Study Of Protein Kinase Detection Based On Gold Nanomaterials-mediated Electrogenerated Chemiluminescence Strategy

Electrochemiluminescencen?ECL?,as a new analytical method,has attracted more and more attention in recent years.New materials exhibit very important roles in the development process of ECL.Nano-materials have been widely used in the field of ECL biosensing,possessing unique photoelectric properties,large surface area and good biocompatibility.Protein phosphorylation is closely related to many life activities,and protein kinase abnormal expression will lead to a variety of diseases and cancer.Therefore,accurate analysis of kinase activity and screening relevant kinase inhibitors are of great significance for disease diagnosis and clinical treatment.In this work,we prepared several kinds of nano-materials using for the fabrication of new ECL biosensor and the detection of protein kinase activity.The main content can be divided into the following three parts:1.In this section,the definition and characteristics of ECL and the classification of luminescent reagents are described.ECL is regarded as an optical analysis method which relate to the electrochemical technology,and has the peculiarity of electrochemical analysis and optical analysis.Recently,graphite phase carbonitride compounds and metal nanomaterials,work as novel materials,have been utilized in the field of ECL with unique structure and properties.Protein kinases,which catalyze the phosphorylation of proteins or peptides,play important regulatory roles in diverse cellular functions.And it has been demonstrated that aberrant protein kinase activity closely relate to severe illnesses.Up to now,several techniques have been developed for protein kinase activity detection.Among them,ECL assay has been proved to be a useful tool for kinase activity detection due to its outstanding advantages.2.Herein,a novel ECL biosensor based on the signal amplification ability of Au NCs for sensitive detection of protein kinase activity was proposed.In this system,graphite-like carbon nitride materials?g-C₃N₄?were used as a cathodic ECL emitter,and gold nanoclusters?Au NCs?were assembled on the phosphorylated peptides modified g-C₃N₄/GCE through Au-S bond in the presence of adenosine 5?-[?-thio] triphosphate cosubstrate and protein kinase A?PKA?.Interestingly,attributed to Au NCs exhibit outstanding catalytic activity in the ECL process,which made the system show significant enhancement of ECL intensity as compared to that without Au NCs.We also found that with the increasing distance between Au NCs and g-C₃N₄,the ECL enhancement effect became weaker.Such enhancement in combination with the intensity of ECL signal and protein kinase activity allows us to construct an ultrasensitive ECL biosensor for the detection of PKA with low background.The proposed biosensor can also be used for quantitative analysis of PKA activity in MCF-7 cell lysates and qualitative screening of kinase inhibitor.3.Herein,we present a ECL sensing approach for monitoring the activity of protein kinases based on the resonance energy transfer?RET?between g-C₃N₄ and Au NPs.In the present of S₂O₈^2-,g-C₃N₄ could emit strong cathodic ECL signal.Peptides were modified onto the electrode through amidation reaction.Then Au NPs were captured onto the thiophosphorylated peptide modified electrode surface through Au-S bond in the presence of ATP-s and PKA.Since the overlaps between the ECL emission spectrum of g-C₃N₄ and the absorption spectrum of Au NPs partially,the energy could transfer from g-C₃N₄ to Au NPs,resulting the decrement of the ECL intensity of g-C₃N₄.The decrease of ECL intensity was logarithmically linear with the concentration of PKA in a wide range from 0.1 U/mL to 80 U/mL,whereby the qualitative and quantitative detection of PKA activity was achieved,and the corresponding detection limit is 0.05 U/mL.This biosensor can also be used for quantitative analysis of PKA activity in serum samples and qualitative screening of kinase inhibition,indicating that the developed method has the potential application in biochemical fundamental research and clinical diagnosis.