Novel Electrochemical Biosensors For Alkaline Phosphatases And Prion Protein Assay

Alkaline phosphatases(AP)are a group of enzymes with low substrate specifility that catalyze the hydrolysis of phosphate at an alkaline pH.The localization of AP in tissues is intracellular.Apart from that,AP has been found in extracellular biological materials.The abnormal level of AP has been proved to be associated with many diseases including breast and prostatic cancer,liver dysfunction,bone disease,and diabetes.The biological function of AP is of great point to human health.Thus,sensitive and selective assay of AP activity is highly desirable.Prion protein(PrP)is thought to be the diseases agent of transmissible spongiform encephalopathy,otherwise known as prion diseases.Prion diseases,a group of transmissible diseases,would cause the disorders in cognition and motor functions and finally lead to death.The detection of PrP is related in early diagnostics of prion diseases.Therefore,it is crucial to find a sensitive method for PrP assay.Electrochemical biosensors are the sensors which contain electrochemical transducers with the high specificity of biological recognition processes.The sensors combine the biological recognition element that selectively reacts with the target analyte of electrochemical transducers and produces an electrical signal.The development of electrochemical biosensors is rapid since their high sensitivity,good selectivity,low cost and quick analysis.Taking advantages of electrochemical biosensors,novel electrochemical DNA biosensors and electrochemical immunosensors have been developed for sensitive and selective detection of AP activity and cellular prion protein(PrPC).(1)Taking TdT-mediated hemin/G-quadruplex DNAzyme nano wires as NADH oxidase and HRP-mimicking DNAzyme,a novel DNA-based electrochemical method has been developed for sensitive and selective assay of alkaline phosphatase(AP)activity.The double-stranded DNA(dsDNA)probe consisted of thiol-functionalized DNA1 and 3’-phosphorylated DNA2,was immobilized on the gold nanoparticles(AuNPs)modified glassy carbon(GC)electrode.In the presence of AP,3’-phosphoryl end of DNA2 was dephosphorylated.Terminal deoxynucletidyl transferase(TdT)catalyzed the sequential addition of deoxynucleotides(dTTPs)at 3’-OH end of DNA2 to extend DNA2 with a poly-T sequence.Then,G-rich DNA3 strand hybridized with the poly-T sequence of DNA2.Upon addition of hemin,the hemin/G-quadruplex DNAzyme was formed.In the presence of NADH,the hemin/G-quadruplex DNAzyme oxidased NADH to NAD+,accompanied by the formation of H2O2 which was further catalyzed by hemin/G-quadruplex DNAzyme(served as a HRP-mimicking DNAzyme)with the thionine(Thi)as electron transfer mediator,leading to the amplified electrochemical signal.Under optimized conditions,the response peak current was linear with the concentration of AP in the range from 0.1 U L-1 to 5 U L-1 with the detection limit of 0.03 U L-1.Also,the developed biosensor possessed good selectivity,reproducibility and stability,and simple sensing structure,showing promising practical applications in AP activity assay.(2)A sensitive electrochemical immunosenor based on HCR has been developed for PrPc assay.Firstly,the Abl was immobilized on the AuNPs modified GC electrode surface.In the presence of PrPC,Ab1 immunoreacts with PrPC.After the incubation with dsDNA labled Ab2,dsDNA is partial complement and contains two HCR primers,the four hairpin DNA(HP1,HP2,HP3,HP4)and electrochemical active component[Ru(NH3)6]Cl3 were added to the electrode.The two trigger DNA of the labled dsDNA could lead to the HCR process,resulting in two long dsDNA structures which could load large amounts of[Ru(NH3)6]Cl3 to produce a large electrochemical response.The developed electrochemical immunosensor shows good sensitivity,selectivity,reproductivity and stability with a linear range of 0.5 pM to 2.5 pM and a detection limit of 0.26 pM.(3)A sensitive electrochemical immunosenor based aptamer and GO/Thi composite has been developed for PrPc detection.Firstly,the Ab1 was immobilized on the AuNPs modified GC electrode surface.In the presence of PrPc,Ab1 immunoreacts with PrPc.After the incubation with the PrPC aptamer(Apt),an oligonucleotide with high affinity to PrPc,the GO/Thi composite dispersions were added to the electrode.The GO/Thi composite interacted with Apt through π-π stacking leading to a large electrochemical response.The developed electrochemical immunosensor shows good sensitivity,selectivity,reproductivity and stability with a linear range of 0.5 pM to 2 pM and a detection limit of 0.12 pM.