Gun-Sik Tae

Expression, purification and improved antigenicity of the Mycobacterium tuberculosis PstS1 antigen for serodiagnosis.

The phosphate-specific transport substrate binding protein-1 (PstS1) is a potential antigen used for the serological diagnosis of tuberculosis. For a highly specific diagnostic result, it is important that the recombinant PstS1 be highly pure and correctly folded. In this study, the PstS1 was expressed as fusion protein with glutathione-S-transferase (PstS1-GST) and Escherichia coli trigger factor (PstS1-TF) and their immunodiagnostic potentials were evaluated. The insoluble PstS1-GST was denatured and refolded to the native conformation by a step-gradient dilution, followed by purification with affinity chromatography on immobilized glutathione whereas the soluble PstS1-TF was directly purified by Ni-NTA affinity and size-exclusion chromatographies. The levels of antibody responses to PstS1-TF and PstS1-GST were measured by enzyme-linked immunosorbent assay (ELISA) in the sera of 22 tuberculosis patients with smear-positive and culture-positive tuberculosis as well as 20 healthy individuals; the antigenicities of the samples were evaluated in terms of sensitivity and specificity. To determine the diagnostic accuracy, receiver operation characteristic (ROC) curves were constructed and then the areas under the ROC curves (AUC) were calculated; the AUC values for PstS1-TF and PstS1-GST were 0.971 and 0.877 with 95% confidence intervals (CI) of 0.927-1.000 and 0.768-0.986, respectively. The specificity of PstS1-TF was reduced from 89.5% to 84.2%, but in case of PstS1-GST it dropped drastically from 78.9% to 26.3% when the sensitivity was raised from 86.4% up to 95.5%. These results indicate that PstS1-TF is capable of producing more accurate and consistent serodiagnostic results than PstS1-GST, possibly due to its conformation being closer to the native state.

Heterogeneous electrochemical immunoassay of hippuric acid on the electrodeposited organic films.

By directly coordinating hippuric acid (HA) to the ferrate (Fe) as an electron transfer mediator, we synthesized a Fe-HA complex, which shows a good electrochemical signal and thus enables the electrochemical immunoanalysis for HA. We electrodeposited organic films containing imidazole groups on the electrode surface and then bonded Ni ion (positive charge) to induce immobilization of Fe-HA (negative charge) through the electrostatic interaction. The heterogeneous competitive immunoassay system relies on the interaction between immobilized Fe-HA antigen conjugate and free HA antigen to its antibody (anti-HA). The electric signal becomes weaker due to the hindered electron transfer reaction when a large-sized HA antibody is bound onto the Fe-HA. However, in the presence of HA, the electric signal increases because free HA competitively reacts with the HA antibody prior to actual reaction and thus prevents the HA antibody from interacting with Fe-HA at the electrode surface. This competition reaction enabled an electrochemical quantitative analysis of HA concentration with a detection limit of 0.5 μg mL−1, and thus allowed us to develop a simple and rapid electrochemical immunosensor.

Simple Electrochemical Immunosensor for the Detection of Hippuric Acid on the Screen-printed Carbon Electrode Modified Gold Nanoparticles

Department of chemistry, Dankook University, Anseo-dong, Cheonan 330-714, Republic of Korea(Received January 25, 2011 : Accepted February 21, 2011)Abstract: This paper describes an electrochemical immunosensor for simple, fast and quanti-tative detection of a urinary hippuric acid which is one of major biological indicator in toluene-exposed humans. The feature of this electrochemical system for immunoassay of hippuric acidis based on the direct conjugation of ferrocene to a hippuric acid. With the competition betweenthe ferrocene-hippuric acid complex and hippuric acid for binding to the anti-hippuric acidmonoclonal antibody coated onto gold nanoparticles, the electrical signals are turned out to beproportional to urinary hippuric acid in the range of 0.01-10 mg/mL, which is enough to beused for the point-of-care. The proposed electrochemical method could extend its applicationsto detect a wide range of different small molecules of antigens in the health care area.Keywords : Immunosensor, Hippuric acid, Ferrocene, Point-of-care

The Electrochemical Studies of Two Osmium Redox Polymer Films and Their Application for Multi-Detecting Biosensor

Screen printed carbon electrodes (SPEs) modified with co-immobilized osmium-based redox polymers can be used to apply multi-detecting biosensors. In this study, we report our initial studies of multi-detecting biosensor concepts using two osmium-based redox polymers for horseradish peroxidase-mediated reduction of coupled to glucose oxidase-mediated oxidation of glucose. We target to synthesize two osmium redox polymers of potentials use, a chloride-containing redox polymer ( + 0.520 vs. Ag/AgCl) and a methoxy-containing redox polymer + 0.150 vs. Ag/AgCl). The former show good catalytic electrical signals with horseradish peroxidase and the latter`s redox polymer is to be an effective redox mediator of glucose oxidation by glucose oxidase.

Simple Electrochemical Immunosensor for the Determination of Rabbit IgG Using Osmium Redox Polymer Films

An amperometric immunosensor for the determination of rabbit IgG is proposed. The immunoassay utilizes a screen-printed carbon electrode on which osmium redox polymer is electrodeposited. This immunoassay detects 0.1 ng/ml of rabbit IgG, which is fold higher than the most sensitive enzyme amplified amperometric immunoassay. The assay utilizes a screen-printed carbon electrode which was pre-coated by a co-electrodeposited film of an electron conducting redox hydrogel and a rabbit IgG. The rabbit IgG in the electron conducting film conjugates captures, when present, the anti-rabbit IgG. The captured anti-rabbit-IgG is labeled with horseradish peroxidase (HRP) which catalyzes the two-electron reduction of to water. Because the redox hydrogel electrically connects HRP reaction centers to the electrode, completion of the sandwich converts the film from non-electrocatalytic to electro-catalytic for the reduction of to when the electrode is poised at 200 mV vs. Ag/AgCl.