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Phosphorylation-Dependent SERS Readout for Activity Assay of Protein Kinase A in Cell Extracts

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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
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Anhui Province Key Laboratory of Condensed Matter Physics at Extreme Conditions, High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei 230031, Anhui, China
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College of Environmental Science and Engineering, North China Electirc Power University, Bejing 102205, China
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Chemistry Department, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia
*
Authors to whom correspondence should be addressed.
Nanomaterials 2020, 10(3), 575; https://doi.org/10.3390/nano10030575
Received: 23 February 2020 / Revised: 17 March 2020 / Accepted: 18 March 2020 / Published: 22 March 2020
(This article belongs to the Special Issue Nanomaterials in Surface-Enhanced Raman Spectroscopy)
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−1 with a detection limit as low as 0.00003 U·μL−1. 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. View Full-Text
Keywords: phosphorylation-dependent SERS readout; spermine-functionalized AgNPs; hot spots; PKA activity; cell extracts phosphorylation-dependent SERS readout; spermine-functionalized AgNPs; hot spots; PKA activity; cell extracts
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Liu, R.; Xie, C.; Yan, Y.; Hu, L.; Wang, S.; Alamry, K.A.; Marwani, H.M.; Chen, L. Phosphorylation-Dependent SERS Readout for Activity Assay of Protein Kinase A in Cell Extracts. Nanomaterials 2020, 10, 575.

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