| Using biosensing techniques to detect the meaningful biomarkers for the accurate and rapid diagnosis of clinical diseases as well as for the field screening and mass monitoring of the epidemic diseases has been a hot topic in the current medical studies. Immunosensing technique, which incorporates high sensitivity of piezoelectric response and high specificity of antibody-antigen immunoreaction, shows an attractive future of potential applications in the biochemical diagnosis of clinical diseases. In this paper, nanotechnique, capillary electrophoresis and flow injection analysis were combined to develop immunological detection methods, which based on the lab previous research. The detail contents are as follows:(1)Immunoassay to rapidly detect bone sialoprotein based on piezoelectric biosensing technique. Bone sialoprotein (BSP) is a highly phosphorylated, glycosylated secretory protein. It is clinically important for bone metastasis and metabolism evaluation to assess the expression of serum BSP. A four-channel chip microarray was designed using AT-cut 10 MHz quartz crystal to detect BSP expression from clinical serum. Au nanopaticles were coated on the surface of gold-membrane electrode by 1,6-Hexanedithiol. Monoclonal antibody of BSP was thiolated by heterobifunctional cross-linkers Sulfo-LC-SPDP, and the hydrosulfuryl-antibody was fixed on the face of gold-membrane electrode via Au nanopaticles modified to make a bio-sensitive membrane, which constructed the piezoelectric immunity nano-chips. The serum BSP was detected after immunoreaction. Comparing with direct self-assembling film of hydrosulfuryl-antibody, the nanocomposite film could immobilize more antibodies with the activity of antibodis well retained. More over, the biosensor using Au nanopaticles as immobilization matrix obtained larger response and better reproducibility. The BSP piezoelectric immunosensor nano-array has simple structure, easy operation, high sensitivity and specificity. The constructed BSP nano-immunosensor array exhibited good linear relationship with serum detection, the linear equation was ?F = 872.66c -58.258(correlation coefficient = 0.9737), and can be used for quantitative detection of serum BSP.(2)Capillary electrophoresis electrochemical immunoassay for determination HBsAg in human serum. Hepatitis b surface antigen (HBsAg) is one of important serological markers of human who infectioned hepatitis b virus. A considerable proportion of patients with low level HBsAg, so it's vital significant to improve the detection sensitivity of HBsAg. Specific immunological reaction was take place at microtiter plate, between the pre-coated anti-HBs, human HBsAg and Ru(bpy)32+-labled anti-HBs, which formed sandwich-typed immunocomplex. Then the target HBsAg was determined by Capillary electrophoresis electrochemical immunoassay. The protocol has been characterized with cyclic voltammogram and potentiostatic method. A good linear relationship between peak current and the HBsAg concentration of 0.08～10 ng/mLwas obtained, the linear equation was y = 0.2840 x + 0.9535 (correlation coefficient=0.9857), and the detection limit was 0.01 ng/mL (3 ). The protocol could realize cheap, rapid, simple, convenient and quantitative determination of low concentration HBsAg in human serum.(3)Electrochemiluminescence system based on flow injection analysis. Using flow injection analysis, the electrochemical behaviors were characterized with cyclic voltammetry and chronoamperometry. Under the optimized conditions, the electrochemiluminescence system based on flow injection analysis offers an excellent amperometric response for tris (2,2′-bipyridine)ruthenium(Ⅱ) with a detection limit of 5×10-8 mol/L and a good linear relationship between 5×10-8～5×10-5 mol/L, which the linear equation was y = 1.3409 x– 77.79 (correlation coefficient = 0.9985), and has specific advantages in application. |
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