Kanamycin triggered nanozyme for homogeneous and amplified colorimetric detection of T4 polynucleotide kinase

It is of significance to develop efficient methods for detecting the activity of T4 polynucleotide kinase (T4 PNK) due to its essential role in the modulation of different life activities. In this work, we constructed a novel nanozyme using Kanamycin (KANA) as a trigger for the [Fe(CN)6]3-coordinated Cu2(OH)3NO3 (Cu2(OH)3NO3/ [Fe(CN)6]3-) nanorods, and designed an amplified colorimetric method to detect T4 PNK. That was, the free KANA efficiently triggered the peroxidase-like activity of Cu2(OH)3NO3/[Fe(CN)6]3-, while the bound KANA by its aptamer lost the stimulative capability for the nanomaterials. On the basis of the bioreaction regulated generation of the KANA aptamer, a highly sensitive colorimetric assay aided by the rolling circle amplification (RCA) reaction for the detection of T4 PNK was realized. Results showed that this assay can detect T4 PNK from 1.0 x 10-3 to 10.0 U/mL, with a limit of detection (LOD) of 1.42 x 10-4 U/mL. The assay also showed acceptable performance in the detection of T4 PNK in serum samples. In addition to the satisfactory sensitivity and selectivity, the displayed T4 PNK assay also presented merits of operational convenience, without labeling or immobilization process and did not require costly instrument. It is expected that the KANA as a stimulator would have extended biosensing applications by coupling various bioreactions that can produce the KANA aptamer.

Automated summary

In this study, a novel nanozyme was successfully constructed to detect the activity of T4 polynucleotide kinase (T4 PNK) using Kanamycin (KANA) as a trigger and an amplified colorimetric method. The assay showed a detection range of 1.0 x 10-3 to 10.0 U/mL and a limit of detection (LOD) of 1.42 x 10-4 U/mL, with satisfactory performance in serum samples. The method also demonstrated the advantages of operational convenience, no labeling or immobilization process, and no costly instrument requirement.