Liu Renyong, Xie Chenggen, Yan Yehan, Hu Lin, Wang Suhua, Alamry Khalid A, Marwani Hadi M, Chen Lijuan
Key Laboratory of Biomimetic Sensor and Detecting Technology of Anhui Province, School of Materials and Chemical Engineering, West Anhui University, Lu'an 237012, Anhui, China.
Anhui Province Key Laboratory of Condensed Matter Physics at Extreme Conditions, High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei 230031, Anhui, China.
Nanomaterials (Basel). 2020 Mar 22;10(3):575. doi: 10.3390/nano10030575.
Protein kinases are key regulators of cell function, the abnormal activity of which may induce several human diseases, including cancers. Therefore, it is of great significance to develop a sensitive and reliable method for assaying protein kinase activities in real biological samples. Here, we report the phosphorylation-dependent surface-enhanced Raman scattering (SERS) readout of spermine-functionalized silver nanoparticles (AgNPs) for protein kinase A (PKA) activity assay in cell extracts. In this assay, the presence of PKA would phosphorylate and alter the net charge states of Raman dye-labeled substrate peptides, and the resulting anionic products could absorb onto the AgNPs with cationic surface charge through electrostatic attraction. Meanwhile, the Raman signals of dyes labeled on peptides were strongly enhanced by the aggregated AgNPs with interparticle hot spots formed in assay buffer. The SERS readout was directly proportional to the PKA activity in a wide range of 0.0001-0.5 U·μL with a detection limit as low as 0.00003 U·μL. Moreover, the proposed SERS-based assay for the PKA activity was successfully applied to monitoring the activity and inhibition of PKA in real biological samples, particularly in cell extracts, which would be beneficial for kinase-related disease diagnostics and inhibitor screening.
蛋白激酶是细胞功能的关键调节因子，其异常活性可能诱发包括癌症在内的多种人类疾病。因此，开发一种灵敏可靠的方法来检测真实生物样品中的蛋白激酶活性具有重要意义。在此，我们报道了用于细胞提取物中蛋白激酶A（PKA）活性检测的精胺功能化银纳米颗粒（AgNPs）的磷酸化依赖性表面增强拉曼散射（SERS）读出法。在该检测方法中，PKA的存在会使拉曼染料标记的底物肽发生磷酸化并改变其净电荷状态，生成的阴离子产物可通过静电吸引吸附到具有阳离子表面电荷的AgNPs上。同时，在检测缓冲液中形成的具有颗粒间热点的聚集AgNPs会强烈增强肽上标记染料的拉曼信号。SERS读出信号在0.0001 - 0.5 U·μL的宽范围内与PKA活性成正比，检测限低至0.00003 U·μL。此外，所提出的基于SERS的PKA活性检测方法成功应用于监测真实生物样品（特别是细胞提取物）中PKA的活性和抑制情况，这将有助于激酶相关疾病的诊断和抑制剂筛选。
