Peroxidase activity of DNA aptamer-pt complexes prepared with cisplatin.

DNA aptamers carrying Pt nanoparticles prepared with cisplatin showed peroxidase enzymatic activity while retaining the specific binding ability of the aptamers. Optimal preparation conditions of DNA-Pt complex prepared with cisplatin were investigated on the synthesis at pH 7-11, a reaction time of 1-18 h and 90 degrees C. The enzymatic reaction of DNA-Pt complex obeyed Michaelis-Menten kinetics. K(M) for the DNA-Pt complex was found to be of the same order as K(M) for hemin and hemin-DNA complex, but one order of magnitude higher than that of horseradish peroxidase. A sandwich type of DNA enzyme-linked aptamer assay (DLAA) using DNA-Pt complex successively detected target protein of thrombin. DLAA using DNA-Pt complex fractioned by ultrafiltration membranes having a molecular weight cut-off of 30 000 and 300 000 showed 1.9-times higher sensitivity than DLAA using DNA-Pt complex without fraction. The DNA-Pt complex having specific size was effective for the sensitive detection of thrombin in DLAA.

Preparation of a DNA aptamer-Pt complex and its use in the colorimetric sensing of thrombin and anti-thrombin antibodies.

DNA aptamers carrying Pt nanoparticles were prepared by the reaction of DNA aptamers (without functionalization with biotin, thiol, or other reactive groups) with K 2[PtCl 4] in solution at 60-90 degrees C. The DNA-Pt complexes possessed peroxidase enzymatic activity while retaining the specific binding ability of the aptamers. The enzymatic reaction of these complexes obeyed Michaelis-Menten kinetics. K M for the DNA-Pt complex was found to be on the same order as K M for hemin and hemin-DNA complex but 1 or 2 orders of magnitude higher than that of horseradish peroxidase. The rate of the reaction catalyzed by the DNA-Pt complex, k cat, was found to be on the same order as that of hemin and hemin-DNA complex but 2 or 3 orders of magnitude lower than that of horseradish peroxidase. Two types of DNAzyme-linked aptamer assays (DLAAs) were developed using these complexes, which successfully detected target proteins, with the sandwich type of DLAA targeting thrombin and the competitive type of DLAA targeting anti-thrombin IgA/G/M in serum. The DNA-Pt complexes retained their peroxidase enzymatic activity even after heat treatment. DLAAs having high thermal stability were developed using these complexes, which were free of animal and plant matter because neither antibodies nor horseradish peroxidase were used in their synthesis.