| Enzyme linked immunoassay( ELISA) which combines the specificity of the antigen-antibody reaction and catalytic amplification of enzyme is the marker staining method by biological enzyme. It maintains the sensitivity of the enzyme catalyzed reaction,but also keeps the specificity of the antigen-antibody reaction. As is known, the disvantages of the natural enzyme are the variability, and not easy store in the practical application. In recent years, gold nanoparticles have been widely used in the field of high performance immunoassay because of its high specific surface, unique biological and optical properties. However, there are some bottlenecks in the use of these materials such as the reduction of the catalytic activity due to the adsorption of biomolecules, the complex process of nano material marking. Thus, in this thesis, we developed a series of new ELISA to solve and improve these problems based on the optical and biological properties of gold nanoparticles. Meanwhile, the low concentration of protein in complex biological samples has been successfully and sensitively detected. The main works in this thesis are:In the first chapter, the ELISA, especially for the gold nanoparticles used in this method was introduced briefly as well as the purpose of this thesis.In the second chapter, an innovative and highly sensitive colorimetric immunoassay was developed based on the formation of catalytic platinum layer on immunogold labels.The immunoassay was implemented in polystyrene microwells based on a classical sandwich-type assay format using gold nanoparticle as label, on which a platinum layer was fromed by the further addition of platinum enhancing solution. The newborn platinum layers could accelerate the oxidation of TMB drastically by H2O2 to generate blue product with a strong absorption around 652 nm, providing a simple way for quantification of captured immunogold labels as well as the amount of target analyte by using microplate readers, spectrophotometers or naked eyes. The experiment results show the absorption at562 nm increased linearly with the concentration of human IgG(a model antigen) in a range from 0.5 to 20 ng/mL. And the detection limit was 0.15 ng/mL. The results also show that it is more sensitive than traditional ELISA, electrochemicalmetalloimmunoassay and the recent deveoped HRP mimic nanomaterial tags-based immunoassay and is comparable with fluorescent immunoassay. Additionally, the eye-readable color change, even for the detection of human IgG with a concentration less than 0.5 ng/mL, also makes the immunoassay more convenient in clinical diagnosis avoiding the use of complex equipments. The successful application of the proposed immunoassay to the assay of human IgG and alpha fetoprotein(an important tumor marker) in real samples indicates that the developed immunoassay has good specificity and is applicable to clinical diagnosis.In the third chapter, a plasmonic ELISA was developed by iodine-mediated etching of gold nanorods(Au NRs), which can be used to dectet the alkaline phosphatase(ALP).Firstly, the sandwich type immunocomplex is formed, and the ALP bound on the polystyrene microwells will hydrolyze ascorbic acid 2-phosphate into ascorbic acid.Subsequently, iodate is reduced into to iodine(a moderate oxidant) which etches Au NRs from rod to sphere in shape. Then, the shape change of Au NRs leads to a blue-shift of longitudinal localized surface plasmon resonance(LSPR). In doing so, the solution of Au NRs changes from blue to red.The proposed plasmonic ELISA has achieved an ultra-low detection limit(100 pg/mL) for human immunoglobulin G(IgG). It was noted that the visual detection limit(3.0 ng/mL) allows the rapid differential diagnosis with naked eye.The further detection of human IgG in fetal bovine serum indicates that the method can be used to determine the low abundance protein in complex biological samples.In the fourth chapter, a plasmonic ELISA was performed by iodine-mediated etching of gold nanorods to dectect the horseradish peroxidase. In this study, horseradish peroxidase catalyzes oxidation of p-lodophenol by H2O2. In the following, the same as mentioned in above, the pruduced iodine etches gold nanorods, which leads to the change of morphology of Au NRs along with a blue shift of longitudinal LSPR absorption peak.The orthogonal experimental design method was use to select the experiment conditions.The results show that the detection limit is 5 ng/mL H-IgG. And this method has been proved to have good anti-interference ability by detecting fetal bovine serum samples added target, with recoveries of 89.9% ?106%. |
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