Detection of T4 polynucleotide kinase based on a MnO2 nanosheet-3,3′,5,5′-tetramethylbenzidine (TMB) colorimetric system

Abstract

Determination of nucleotide kinase activity is valuable due to its importance in regulating nucleic acid metabolism. In this work, a novel and simple colorimetric method for discriminative detection of T4 polynucleotide kinase (PNK) through a MnO₂ nanosheet-3,3′,5,5′-tetramethylbenzidine (TMB) colorimetric system is developed. In aqueous solution at pH 7.4, the MnO₂ nanosheet, a mimic enzyme, rapidly converts colourless TMB to a blue oxidized form. In the presence of T4PNK, a hairpin DNA could be phosphorylated and further cleaved by λ exonuclease (λ exo) and the ssDNA fragments would adsorb on the surface of the MnO₂ nanosheet, resulting in the inhibition of the catalytic effect of the MnO₂ nanosheet to the oxidation of TMB and weakening of the blue color of oxidized TMB with the absorption peak centred at 652 nm. The presence of T4PNK can in this way easily be detected with the naked eye, based on the obvious colour change. The experimental results revealed that the advanced strategy was sensitive for detecting T4PNK in the concentration range from 0.005 to 10 U mL⁻¹ with a detection limit of 0.005 U mL⁻¹. In addition, the relative standard deviation was 3.92% in 3 repetitive assays of 10 U mL⁻¹ T4PNK, indicating that the reproducibility of this strategy was acceptable. By comparison with other relevant fluorogenic assays, the sensitivity of the developed chromogenic assays was satisfactory. The present approach opens the possibility for the design of simple and sensitive colorimetric assays for polynucleotide kinase using inexpensive and available TMB as a universal chromogenic compound. Therefore, besides desirable sensitivity and selectivity, due to the colorimetric system, the developed method for T4PNK detection also showed cost-effective, reliable and simplified operations and strategy, which has considerable potential for biological process research.