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.

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.

Microfluidic chip-based electrochemical immunoassay for hippuric acid.

Urinary hippuric acid (HA), of molecular weight 180 Da, is one of the major metabolites in toluene-exposed humans and is a major biological indicator. Simple and ubiquitous monitoring of exposure to toluene is very important in occupational health care, and a microfluidic chip-based electrochemical immunoassay for rapid and quantitative detection of HA in human urine is proposed in this paper. The system employs a conjugate of ferrocene (Fc) and hippuric acid (HA). The competition between hippuric acid (HA) and the ferrocene-hippuric acid complex (Fc-Lys-HA) to bind with a HA antibody coated onto polybeads generated electrical signals proportional to the HA concentration in the range of 0-40 mg mL(-1). All the complicated HA detection processes were integrated on the single microfluidic platform. The quantitative advantages of our HA detection chip are as follows: (1) the total chip size was reduced to 3.0 x 2.0 x 0.5 cm and is small enough to be portable, (2) the assay time took 1 min, and is shorter than that of conventional electrochemical HA immunoassay systems (about 20 min) and (3) 40 microL of the sample solution was enough to detect HA in the range of 0-40 mg mL(-1), which is enough range to be used for the point-of-care system. In addition, we suggest the improved chip-based HA assay method by the combination of electrochemical and enzymatic amplification processes for the detection of greater electrical signals. The sensitivity of the combined method was increased about three times compared to that of the non-enzymatic process.

Immunochromatographic analysis of hippuric acid in urine.

Toluene, a clear, colorless liquid with a distinctive smell, is the most commonly used industrial organic solvent. The adverse effects of chronic toluene exposure have been reported. The abuse of volatile substances is practiced mainly by adolescents and young adults. Chronic toluene abuse causes permanent changes in brain structure correlated with brain dysfunction; therefore, it is important to monitor the level of toluene exposure to prevent neurological damages. In this study, immunochromatographic analysis was performed to measure a level of the exposed toluene easily and accurately in urine. Inhaled toluene is metabolized to hippuric acid (HA) in the liver and secreted in urine. Therefore, the monoclonal antibodies against HA were generated and characterized by indirect competitive ELISA. The sensitivity was then monitored in order to adjust the cutoff concentration to 2 mg of HA/mL of urine. Using these monoclonal antibodies as raw materials, the immunochromatographic device was manufactured with the lateral flow system. The clinical studies were performed with suspected users' urine samples, and the results were confirmed by high-performance liquid chromatography.

The enigmatic cytochrome b-559 of oxygenic photosynthesis.

The ubiquitous and obligatory association of cytochrome b-559 with the photosystem II reaction center of oxygenic photosynthesis is a conundrum since it seems not to have a function in the primary electron transport pathway of oxygen evolution. A model for the cytochrome structure that satisfies the cis-positive rule for membrane protein assembly consists of two short, non-identical hydrophobic membrane-spanning polypeptides (α and ß), each containing a single histidine residue, as ligands for the bridging heme prosthetic group that is on the side of the membrane opposite to the water splitting apparatus. The ability of the heterodimer, but not the single α-subunit, to satisfy the cis-positive rule implies that the cytochrome inserts into the membrane as a heterodimer, with some evidence implicating it as the first membrane inserted unit of the assembling reaction center. The very positive redox potential of the cytochrome can be explained by a position for the heme in a hydrophobic niche near the stromal aqueous interface where it is also influenced by the large positive dipole potential of the parallel α-helices of the cytochrome. The requirement for the cytochrome in oxygenic photosynthesis may be a consequence of the presence of the strongly oxidizing reaction center needed for H2 O-splitting. This may lead to the need, under conditions of stress or plastid development, for an alternate source of electrons when the H2 O-splitting system is not operative as a source of reductant for the reaction center.