RESUMEN
The present study described a new strategy to examine the interaction between the targeted ultrasound contrast agent (USCA) and its target under flow conditions with a surface acoustic wave (SAW) transducer. The sensing principle is based on the measurement of the phase change on the sensing element upon the binding of specific biomolecules. Love-wave biosensor array was consisting of sensor elements and reference elements. The sensor elements have been prepared by coating the sensor surface with tumor marker EDB-fibronectin by means of SAM technique and carbodiimide chemistry. Reference elements were left blank or coated with fibronectin and used to eliminate thermal drift, unspecific binding, and turbulence from injection of liquids by calculating the differential phase shift with respect to the sensor elements. The binding of targeted USCA to the sensor surface was constantly recorded by monitoring the phase shift on the sensor element. The binding of targeted USCA generated a high phase shift on the sensor elements, but almost no change on the reference elements. Control experiments using non-targeted and isotype-targeted USCA confirmed the specificity of binding due to anti-EDB-fibronectin scFv-antibody-fragment-EDB-fibronectin antigen interaction. The suitability of the SAW technique to monitor the specific binding behavior of targeted micron-sized USCA in real time has been well established.
Asunto(s)
Técnicas Biosensibles/instrumentación , Materiales Biocompatibles Revestidos/química , Medios de Contraste , Sistemas de Liberación de Medicamentos/métodos , Fibronectinas/química , Ultrasonografía/instrumentación , Técnicas Biosensibles/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Estudios de Factibilidad , Fibronectinas/análisis , Ensayo de Materiales , Ultrasonografía/métodosRESUMEN
PURPOSE: To develop an in vitro assay for studying the feasibility of specific targeting of ultrasound contrast agents (USCAs) for ultrasound diagnostics by employing the parallel plate flow chamber, which provides an environment that mimics some aspects of the in vivo conditions like shear rate and flow effects. METHODS: USCAs based on air-filled microparticles (MP) were functionalized with specific antibodies using carbodiimide coupling chemistry and characterized by fluorescence activated cell sorter (FACS). The binding experiments were done by subjecting the MP to shear stress as they interact with the target-coated surface of the flow chamber. RESULTS: A successive modification of MP with antibody and the glass surface with antigen was achieved and quantified. The binding studies showed specific attachment of targeted MP to EDB-FN (EDB domain of fibronectin) surface. The binding of MP via nonspecific interactions was minimal. The binding efficiency of antibody-loaded MP is dependent on the applied shear stress. An increase in the wall shear stress resulted in a decrease in binding efficiency. Binding efficiency was found to be correlated with the antibody density and antigen density on the interacting surfaces. CONCLUSIONS: The results indicate that the test system developed is reliable for characterizing targeted MP without any additional labeling and can be used as a functionality assay for studying the binding characteristic of USCA with respect to different parameters like density of targeting antibodies on the microparticle surface and of target protein. In addition, the microparticles can be studied in detail under different shear rates and flow conditions. Further studies concerning the in vitro-in vivo correlation will be necessary to further increase the value of this in vitro method.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Microburbujas , Tecnología Farmacéutica/métodos , Tecnología Farmacéutica/tendencias , Biotina/química , Ensayo de Inmunoadsorción Enzimática , Fibronectinas/química , Citometría de Flujo/instrumentación , Citometría de Flujo/métodos , Fluorescencia , Fragmentos de Inmunoglobulinas/química , Ligandos , Fragmentos de Péptidos/química , Estreptavidina/química , Estrés MecánicoRESUMEN
We developed a biosensor based on the redox properties of human CYP3A4 to directly monitor electron transfer to the heme protein. Enzyme films were assembled on gold electrodes by alternate adsorption of a CYP3A4 layer on top of a polycation layer. Direct, reversible electron transfer between the electrode and CYP3A4 was observed with voltammetry under anaerobic conditions. In the presence of oxygen, the oxidation peak of the hemoprotein disappeared, and the reduction peak increased 2- to 3-fold. Addition of CYP3A4 substrates (verapamil, midazolam, quinidine, and progesterone) to the oxygenated solution caused a concentration-dependent increase in the reduction current in cyclic voltammetric and amperometric experiments. Product analyses after electrolysis with the enzyme film showed catalytic activity of the biosensor depending on substrate concentration, its inhibition by ketoconazole, and a minor contribution of H(2)O(2) to the catalytic cycle. These results suggest that electron exchange between the electrode and the immobilized CYP3A4 occurred, and that metabolic activity of the enzyme was maintained. Thus, important requirements for the application of human CYP biosensors in order to identify drugs or drug candidates as substrates or inhibitors to the attached enzyme are fulfilled.