Abstract
A novel electrochemical biosensing method for protein kinase (PKA) activity was demonstrated by using a reduced graphene oxide-polydopamine-silver nanoparticle-Ti4+ (rGO-PDA-AgNPs-Ti4+) nanocomposite. The obtained nanocomposite possessed an integral capability for phosphopeptide recognition and signal readout. The polydopamine modified reduced graphene oxide (rGO-PDA) was firstly prepared based on a self-polymerization method of dopamine. The silver ions were adsorbed onto polydopamine (PDA) layer and directly reduced into silver nanoparticles (AgNPs), which was used for electrochemical signal reporting. Then, the Ti4+ cations were attached onto the PDA layer for phosphopeptide recognition according to the strong coordination ability of PDA with Ti4+ and phosphate group. The prepared rGO-PDA-AgNPs-Ti4+ nanocomposites were characterized with different methods. The developed rGO-PDA-AgNPs-Ti4+ nanocomposites were then employed for electrochemical analysis of PKA-catalyzed kemptide phosphorylation. The sensitive detection toward PKA activity was realized with an experimental detection limit of about 0.01 U/mL. It may be also extended for the inhibitor evaluation. Thus, it provided a facile and sensitive means for electrochemical analysis of PKA activity and inhibitor screening.
Highlights
Protein kinase (PKA)-catalyzed protein phosphorylation is an important protein post-translational modification event that plays a crucial role in cellular signaling pathways (Johnson and Lewis, 2001; Manning et al, 2002; Tarrant and Cole, 2009)
The rGO-PDAAgNPs-Ti4+ nanocomposites were used for electrochemical investigation of protein kinase (PKA)-catalyzed kemptide phosphorylation
The phosphorylated kemptide was directly captured by the Reduced Graphene Oxide-Polydopamine Nanocomposite (rGO-PDA)-AgNPs-Ti4+ nanocomposite based on the multi-coordinative interaction between Ti4+ and the phosphate group, generating an electrochemical response toward PKA activity
Summary
Protein kinase (PKA)-catalyzed protein phosphorylation is an important protein post-translational modification event that plays a crucial role in cellular signaling pathways (Johnson and Lewis, 2001; Manning et al, 2002; Tarrant and Cole, 2009). The immobilized kemptide on the electrode could be phosphorylated by PKA and the phosphorylated kemptide was captured by the rGO-PDA-AgNPs-Ti4+ nanocomposite for electrochemical responses related with PKA activity. The kemptide phosphorylation process was operated in 50 mM Tris-HCl buffer (pH 7.4, 20 mM MgCl2) that contained different concentrations of PKA and 100 μM ATP. The phosphorylated electrode was incubated into the 50 μL above rGO-PDA-AgNPs-Ti4+ nanocomposite suspension for 1 h (room temperature) to accomplish the recognition of nanocomposites with the phosphorylated kemptide. After washing by Tris-HCl buffer, the electrode was incubated into the 50 μL rGO-PDA-AgNPs-Ti4+ nanocomposite suspension for 1 h at room temperature and electrochemical interrogation was conducted. The modified gold electrode, platinum wire and Ag/AgCl electrode were used as the working, auxiliary and reference electrodes, respectively
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