An Amplificated Electrochemical Biosensor Strategy For Effective Detection Of Kinase

Kinase-catalyzed phosphorylation plays a significant role in many vital biological processes,including metabolism,cellular signal transduction,cell proliferation and differentiation,and so on.Over 2000 kinase genes are contained in human body,and aberrant phosphorylation and kinase activity are linked with many common diseases,such as cancer,Alzheimer’s disease and diabetes.Towards this end,the accurate identification of phosphorylation and kinase activity has aroused the interests of many people.Electrochemical biosensors,possessing several advantages such as high sensitivity,simple operation,avoid separation,good selectivity,and need no sample preparation,have widely attracted the interests of researchers and explored for the dection of various targets,expcicaily for kinase and phosphorylation.In this paper,utilizing the nanomaterial and self-assembled of nucleic acid assisted signal amplification,we constructed a series of heterogeneous and homogeneous electrochemical sensors to sensitively detect the kinase activity.The main works are summarized as follows:1.Amplified electrochemical detection of protein kinase activity based on gold nanoparticles/multi-walled carbon nanotubes nanohybridsA sensitive and simple electrochemical strategy has been developed for detection of protein kinase A(PKA)activity and inhibition using gold nanoparticles/multi-walled carbon nanotubes(AuNPs/MWNTs)nanohybrids.Key features of this detection included intrinsic peroxidase-like activity of positively-charged gold nanoparticles(AuNPs)and signal transduction and amplification of multi-walled carbon nanotubes(MWNTs).In this electrochemical strategy,an N-terminally cysteine-containing peptide was self-assembled onto the gold electrode via Au-S bonding and used as substrate for PKA,and adenosine-50-(y-thio)-triphosphate was used as co-substrate.Upon thiophosphorylation in the presence of PKA,the AuNPs/MWNTs nanohybrids would be fixed onto the peptides via Au-S bond.The conjugated AuNPs/MWNTs nanohybrids could catalyze the 3,3,5,5-Tetramethylbenzidine(TMB)oxidation by H2O2 to form TMB oxidation product,which was reduced at the electrode surface to generate an electrochemical current.It was eT on state.The current signal intensity is proportional to the activity of PKA.Here,the presence of MWNTs not only increased the surface area for accumulation of AuNPs but also could promote electron-transfer reaction.It was found that the electrochemical strategy can be employed to assay PKA activity with a low detection limit of 0.09 U/mL.The linear range of the detection for PKA enzymatic unit/ml was 0.10-1 U/mL.Furthermore,the interferences experiments of T4 polynucleotide kinase(T4 PNK)and Casein kinase II(CK2),and inhibition of PKA,have also been studied by using this strategy.The developed method would provide a diversified platform for kinase activity and inhibition monitoring.2.A highly sensitive electrochemiluminescence detection for protein kinase based on double-quenching of graphene quantum dots by G-quadruplex-hemin and gold nanoparticlesA highly sensitive electrochemiluminescence(ECL)strategy was developed for the protein kinase A(PKA)activity and inhibition detection based on double-quenching of graphene quantum dots(GQDs)ECL by G-quadruplex-hemin DNAzyme and gold nanoparticles(AuNPs).In this strategy,the GQDs were modified onto the indium-tin oxide(ITO)electrode and further assembled with substrate peptide of target protein kinase through covalent coupling,which can exhibit high and stable ECL signal.The AuNPs,functionalized with the phosphorylated DNA and G-quadruplex-hemin DNAzyme via Au-S chemistry,were selected as quenching probes.In the presence of PKA,the peptide on the electrode was phosphorylated and the AuNPs functionalized with the phosphorylated DNA and G-quadruplex-hemin DNAzyme were subsequently integrated onto the phosphorylated peptide by Zr4+.Owing to the reduction of coreactant H2O2 resulting from G-quadruplex-hemin DNAzyme catalytic reaction and the ECL energy transfer(ECL-RET)between AuNPs and GQDs,the ECL intensity of GQDs was significantly decreased.By taking advantage of the double-quenching effect,this assay can detect PKA with a linear range of 0.05 to 5 U/mL and a detection limit of 0.04 U/mL.In addition,the PKA inhibition detection and interferences experiments of CK2 and T4 PNK have been studied respectively.This ECL strategy was also successfully applied to PKA detection in serum samples and cell lysates,indicating that the developed method have the potential applications in protein kinase-related biochemical fundamental research and clinical diagnosis.3.Homogeneous electrochemical strategy for T4 polynucleotide kinase activity assay based on three-way DNA junction assisted recycling amplificationThe phosphorylation of nucleic acid with 5’-OH termini catalyzed by T4 polynucleotide kinase(T4 PNK)involves several significant cellular events.Here,a simple,homogeneous and generic method for detecting T4 PNK is developed based on lambda exonuclease(X exo)-assisted dynamic strand displacement and three-way-DNA junction architecture.In this strategy,four hairpin DNA probes were designed which were kinetically handicapped from cross-opening in the absence of the T4 PNK.In the presence of T4 PNK,the hairpin DNA T4 HP was phosphorylated and then cleaved by λ exonuclease from 5’ to 3’ direction to generate a single-stranded initiator which can successively open hairpins HP1,HP2 and HP3 to trigger an autonomous assembly of three-way-DNA junction architecture.At the same time,an intermolecular G-quadruplex could be formed between HP1,HP2 and HP3.Thus,with the electrochemical indicator methylene blue(MB)selectively intercalated into the duplex DNA chain and the multiple G-quadruplexes,a significant electrochemical signal drop is observed,which is dependent on the concentration of the T4 PNK.This interesting design has led to the development of an ultrasensitive detection for T4 PNK with a detection limit of 0.09 U/mL.More importantly,its successful application for detectingT4 PNK activity in a complex biological matrix and studying the inhibition effects of T4 PNK inhibitors demonstrated that it provides a promising platform for screening T4 PNK inhibitors as well as detecting PNK activity.Therefore,it is a highly sensitive,specific,reliable and cost-effective strategy which shows great potential for biological process research,drug discovery,and clinical diagnostics.4.A label-free homogeneous electrochemical sensing platform for protein kinase detection based on carboxypeptidase Y-assisted peptide cleavage and vertically ordered mesoporous silica filmsHerein,a simple,robust,and label-free homogeneous electrochemical sensing platform was constructed for the detection of protein kinase activity and inhibition by integration of carboxypeptidase Y(CPY)-assisted selective peptide cleavage reaction and vertically ordered mesoporous silica films(MSFs)grown in suit of indium-tin oxide(ITO)electrode.In this sensing platform,the substrate peptide composed of a kinase specific recognized sequence and multiple positively charged arginine(R)residues has been ingeniously designed.In the presence of protein kinase,the substrate peptide was phosphorylated and then immediately resisted CPY cleavage.The phosphorylated peptide could be effectively adsorbed on the negatively charged surface of MSFs modified ITO electrode(MSFs/ITO)by noncovalent electrostatic attraction and subsequently used as a hamper to prevent the diffusion of electroactive probe(FcMeOH)to the electrode surface through the vertically aligned nanopores,resulting in a detectable reduced of electrochemical signal.As test for the feasibility and universality of this sensing platform,both of protein kinase A(PKA)and casein kinase Ⅱ(CK2)were selected as the models,and the detection limits were determined to be 0.08 UmL-1 and 0.09 UmL-1,respectively.This sensing platform has the merits of simplicity,easy to implement and improved phosphorylation and cleavage efficiency,which benefits from homogeneous solution reactions without sophisticated modification or immobilization procedures.In addition,given the key role of inhibition and protein kinase activity detection in cell lysates,this proposed sensing platform showed great potential in kinase-related biochemical fundamental and clinical diagnostics.5.Vertically ordered mesoporous silica film-assisted label-free and universal electrochemiluminescence aptasensor platformHerein,a simple,facile,and label-free electrochemiluminescence(ECL)aptasensor platform was constructed by integration of aptamer-gated systems and vertically ordered mesoporous silica films(MSFs)grown in suit of indium-tin oxide(ITO)electrode.In this aptasensor platform,aptamer could be effectively adsorbed on the surface of aminated MSFs by noncovalent electrostatic attraction and then employed as an ideal gate material to control the blocking and releasing of luminescence reagents(Ru(bipy)32+)entrapped within the pores of MSFs.In the presence of target,the specific aptamer-target binding could trigger the uncapping the pores,releasing the Ru(bipy)32+ with detectable reduced of ECL signal.The feasibility and universality of this design was validated by employing three aptamers that bind to lysozyme,adenosine,and K+ as gate materials,and the detection limits were determined to be 0.06 nM,0.75 nM,and 0.50 μM,respectively.This ECL aptasensor,based on the simple competitive procedure,was simple design,undemanding,and fast in operation.In addition,no other chemical modification of the aptamer was required,suggesting that this ECL aptasensor could be applied to many other target detections just by altering the aptamer sequence.