Immunological Recognition Combined With Nucleic Acid Amplification Reaction:An Ultrasensitive Approach For Determination Of Tumor Marker

Current research shows that the incidence of cancer is increasing,which has seriously threatened human health.The early onset of cancer is relatively simple,but it is easy to be multiple and metastasized to other organs in the middle and late stages,and the cure rate is greatly reduced.Therefore,early detection and early diagnosis is the best way to cure cancer.When cancer occurs,the concentration of its corresponding tumor marker in the blood will change greatly.Therefore,tumor markers can be used as one of the most valuable tools for early diagnosis and treatment of cancer.Protein,as a kind of important biomarker,is of great significance in the research of life science.MUC1,a highly glycosylated tumor marker,is highly expressed in malignancy related diseases such as breast,lung,ovarian and other female cancers,and plays an important role in the early diagnosis of cancer.Nucleic acid amplification technology refers to the detection of amplification of signals by hybridization or enzymatic reaction to generate a large number of copied sequences or to convert the generated test objects into other signals.Such signal amplification processes normally render the detection more sensitive and selective.In this thesis,MUC1 was used as the target protein.Based on the immunologic specificity and nucleic acid amplification principle,a new tumor marker detection method with high specificity,sensitivity and simple operation was established.The main work of my thesis will be written in details in the following parts.1.According to the contents of this paper,the significance of tumor markers in clinical diagnosis,immunological detection of tumor markers and nucleic acid amplification technology were reviewed.Firstly,the incidence of common tumors and what is MUC1 were introduced,and the significance of early detection of tumor markers in biological fluids,circulating tumor cells and tumor tissues were discussed.Secondly,the detection methods of tumor markers,mainly immunoassay methods,were introduced.Then,a review of several commonly nucleic acid amplification technology,including polymerase chain reaction?PCR?technology,rolling circle amplification?RCA?technology,loop-mediated isothermal amplification?LAMP?technology and isothermal exponential amplification reaction?EXPAR?technology were partially summarized.Finally,the applications of nanomaterials in biological analysis,including carbon nanotubes,graphene,metal nanoparticles and quantum dots,were reviewed,in which gold nanoparticles are widely used in biological analysis because of their good compatibility in biological analysis.2.A simple and rapidness signal amplification method was proposed.the primers labeled secondary MUC1-antibodies were prepared from biotinylated antibodies?B-Ab₂?and biotinylated primer through reaction with streptavidin.PCR plates were fabricated by immobilization of the primary MUC1-antibodies.In the presence of MUC1,sandwich-type compositions were formed.After specifically initiation of the EXPAR,a large number of primer replica were produced in minutes,which was fluorescently monitored in the presence of SYBR Green I on a real-time quantitative PCR.The proposed strategy has a highly sensitivity and specificity,and the detection of limit is two orders of magnitude lower than those of other methods.This method is rapid,simple,and can be applied to the detection of various proteins.3.A primer probe amplification method based on gold nanoparticle double-labeled antibody and oligonucleotide nucleic acid was developed for the detection of tumor marker MUC1.First,gold nanoparticles were synthesized,and the antibody-labeled gold nanoparticles were generated by using the antibody-adsorption effect on the surface of gold nanoparticles.Then,by using the strong affinity of Au-S bond,the thiol labeled amplified primer probe was labeled on the surface of gold nanoparticles,and the double-functionalized gold nanoparticles were prepared.Finally,in the presence of MUC1,sandwich-type compositions were formed and after specifically initiation of the EXPAR,a large number of primer sequence were produced in minutes,molecular beacons with complementary sequences were added for hybridization.When the primer was absent,the MB was in a hairpin structure,the fluorescence group was close to the quenching group,the fluorescence was quenched.When the primer was presented,the primer binds to the MB,the hairpin structure was opened,the fluorescent group was away from the quenching group,and the fluorescence was released.The method has high sensitivity,wide detection range and low detection limit.The proposed strategy provides a potential universal platform for the detection of tumor marker by assembling different antibodies on gold nanoparticles.