presence of hydrogen peroxide?H₂O₂?,DNA-Cu???complexes have the capability to catalyze the oxidation of 3',3',5',5'-tetramethyl benzidine?TMB?at acidic pH,dopamine?DA?at neutral pH and methylene blue?MB?at alkaline pH.Importantly,S1 nuclease significantly inhibits the enzymatic activity of DNA-Cu???through digesting DNA scaffolds into small fragments.Therefore,a colorimetric assay of S1 nuclease was developed,with the limit of detection of 0.017U/mL and the linear response of 0.001⁰.1 U/mL.Next,a series of oligonucleotides with different composition?A,T,G,C?and strand length were designed,and utilized to catalyze H₂O₂-mediated oxidation of TMB in the presence of acetic acid.Guanine?G?-rich sequences are capable of catalyzing the formation of peracetic acid?PAA?between acetic acid and H₂O₂.In the meanwhile,DNA molecules can recognize organic substrate TMB,which can be in situ oxidized by generated PAA.It is found that the strand length of oligonucleotides plays a critical role on the generation of PAA,and the formation of secondary structures retards the catalytic activity of DNA.At 25 ^oC,enzyme kinetic parameters of TG₃T and?TG₃T?₂ were measured in 10 mM and 30 mM sodium acetate?NaAc?,respectively.The affinity of TG₃T/?TG₃T?₂ toward H₂O₂ in 30 mM NaAc was 3.3-times higher than that in 10 mM NaAc.Moreover,the affinity of?TG₃T?₂ toward TMB was 2-fold higher than that of TG₃T.In addition,phosphoric acid was found to effectively inhibit the catalytic activity of DNA in the oxidation of TMB.Thus,both 3'and 5'-phosphorylated?TG₃T?₂ was chosen to develop a colorimetric detection of alkaline phosphatase?ALP?with the limit of detectionof is0.057 mU/mL and the linear range of 1⁴0 mU/mL.The proposed method has strong anti-interference ability against other proteins in human blood.