| Tuberculosis is the most frequent cause of infection-related death worldwide.Overexpressed IFN-γ production in biological samples is generally regarded as a good indicator of tuberculosis infection. At the present time, enzyme-linked immunosorbent method(ELISA) is mostly used for the detection of IFN-γ in clinical biological samples. Although the sensitivities of these detection kits are satisfactory, the assays are still cumbersome, expensive, and time-consuming, involving multiple washing steps and requiring large sample volumes for analysis. At present, electrochemical biosensor based on glassy carbon electrode modified with gold nanoparticle(AuNPs) are commonly used. Although the sensitivity and stability are satisfactory, the method are still not be widely used, due to its high cost. In this thesis, based on the current technical limitation for IFN-γ detecting, firstly, indium tin oxide(ITO)conductive glass was modified with gold nanocomposites, then loaded with antibodies, and antigens to obtain disposable probe. Then, fixed it on the platinum electrode holder to obtain working electrode(WE). And two kinds of electrochemical biosensor were constructed for highly sensitive and rapid detection of IFN-γ. The structure and performance were researched.The main research contents of this thesis were as follow:1. We used sodium citrate reduction method to prepare the gold particles, the gold particles obtained was spherical, uniform distribution, and the average particle size was 25 nm. ITO modified with poly diallyldimethylammonium chloride(PDDA) was covered with squamaceous flim, after electrostatic interactions with gold nanoparticles(AuNPs), spherical particles were uniform distribution on the surface of the AuNPs/PDDA/ITO composite. Homemade screen printing ITO electrode modified with graphene-chitosan(Gr-Chit) was covered with a layer of wrinkled film, after electrostatic interactions with glutathione-modified gold nanoparticles(GSH-AuNPs), spherical particles were uniform distribution on the surface of the GSH-AuNPs/Gr-Chit/ITO composite.2. The disposable probe was preparaed by loading antibody on AuNPs/PDDA/ITO composite, using BSA to remove nonspecific adsorption, then based on the specific reaction to connect IFN-γ, and at last modified with HRP-Ab2-AuNPs, and then working electrode was assembled and the biosensor was constructed. The structure and performance were researched.The results showed that the disposable probe(HRP-Ab2-AuNPs/IFN-γ/BSA/Ab1/AuNPs/PDDA/ITO) we prepared presented honeycombed morphology. Under optimized conditions the proposed sensor showed a high sensitivity and a linear range of 0.1-10000 pg/mL with a detection limit of 0.048 pg/mL(S/N=3). The biosensor shows excellent detection reproducibility,good specificity, and stability. The assay results of clinical serum samples obtained by the biosensor were in acceptable agreement with the reference values.3. The disposable probe was preparaed by loading antibody on GSH-AuNPs/Gr-Chit/ITO composite, using BSA to remove nonspecific adsorption, then, based on the specific reaction to connect IFN-γ. Then working electrode was assembled and the biosensor was constructed. The structure and performance were researched. The results indicated that the disposable probe(IFN-γ/BSA/Ab1/GSH-AuNPs/Gr-Chit/ITO) we prepared presented honeycombed morphology.Under optimum conditions the relative increased differential pulse voltammetry(DPV) peak current values were proportional to the logarithmic value of IFN-γ concentrations in a wide range of 5 to 4000 pg/mL with a low detection limit of 0.5 pg/mL(S/N=3).The resulting IFN-γbiosensor shows excellent detection reproducibility, good specificity, and stability. In addition,for the detection of clinical serum samples, it is well consistent with the standard enzyme-linked immunosorbent assay(ELISA) tests. |
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