Photoelectrochemical Biosensor For Protein Kinase A Activity Detection Based On G-C₃N₄

Protein phosphorylation is an important biological metabolism process which is catalyzed by protein kinase.It also plays crucial role in regulation of signal transduction,cellar proliferation,hormone secretion,cell differentiation,gene expression and apoptosis.It has been reported that the aberrant protein kinase activity and abnormal protein phosphorylation have related to many diseases,such as immune-deficiencies,cancer,diabetes and Alzheimer’s diseases.Protein kinase activity might be a kind of new biomarker for early detection of disease and evaluation of therapeutic efficacy.Thus,it is important for sensitively detecting protein kinase activity.The current widely used standard methods for protein kinase activity detection are fluorescence,photoluminescence,electrochemiluminescence,Raman spectroscopy,mass spectrometry and resonance light scattering.However,these methods suffer from the disadvantages of complicated and expensive instruments,time-consuming,tedious sample treatment and sophisticated operation procedure.Thus,the development of fast,specific,sensitive and convenient methods for protein kinase activity detection is urgently needed.（1）In this work,we develop a simple and sensitive photoelectrochemical strategy for the detection of protein kinase activity based on the bond between phosphorylated peptide and phosphorylated graphite-like carbon nitride（P-g-C₃N₄）conjugates triggered by Zr4+ ion coordination.Under optimal conditions,the increased photocurrent is proportional to PKA concentration ranging from 0.05 to 50 U/mL with a detection limit of 0.077 U/mL.Moreover,this photoelectrochemical assay can be also applied to quantitative analysis of kinase inhibition.The results indicated that the IC50 value（inhibitor concentration producing 50% inhibitor）for ellagic acid was 9.1 μM.Moreover,the developed method is further applied to detect PKA activity in real samples,which contains serum from healthy person and gastric cancer patients and breast tissue from healthy person and breast cancer patients.Therefore,the established protocol provides a new and simple tool for assay of kinase activity and its inhibitors with low cost and high sensitivity.（2）Taking advantage of the CdS quantum dots to enhanced g-C₃N₄ photoelectrochemical signal,a simple and sensitive PEC biosensor has been proposed.First,the composite materials Au/g-C₃N₄ was synthetic and then matrix peptides were assembled onto electrode through a Au-S bond,followed by catalyzing with PKA in the presence of ATP-S.As a result,phosphate groups containing –SH were introduced into matrix peptides.Subsequently,CdS quantum dots were captured on electrode through the specific recognition between CdS quantum dots and –SH.Under the visible light irradiation,with increasing the amounts of CdS quantum dots,the photocurrent increased gradually,which could be ascribed to CdS quantum dots strengthening the photoelectric ability of g-C₃N₄.These changes of photocurrent could verify that the PKA activity was successfully detected.The applicability of this PEC biosensor was further testified by the evaluation of PKA inhibition by ellagic acid.Moreover,the developed strategy is further applied to detect PKA activity in real samples,which contains serum from healthy person and gastric cancer patients and breast tissue from healthy person and breast cancer patients.Therefore,the developed PEC method has great potential in screening of kinase inhibitors and the detection of kinase activity.（3）In this work,we applied visible-light active g-C₃N₄ and metal-organic frameworks（MOFs）as a porous matrix to encapsulate AuNPs for sensitive detection of activity of PKA.On the one hand,as we all know,titanium dioxide（TiO2）can specifically bind with phosphate groups.Thus,the composite materials TiO2/g-C₃N₄ was synthetic.On the other hand,the specific construction of Au-ZIF-8 brings two advantages.（1）The existed cages in ZIF-8 sterically limit the growth of particles and confine the size of AuNPs in a distribution of ⁵ nm.（2）The specific construction of the hybrid,in which AuNPs are uniformly dispersed in ZIF-8,made fully use of the large surface area of ZIF-8 and provided sufficient active sites for combining with matrix peptides.The matrix peptides were assembled on the electrode through the Au-S bond between Au-ZIF-8 and –SH of the peptides and then the immobilized peptides are phosphorylated under PKA catalysis.Subsequently,TiO2/g-C₃N₄ was captured on the electrode because of TiO2 can specifically bind with phosphate groups.With the good photoactivity of g-C₃N₄ and ascorbic acid providing electrons,the fabricated biosensor presents high sensitivity and low detection limit（0.02 U/mL,S/N=3）for PKA detection,whose linear equation is I（nA）=259.298+136.89 logc.The applicability of this PEC biosensor was further testified by the evaluation of PKA inhibition by ellagic acid.Therefore,the developed PEC method has great potential in screening of kinase inhibitors and highly sensitive and selective detection of kinase activity.（4）A novel photoelectrochemical（PEC）assay is developed for sensitive detection of PKA activity based on PKA-catalyzed phosphorylation reaction in solution and signal amplification strategy triggered by PAMAM dendrimer and ALP.In this strategy,it is noteworthy at this point that PKA phosphorylation was achieved in solution instead of on the surface of the electrode,which has advantages of the good contact in reactants and simple experimental procedure.For immobilizing the phosphorylated peptide（P-peptide）on electrode surface,g-C₃N₄ and TiO2 complex is synthesized and characterized,which plays a significant role for TiO2 conjugating phosphate groups and g-C₃N₄ providing PEC signal.Subsequently,PAMAM dendrimer and ALP can be captured on P-peptide and TiO2/g-C₃N₄ modified ITO electrode via interaction between the-COOH groups on the surface of PAMAM dendrimer and the –NH2 groups of peptide and ALP,which can lead to the increase of ALP amount on the modified electrode surface assisted with the PAMAM dendrimer.As a result,the amount of ALP catalyzes of AAP to produce electron donor of AA,resulting an increased photocurrent.The proposed detection assay displays high selectivity and low detection limit of 0.048 U/mL（S/N = 3）for PKA activity.This biosensor can also be applied for the evaluation of PKA inhibition.Therefore,the fabricated PEC biosensor is well potential in PKA activity detection and inhibitor screening.