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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Acknowledgments
This work is supported by the National Natural Science Foundation of China with the Grant 20735003 and 20890020, 973 projects 2009CB930100 and 2010CB933600, and the Chinese Academy of Sciences KJCX2.YW.H11.
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Zhu, J., Li, T., Hu, J. et al. A novel dot-blot DNAzyme-linked aptamer assay for protein detection. Anal Bioanal Chem 397, 2923–2927 (2010). https://doi.org/10.1007/s00216-010-3802-9
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DOI: https://doi.org/10.1007/s00216-010-3802-9