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Anal Chim Acta. 2013 Jul 25;788:171-6. doi: 10.1016/j.aca.2013.06.029. Epub 2013 Jun 24.

A colorimetric method for protein assay via exonuclease III-assisted signal attenuation strategy and specific DNA-protein interaction.

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Department of Biochemistry and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China.


Taking advantage of exonuclease III (Exo III)-assisted signal attenuation strategy and the protection of DNA from Exo III-mediated digestion by specific DNA-protein interaction, a colorimetric method is proposed in this paper for protein assay. Specifically, in the absence of target protein, Exo III-assisted signal attenuation can be achieved by digesting the report DNA in a complex formed by the hybridization of a report DNA and a probe DNA. Nevertheless, in the presence of target protein, the binding of the analyte to the probe DNA will inhibit the Exo III-assisted nucleotides cleavage, so that cyclic signal attenuation is blocked. Therefore, a bridge can be established between the concentration of target protein and the degree of the attenuation of the obtained signal, and the relationship can be shown by the surface plasmon changes caused by the report DNA-induced aggregation of DNA-modified gold nanoparticles (AuNPs). Our method can also have considerable sensitivity and selectivity, which has been demonstrated by the assay of human α-thrombin. Furthermore, by simply changing the sequence of the probe DNA, we can expand the application of our method to not only aptamer binding proteins but also DNA binding proteins, thus we have also used this method to analyze a specific serological marker for systemic lupus erythematosus (SLE) in this study. With a broad detection range of 1.3-133 nM and a detection limit of 0.61 nM (S/N=3), it may hold great promise for clinical application.


Anti-double-stranded DNA antibody; DNA–protein interaction; Exonuclease III; Gold nanoparticles; Human α-thrombin

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