Study On Signal Enhancing Electrochemical Immunoassay

The enhancement of immunoassay signal is always important in immunoassay study, which is vital to improvement of the assay sensitivity. Electrochemical apparatus are simple, sensitive and easy to be miniaturized. So it is undoubtable that the study of new electrochemical immunoassay with signal enhancement can accelerate the devolpment of immunoassay. Therefore, this research focuses on the enhancement of the signal of immunoassay, especially on the enhancement of the signal of electrochemical immunoassay. The detail contents are as follows:1. A new immunosensor was proposed based on the receptor protein adsorbed directly on a porous gold film. The film was produced electrochemically on a glassy carbon electrode in 0.08 M hydrogen tetrachloroaurate solution containing 0.004 M lead acetate with applied potential of -0.5V (versus Ag/AgCl) for 50 s. The assay was carried out based on a sandwich procedure. The impedance signals amplified by precipitation of an insoluble product on the electrode showed good linearity with the content of IgG in the range of 0.011–11 ng/mL with a detection limit of 0.009 ng/mL.2. We have reported an electrochemical amplification immunoassay using biocatalytic metal deposition coupled with anodic stripping voltammetric detection. In this method, the capture antibody was first immobilized onto a gold electrode via a self-assembled layer. After a sandwich immunoreaction, alkaline phosphatase labeled second antibody was bound to the gold electrode. The alkaline phosphatase on the electrode initiates the hydrolysis of ascorbic acid 2-phosphate to produce ascorbic acid. The latter, in turn, reduces silver ions on the electrode surface, leading to deposition of the metal onto protein modified electrode surface. The deposited metal was electrochemically stripped into solution and was then measured by anodic stripping voltammmetry. Compared with the direct voltammetry detection of ascorbic acid, anodic stripping voltammetry detection of metal ions is more sensitive. For the amount of deposited silver relates to the amount of enzyme-generated ascorbic acid, which was controlled by the amount of enzyme bond on the electrode surface, the stripping current signal reflects the amount of target protein. The utilization of the high catalysis activity of enzyme and the sensitive anodic stripping voltammetry to detect metal ions dramatically enhanced the sensitivity in immunoassay.3. An electrochemical immunoassay technique has been developed based on thesensitive detection of the enzyme-generated product with a bi-electrode signal transduction system. The system uses two separate electrodes, an immunoelectrode and a detection electrode to form a galvanic cell to implement the red/ox reactions on two different electrodes. That is the enzyme-generated reductant in the anode region is electrochemically oxidized by an oxidant (silver ions) in the cathode apartment. Based on a sandwich procedure, after immunoelectrode with antibody immobilized on its surface bound with the corresponding antigen and alkaline phosphatase conjugated antibody successively, the immunoelectrode was placed in enzyme reaction solution and wired to the detection electrode which was immerged into a silver deposition solution. These two solutions are connected with a salt bridge. Thus a bi-electrode signal transduction system is constructed in which the immunoelectrode acts as anode and the detection electrode serves as cathode. The enzyme bound on the anode surface initiates the hydrolysis of ascorbic acid 2- phosphate to produce ascorbic acid in the anode region. The ascorbic acid produced in the anodic apartment is electrochemically oxidized by silver ions coupled with the deposition of silver metal on the cathode. Via a period of 30 min deposition, silver will deposited on the detection electrode in an amount corresponding to the quantity of ascorbic acid produced, leading to a great enhancement in the electrochemical stripping signal due to the accumulation of metallic silver by enzyme-generated product. Compared with the method using chemical deposition of silver, the electrochemical deposition of silver on a separate detection electrode apartment avoids the possible influence of silver deposition on the enzyme activity.4. A novel electrochemical immunosensor with amplification effect based on the enzyme inhibition of silver deposition was proposed. In this method, the capture antibody was first immobilized onto a gold electrode via a self-assembled layer. After a sandwich immunoreaction, HRP labeled antibody was bound to the gold electrode. The HRP on the electrode inhibited silver deposition when the electrode was incubated in hydroquinone-H2O2 solution and silver ion solution. The linear sweep voltammetry was chosen to detect the deposited silver and the result showed that the peak current was linearly proportional to the content of IgG in the range of 50 to 2500 ng/mL with a detection limit of 35 ng/mL.5. A successive signal amplification electrochemical immunoassay based on the biocatalytic precipitation of metal nanoparticles and the nanoparticles produced in turn caused catalytic precipitation of metal onto themselves, leading to the nanoparticles enlarged further. The immunoassay was carried out based on a sandwich procedureusing polystyrene microwells to immobilize antibody. After all the procedures including immunoreaction and metal precipitation were completed, the metal on polystyrene microwells was dissolved and was measured by anodic stripping voltammetry. For the method had coupled enzyme catalysis with the catalysis of nanoparticles to successively amplify the immunoassay signal, the sensitivity was enhanced dramatically. The anodic stripping peak current was proportional to the concentration of human IgG in a dynamic range of 0.1 to 10 ng/ml with a detection limit of 0.06 ng/ml. So it is obviously that the utilization of the high catalysis activity of enzyme and of nanoparticles is a promising method to improve the sensitivity of immunoassay.6. A sensitive immunosensor using colloidal gold as electrochemical label is described. In this method, the capture protein was first immobilized on a carbon paste electrode surface through passive adsorption to bind quantitatively with corresponding antigen and colloidal gold labeled second antibody to perform a sandwich assay. To detect the amount of the colloidal gold captured on the electrode surface, the colloid was first oxidized electrochemically to produce AuCl4- which was adsorbed strongly on the electrode surface. Adsorptive voltammetry was employed for the determination of AuCl4- adsorbed on the electrode surface. A linear relationship between peak current and the antigen concentration (human IgG) from 10 to 500 ng/ml is obtained with a detection limit of 4.0 ng/ml.7. Besides the the signal enhancing electrochemical immunoassay, we used electrochemical and other tools to investigate a novel, simple and sensitive immunoassay using fluorescence quenching caused by gold nanoparticles coated with monoclonal antibody. The method is based on a noncompetitive heterogeneous immunoassay of human IgG conducted by the typical procedure of sandwich immunocomplex formation. Goat anti-human IgG was first adsorbed on polystyrene microwells, and human IgG analyte was captured by the primary antibody and then sandwiched by secondary antibody labeled with gold nanoparticles. The sandwich-type immunocomplex were subsequently dissociated by the mixing solution of sodium hydroxide and trisodium citrate, the solution containing gold nanoparticles coated with antibody obtained was used to quench fluorescence. The fluorescence intensity of fluorescein at 517nm was reversibly proportional to the logarithm of the concentration of human IgG in the dynamic range from 10 to 5000 ng/mL with a detection limit of 4.7 ng/mL. The electrochemical experiments and the UV-Vis measurements were applied to demonstrate whether the immunoglod was dissociated completely andwhether the gold nanoparticles aggregated after being dissociated, respectively. The proposed system can be extended to detect target molecules such as other kinds of antigen, DNA strands and has broad potential applications in disease diagnosis.