Construction Of Electrochemiluminescence Sensor And Its Application In Rabies Virus Detection

Rabies is a zoonotic infectious disease lethal encephalitis caused by rabies virus infecting the central nervous system.The mortality rate up to 100%,which poses a serious threat on the health of human.Early diagnosis is essential for rabies control.Currently,methods for diagnosing rabies have certain limitations such as low sensitivity,cumbersome procedures and time-consuming.Therefore,it is urgent to develop sensitive,reliable,simple,and rapid detection methods to achieve early diagnosis of rabies.Electrochemiluminescence(ECL)is an emerging and powerful analytical technique that combines superiorities of electrochemistry and optical detection.Improving the sensitivity of the detection method is a necessary condition for the early diagnosis of rabies virus.Nano-material signal amplification strategy has become an effective means to improve the sensitivity of the ECL biosensor.In addition,the combination of isothermal nucleic acid amplification strategy and ECL sensor can effectively improve the sensitivity of detection,while having excellent reliability.In this thesis,novel and efficient ECL signal probes combined with an isothermal nucleic acid amplification strategy were introduced to construct two ECL sensors to achieve rapid and sensitive detection of rabies virus.The detailed research contents are as follows:1.Construction of Ru@DMSNs ECL sensor and its application in rabies virus nucleoprotein detectionDendritic mesoporous silica nanoparticles(DMSNs)with high specific surface area,loading capacity and adjustable pore structure were used as carriers to load a large number of Ru(bpy)₃²⁺signal molecules.By loading many Ru(bpy)₃²⁺signaling molecules in the pores.Ru@DMSNs were synthesized as novel and efficient ECL luminors,and a turn-off mode ECL sensor for sensitive detection of rabies virus nucleoprotein was constructed.The specific construction processes were as follows:the antibody was connected to the surface of Ru@DMSNs through covalent cross-linking of carboxyl and amino groups,and loaded on the surface of the electrode to construct the rabies virus nucleoprotein detection interface.Through antigen-antibody specific recognition,the target rabies virus nucleoprotein was captured on the electrode surface.Due to the blocking effect of the steric hindrance between biomolecules on the electron transfer at the electrode interface,the ECL intensity gradually decreased as improving rabies virus nucleoprotein concentrations,thereby realizing the sensitive detection of rabies virus nucleoprotein.The detection linear range of this method was 1 pg/m L?50 ng/m L.The detection limit was 0.16 pg/m L,and the recovery rate of standard addition ranged from 95.17%to 103.89%.2.Construction of porphyrin Zr-metal-organic framework PCN-224 ECL sensor and its application in detecting rabies virus oligonucleotidesIn this study,an ECL sensor was constructed by combining new and efficient ECL material porphyrin Zr-metal organic framework PCN-224 with nucleic acid isothermal amplification technology to detect the oligonucleotides of rabies virus oligonucleotide.The specific construction process was as follows:DNA was coupled to the surface of PCN-224nanoparticles through amide bond cross-linking to prepare ECL signal probes.The electrode surface modified with sulfhydryl DNA was used as the sensing interface,the introduction of isothermal nucleic acid amplification strategy can convert a small amount of rabies virus oligonucleotides into numerous output DNA which fixed to the surface of the electrode through part of the base complementation and acted as a bridging molecule to capture the PCN-224 ECL signal probe on the electrode surface.The ECL signal increased with the increase of the output DNA concentration,that is,indirectly corresponded to the concentration of the rabies virus oligonucleotide.Moreover,the ECL sensor had a good linear relationship with rabies virus oligonucleotide in the range of 1.0×10^-14 mol/L?1.0×10^-9 mol/L.The detection limit was 1.4×10^-15 mol/L,and the standard recovery rate was 95.49%?105.37%.