A novel dot-blot DNAzyme-linked aptamer assay for protein detection |
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Authors: | Jinbo Zhu Tao Li Jiming Hu Erkang Wang |
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Affiliation: | (1) State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, China;(2) Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072, China; |
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Abstract: | 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 H2O2-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 μM. Furthermore, this assay has been applied successfully to the detection of thrombin in biological samples (e.g., human serum), indicating its practicality for bioanalysis. |
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