Copper nanoclusters/polydopamine nanospheres based fluorescence aptasensor for protein kinase activity determination

Highlights: • A CuNCs/PDANS based aptasensor for PKA activity detection was constructed. • The interactions between ATP aptamer of apt-CuNCs and PDANS resulted in the FRET. • ATP-aptamer complex with less affinity to PDANS was formed with ATP addition and destroyed by PKA changing ATP to ADP. • This method was further exploited to establish a model for PKA inhibitor screening. • The aptasensor was applied for PKA detection in HepG2 cell lysates with satisfactory results. - Abstract: A fluorescence aptasensor was constructed for protein kinase (PKA) activity detection by utilizing copper nanoclusters (CuNCs) and polydopamine nanospheres (PDANS). Through the p-p stacking interactions between adenosine triphosphate (ATP) aptamer and PDANS, the ATP aptamer modified CuNCs (apt-CuNCs) were absorbed onto PDANS surface, thus the fluorescence of apt-CuNCs were quenched through fluorescence resonance energy transfer (FRET) from apt-CuNCs to PDANS. In the presence of ATP, ATP specifically bound to aptamer, causing the dissociation of apt-CuNCs from PDANS surface and restoring the fluorescence of apt-CuNCs. However, PKA translated ATP into adenosine diphosphate (ADP), and ADP had no competence to combine with ATP aptamer, thus, apt-CuNCs were released and absorbed onto the PDANS surface to cause the fluorescence quenching of apt-CuNCs again. Therefore, PKA activity was conveniently detected via the fluorescence signal change. Under the optimal conditions, PKA activity was detected in the range of 0.05–4.5 U mL^-1 with a detection limit of 0.021 U mL^-1. Furthermore, the feasibility of the aptasensor for kinase inhibitor screening was explored via assessment of kinase inhibitor H-89 as one model. This aptasensor was also performed for PKA activity determination in HepG2 cell lysates with satisfactory results.