Nano-impact Electrochemistry:Data Analysis And Protein Kinase Activity Detection

In recent years,nano-impact electrochemistry has aroused a wide attention as an emerging electroanalytical approach towards the physical and chemical properties of single nanoparticles in solution.The nanoparticles diffuse freely in solution in the form of Brownian motion,and when impact to the ultramicroelectrode(UME)surface randomly,they may undergo electrolysis themselves or catalyze the electrochemical reactions of redox-active molecules in solution on the basic of different potential.According to the transient currents recorded in the current-time curves under the constant potential,the researchers can learn the information such as collision behavior and electrochemical kinetics at the individual particle level.The quantification of the transient currents on the current-time traces can get the concentration,size distribution,aggregation state,sticking coefficient and catalytic properties of the single nanoparticles.These studies are always based on collection and analysis of numerous transient currents,so that the accurate analysis of the large volume of the experimental data is of great significance in improving the reliability and efficiency of this method.Compared with traditional electrochemical methods,the limit of detection(LOD)of nanoimpact electrochemisty expected to reach the single entity level and it allows in-situ detection of analytes with simplicity,fast and high throughput.In addition,since nanoparticles are easy to modify and engineer,and different nanoparticle has its characteristic potential,the simultaneous detection of multiple markers can be performed by potential control.The application of nano-impact electrochemistry has also been extended to the detection of DNA,RNA,enzymes,bacteria,vesicles,cells,and proteins.Protein phosphorylation is one of the most significant post-translational modifications and it is important for intracellular signal communications,gene transcription,cell proliferation,differentiation,apoptosis,etc.Protein phosphorylation is catalyzed by protein kinases,which transfers the γ phosphate of ATP to the hydroxyl groups of the serine,threonine or tyrosine on protein.The aberrant activities of protein kinases are involved in many diseases,so protein kinase activity assay is significant,not only to the biochemical research,but also to the diagnosis and therapy.Up to now,numerous methods have been reported for protein kinase activity detection,such as radiometric,colorimetric,mass spectrometry electrochemical,fluorescent approaches.However,these assays have the disadvantages of low sensitivity,complex procedure and expensive instruments,which cannot be possible for lable-free and insite analysis.We proposed a spike detection algorithm for automatically processing the data from the nano-impact experiments,including baseline extraction,spike identification,threshold adjustment and spike area integration.By using of this algorithm to processing the data,we detected the activity of protein kinase by using the different impact frequency caused by different degrees of AgNPs aggregation before and after phosphorylation.The specifics are discussed below.(1)Agnanoparticles(AgNPs)were successful synthesized,nano-impact experiments were carried out to collect the experimental data points.The baseline was extracted by a moving average filter,which can minimize the interference of high-frequency noises.The threshold was chosen according to the discreteness of data.All the data points in the whole current-time trace were indentified,which the corresponding current value is greater than a threshold to be considered as a spike point.And the data points near the threshold were reevaluated to restore the shape of the spike as much as possible.Trapezoidal numerical integration was adopted to calculate the spike area,which is the charge of single impact event.The resulting size distribution of AgNPs calculated by Faraday laws is found to agree very well with that from transmission electron microscopy(TEM),showing that the current algorithm is promising for automated analysis of nano-impact electrochemical data with high efficiency and accuracy.(2)Since phosphorylation changed the charge of the substrate peptide,the electrostatic interaction between the peptide and the negatively charged AgNPs has changed.Therefore,different concentrations of S-peptide and P-peptide induce different aggregation degrees of AgNPs,which was proved by Ultraviolet-visible spectrophotometer(UV-Vis),dynamic light scattering(DLS)and scanning electron microscope SEM.The concentration of the substrate peptide was optimized by nano-impact electrochemistry.The feasibility of protein kinase activity detection was proved by different ratios of S-peptide and P-peptide mixture with AgNPs.In addition,the interference of enzyme co-reactants on protein kinase assays was separately excluded.Finally,the change in the impact frequency of AgNPs induced by different concentrations of protein kinase was quantified using the automated data processing algorithm.Under optimal conditions,the increased impact frequency is proportional to the protein kinase A(PKA)concentration ranging from 0.001 to 0.01 U/μL with a detection limit of 0.0046 U/μL.And the specificity detection of PKA is realized.This work provides a new method for the insitu detection of phosphorylation with high sensitivity and specificity.