DNA-hosted copper nanoclusters/graphene oxide based fluorescent biosensor for protein kinase activity detection

Highlights: • A novel biosensor for convenient detection of PKA activity and its inhibitor was developed. • The affinity of ATP aptamer sequence of dsDNA-CuNCs to GO caused the FRET. • ATP broke the affinity of ATP aptamer to GO by forming ATP-ATP aptamer complexes. • ATP aptamer was released and absorbed onto GO as soon as PKA translated ATP into ADP. • The biosensor was performed for PKA detection in cell lysates with satisfactory results. - Abstract: A novel fluorescent biosensor for protein kinase activity (PKA) detection was designed by applying double-strands DNA-hosted copper nanoclusters (dsDNA-CuNCs) and graphene oxide (GO). One DNA strand of the dsDNA consisted of two domains, one domain can hybridize with another complementary DNA strand to stabilize the fluorescent CuNCs and another domain was adenosine 5'-triphosphate (ATP) aptamer. ATP aptamer of the dsDNA-CuNCs would be spontaneously absorbed onto the GO surface through p-p stacking interactions. Thus GO can efficiently quench the fluorescence (FL) of dsDNA-CuNCs through fluorescence resonance energy transfer (FRET). In the present of ATP, ATP specifically combined with ATP aptamer to form ATP-ATP aptamer binding complexes, which had much less affinity to GO, resulting in the fluorescence recovery of the system. Nevertheless, in the presence of PKA, ATP could be translated into ADP and ADP could not combine with ATP aptamer resulting in the fluorescence quenching of dsDNA-CuNCs again. According to the change of the fluorescence signal, PKA activity could be successfully monitored in the range of 0.1–5.0 U mL^-1 with a detection limit (LOD) of 0.039 U mL^-1. Besides, the inhibitory effect of H-89 on PKA activity was studied. The sensor was performed for PKA activity detection in cell lysates with satisfactory results.