Phosphate-guanidine interaction based fluorometric strategy for protein kinase activity sensing

Abstract

In this paper, a fluorometric strategy for protein kinase A (PKA) activity detection was proposed based on the recognition of phosphate and guanidine groups. Two artificial substrate peptides (T1 and T2) were specially designed, T1, containing PKA specific recognition domain, was self-assembled on gold nanoclusters (T1-AuNCs). T2, possessing guanidine groups at arginine residues for the interaction with phosphorylation site, was used for modifying gold nanoparticles (T2-AuNPs). In the presence of adenosine triphosphate (ATP) and PKA, T1 was phosphorylated and then the phosphorylated T1 specially combined with T2 via phosphate-guanidine recognition. So, T1-AuNCs and T2-AuNPs were brought in close distance and fluorescence resonance energy transfer (FRET) process from T1-AuNCs to T2-AuNPs was triggered to result in the fluorescence quenching of T1-AuNCs. Accordingly, PKA activity was efficiently determined depending on the fluorescent signal changes within a good linearity from 0.04 to 8.0 U mL−1 with the detection limit down to 0.013 U mL−1. Moreover, this sensing platform was used to investigate the inhibition effect of PKA inhibitor (H-89) with good performance. Also, the practical application of this strategy for analysis of PKA activity in complicated biologic sample was demonstrated in HepG2 cell lysates.