Rapid synthesis of dual proteins co-functionalized gold nanoclusters for ratiometric fluorescence sensing of polynucleotide kinase activity

Abstract

Herein, we reported a facile strategy for rapidly synthesizing gold nanoclusters co-functionalized by dual proteins (bovine serum albumin and lysozyme, BL-AuNCs). The BL-AuNCs exhibited intense fluorescence emission at 640 nm and good biochemical stability. And a sensitive and simple ratio fluorescent method was constructed for polynucleotide kinase (PNK) activity analysis by utilizing BL-AuNCs and dsDNA-intercalating dye-SYBR Green I (SGI) as sensing receptors for the first time. BL-AuNCs were modified with specific designed double-stranded DNA (dsDNA) (BL-AuNCs-dsDNA). SGI, a specific intercalator of dsDNA, displayed strong fluorescence emission at 526 nm when intercalated into the dsDNA on BL-AuNCs. With adenosine triphosphate (ATP), PNK can catalyze phosphorylation of dsDNA at 5′-terminus. Phosphorylated dsDNA was specially recognized and degraded by λ exonuclease (λ exo) to produce single-stranded DNA and mononucleotides, which lacked ability to combine with SGI. Therefore, the fluorescent signal at 526 nm would decrease, while the BL-AuNCs fluorescence at 640 nm kept unaffected and was employed as the reference signal. The PNK activity was detected by monitoring variation of the ratio of fluorescence intensity (F526/F640) with a low detection limit of 0.012 U mL−1. This ratio fluorescent strategy was also applied for PNK determination in cell lysates with satisfying results.