Surface attachment of protein fibrils via covalent modification strategies.

Chemical control of surface functionality and topography is an essential requirement for many technological purposes. In particular, the covalent attachment of monomeric proteins to surfaces has been the object of intense studies in recent years, for applications as varied as electrochemistry, immuno-sensing, and the production of biocompatible coatings. Little is known, however, about the characteristics and requirements underlying surface attachment of supramolecular protein nanostructures. Amyloid fibrils formed by the self-assembly of peptide and protein molecules represent one important class of such structures. These highly organized beta-sheet-rich assemblies are a hallmark of a range of neurodegenerative disorders, including Alzheimer's disease and type II diabetes, but recent findings suggest that they have much broader significance, potentially representing the global free energy minima of the energy landscapes of proteins and having potential applications in material science. In this paper, we describe strategies for attaching amyloid fibrils formed from different proteins to gold surfaces under different solution conditions. Our methods involve the reaction of sulfur containing small molecules (cystamine and 2-iminothiolane) with the amyloid fibrils, enabling their covalent linkage to gold surfaces. We demonstrate that irreversible attachment using these approaches makes possible quantitative analysis of experiments using biosensor techniques, such as quartz crystal microbalance (QCM) assays that are revolutionizing our understanding of the mechanisms of amyloid growth and the factors that determine its kinetic behavior. Moreover, our results shed light on the nature and relative importance of covalent versus noncovalent forces acting on protein superstructures at metal surfaces.

Feasibility of Protein a for the Oriented Immobilization of Immunoglobulin on Silicon Surface for a Biosensor with Imaging Ellipsometry

The feasibility of using protein A to immobilize antibody on silicon surface for a biosensor with imaging ellipsometry was presented in this study. The amount of human IgG bound with anti-IgG immobilized by the protein A on silicon surface was much more than that bound with anti-IgG immobilized by physical adsorption. The result indicated that the protein A could be used to immobilize antibody molecules in a highly oriented manner and maintain antibody molecular functional configuration on the silicon surface. High reproducibility of the amount of antibody immobilization and homogenous antibody adsorption layer on surfaces could be obtained by this immobilization method. Imaging ellipsometry has been proven to be a fast and reliable detection method and sensitive enough to detect small changes in a molecular monolayer level. The combination of imaging ellipsometry and surface modification with protein A has the potential to be further developed into an efficient immunoassay protein chip.

Application of Optical Protein-chip in Detecting Phage M13KO7

Abstract: Avidin layer was bound on the substrate surface of Silicon wafer modified with aldehyde. The interaction between avidin and biotin was adopted for the immobilization of mouse monoclonal biotin-anti-M13 (antibody GP3)-labeled biotin. The surface was incubated in a solution containing phage M13KO7, which was trapped by the antibody GP3 with the interaction between phage M13KO7 and antibody GP3, resulting in a variation of layer thickness that was detected by imaging ellipsometry. The results showed a saturated layer of antibody GP3 with a thickness about 6.9 nm on the surface of the silicon wafer. The specific interaction between phage M13KO7 and antibody GP3 resulted in a variation of layer thickness. The layer of phage M13KO7 bound with antibody GP3 was 17.5 nm in the concentration of 1.1×1010 pfu/mL. Each variation of the layer thickness corresponded to a concentration of phage M13KO7 in the range of 0.1×1010–2.5×1010 pfu/mL, with the sensitivity of 109 pfu/mL. Compared with other methods, the optical protein-chip, requiring only short measurement time, label free, is a quantitative test, and can be visualized. This study could be significant on the interactions between the antibody and the virus, showing potential in the early diagnosis of virosis.

Competitive protein adsorption studied with atomic force microscopy and imaging ellipsometry

The adsorption and competitive adsorption of collagen and bovine serum albumin (BSA) were directly visualized and quantified using atomic force microscopy (AFM) and imaging ellipsometry. Chemically modified silicon surfaces were used as hydrophilic and hydrophobic substrates. The results showed that collagen and BSA in single component solution adsorbed onto a hydrophobic surface two times more than that onto a hydrophilic surface. The competitive adsorption between collagen and BSA showed that serum albumin preferentially adsorbed onto a hydrophobic surface, while collagen on a hydrophilic surface. In the binary solution of BSA (1 mg/ml BSA) and collagen (0.1 mg/ml), nearly 100% of the protein adsorbed onto the hydrophobic surface was BSA, but on the hydrophilic surface only about 6% was BSA. Surface affinity was the main factor controlling the competitive adsorption.

Quantitative approaches for characterising fibrillar protein nanostructures

Polypeptide sequences have an inherent tendency to self-assemble into filamentous nanostructures commonly known as amyloid fibrils. Such self-assembly is used in nature to generate a variety of functional materials ranging from protective coatings in bacteria to catalytic scaffolds in mammals. The aberrant self-assembly of misfolded peptides and proteins is also, however, implicated in a range of disease states including neurodegenerative conditions such as Alzheimer's and Parkinson's diseases. It is increasingly evident that the intrinsic material properties of these structures are crucial for understanding the thermodynamics and kinetics of the pathological deposition of proteins, particularly as the mechanical fragmentation of aggregates enhances the rate of protein deposition by exposing new fibril ends which can promote further growth. We discuss here recent advances in physical techniques that are able to characterise the hierarchical self-assembly of misfolded protein molecnles and define their properties. © 2010 Materials Research Society.

A Label-Free Protein Microfluidic Array for Parallel Immunoassays

A label-free protein microfluidic array for immunoassays based on the combination of imaging ellipsometry and an integrated microfluidic system is presented. Proteins can be patterned homogeneously on substrate in array format by the microfluidic system simultaneously. After preparation, the protein array can be packed in the microfluidic system which is full of buffer so that proteins are not exposed to denaturing conditions. With simple microfluidic channel junction, the protein microfluidic array can be used in serial or parallel format to analyze single or multiple samples simultaneously. Imaging ellipsometry is used for the protein array reading with a label-free format. The biological and medical applications of the label-free protein microfluidic array are demonstrated by screening for antibody–antigen interactions, measuring the concentration of the protein solution and detecting five markers of hepatitis B.

Micro-systems for Optical Protein-Chip

Protein-Chip as micro-assays for the determination of protein interaction, the analysis, the identification and the purification of proteins has large potential applications. The Optical Protein-Chip is able to detect the multi-interaction of proteins and multi-bio-activities of molecules directly and simultaneously with no labeling. The chip is a small matrix on solid substrate containing multi-micro-area prepared by microfabrication with photolithography or soft lithography for surface patterning, and processed with surface modification which includes the physical, chemical, and bio-chemical modifications, etc. The ligand immobilization, such as protein immobilization, especially the oriented immobilization with low steric hindrance and high bio-specific binding activity between ligand and receptor is used to form a sensing surface. Each area of the pattern is corresponding to only one bioactivity. The interval between the areas is non-bioactive and optically extinctive. The affinity between proteins is used to realize non-labeling microassays for the determination of protein identification and protein interaction. The sampling of the chip is non-disturbing, performed with imaging ellipsometry and image processing on a database of proteins.

Evaluacion de alternativas quimicas y botanicas para el manejo del acaro blanco (Poliphagotarsonemus latus, Bank) en chiltoma (Capsicum annum L.) Tisma, Masaya

El ácaro blanco ( Poliphagotarsonemus latus, Bank) se ha convertido en un severo proble ma fitosanitario y socioeconómico para los productores de chiltoma ( Capsicum annum L.) del municipio de Tisma, Masaya. Esta plaga ha provocado grandes e importantes pérdidas económicas al reducir los rendimientos hasta en un 90%, disminuyendo así la calid ad y aumentando los costos de producción de la chiltoma. Ante la problemática existente en el municipio de Tisma y con el objetivo de encontrar una solución, se realizó un estudio en el periodo comprendido entre el mes de Agosto del 2007 a Enero del 2008, donde se evaluaron cuatro alternativas químicas y una botánica para el manejo del acaro blanco. Las alternativas evaluadas fueron: Oberón, Caldo sulfacálcico, Nim, Azufre y Vertimec en comparación con el testigo (aplicación de agua). Las variables evaluada s en el estudio fueron: Identificación del acaro blanco y sus daños en las plantas de chiltoma, Acaro blanco ( Poliphagotarsonemus latus, Bank ) por planta, Incidencia y Severidad del daño de acaro blanco, rendimiento en (kg ha - 1 ), altura de la planta (cm). Para decidir el momento de las aplicaciones de los productos se realizaron monitoreos semanales con una lupa de 16x tomando como nivel crítico un acaro por hoja tierna. Los resultados obtenidos en el estudio indican que el tratamiento Oberón fue el que pr esentó las poblaciones más bajas del acaro blanco, además de ser el más efectivo en el manejo, ya que las plantas tratadas con este producto presentaron el porcentaje de severidad más bajo en comparación con los demás tratamientos en estudio. El análisis e conómico realizado en este estudio determinó que el tratamiento Oberón fue el que presentó el mayor rendimiento, menor costo variable y fue el que obtuvo el mayor beneficio neto en comparación con los demás tratamientos en estudio

Probing protein aggregation with quartz crystal microbalances.

The supra-molecular self-assembly of peptides and proteins is a process which underlies a range of normal and aberrant biological pathways in nature, but one which remains challenging to monitor in a quantitative way. We discuss the experimental details of an approach to this problem which involves the direct measurement in vitro of mass changes of the aggregates as new molecules attach to them. The required mass sensitivity can be achieved by the use of a quartz crystal transducer-based microbalance. The technique should be broadly applicable to the study of protein aggregation, as well as to the identification and characterisation of inhibitors and modulators of this process.

Observation Of Diffusion Process Of A Water Droplet Immersed In Protein Solution In Microgravity

Pure liquid - liquid diffusion driven by concentration gradients is hard to study in a normal gravity environment since convection and sedimentation also contribute to the mass transfer process. We employ a Mach - Zehnder interferometer to monitor the mass transfer process of a water droplet in EAFP protein solution under microgravity condition provided by the Satellite Shi Jian No 8. A series of the evolution charts of mass distribution during the diffusion process of the liquid droplet are presented and the relevant diffusion coefficient is determined.

Evaluación de alternativas botánicas y químicas para el manejo del acaro blanco (Poliphagotarsonemus latus, Bank)(Acarina: Tarsonemidae) e insectos plagas en el cultivo de chiltoma (Capsicum annuum L.) Tisma, Masaya

El ácaro blanco (Poliphagotarsonemus latus , Bank), es uno de los problemas fitosanitarios más severos para los productores de chiltoma ( Capsicum annuum , L.) en el municipio de Tisma, Masaya. Esta plaga ha provocado importantes pérdidas económicas, disminuyendo así la calidad de los frutos y aumentando los costos de producción. En vista de este problema en el municipio de Tisma, se realizó un estudio en el periodo comprendido entre los meses de Septiembre a Diciembre del año 2009, donde se evaluaron algunas alternativas botánicas y químicas para el manejo del acaro blanco. Las alternativas evaluadas fueron: Oberon, Chile+Ajo+Jabón, Rienda, Vertimec, Sulfato de Amonio. Los resultados obtenidos en el estudio indican que el tratamiento Rienda fue el tratamiento con mejor control en el manejo del acaro blanco (39%) comparado con el testigo, ya que las plantas tratadas con este producto presentaron el menor número de ácaros por planta (2.73), menor porcentaje de incidencia (33%) y severidad (19%) en comparación con los demás tratamientos evaluados. Los tratamientos que obtuvieron los mejores rendimientos en k/Ha fueron Vertimec con (12237) y Oberon con (10944), Sulfato de Amonio obtuvo (9353). En el análisis de tasa de retorno marginal el tratamiento Vertimec es el que obtuvo mejor resultado con 659.53%.

Evaluación de productos botánicos y químicos para el manejo del acaro blanco (Poliphagotarsonemus latus, Bank) y otras plagas claves en el cultivo de chiltoma (Capsicum annuum L.) y su efecto en los enemigos naturales en Tisma, Masaya

El ácaro blanco ( Poliphagotarsonemus latus , Bank), es uno de los problemas fitosanitarios más severos para los productores de chiltoma ( Capsicum annuum , L.) en el municipio de Tisma, Masaya. Esta plaga ha ocasionado importantes pérdidas económicas, ya que reduce los rendimientos, disminuyendo así la calidad de los frutos y aumenta los costos de producción. En vista de este problema en el municipio de Tisma, se realizó un estudio con en el objetivo de evaluar productos botánicos y químicos para el manejo del ácaro blanco en chiltoma en el período comprendido entre los meses de Julio a Octubre del año 2009, donde se evaluaron algunos productos botánicos y químicos para el manejo del ácaro blanco. Los productos evaluados fueron: Chile + Jabón, Oberón, Neem, Vertimec, Ajo + jabón en comparación con el Testigo. Las variables evaluadas fueron: Fluctuación poblacional del ácaro blanco , Incidencia y Severidad de daño de ácaro por planta, rendimiento (Kg/ha-1) y fluctuación poblacional de organismos plagas y benéficos asociados al cultivo de la chiltoma como: Mosca blanca ( Bemisia tabaci), Áfidos (Aphis gossypii ), Minador de la hoja ( Liriomyza sp ), Mariquitas ( Coccinella sp ), Hormigas ( Atta sp ) y Arañas. Los resultados obtenidos en el estudio indican que el tratamiento Oberón presento la menor fluctuación poblacional de ácaro blanco seguido por los tratamientos Vertimec y Chile + jabón, el tratamiento Oberón fue el tratamiento con menor incidencia y severidad del daño de ácaro. El análisis económico realizado en este estudio determinó que el tratamiento Oberón fue el que presentó el mayor rendimiento y obtuvo el mayor beneficio neto, por el contrario el tratamiento que presentó los mayores costos variables fue el tratamiento Vertimec, seguido por el Neem. Además en este estudio se muestrearon las fluctuaciones poblacionales de otros organismos plagas y benéficos relacionados al cultivo de la chiltoma, donde resultados de este estudio demuestran que los productos utilizados para el manejo del ácaro blanco no tienen ningún efecto en las poblaciones de estos insectos.