The authors describe a fluorometric method for the determination of the activity and inhibition of protein kinase A (PKA). In the presence of ATP, PKA catalyzes the transfer of phosphate groups from ATP to a peptide, and the generated phosphorylated peptide quenches the fluorescence (measured at excitation/emission peaks of 340/440 nm) of the hydroxyapatite nanoparticles (HAP-NPs). A linear logarithmic relationship of PKA concentrations with fluorescence intensity in the range from 1 to 50 U·L −1 was obtained, and the lower limit of detection (LOD) is 0.5 U·L −1 . This is much lower than LODs reported in the literature. The PKA inhibitor H-89 was studied, and the inhibition plot has a sigmoidal shape with a half-maximal inhibitory concentration of around 750 nM of H-89. At a 4.5 nM level of H-89, fluorescence of HAP-NPs fell to levels of no PKA controls, demonstrating that the assay is a viable tool to screen for kinase inhibitors. An assay with Hela cell lysates in combination with forskolin (an activator of adenylyl cyclase) and IBMX (a phosphodiesterase inhibitor used to activate the cellular activity of PKA) resulted in decreased fluorescence of HAP-NPs. This suggests that the assay can be applied for testing in vitro cell kinase activity. In our perception, this method will enable high-throughput screening for kinase-related drugs and fluorometric enzymatic detection in various areas.
Fluorescence assay based on hydroxyapatite nanoparticles (HAP) fluorescence quenching was developed for analysis of the activity and inhibition of protein kinase A (PKA).
In the classical heterogeneous electrochemical assay, phosphorylation of peptide substrate is usually performed on the solid-liquid surface. However, immobilization of probe on the solid surface may limit the interaction between the reaction site of probe and the active center of kinase due to the steric hindrance effect. In this work, we proposed a heterogeneous electrochemical method for kinase detection, in which the probe is immobilization-free during the phosphorylation reaction. A biotinylated peptide was used as the kinase substrate. After phosphorylation, the biotinylated phosphopeptide was captured by the neutravidin (NA)-modified electrode through the avidin-biotin interaction. The phosphate groups on the electrode surface were then recognized by the conjugates preformed between biotinylated Phos-tag™ (Bio-tag-Phos) and ferrocene (Fc)-capped NA-modified gold nanoparticle (Fc-AuNP-NA). The method integrates the advantages of homogeneous reaction and heterogeneous detection with high simplicity, sensitivity and specificity. The strategy can be applied to design other heterogeneous biosensors without the immobilization of probe during the enzyme catalyzed reaction .
Zhiqiang Chen,Ying Liu,Lijie Hao,Zhencai Zhu,Fang Li,Shufeng Liu
Protein kinase activity analysis is essential and important for elucidation of many fundamental biological processes, disease diagnosis, and drug discovery. Herein, a novel electrochemical biosensing method for protein kinase (PKA) activity was demonstrated by the use of a reduced graphene oxide-zirconium dioxide–thionine (rGO-ZrO2–Thi) nanocomposite, which interestingly served as an integral phosphopeptide-recognizing, signal amplifying and reporting platform. The ZrO2 nanoparticle-decorated reduced graphene oxide (rGO-ZrO2) was first prepared by a hydrothermal reaction route, and then the thionine was conjugated onto the rGO surface via π–π stacking as an excellent electrochemical probe. The prepared rGO-ZrO2–Thi nanocomposites were well-characterized by various techniques. With the full advantage of specific recognition of ZrO2 nanoparticles for the phosphate group, signal amplification, and transduction of abundant thionines onto the rGO surface, and excellent conductivity of rGO, the rGO-ZrO2–Thi nanocomposite endowed a label-free and one-step electrochemical analysis of kemptide phosphorylation catalyzed by PKA. The detection limit for PKA activity was experimentally achieved as 0.005 U/mL, which was evidently lower than most of the reported methods. The proposed sensing strategy could be also applied for an efficient inhibitor evaluation. Therefore, it offered an excellent pathway for a generic and sensitive electrochemical assay of PKA activity and inhibitor.
Yong Chang,Fengli Gao,Tong Wu,Qiuzhi Pan,Lin Liu,Qijun Song
Protein kinases play a vital regulatory role in various biological processes. In this work, we developed an electrochemical biosensor for the detection of protein kinases using methylene blue (MB)-functionalized Zr-based metal-organic frameworks (MOFs, UiO-66-NH 2 ) as the signal labels. The results of UV-Vis spectroscopy, X-ray diffraction and N 2 adsorption–desorption experiments confirmed the successful encapsulation of MB into UiO-66-NH 2 . The phosphate groups in phosphorylated peptides were recognized by UiO-66-NH 2 via the robust coordination with Zr-O clusters on the surface of MOFs. The encapsulated MB molecules in UiO-66-NH 2 produced a strong electrochemical signal for highly sensitive detection of protein kinases, which was recorded by square wave voltammetry (SWV). With protein kinase A (PKA) as a model target, the biosensor achieved a detection limit of 0.02 U/mL and a linear range from 0.05 to 20 U/mL. The proposed biosensor was utilized to screen potential inhibitors and determine PKA in serum samples with satisfactory results. This work provided an effective strategy for electrochemical assays of phosphorylation-related biological events in clinical diagnosis.
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