RESUMEN
Here, we describe a highly sensitive method that allows for the correct quantification of inhibition effect with a higher degree of accuracy directly at the molecular level. The protocol involves two stages, namely serological virus titration in comparison with the same procedure for virus-effector mixture. Owing to the robustness of the analysis this assay can be performed on crude cellular and plant extracts, and therefore it may be suitable for the routine analysis of clinical samples, or in the field. The efficiency of the approach to the quantification of the inhibition effect of polysaccharide glucuronoxylomannan (GXM) on the infection efficiency of the tobacco mosaic virus (TMV) was investigated using advanced serological approaches based on label-free surface plasmon resonance technique. It was shown that GXM drastically decreases the efficiency of TMV infection by blocking up to 70% of the virus shell. The obtained results are in conformity with the method of indicator plant infection.
Asunto(s)
Antivirales/farmacología , Polisacáridos/farmacología , Virus del Mosaico del Tabaco/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Extractos Celulares , Enfermedades de las Plantas/virología , Extractos Vegetales , Hojas de la Planta/virología , Resonancia por Plasmón de Superficie , Nicotiana/virología , Virus del Mosaico del Tabaco/fisiología , Carga ViralRESUMEN
The green synthesis of irregular-shaped nanomaterials used for various applications in nanoplasmonics, medicine, and biotechnology creates an economical and environmental challenge. We describe the rapid wet-chemical approach to synthesis of stable and water-soluble gold nanostructues at room temperature. In addition to spherical and road-like nanoparticles, gold decahedra and triangular plates were grown using the one-step synthesis process of HAuCl(4) in the presence of honey, in which main components act as reducing (glucose) and stabilizing (fructose) agents; the mechanism of the process is discussed in details. The requirements for anisotropic phase boundaries for generation of polyhedral gold nanocrystals in solutions are highlighted. The synthesis, morphology, and separation procedure of gold nanoparticles are examined using the techniques of optical spectroscopy, transmission electron microscopy, and atomic force microscopy. We demonstrate that centrifugation can be used for efficient separation of nanoparticles with different shapes from a mixture. It was found that while centrifuging, the spheres sediment at the bottom of the tube, segregating from rods that form a deposit on the side wall, whereas polygons remain in the solution.
Asunto(s)
Oro/química , Miel , Nanopartículas del Metal/química , Nanotecnología/métodos , Polisacáridos/química , Microscopía Electrónica de Transmisión , Agua/químicaRESUMEN
Traditional methods of analytical chemistry to detect an interaction between certain proteins in multicomponent mixtures (e.g. cell lysates, etc.) have limitations. This is due to difficulties in identification of a specific signal of an analyte (a molecule to be detected) against the background. In the present work, we propose the new analytical protocol for transducer-based sensors with a restricted sensitive area. It uses a combination of analyte-receptor complex precipitation with serial additions of the receptor (CARSAR). To test this new analytical strategy, we used a surface plasmon resonance technique to confirm an interaction between the Epstein-Barr virus-encoded nuclear antigen 6 and the mitochondrial ribosomal protein MRPS18-2.
Asunto(s)
Resonancia por Plasmón de Superficie/métodos , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Unión Proteica , Proteínas Ribosómicas/metabolismoRESUMEN
The sensitivity of surface plasmon resonance (SPR) transducers depends on the thickness and spatial organization of interfacial structures at their surfaces. This is because the response of the SPR sensor is determined by integrating the distance-dependent refractive index (spatial interfacial architectures), weighted by the square of the electromagnetic field, from zero to infinite distance. The effect of SPR transducer sensitivity variation on the accuracy of SPR analysis is considered. Our quantitative estimation (based on the results of refractometric studies) gave a value for sensitivity variation of about 3% for the formation of a self-assembled thiocyanate layer or a trypsin-soybean trypsin inhibitor surface complex. The estimated accuracy in measured variation (i.e., by 0.01) for the refractive index of the external medium was 3x10(-4). This restriction, which follows immediately from the physical mechanism of the SPR phenomenon, should be taken into account when analyzing data obtained with the above technique.