Signal Enhancing Bioassay Methods Using Nanoparticles And Enzymes

Developing of sensitive immunoassay technology is one of the important tasks in immunoassay, which is crucial for realizing the detection of some vital diseases. 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. Moreover, Many pathogenic and genetic diseases are associated with changes in the sequence of particular genes. Among these changes, the point mutation, i.e. single nucleotide polymorphisms (SNPs), are the most abundant form of genetic variation. Achieving early, accurate, simple and rapid identification to these single-base mutations is of particular importance for early diagnosis and therapy of corresponding diseases. Up to now, many techniques have been developed for SNP detection. However, these conventional procedures are applied to date in very few laboratories as they are generally time-consuming, not quantitative, complicated, and requiring expensive instrumentation and technical skills. Accordingly, exploring some simple, cost-effective, accurate and easy to be clinically popularized detection methods for SNPs is still of considerable interest. Therefore, this research focuses on the sensitivity enhancement of electrochemical immunoassay and the development of electrochemical DNA quantification. The detail contents are as follows:1. A new method based on cyclic accumulation of gold nanoparticles has been proposed for determining human immunoglobulin G (IgG) by anodic stripping voltammetry. The dissociation reaction between dethiobiotin and avidin in the presence of biotin provides efficient means for the cyclic accumulation of gold nanoparticles used for the final analytical quantification. The immunoassay was conducted by following the typical procedure for sandwich-type immunoreaction. Goat anti-human IgG is immobilized on the wells of microtiter plates. The human IgG analyte is first captured by the primary antibody and then sandwiched by secondary antibody labeled with dethiobiotin. Avidin-Au solution was introduced to react with dethiobiotinylated antibody followed by adding of bioitin solution to wash it down. The dissociation of avidin-Au from dethiobiotinylated antibody was caused by displacing function of biotin to dethiobiotin. Making use of the ability of dethiobiotinylated antibody to keep its activity after the association/dissociation reaction, the alternatively treatment with avidin-Au and biotin solutions could realize cyclic accumulation of gold nanoparticles for ASV quantification. The anodic peak current increases gradually with the increasing accumulation cycles. Five cycles of accumulation are sufficient for the assay. The experiment conducted with a blank solution without addition of the analyte showed an extremely low background even with the number of accumulation cycles reached 10. The low background of the proposed method is a distinguished advantage providing a possibility for determination of down to 0.1 ng/ml human IgG. The RSD of the method is 9.57% for eight parallel determinations of 1 ng/ml human IgG under the same conditions.2. A new method based on accumulation of gold nanoparticles using magnetic particles has been proposed for determining human IgG by anodic stripping voltammetry. The feasibility of the approach was investigated for a noncompetitive heterogeneous immunoassay of human immunoglobulin G as a model system. Goat anti human IgG is immobilized on the wells of microtiter plates. The human IgG analyte is first captured by the primary antibody and then sandwiched by secondary antibody labeled with dethiobiotin. The conjugate of biotin modified magnetic particles captured avidin functionalized gold nanoparticles (avidin-Au) was introduced to react with dethiobiotinylated antibody followed by adding of bioitin solution to wash it down. The dissociation of the conjugates from the dethiobiotinylated antibody was caused by displacing function of biotin to dethiobiotin. Making use of the ability of the modified magnetic particles capturing avidin-Au, the following treatment with biotin solutions could realize release of the conjugates for gold ASV quantification. The experiment performed with a blank solution without addition of the analyte showed a low background which is a distinguished advantage providing a possibility for determination of down to 0.1 ng/ml human IgG.3. A new method based on cyclic accumulation of HRP enzyme has been proposed for determinating human IgG by fluorescence. The dissociation reaction between dethiobiotin and avidin in the presence of biotin provides efficient means for the cyclic accumulation of HRP enzymes used for the final analytical quantification. The experiment conducted with a blank solution without addition of the analyte showed an extremely low background even with the number of accumulation cycles reached seven. The relatively low background of the proposed method is a distinguished advantage providing a possibility for determination of down to 0.05 ng/ml human IgG.4. 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.5. 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. The immunoassay was conducted by following the typical procedure for sandwich-type immunoreaction. Goat anti-human IgG was immobilized on a glass carbon electrode. 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. 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.6. We demonstrated herein a novel electrochemical immunoassay protocol based on agarose beads and biometallization. Agarose beads were modified with avidin. The biotin labeled goat anti-human IgG would be captured on the beads through the reaction of avidin with biotin. The beads captured goat anti-human IgG antibodies was used as probe beads. During the immunoassay, certain quantities of the probe beads, alkaline phosphatase labeled antibodies and target human IgG were mixed to react. After washed for several times with buffer solution and ultra pure water, ascorbic acid phosphate and silver ions solution were added to react for 30 minutes in dark. After washed for eight times with ultra pure water, the silver which had been deposited was dissolved with concentrated HNO3. The quantification would be realized by ASV. 7. We developed herein a novel electrochemical biosensor for quantification of DNA based on conformational change of molecular beacon caused by hybridization with subsequently enzymatic amplification quantification. 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.