The Preparation Of C-Reactive Protein And Single Nucleotide Polymorphism Sensor Based On Organic Porous Materials And Conductive Hybrid Nanomaterials

In recent years,research on new materials continues to become a hot spot,and a variety of high-performance materials with specific functions have emerged.Among the porous organic polymers（POPs）,covalent-organic frameworks（COFs）and metal-organic frameworks（MOFs）are two newly developed materials in recent years.Due to their structural diversity and flexible operability,as well as their low skeleton density and strong chemical modifiability,they have considerable application prospects in biomedical sensing and cancer diagnosis and treatment.Conductive hybrid nanomaterials are a combination of several nanomaterials that makes it superior to each nanomaterial alone.Conductive hybrid nanomaterials have many attractive advantages in electrochemical immunoassays due to their large area specific interface and good electrical conductivity.According to the characteristics of the above materials,composite materials such as Au NPs@COF-TpPa-1,Au NPs@COF-LZU8,Cd-MOF,Co-MOF,Au NPs@g-C₃N₄,IL-MoS₂,Ir NPs@GO-DN were synthesized.They were used to construct C-reactive protein sensors and single nucleotide polymorphism DNA sensors.（1）Nano-mimetic enzymes have attracted much attention as a promising functional material.Co₃O₄ has the properties of mimicking catalase and has a good catalytic effect on the reduction of H₂O₂.Au NPs@COF-TpPa-1 has a large specific surface area and good protein adsorption.In this experiment,Au NPs@COF-TpPa-1was used to immobilize C-reactive protein antibody.NH₂-Co₃O₄ was used to label C-reactive protein antibody.A novel sandwich C-reactive protein sensor was constructed.The reduction current of H₂O₂ was detected by chronoamperometry（i-t）.The response current and CRP concentration had a good linear relationship in the range of 0.05-80 ng/mL.The correlation coefficient was 0.9955 with detection limit of 16.7 pg/mL.The sensor has good responsiveness and sensitivity.（2）A fast,sensitive,label-free electrochemical immunosensor was developed to measure C-reactive protein（CRP）in actual serum samples.Since Au NPs@COF-LZU8 has high conductivity,this experiment used it as a substrate to immobilize CRP antibodies.When CRP binds with CRP antibody,the electric signal of Fe（CN）₆^4-/3-was obstructed by the formed immuno complex,and the detection of CRP can be realized.In addition,Au NPs@COF-LZU8 has rich adsorption site and large specific surface area,which can greatly increase the loading amount of antibody and significantly improve the performance of the immunosensor.Under the optimal experimental conditions,the linear range of the electrochemical immunosensor ranged from 0.1 ng/mL to 200 ng/mL,the correlation coefficient was 0.9913 with the detection limit of 33.3 pg/mL.The immunosensor was used in human serum samples.The detection of CRP is satisfactory,and this strategy can also be used for the analysis of other proteins.（3）The conductive hybrid nano-material Au NPs@IL-MoS₂ has a large specific surface area and good conductivity.The Ir NPs@GO-DN composite has a good catalytic effect on the reduction of H₂O₂.Based on the above points,a new sandwich CRP immunosensor was constructed by immobilization of CRP antibody with Au NPs@IL-MoS₂ and labeling antibody by Ir NPs@GO-DN.The electrochemical immunosensor has a linear range of 0.01-100 ng/mL with a correlation coefficient of0.9931,and a detection limit of 3.3 pg/mL.This method shows good sensitivity and provides a new method for the detection of C-reactive protein.（4）In the human genome of 3 billion bases,about one base per 1000 bases may be mutated,which may cause abnormalities in the structure and function of the protein it encodes,leading to disease.Therefore,the detection of specific sequence DNA and the effective recognition of mutated bases,that is,single nucleotide polymorphism（single nucleotide polymorphism,SNP）detection technique,are of great significance.In the fifth chapter,a single nucleotide polymorphism（SNP）DNA sensor based on functionalized metal organic framework materials was constructed using the hybridization chain reaction to amplify the signal to increase the sensitivity of the sensor.This work utilizes the large specific surface area of Au NPs@GO to immobilize the capture probe with a thiol group at the 3’end by the binding force of the Au-S bond on the modified Au NPs@GO electrode.The cadmium metal organic framework material（Cd-MOF）was prepared and the metal nanoparticles（Au NPs）and avidin（SA）were modified on its surface.The synthesized SA/Au NPs/Cd-MOF complex material was assembled on the electrode surface by biotin-modified signal DNA.Moreover,the synthesized Cd-MOF has electrochemical activity and can output an electrochemical signal as a signal tag.Differential pulse voltammetry（DPV）was used to detect the current signal peak of cadmium ions,thereby achieving quantitative detection of target DNA S1.The electrochemical SNP sensor has a linear range of 0.001-10 pM with a correlation coefficient of 0.9790,and a detection limit of S1 of 0.33 fM.This method shows good sensitivity and has potential for clinical application.（5）SNP is considered not only as a genetic marker for cancer-related drug metabolism or responsiveness,but also as a basic tool for identifying hereditary pathogenic genes.In Chapter 6,in order to further improve the sensitivity of SNP detection for clinical disease diagnosis,a signal amplification SNP DNA sensor based on DNA Walkers,a nano-machine of DNA,was constructed in this experiment.DNA Walkers were constructed using functionalized Au NPs@CoFe₂O₄ and well-designed nucleotide sequences.In the preparation of the sensor,the capture probe with a thiol at 3’-end was immobilized on the electrode by using large specific surface area and a binding force of Au-S bond of Au NPs@g-C₃N₄.In addition,a cobalt metal organic framework material（Co-MOF）was prepared which has a large surface area and electrochemical activity,and can be used as a signal tag to output an electrochemical signal.This work designed a signal amplification strategy,which can achieve signal amplification through the signal probe and the auxiliary probe complementary hybridization cycle.The electrochemical SNP sensor has a linear range of 0.0001-0.1fM with a correlation coefficient of 0.9796,and a target detection limit of 0.033 aM.