Construction Of A Novel Nano-fluorescence Immunosensor And Its Application In Detection Of Carcinoembryonic Antigen

Cancer is a complex disease characterized by infinite proliferation and diffusion of abnormal cells,has always been the main cause of death for human beings all over the world,which caused great harm to human life and health.Tumor markers have important reference value for early diagnosis,monitoring and prognostic treatment of cancer,and have become a reliable tool for predicting various tumor behaviors.Therefore,timely and accurate detection of tumor markers is highly important for clinical diagnosis,monitoring and prognostic treatment of cancer.At present,enzyme linked immunosorbent assay（ELISA）is commonly used in clinical quantitative analysis of tumor markers,but this method has some limitations in the accurate determination of various tumor markers due to its high cost,low sensitivity and the need for professional technical knowledge.The fluorescence immunoassay is one of the most promising immunoassays,which has the advantages of simple operation,fast response and high stability.Traditional fluorescent immunosensors often use enzyme-labeled antibodies as recognition units and organic dyes as fluorescent probes,which are susceptible to environmental factors and have low sensitivity.Nanomaterials have unique photostability,catalytic properties and biocompatibility,which opens up a new way to construct stable and sensitive fluorescent immunosensors.Combining the developed the lack of fluorescence immunosensor and the actual needs of practical application,this thesis is mainly based on functional nanomaterials to build a series of simple and sensitive fluorescence immunosensors to detect tumor markers.The main research contents are as follows:1.DNA functionalized gold nanoparticles（Au NPs）were synthesized for the sensitive detection of carcinoembryonic antigen（CEA）by fluorescence immunosensing platform with HCR signal amplification.In this study,the antibody was fixed to the carboxyl-functionalized magnetic beads to form magnetically controlled immune probes（MB-Ab）,and the CEA aptamer chain and S₀initiator chain were modified to the Au NPs surface by Au-SH bond to form functionalized Au NPs（Apt/Au NPs/S₀）.In the presence of CEA,Apt/Au NPs/S₀and MB-Ab specifically recognized CEA antigen to form sandwich immune complex.Then introduced hairpin chain S₁and S₂,S₀in sandwich immune complex triggers HCR reaction of S₁and S₂chains,and a large number of long double-stranded DNA with G-quadruplex was formed on the surface of Au NPs.Subsequently,methylene blue solution was added,and methylene blue was embedded into G-quadruplex and double-stranded DNA skeleton throughπ-πstacking interaction,resulting in a significant reduction in methylene blue and its fluorescence intensity in the solution after the magnetic separation.CEA was quantified by detecting the fluorescence intensity of methylene blue in the solution.The HCR signal amplification fluorescence immunosensor constructed in this work can effectively detect CEA with a linear detection range of 0.1～80 ng m L^-1and a detection limit of 0.075 ng m L^-1,owing to the separation and enrichment capacity of MB-Ab,the surface loading capacity of Au NPs and the amplification of HCR signal.In addition,the selectivity of fluorescence immunosensor was investigated and successfully applied to the determination of CEA in human serum samples,providing an effective way for early clinical diagnosis.2.A multifunctional three-dimensional liposome network structure was designed by using immunomagnetic beads,liposome embedded gold clusters and streptavidin-biotin recognition.Based on this multifunctional network structure,a magneto-controlled fluorescent immunosensor was established for selective and sensitive detection of CEA.The sandwich immune complex was formed by adding CEA,MB-Ab and CEA aptamer modified liposome embedded gold cluster complex（Biotin-Lip-Au NCs）.Then streptavidin and Biotin-Lip-Au NCs were introduced to form three-dimensional liposome network structure.Triton X-100 was used to break the liposome to release Au NCs in the three-dimensional liposome network,and a large number of released Au NCs were obtained by magnetic separation,and the fluorescence of abundant Au NCs act as signal output.The designed magneto-controlled immunosensor has good selectivity and sensitivity,which can effectively detect CEA.The linear detection range is 0.05～40 ng m L^-1,and a low limit of detection is 13.2 pg m L^-1.The immunosensor can sensitively detect CEA in serum samples,providing an effective way to simplify the experimental process in the development of onsite analysis equipment.3.Polydopamine-functionalized copper peroxide/zeolite imidazole framework（CP/ZIF-8/PDA）nanoparticles were synthesized and self-supplied with H₂O₂for the determination of CEA by fluorescence immunosorbent assay（FLISA）.In this study,CEA aptamer was modified on the surface of CP/ZIF-8/PDA to form a signal probe.In the presence of CEA,the signal probe could be fixed to the 96-well plate through sandwich immune reaction,which corroded PDA and ZIF-8 under acidic conditions and released CP.CP generated Cu²⁺and H₂O₂at the same time under acidic conditions,followed by fenton-like reaction of Cu²⁺and H₂O₂to generate hydroxyl radical（·OH）.The·OH oxidized o-phenylenediamine（OPD）to 2,3-diaminophenazine（DPA）through its strong oxidability,and the fluorescence of DPA acted as effective signal output.In this work,acid was used to trigger the cascade reaction,and Cu²⁺and H₂O₂were generated in the same place at the same time,which was equivalented to rich concentration in a solution somewhere,so as to carry out efficient Fenton-like reaction to generate a large amount of·OH to promote OPD oxidation.Through two rounds of efficient reaction,the fluorescence signal could be effectively amplified and the sensitivity of fluorescence immunosensor could be improved.The method was simple to operate,and efficient Fenton-like reactions could occur without the involvement of enzymes and the addition of metal ions or H₂O₂.The sensitive detection of CEA was realized,the linear detection range was 0.01～20 ng m L^-1,and the detection limit was 7.6 pg m L^-1.