Preparation Of Novel Label-Free Electrochemical Immunosensor For Tumor Markers Detection

Taking advantage of the merits of rapid and simple label-free immunoassay and high sensitivity of electrochemical detection,label-free electrochemical immunosensor has gradually become one of the research hotspots in the field of immunoassay.Nanomaterials have excellent biocompatibility,large specific surface area and catalytic activity,which improve the load capacity,biological activity,and stability of bioactive molecules.Moreover,the analytical performance of biosensors such as detection sensitivity can be further improved.This thesis focuses on preparing a series of label-free immunosensor based on effective electrode materials with unique properties.Moreover,peroxidase-like materials were introduced into the field of label-free electrochemical immunoassay.It provides different label-free immunoassay methods for different properties of electrode materials,so as to detect tumor markers quickly,sensitively and cheaply.The main research was carried out as follows:(1)Reduced graphene oxide-coated polystyrene nanospheres(rGO@PS NSs)were synthesized by electrostatic interaction,which solved the problems of easy agglomeration and poor dispersion of graphene.The prepared rGO@PS NSs demonstrated excellent conductivity,good hydrophilicity,large specific surface area,and high antibody loading capacity.After being functionalized with streptavidin,biotinylated antibodies can be efficiently immobilized on the electrode through the biotin-streptavidin system.After one-step incubation process,the immune complexes formed at the sensor interface.In the detection system of potassium ferricyanide/potassium ferrocyanide(Fe(CN)63-/4-),the immune complexes will effectively block electron transfer on electrode surface,thus causing the decrease of the electrochemical signal.It can be realizing the rapid label-free electrochemical detection of antigen molecules according to the linear relationship between the change of electrochemical signal and the concentration of target antigen.Using fetoprotein(AFP)as a model analyte,the label-free electrochemical immunosensor showed wide linear range(0.1-100 ng/mL)and low detection limit(0.03 ng/mL,S/N=3).In addition,the rGO@PS NSs based label-free electrochemical immunosensor has high specificity,good reproducibility,and stability,and can be used for the detection of tumor marker and early cancer screening.(2)Platinum nanoparticles@reduced graphene oxide-coated polystyrene nanospheres(PtNPs@rGO@PS NSs)were synthesized by microwave radiation heating method.The dispersion of graphene was further improved by loading PtNPs on the surface of rGO@PS NSs.The synthesized PtNPs@rGO@PS NSs demonstrated excellent electrical conductivity,good hydrophilicity,large specific surface area,and high antibody loading capacity.After the functionalization of the PtNPs@rGO@PS NSs material by the streptavidin,the biotinylated antibody was captured at the sensing interface.In the detection system of potassium ferricyanide/potassium ferrocyanide(Fe(CN)63-/4-),the immune complexes formed by the antigen-antibody specific reaction could effectively block electron transfer on electrode surface,thus causing the decrease of the electrochemical signal.It can be realizing the rapid label-free electrochemical quantitative detection of the tumor marker according to the linear relationship between the decrease of electrochemical signal and the concentration of target antigen.The introduction of PtNPs greatly improves the response and analysis sensitivity of the sensor.Using Carcinoembryonic antigen(CEA)as the model analyte,the label-free electrochemical immunosensor showed a wide linear range(0.05-70 ng/mL)with a detection limit of 0.01 ng/mL(S/N=3).In addition,the PtNPs@rGO@PS NSs based label-free electrochemical immunosensor has high specificity,good reproducibility and stability,and has been successfully applied for the detection of CEA in real samples.(3)A novel label-free electrochemical immunoassay method based on three-dimensional porous structure of Cu@Cu2O gel.The Cu@Cu2O gel showed high peroxidase activity.In acidic medium,Cu@Cu2O can catalyze the polymerization of aniline to produce polyaniline in aniline-hydrogen peroxide(Aniline/H2O2)system.The electrochemical signal of polyaniline can be obtained for the immunoassay method.After the functionalization of the Cu@Cu2O material by the streptavidin,the biotinylated antibody was fixed at the sensing interface.The immune complexes formed by the antigen-antibody specific reaction could inhibit the catalytic polymerization reaction,leading to the decrease of the electrochemical signal of polyaniline.It can be realizing the rapid label-free electrochemical quantitative detection of the tumor marker according to the linear relationship between the decrease of electrochemical signal and the concentration of target antigen.Using carbohydrate antigen 125(CA125)as the model analyte,the label-free electrochemical immunosensor showed a wide linear range(0.050-100 U/mL)with a detection limit of 0.022 U/mL(S/N=3).This research provides new strategy and platform for immunoassay and early diagnosis of diseases(4)An iron-based metal-organic framework(Fe-MOF)was prepared with 2,5-dihydroxyterephthalic acid organic ligand and iron ions through hydrothermal synthesis method.It was found that Fe-MOF exhibit high-efficiency and stable peroxidase activity.A label-free electrochemical immunosensor was constructed in aniline/H2O2 system using the Fe-MOF as the modified electrode material to detect tumor markers.Fe-MOF can catalyze the polymerization of aniline to produce electrochemical signal of polyaniline.The immune complexes formed on the sensing interface could inhibit the catalytic polymerization reaction,leading to the decrease of the electrochemical signal.It can be realizing the rapid label-free electrochemical quantitative detection of the tumor marker according to the linear relationship between the decrease of electrochemical signal and the concentration of target antigen.Using carbohydrate antigen 125(CA125)as the model analyte,the label-free electrochemical immunosensor was able to detect CA125 in the range of 0.050-140 U/mL with a detection limit as low as 0.015 U/mL(S/N=3).This label-free electrochemical immunosensor based on Fe-MOF can realize rapid,inexpensive,and highly sensitive detection of tumor markers,and was successfully applied for the detection of CA125 in real samples.The label-free electrochemical immunosensor can be used for early cancer screening and clinical diagnosis applications.(5)A Zr-MOF framework material(Zr-MOF)MOF-808 was synthesized with 1,3,5-benzoic acid organic ligand and zirconium ions through hydrothermal method.The prepared MOF-808 material not only shows good stability but also exhibits high peroxidase-like properties at neutral pH medium.Using MOF-808 as the electrode substrate material,a new label-free electrochemical immunosensor was developed for the detection of tumor markers.After the functionalization of the MOF-808 material by the streptavidin,the biotinylated antibody was fixed at the sensing interface.MOF-808 catalyzes hydroquinone to produce electrochemical signals in hydroquinone/hydrogen peroxide(HQ/H2O2)system.The immune complexes formed by the antigen-antibody specific reaction could inhibit the MOF-808 catalytic reaction,leading to the decrease of the electrochemical signal.It can be realizing the rapid label-free electrochemical quantitative detection of the tumor marker according to the linear relationship between the decrease of electrochemical signal and the concentration of target antigen.Using carbohydrate antigen 125(CA125)as the model analyte,the label-free electrochemical immunosensor showed a wide linear range(0.10-150 U/mL)with a detection limit of 0.043 U/mL(S/N=3).In addition,the MOF-808 based label-free electrochemical immunosensor has high specificity,good reproducibility and stability,and has been successfully applied for the detection of CA125 in real samples.(6)A Cu-MOF material was synthesized at room temperature,and Au nanoparticles were in situ grown on the Cu-MOF surface to obtain the Au@Cu-MOF composite material.Due to the synergistic effect of between Au and Cu-MOF,the Au@Cu-MOF composite exhibits better electrochemical performance and higher peroxidase activity,which can amplify the electrochemical detection signal.Based on the Au@Cu-MOF composite,a new label-free electrochemical immunosensor was proposed for the detection of tumor markers.The immune complexes formed on the sensing interface could inhibit Au@Cu-MOF catalyzed reaction of hydroquinone/hydrogen peroxide(HQ/H2O2),leading to the decrease of the electrochemical signal.It can be realizing the rapid label-free electrochemical detection of antigen molecules according to the linear relationship between the change of electrochemical signal and the concentration of target antigen.Using the carbohydrate antigen 125(CA125)as a model analyte,the immunosensor showed wide linear range(0.05-300 U/mL)and low detection limit(0.02 U/mL,S/N=3)This proposed Au@Cu-MOF composites based label-free electrochemical immunosensor is simple,ultrasensitive,low consumption.It provides a new strategy and platform for large-scale cancer screening.