| Prion protein is one kind of proteins existing in the nervous system of human or animals which is closely related to neurodegenerative diseases such as infectious spongiform encephalopathy?TSE?.Prion disease is mainly caused by the incorrect folding of normal cell type prion protein?PrPC?into pathogenic prion protein(PrPSc).The pathogenic prion protein accumulates in organisms,which infects the central nerve cells and eventually leads to the death of the organisms.In the early stage of the disease,the pathogenic prion protein(PrPSc)in the body is very low and difficult to be detected.The concentration of PrPScincreases sharply when the disease occurs,causing irreversible damage to the central nervous system.Therefore,it is of great significance to develop a rapid and sensitive method for the detection of prion protein in early diagnosis and prevention of prion disease.Nowaday,in the studies of prion protein,cell prion protein?PrPC?is generally used to replace the pathogenic prion protein(PrPSc).This is because the pathogenic prion protein(PrPSc)is extremely infectious and poses a threat to the health of researchers.Therefore,cell prion protein?PrPC?is used as the detection target in this dissertation.The electrochemical biosensor has many advantages,such as simple design,fast response,high sensitivity and low detection cost.It has been widely used in analytical chemistry.It can be used not only in the detection of heavy metal ion concentration,but also for the detection of nucleic acid?DNA or RNA?,tumor markers,small biological molecules and targeted drugs.Based on the advantages of electrochemical biosensors,a biosensor platform is constructed by using a variety of signal strategies and successfully used for the detection of prion protein.The main work of this dissertation is as follows:?1??-cyclodextrin is electropolymerized onto the surface of gold nanoparticles modified glassy carbon electrode.P?-CD film provided lots of?-CD units to capture the prion antibody by host-guest interaction.In the presence of prion,the specific interaction between prion and its antibody brought prion protein to the electrode surface to block the electron transfer channel of[Fe?CN?6]3-/4-ions provided by the inner cavity of?-CD,resulting in the decrease of the redox currents of[Fe?CN?6]3-/4-ions.Based on this kind of“blocking effect”,prion protein was detected sensitively.Under the optimal conditions,the linear detection range of the immunosensor for PrPC is from 15 fM(0.378 pg mL-1)to 1500 fM(37.8 pg mL-1),and the detection limit is as low as 2.1 fM(3?,0.052 pg mL-1).The sensor has simple design structure,high sensitivity,good selectivity and low cost.By changing the types of antibodies,the constructed sensor can be used for detection of pathogenic prion protein(PrPSc)or other tumor markers.?2?A new electrochemical immunosensor was developed for prion protein detection based on hybridization chain reaction?HCR?with hemin/G-quadruplex DNAzyme as signal amplification strategy of catalytic enzyme-like catalytic redox reaction.The antibody was immobilized on the surface of the electrode by Au-NH2bond.The specific immune reaction occurs with the primary antibody?Ab1?when the target exists,and the secondary antibody?Ab2?modified DNA single strand serves as an initiating chain to induce the complementary S1 and S2 on the surface of the electrode to generate hybridization chain reaction.The addition of hemin makes the bases rich in guanine?G?form a G-quadruplex structure.G-quadruplex can catalyze the reduction of hydrogen peroxide and the oxidation of L-cysteine,accelerate the electron conduction,amplify the signal of the sensing platform and detect concentration of the prion protein?PrPC?.The linear range of the immunosensor for detecting prion protein is from 0.5 pg mL-11 to 100 ng mL-1,and the detection limit is0.38 pg mL-1?3??.At the same time,the sensor has desirable selectivity,reproducibility and stability,and can be widely used in biological analysis and early diagnosis and treatment of diseases.?3?A smple and new electrochemical sensor for prion protein was constructed based on the strategy of hybrid chain reaction and precipitation reaction.The sandwich-type sensing platform is constructed by antigen and antibody specific immune reaction,wherein the Ab2 labeled DNA1 chain is used as an initiating chain and DNA2 partial complementary base pairing hybridization,and then four segments of DNA chains are added,ds-DNA of the two initiating chains labeled on the Ab2 can trigger S1,S2 and S3,S4 to HCR processes to form a double long chain DNA structure.The addition of hemin can make the base rich in guanine?G?form a G-quadruplex structure,catalyze the oxidation of 4-chloro-1-naphthol to generate precipitates and adhere to the surface of electrode,resulting in the decrease of[Fe?CN?6]3-/4-electron transfer.The experimental results show that the sensor has excellent selectivity and reproducibility,the linear range is from 0.5 pg mL-11 to 100 ng mL-1with a detection limit of 0.31 pg mL-1?3??.?4?A novel electrochemical immunosensor was prepared for prion protein detection based on conductive polymer and gold nanoparticle complex.The target was immobilized on the electrode by antigen-antibody immune reaction.The aptamer end was specifically bound to the antigen,and the labeled sulfhydryl group end and PMB-AuNPs were introduced onto the sensing platform through Au-S bond.The platform could produce obvious DPV response current due to its good electrochemical activity,thus realizing the sensitive detection of prion protein.The linear range of PrPC detection is from 0.5 pg mL-11 to 100 ng mL-11 and the detection limit is0.45 pg mL-1?3??,which can be widely used in genetic analysis,diagnosis and treatment of genetic diseases.?5?A dual-signal prion electrochemical immunosensor was developed based on conductive polymer dye molecules and gold nanoparticles.The conductive polymer of polyphenyl-p-phenylenediamine?PPP?-AuNPs was prepared to adsorb masses of MB molecules,a sensing platform was constructed through the specific reaction of antigen-antibody aptamer to introduce PPP-MB-AuNPs complex onto the electrode surface.Both PPP and MB have electrochemical activity,two reduction peak currents were measured by DPV and the difference between the two peak potentials was more than 200 mV.By calculating the sum of two peak currents?PPP and MB?,the peak current is linearly related to the concentration of prion protein in the range of1 pg mL-1-100 ng mL-11 and the detection limit is 0.41 pg mL-1?3??.The dual-signal immunosensor shows excellent selectivity to the actual serum samples and can be used for the analysis of clinical samples.?6?A novel electrochemical immunosensor based on hybrid chain reaction and dye molecules was prepared for the special detection of prion protein.The primary antibody was immobilized on the electrode surface by Au-NH2 bond.The target prion protein and Ab2-S0 complex formed sandwich immune structure with the primary antibody.The DNA tandem was prepared by HCR process,and the electroactive MB molecule was embedded into the double-stranded DNA groove.Finally,the signal probe was used as a carrier to record electrochemical signals by using square wave voltammetry?SWV?.The results demonstrated that the developed method could be used in sensitive detection of prion protein.The linear range of detection is from0.5 pg mL-11 to 10 ng mL-11 with a detection limit of 0.17 pg mL-1?3??.The immunosensor can be used for early screening and diagnosis of diseases,and has a strong practical significance. |
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