Ultrasensitive Electrochemical Immunosensor Based On Quantum Dots For The Detection Of Tuberculosis Infection

Objective:1. To design and synthesis a number of ESAT-6, CFP-10 and Ag85 B antigen peptides, and to select several tuberculosis antigen peptides with high specificity.2. To develop a novel ultra-sensitive nano electrochemical immunosensor and to evaluate its value in the diagnosis of tuberculosis infections.Methods:1. Designed mycobacterium tuberculosis specific antigen polypeptide by consulting a large number of references and using the antigen epitope analysis software. Single blood mononuclear cells(PBMC) were collected from 189 pulmonary tuberculosis patients, and cultured with tuberculosis specific antigen polypeptide ESAT-6, CFP-10 and Ag85 B for 72 h respectively. IFN-γ secreted in the cell culture was determined by enzyme-linked immunosorbent assay(ELISA). The polypeptides which could stimulate PBMC to secrete more IFN-γ were chose.2. Single blood mononuclear cells(PBMC) were collected from 151 pulmonary tuberculosis patients, and cultured with ESAT-6, CFP-10 and Ag85 B polypeptides early chose for 22 h alone or in different combinations respectively. ELISPOT assay was used to detect the number of spot forming cells(SFC). The polypeptides which could stimulate PBMC to have more SFC were chose.3. Quantum dots(QD) and secondary antibodies(Ab2) were covered on the surface of magnetic beads(MB). The new ultra-sensitive nano electrochemical immunosensor was constructed using the MB-QDs-Ab2 as immune probe. The antigen peptides were used to stimulate PBMC of TB patients for secreting IFN-γ. The IFN-γ levels were determined by sandwich immune reaction and electrochemical square wave stripping voltammetry. Results:1. 10 ESAT-6 polypeptides(numbered E1-E10), 10 CFP-10 polypeptides(numbered C1-C10) and 9 Ag85 B polypeptides(numbered A1-A9) were designed and synthesized.2. Three ESAT-6 polypeptides(E3, E6, E9), three CFP-10 polypeptides(C1, C6, C8) and three Ag85 B polypeptides(A2, A5, A9) were chose by the ELISA method, and the advantage combination of different polypeptides were chose by the ELISPOT method.3. Ultra-sensitive electrochemical immunosensor based on quantum dots was constructed successfully. The measured current responses were proportional to the concentration of IFN-γ. Under optimal conditions, the voltammetric response was linear over the range of 0.01pg/m L-10ng/m L of IFN-γ(R2=0.966). The immunosensor could be used for the detection of real sample with consistent results in comparison with those obtained by the ELISA method. Conclusion:Tuberculosis specific antigen polypeptides E3, E6, E9, C1, C6, C8, A2, A5, A9 and the different combinations of them have good specificity and important application value in the IFN-γ release test. The new ultra-sensitive electrochemical immunosensor based on quantum dots showed high sensitivity, good stability, quick response, low price, easy to operate, and many other advantages. The nano electrochemical immunosensor is expected to be used in clinical TB infection diagnosis and other pathogenic microorganism detection and other disease diagnosis.