A novel dot-blot DNAzyme-linked aptamer assay for protein detection.
In this work, a novel dot-blot DNAzyme-linked aptamer assay (DLAA) for protein detection is developed with thrombin as a model protein. A peroxidase-like DNAzyme which serves as the catalytic label is tethered to a 15-mer thrombin-binding aptamer to form a label-free DNAzyme-linked aptamer probe. Based on specific interaction of the aptamer with target protein immobilized on nitrocellulose membrane, a DNAzyme layer is introduced onto the membrane. The DNAzyme can catalyze the H(2)O(2)-mediated oxidation of 3,3',5,5'-tetramethylbenzidine to produce a colored insoluble product that is apt to be adsorbed onto the nitrocellulose membrane. As a result, blue dots appear on the membrane, in contrast to the colorless background. As the concentration of thrombin increases, the color of dots gets deep. Such a protein concentration-dependent color change can be quantified via an image-processing software, with a detection limit of 0.6 microM. Furthermore, this assay has been applied successfully to the detection of thrombin in biological samples (e.g., human serum), indicating its practicality for bioanalysis.
Zhu
Jinbo
J
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China.
Li
Tao
T
Hu
Jiming
J
Wang
Erkang
E
D052157:Aptamers, Nucleotide / Q000032:analysis*; D021881:DNA, Catalytic / Q000032:analysis*; D006801:Humans; D057230:Limit of Detection; D052156:SELEX Aptamer Technique / Q000379:methods*; D013917:Thrombin / Q000032:analysis*
D052157:Aptamers, Nucleotide; D021881:DNA, Catalytic; D013917:Thrombin