Electrochemical Biosensing Technology For Genetyping And Immunoassay

The detection of proteins and DNA plays essential roles in the fields of biomedical research and clinical diagnosis. Hence, it's an important domain that high sensitive assay methods were developed for the detection of proteins and DNA. Electrochemical apparatus are simple, sensitive and easy to be minisized,it is undoubted that the study of new electrochemical techniques with signal enhancement can accelerate the development of biosensor. Therefore, several new electrochemical techniques for genotyping or detection of protein have been developed in the presented paper and described as follows:(1) We developed a novel electrochemical genotyping technique based on gap ligation reaction with surface hybridization of ferrocene-tagged probe for the detection of a single-base mutation in -28 site (A→G) forβ-thalassemia gene. In this strategy, we design a pair of detection probes complementary to target DNA flanking the SNP locus with the polymorphic nucleotide left as a gap between the probe ends. Then, a nucleotide that complement to the single-base gap was filled and ligated with the presence of polymerase and ligase, The ligation products were then released from the hybrids and folded into a hairpin structure through intramolecular hybridization of two pre-designed complementary segments in probes. The hairpin-structured products can be captured to the electrode surface by hybridized with capture probes immobilized on electrode. There by presenting Fc-tags adjacent to electrode with redox current readily detected. This technique affords a robust, specific and sensitive platform for enzymatic SNP typing.(2) A novel electrochemical method based on allele-specific extension in detection of SNP genetyping has been proposed. Briefly, allele-specific primer perfectly matching with template can be extended to result in an increase of current, whereas allele-specific primer mismatching with the template at 3' terminal bases cannot be extended to result in an unchanged of the current. The present approach has been demonstrated with the identification of single-base mutation in -28 site (A to G) forβ-thalassemia gene and the wild type and mutant type were successfully scored.(3) A new method was developed for determination of platelet-derived growth factor BB (PDGF-BB) using a label-free electrochemical immunosensor with aptamer primed long-strand circular detection probe. Rabbit anti-human PDGF-B polyclonal antibody was used as the capture antibody immobilized on the electrode. The detection probe was pre-synthesized via polymerase extension along a single-stranded circular plasmid DNA template with a primer headed by anti-PDGF-B aptamer. In the presence of analyte, the aptamer primed circular probe was captured on the electrode via the formation of antibody/PDGF-BB/aptamer sandwiched complex. Electroactive indicator methylene blue (MB) could be adsorbed on the electrode surface via the analyte-sandwiched complex with long-strand circular DNA, thus yielding a strong electrochemical signal for the quantification of PDGF-BB. This strategy allowed label-free electrochemical detection with enormous signal amplification arising from the long-strand localized circular probe. It was observed that the oxidation peak current of MB in square wave voltammetric measurements showed a linear dependency upon the concentration of PDGF-BB in the range from 50 pg mL-1 to 500 ng mL-1.(4) A novel ultrasensitive electrical immunosensor using a microgapped interdigitated electrode array (MGIDEA) was proposed based on an enzyme-linked immunoassay format with enzymatic silver deposition for the detection of a tumor biomarker of prostate specific antigen (PSA). The capture antibody was covalently immobilized on the microgaps through a silanization layer. The presence of PSA resulted in the formation of a sandwiched complex of PSA with the capture antibody and the detection antibody conjugated with alkaline phosphatase, which could catalyze the production of a reductive agent ascorbic acid. The reduction of Ag+ ions by ascorbic acid yielded dispersed silver deposits over the microgaps, allowing the interdigitated electrodes to be electrically connected with a readily detectable increase in electrical conductance. This strategy was the first demonstration of incorporating enzymatic deposition of conductive materials in MGIDEA-based electrical detection for the development of biosensors. Such enzyme-based transformation was analyte-specific, highly efficient, and easily controlled, thereby offering the possibilities of enormous signal amplification without side effects from interfering enhancement reactions. The results revealed that the electrical conductance signal showed linear dependency on PSA concentration over a six-decade range from 1.0 fg mL-1 to 1.0 ng mL-1.(5) A novel electrochemical immunosensor was developed based on enzymatic deposition of copper onto platinum (Pt) nanoparticle modified electrode that inhibited the electrocatalytic reduction of protons to hydrogen in acidic medium by Pt. The method was implemented for the determination of a model target, human immunoglobulin G (hIgG), using a microtiter-based sandwiched immunoassay with alkaline phosphatase (ALP)-antibody conjugate as the detection probe. The binding of ALP on the microtiter interface due to the presence of target hIgG catalyzed the hydrolysis of a substrate ascorbic acid 2-phosphatase (AAP), producing a reductive product ascorbic acid that mediated the deposition of copper on a Pt nanoparticle modified electrode. A negative shift of hydrogen evolution potential was thus obtained at the Pt nanoparticle modified electrode, which could be determined using linear sweep voltammetry in 0.1 M HCl. The influence of experimental variables including the concentrations of H2PtCl6, Cu2+ and AAP as well as the reaction time of enzymatic copper deposition upon the potential shift was investigated. Under optimized conditions, the potential shift was observed to show linear dependency on hIgG concentration over a range from 10 pg mL-1 to 1.0μg mL-1.(6) A sensitive electrochemical immunoassay method was proposed based on gold nanoparticle mediated biocatalytic deposition of platinum followed by stripping voltammetric determination. The feasibility of the approach was investigated using a"sandwich"immunoassay format with hIgG as the analyte. HIgG was firstly captured by primary goat anti-human IgG polyclonal antibody (hIgG Ab) immobilized on polystyrene microwells. Gold nanopartcile- labeled ALP-hIgG Ab was then bound to the microwells through sandwiched hIgG. The surface-bound alkaline phosphate catalyzed the generation of ascorbic acid, which, in turn, reduced platinum ions into its metal form in the presence of gold nanoparticles. The deposited metal was released in aqua regia [V(HNO3):V(HCl)=1:3], and reduced on glassy-carbon electrode, which generated a significant cathodic current due to the platinum-catalyzed hydrogen evolution. The cathodic current was observed to show linear correlation to logarithmic hIgG concentration over the range from 100 ng mL-1 to 2μg mL-1 and from 100 pg mL-1 to 100 ng mL-1.