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The optimization of experimental conditions for the chemiluminescent determination of peroxidase-mimicking DNAzyme formed at interaction of hemin and its aptamer EAD2 was performed. The effect of concentrations of hydrogen peroxide and luminol, acidity of the substrate solution and chemical nature and concentration of used buffer was estimated. Under optimized conditions a value of detection limit for the DNAzyme was 80 nM. Comparison of the conditions determined in this work with those reported previously showed that the optimization of the composition of substrate solution improved the sensitivity of the chemiluminescent determination of the DNAzyme. The obtained results open up promising perspectives for using the proposed method to improve the sensitivity of DNAzyme-based assays. Acknowledgement. The authors thank the Russian Foundation for Basic Research (NK-13-04-91164/13 and 13-04-91164_GFEN_a) and the National Natural Science Foundations of China (Grant No. 21311120056) for financial support.