RESUMO
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.
Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Hipuratos/isolamento & purificação , Imunoensaio/métodos , Ferro/química , Anticorpos/química , Anticorpos/imunologia , Antígenos/imunologia , Antígenos/isolamento & purificação , Hipuratos/química , Hipuratos/imunologia , Humanos , Limite de Detecção , Níquel/química , Tolueno/químicaRESUMO
A homogeneous electrochemical immunoassay is based on the interaction of osmium-antigen conjugate with its antibody. The novelty presented herein is the direct conjugation of the osmium complex to a small antigen and the application of the quantitative analysis of the antigen and its antibody as the electrical signal for homogeneous immunoassay. The small antigen chosen is hippuric acid (HA), a major urinary metabolite in toluene-exposed humans. As a redox mediator, [Os(4,4'-dimethoxy-2,2'-bipyridine)2(4-aminomethylpyridine-HA)Cl](+/2+) (Os-HA antigen) has been synthesized and characterized on screen-printed carbon electrodes. The synthesized Os-HA antigen shows reversible redox peaks at E(½)=0.056 V versus Ag/AgCl. The homogeneous competitive immunoassay relies on the interaction between Os-HA antigen conjugate and free antigen to its antibody, which can generate electrical signals linearly proportional to the free antigen monitored by cyclic voltammetry and differential pulse voltammetry in the range of 10 µg mL(-1) to 5.12 mg mL(-1). The cutoff concentration of HA in urine samples is 2.0 mg mL(-1), so the method can be used to develop a HA immunosensor. Moreover, the proposed homogeneous electrochemical immunoassay method can be applied to detect low concentrations of small antigens found in the healthcare area.
Assuntos
Técnicas Eletroquímicas , Hipuratos/urina , Imunoensaio , Compostos Organometálicos/química , Osmio/química , Animais , Reações Antígeno-Anticorpo , Antígenos/imunologia , Antígenos/urina , Carbono/química , Eletrodos , Hipuratos/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Compostos Organometálicos/síntese química , Compostos Organometálicos/imunologia , Osmio/imunologia , Propriedades de SuperfícieRESUMO
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.
Assuntos
Anticorpos Monoclonais/análise , Hipuratos/urina , Tolueno/metabolismo , Adulto , Poluentes Ocupacionais do Ar/metabolismo , Cromatografia/métodos , Ensaio de Imunoadsorção Enzimática , Feminino , Hipuratos/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Solventes/metabolismo , Detecção do Abuso de Substâncias/métodosRESUMO
A novel monoclonal antibody (MAb) to hippuric acid (HA) was prepared using an HA analog, N-alpha-benzoyl-lysine, as an immunogen. An enzyme-linked immunosorbent assay (ELISA) for HA was established using the anti-HA MAb named HA01BL. When the specificity of the MAb was analyzed by the ELISA system, the MAb was revealed to be less reactive to methylhippuric acids and to be more specific to HA than previously reported polyclonal antibodies. The detection limit of HA by the ELISA was approximately 1 microg/mL. The urinary HA concentration determined by the ELISA system correlated well with that obtained by high performance liquid chromatography.
