Novel Photoelectrochemical Biosensors For Prion Protein And Nucleic Acids Assay

Prion protein（PrP）is mainly distributed in the central nervous system of mammals,which is closely related to the disease of infectious cavernous encephalopathy（TSE）,such as Creutzfeldt-Jakob disease in humans（CJD）,bovine spongiform encephalopathy in cattle（BSE）and scrapie in sheep.Prion diseases can cause the permanent degeneration of human and animal’s nerve system tissue until death.And the conformational change from the normal cellular form（PrP^C）to its infectious isoform(PrP^Sc)is thought to be the crucial step in the propagation of prion disease.Therefore,it is very crucial to find a sensitive method for prion protein assay in living organisms.Nucleic acid is the main carrier of genetic information,which determines a series of important processes in life,such as growth,development,reproduction,heredity and variation of organisms.The selective detection of nucleic acid is wide ly used in numerous fields,such as genetic analysis,diagnosis of genetic diseases and pathological study.Therefore,it is rather significant and valuable to develop some simple,cheap,efficient,sensitive,and easily miniaturized biosensors for nucleic acid assay.Photoelectrochemical（PEC）biosensor is a new developed biosensor that derived from the electrochemical biosensor,which has attracted substantial attention benefiting from its advantages such as simple instrument,easy operation,low cost,rapid response,and high sensitivity.In this paper,photoelectrochemical biosensors have been used for the sensitive detection of prion protein and nucleic acid.（1）A simple and novel photoelectrochemical aptamer sensor has been developed for prion protein assay based onβ-CD and Rhodamine B.Au-TiO₂ composite was selected as the photoelectric materials,and HS-β-CD was immobilized on the Au-TiO₂/ITO electrode surface through Au-S bond.Then Rhodamine B（RhB）labeled prion-aptamer was introduced to the HS-β-CD/Au-TiO₂/ITO electrode based on the host-guest interaction between HS-β-CD and RhB,and BSA was used to block the nonspecific sites of the electrode surface.When BSA/aptamer/β-CD/Au-TiO₂/ITO electrode was incubated with prion protein,the specific interaction between prion and its aptamer could block the cavity of HS-β-CD and hinder the charge transfer between the electron donor（AA）and the surface of the electrode,resulting in an obvious decrease of photocurrent.The PEC aptasensor shows a linear range from 200 fM to2000 fM for prion protein assay,and the detection limit is 50.9 fM.This strategy shows simple sensing structure,low cost,satisfactory reproducibility,good selectivity,and perfect storage stability.Also,by changing the specific apt amer,this strategy could be easily extended to detect the infectious isoform of prion(PrP^Sc)and other proteins.（2）A simple“signal-on”photochemical biosensor for nucleic acid assay was developed by combining catalytic hairpin assembly（CHA）and the electrochemical active component[Ru（NH₃）₆]Cl₃.Firstly,CdS was immobilized on the TiO₂/ITO electrode surface by the successive ionic layer adsorption and reaction（SILAR）method,then C-DNA and MCH were introduced to the CdS/TiO₂/ITO electrode surface through Cd-S bond.After that,the mixture solution of Au-HP1,HP2 and T-DNA were incubated for the CHA reaction to obtain large numbers of Au-HP1:HP2complexes.ThenAu-HP1:HP2complexwasintroducedtothe MCH/C-DNA/CdS/TiO₂/ITO electrode surface by the chain hybridization reaction between HP1 and C-DNA.Finally,large amounts of Ru（NH₃）₆³⁺interacted with the DNA phosphate backbone,and led to an obvious increase of photocurrent.Under optimized conditions,the photoelectrochemical biosensor shows a good linear relationship between photocurrent intensity and the nucleic acid concentration in the range of 10 fM to 1500 fM,and the detection limit is 6.19 fM.This method could be an attractive candidate in bioanalysis,clinical biomedicine,and disease diagnos tics.（3）An ultrasensitive PEC biosensor has been developed for nucleic acid assay based on three-stage cascade signal amplification strategy by combining CHA,hybridization chain reaction（HCR）and enzyme catalytic amplification methods.Firstly,the MCH/C-DNA/CdS:Mn/TiO₂/ITO electrode was assembled layer by layer.Then the mixture solution of CHA-HP1,CHA-HP2 and T-DNA were incubated for CHA reaction to obtain large numbers of CHA-HP1:HP2 complexes.After that,CHA-HP1:HP2 complex was introduced to the MCH/C-DNA/CdS:Mn/TiO₂/ITO electrode surface by the chain hybridization reaction between HP1 and C-DNA,and one end of HP2 was used as the initiator of dual-biotin labeled HCR hairpin DNA1and DNA2（HCR-HP1-Bio,HCR-HP2-Bio）to initiate the HCR rection.After the HCR rection,long nicked duplex DNA structures were formed in the electrode surface,and the dual-biotin modified HCR-hairpins can anchor more streptavidin labeled alkaline phosphatase（SA-ALP）to catalyze 2-phospho-L-ascorbic acid trisodium salt（AAP）to yield more AA,leading to larger PEC response.The proposed PEC biosensor shows superior analytical performance for T-DNA detection with a linear response range from 0.1 fM to 100 pM and a detection limit of 0.052 fM.