Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun 130012, China.
Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun 130012, China; Department of Respiratory, China-Japan Union Hospital of Jilin University, Changchun 130031, Jilin, China.
Anal Chim Acta. 2018 Jul 12;1013:71-78. doi: 10.1016/j.aca.2018.01.053. Epub 2018 Feb 3.
Protein kinase (PKA) can regulate many cellular biological processes by phosphorylation substrate peptide or protein. A new fluorescent biosensing method for the detection of PKA activity was developed by using 11-mercaptoundecanoic acid-capped gold nanoclusters (MUA-Au NCs) and graphene oxide (GO) with low background noise. In this strategy, the special designed peptide could be anchored on the surface of MUA-Au NCs by the Au-S bond and also adsorbed on the surface of GO owing to the electrostatic interaction. As a result, the fluorescence of MUA-Au NCs was quenched leading to low background fluorescence due to the forster resonance energy transfer (FRET) between MUA-Au NCs and GO via peptide as a bridge. However, when the substrate peptide was phosphorylated by PKA, the FRET between GO and MUA-Au NCs was disrupted because of the weakened interaction between the phosphorylated peptide and the GO, resulting in recovery of the fluorescence intensity. The developed label-free fluorescence "turn-off-on" method can detect protein kinase activity in the range of 0.6-2.0 U mL with a detection limit of 0.17 U mL (3σ). The feasibility of this present method for kinase inhibitor screening was also studied by assessment of H-89 kinase inhibition with an IC value of 0.049 μmol L.
蛋白激酶（PKA）可以通过磷酸化底物肽或蛋白质来调节许多细胞生物过程。本研究利用具有低背景噪声的 11-巯基十一酸修饰的金纳米簇（MUA-Au NCs）和氧化石墨烯（GO），开发了一种用于检测 PKA 活性的新型荧光生物传感方法。在该策略中，通过 Au-S 键将特殊设计的肽锚定在 MUA-Au NCs 的表面上，并且由于静电相互作用也吸附在 GO 的表面上。结果，由于 MUA-Au NCs 和 GO 之间通过肽作为桥发生Förster 共振能量转移（FRET），导致 MUA-Au NCs 的荧光猝灭，从而产生低背景荧光。然而，当底物肽被 PKA 磷酸化时，由于磷酸化肽与 GO 之间的相互作用减弱，GO 和 MUA-Au NCs 之间的 FRET 被破坏，导致荧光强度恢复。所开发的无标记荧光“关闭-开启”方法可以在 0.6-2.0 U/mL 的范围内检测蛋白激酶活性，检测限为 0.17 U/mL（3σ）。还通过评估 H-89 激酶抑制作用来研究该方法用于激酶抑制剂筛选的可行性，其 IC 值为 0.049 μmol/L。
