Amplified electrochemical detection of protein kinase activity based on gold nanoparticles/multi-walled carbon nanotubes nanohybrids

Abstract

A sensitive and simple electrochemical strategy has been developed for assay of protein kinase A (PKA) activity and inhibition using gold nanoparticles/multi-walled carbon nanotubes (AuNPs/MWNTs) nanohybrids. Key features of this assay included intrinsic peroxidase-like activity of positively-charged gold nanoparticles (+AuNPs) and signal transduction and amplification of multi-walled carbon nanotubes (MWNTs). In this assay, an N-terminally cysteine-containing peptide was self-assembled onto the gold electrode via Au–S bonding and used as substrate for PKA, and adenosine-5′-(γ-thio)-triphosphate was used as co-substrate. Upon thiophosphorylation in the presence of PKA, the AuNPs/MWNTs nanohybrids would be fixed onto the peptides via Au–S bond. The conjugated AuNPs/MWNTs nanohybrids could catalyze the 3, 3′, 5, 5′-Tetramethylbenzidine (TMB) oxidation by H2O2 to form TMB oxidation product, which was reduced at the electrode surface to generate an electrochemical current. It was eT on state. The current signal intensity is proportional to the activity of PKA. Here, the presence of MWNTs not only increased the surface area for accumulation of +AuNPs but also could promote electron-transfer reaction. It was found that the electrochemical strategy can be employed to assay PKA activity with a low detection limit of 0.09U/mL. The linear range of the assay for PKA enzymatic unit/ml was 0.1–1U/mL. Furthermore, the interferences experiments of T4 polynucleotide kinase (T4 PNK) and Casein kinase II (CK2), and inhibition of PKA, have also been studied by using this strategy. The developed method would provide a diversified platform for kinase activity and inhibition monitoring.