Signal Enhancing Bioassay Methods Based On Catalytic Metal Deposition

The enhancement of immunoassay signal is always important in immunoassay study, which is crucial for improving the assay sensitivity and realizing the detection of some vital diseases. Enzyme and nanoparticles, because of their special properties, have promising potential clinical application. Moreover, exploring some simple, cost-effective, accurate and easy to be clinically popularized detection methods for DNA is still of considerable interest. Therefore, this research focuses on the enhancement of immunoassay signal and the development of electrochemical DNA quantification. The detail contents are as follows:1. Signal enhancing immunoassay based on biometallization (enzymatic silver deposition)In chapter 2, we have developed two simple, rapid and sensitive immunoassay protocols, which coupled the special amplified properties of biometallization with the electrochemical or SERS-based detection for quantification of human IgG. The biometallization holds great promise for the enhancement of immunoassay signal in biosensing.(1) A novel, sensitive electrochemical immunoassay in a homogeneously dispersed medium is described herein based on the unique features of agarose beads and the special amplified properties of biometallization. The immunochemical recognition event between human immunoglobulin G (IgG) and goat anti-human IgG antibody is chosen as the model system to demonstrate the proposed immunoassay approach. Avidin-agarose beads rapidly react with the biotinylated goat anti-human IgG antibody to form agarose beads-goat anti-human IgG conjugate (agarose bead-Ab). Agarose bead-Ab, alkaline phosphatase conjugated goat anti-human IgG antibody (ALP-Ab) and the human IgG analyte are mixed to form sandwich-type immunocomplex followed by the addition of the enzymatic silver deposition solution to deposit silver onto the surface of proteins and agarose beads. The silver deposited are dissolved and quantified by anodic stripping voltammetry. The logarithm of the anodic stripping peak current depended linearly on the logarithm of the concentration of human IgG in the range from 1 to 1000 ng/mL. A detection limit as low as 0.5 ng/mL human IgG was attained by 3σ-rule. The R.S.D. of the approach is 9.65% for eight times determination of 10 ng/mL human IgG under same conditions. Optical microscope and TEM graphs were also utilized to characterize agarose beads and silver nanoparticles formed.(2) We have reported on a novel enzyme immunoassay method for the detection of protein using biocatalytic silver nanoparticles as an enhanced substrate based on surface-enhanced Raman scattering (SERS). First, ascorbic acid was converted from ascorbic acid 2-phosphate by alkaline phosphatase immobilized on polystyrene microwells after a typical sandwich immunoreaction. Then Ag(I) ions were reduced to silver nanoparticles by the obtained ascorbic acid, which would result in a SERS signal when Raman dyes were absorbed. Using human IgG as a model protein, a wide linear dynamic range (1 to 100 ng/mL) was reached with a low detection limit (0.02 ng/mL) under the optimized assay conditions. Moreover, the production of an enhanced substrate was chosen as the signaling element in this method, which demonstrates a new way for SERS-based quantitative detection.2. Electrochemical protocol and immunosensor based on the precipitation of copper on gold nanoparticle tagsIn chapter 3, a novel electrochemical protocol for quantification of human IgG is demonstrated herein based on the precipitation of copper on gold nanoparticle tags, and a simple electrochemical immunosensor for quantification of human IgG is developed based on precipitation of copper on gold nanoparticle tags later.(1) We demonstrate herein a novel electrochemical protocol for quantification of human IgG based on the precipitation of copper on gold nanoparticle tags and a subsequent electrochemical stripping detection of the dissolved copper. The immunoassay was conducted by following the typical procedure for sandwich-type immunoreaction. Goat anti-human IgG was immobilized on the wells of microtiter plates. The human IgG analyte was first captured by the primary antibody and then sandwiched by secondary antibody labeled with gold nanoparticles. The copper enhancer solution was then added to deposite copper on the gold nanoparticle tags. After dissolved with HNO3, the released copper ions were then quantified by ASV. The detection limit is 0.5 ng/mL by 3σrule. In order to investigate the feasibility of the newly developed technique to be applied for clinical analysis, several standard human IgG serum specimens were also examined by the method.(2) We developed herein a novel electrochemical immunosensor for quantification of human IgG based on precipitation of copper on gold nanoparticle tags with subsequent electrochemical detection of copper. First, goat anti-human IgG was immobilized on a glass carbon electrode. Then the human IgG analyte was captured by the primary antibody and sandwiched by secondary antibody labeled with gold nanoparticles. The copper enhancer solution was then added to deposite copper on the gold nanoparticle tags. The quantification of copper was performed in diluted HNO3 by using cyclic voltammetry. The elimination of the acid dissolution and metal accumulation steps greatly simplifies and shortens particle-based electrochemical bioassays, and eliminates background contributions from electrostatically bound metal ions which would otherwise be released through the acid dissolution. The detection limit is 0.075 ng/mL by 3σrule. In order to investigate the feasibility of the newly developed technique to be applied for clinical analysis, several standard human IgG serum specimens were analyzed by the method. To our knowledge, the copper enhancing procedure is the first time to be developed for immunsensors.3. Enzymatic amplification detection of DNA based on"molecular beacon"biosensorsIn chapter 4, we have described a novel electrochemical DNA biosensor based on enzymatic amplification protocol and molecular beacon (MB) probe. Molecular beacon (MB) modified with a thiol at its 5′end and a biotin group at its 3′end was immobilized on the gold electrode through mixed self-assembling with mercaptopropionic acid. In the absence of complementary target, streptavidin-HRP could not be captured by biotin groups labeled on MB for the steric blocking of the hairpin structure and mercaptopronoic acid. The biotin group would be released by hybridization reaction in the presence of complementary target, thus streptavidin-HRP would ease to reacte with biotin groups. The subsequent quantification of DNA would realize by electrochemical enzymatic amplification reaction. The detection limit as defined by 3σrule is 0.1 nM. In order to investigate the feasibility of the newly developed technique to be applied for mismatch discrimination, an oligonucleotide with one G-G mismatch compared with the complementary sequence were employed. Results indicated this novel protocol we advanced could discriminate single mismatch efficiently.