Journal
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
Volume 397, Issue 7, Pages 2923-2927Publisher
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-010-3802-9
Keywords
DNAzyme; Aptamer; Thrombin; Dot-blot assay
Funding
- National Natural Science Foundation of China [20735003, 20890020]
- 973 projects [2009CB930100, 2010CB933600]
- Chinese Academy of Sciences [KJCX2.YW.H11]
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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 mu M. 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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