Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun 130012, China.
Department of Chemistry and Chemical Engineering, Changchun University of Science and Technology, Changchun 130022, China.
Anal Chim Acta. 2018 Jul 5;1012:66-73. doi: 10.1016/j.aca.2018.01.029. Epub 2018 Jan 20.
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 π-π 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 with a detection limit (LOD) of 0.039 U mL. 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.
一种用于检测蛋白激酶活性（PKA）的新型荧光生物传感器，是通过应用双链 DNA 负载的铜纳米簇（dsDNA-CuNCs）和氧化石墨烯（GO）设计的。dsDNA 的一条链由两个域组成，一个域可以与另一个互补的 DNA 链杂交，以稳定荧光 CuNCs，另一个域是腺苷 5'-三磷酸（ATP）适体。dsDNA-CuNCs 的 ATP 适体通过π-π 堆积相互作用自发地被吸附到 GO 表面。因此，GO 可以通过荧光共振能量转移（FRET）有效地猝灭 dsDNA-CuNCs 的荧光（FL）。在存在 ATP 的情况下，ATP 特异性地与 ATP 适体结合形成 ATP-ATP 适体结合复合物，该复合物与 GO 的亲和力较弱，导致体系的荧光恢复。然而，在存在 PKA 的情况下，ATP 可被转化为 ADP，ADP 不能与 ATP 适体结合，导致 dsDNA-CuNCs 的荧光再次猝灭。根据荧光信号的变化，可以在 0.1-5.0 U/mL 的范围内成功监测 PKA 活性，检测限（LOD）为 0.039 U/mL。此外，还研究了 H-89 对 PKA 活性的抑制作用。该传感器用于细胞裂解物中 PKA 活性的检测，结果令人满意。
