Studies On Electrochemical Immunosensors Based On Nanomaterials For Human Chorionic Gonadotrophin

Human chorionic gonadotropin (HCG), secreted by the trophoblastic cells of the placenta chorionic vesicle, is an important diagnostic marker of pregnancy and carbohydrate tumor. Increasing levels of HCG in serum or urine are related to early pregnancy, ectopic pregnancy, miscarriage, prostate cancer, hydatid pregnancy, germ cell tumors, choriocarcinoma and trophoblastic cancer. At present, many assays for detection of HCG have been reported, including radioimmunoassay, enzyme-linked immunosorbent assay, colloidal gold-labeled test paper card assay, fluorescence-labeled antibody immunoassay, enzyme-labeled electrophoretic method and electrochemical immunoassay. Among various immunoassays, electrochemical immunosensors have drawn extensive attention due to their portability, low cost and easy manipulation. The present study emphasize that how to effectivly immobilize immunoprotein molecules, improve the sensitivity and other performance of the immunosensor.Nanomaterials are often used as the satisfactory matrix for the immunosensor because of their large specific surface area, favorable biocompatibility and high surface reaction activity, which could increase the amount of immobilized biomolecules and keep their activity well. In this thesis, we have constructed three immunosensors for detection of HCG based on different nanomaterials and using covalent bonding, electrodepositing and electrostatic interaction.The main works and conclusions are included as follows:1. Electrochemical immunoassay for human chorionic gonadotrophin based on Pt hollow nanospheres and silver/titanium dioxide nanocomposite matrixA new electrochemical immunosensor for HCG assay was developed based on hollow Pt nanospheres (HPtNPs) and silver-chitosan-titanium dioxide nanocomposites (Ag-TiO2-CS). The Ag-TiO2-CS nanocomposites have good redox electrochemical activity and excellent film-forming ability. Moreover, the free amino groups of CS could be used to attach HPtNPs for the immobilization of human chorionic gonadotropin antibody (anti-HCG). Cyclic voltammetry and electrochemical impedance spectroscopy (EIS) confirmed consecutive growth of the multilayer films, and TEM described the microstructure of the nanoparticles. Under optimized conditions, the immunosensor exhibited a wide linear range from 0.5 to 250 mIU·mL-1 with a relatively low detection limit of 0.26 mIU·mL-1 at 3 times back ground noise.2. Electrochemically deposited nanocomposites of carbon nanotubes and chitosan for detection of human chorionic gonadotrophinA new label-free amperometric immunosensor was developed for detection of HCG based on multiwall carbon nanotubes-chitosan (CNTs-CS) complex film and three-dimensional Au-TiO2 hybrids. Firstly, CNTs-CS film was deposited on a glassy carbon electrode (GCE) by a simple and controllable electrodeposition method. Next, thionine (Thi), as a redox probe, was covalently bound onto the CNTs-CS film with glutaraldehyde (GA) to obtain the Thi/CNTs-CS film. The free amino groups of the composite membrane were used to adsorb Au-TiO2 for immobilizing anti-HCG because of its large surface area and satisfactory biocompatibility. At last, BSA was employed to block possible remaining active sites. Under optimized conditions, the immunosensor displayed high sensitivity, good reproducibility, and a low detection limit of 0.08 mIU·mL-1 at 3 times the background noise. The promising feature of biocomposite and the ease of non-manual technique overcame the irreproducibility for preparing CNTs-based immunosensor systems and the proposed immunosensor displayed a satisfactory reproducibility and stability.3. Electrochemical immunosensor for human chorionic gonadotropin based on horseradish peroxidase-functionalized Prussian blue-carbon nanotubes/gold nanocomposites as labels for signal amplificationA new strategy for the sensitive detection of biomarker using Prussian blue-carbon nanotubes/gold nanocomposites (AuNPs-PB-CNTs) as labels and horseradish peroxidase (HRP) as an enhancer is presented herein. Chitosan hydrogel and TiO2 nanocomposites were first coated onto a GCE surface, which provided a biocompatible interface with abundant-NH2 groups for the immobilization of primary antibodies (Ab1). The detectable signal was recorded and amplified based on a sandwich-type immunoassay by the employment of HRP-labeled secondary antibodies (HRP-Ab2) and AuNPs-PB-CNTs bioconjugates according to the electrocatalytic reduction of H2O2 by bound HRP in the presence of PB. Using human chorionic gonadotropin (HCG) as a model analyte, experimental results showed that the prepared HRP-Ab2-AuNPs-PB-CNTs bioconjugates exhibited satisfactory redox activity and high efficiency of enzyme catalysis. Under optimized conditions, the immunosensor showed a low detection limit of 0.023 mIU·mL-1 HCG, with a linear range from 0.05 to 150 mIU·mL-1 at 3 times the background noise.