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At least 919 records · Page 51Linked to original sources
PubMed2001

Fabrication of nanometer-sized protein patterns using atomic force microscopy and selective immobilization.

A new methodology is introduced to produce nanometer-sized protein patterns. The approach includes two main steps, nanopatterning of self-assembled monolayers using atomic force microscopy (AFM)-based nanolithography and subsequent selective immobilization of proteins on the patterned monolayers. The resulting templates and protein patterns are characterized in situ using AFM. Compared with conventional protein fabrication methods, this approach is able to produce smaller patterns with higher spatial precision. In addition, fabrication and characterization are completed in near physiological conditions. The adsorption configuration and bioreactivity of the proteins within the nanopatterns are also studied in situ.

Adsorption↗
PubMed2005

A study of sulfamerazine single crystals using atomic force microscopy, transmission light microscopy, and Raman spectroscopy.

Sulfamerazine (SMZ) Form I and II single crystals were prepared from aqueous dispersions of SMZ bulk samples and studied using several microscopic and spectroscopic techniques. Transmission light microscopy and Raman spectroscopy were used to observe and identify single crystals. The results indicated that Form I single crystals tended to be rectangular laths while Form II ones tended to be hexagonal laths. Surface morphology of individual single crystals was further investigated by atomic force microscopy (AFM). AFM images revealed a smooth top surface, a uniform height, and sharp edges for both forms of single crystals. Both height and phase images showed crystalline terraces with different step heights for the top surface of Form I. Surface properties of single crystals were evaluated using AFM force measurements. Experimental results indicated that the top surface of Form I single crystals was more hydrophilic than that of Form II. Theoretical calculations predicted a dominant crystal face of (020) for the Form I single crystals and (002) for the Form II ones. The correlations between calculation predictions and experimental results were discussed.

Anti-Infective Agents↗
PubMed2004

Biofunctionalization of carbon nanotubes for atomic force microscopy imaging.

The study of biological processes relies increasingly on methods for probing structure and function of biochemical machinery (proteins, nucleic acids, and so on) with submolecular resolution. Atomic force microscopy (AFM) has recently emerged as a promising approach for imaging biological structures with resolution approaching the nanometer scale. Two important limitations of AFM in biological imaging are (1) resolution is constrained by probe tip dimensions, and (2) typical probe tips lack chemical specificity to differentiate between functional groups in biological structures. Single-walled carbon nanotubes (SWNTs) offer an intriguing possibility for providing both high resolution and chemical selectivity in AFM imaging, thus overcoming the enumerated limitations. Procedures for generating SWNT tips for AFM will be described. Carboxylic acid functional groups at the SWNT ends can be functionalized using covalent coupling chemistry to attach biological moieties via primary amine groups. Herein, the focus will be on describing methods for attaching biotin to SWNT tips and probing streptavidin on surfaces; importantly, this same coupling chemistry can also be applied to other biomolecules possessing primary amine groups. Underivatized SWNT tips can also provide high-resolution AFM images of DNA. Biofunctionalization of SWNT AFM tips offers great potential to enable high-resolution, chemically selective imaging of biological structures.

Biotin↗
PubMed2002

Size distribution measurement of vesicles by atomic force microscopy.

Vesicles have been utilized as nanoscale vehicles for reagents including potential drug delivery systems. When used to deliver drugs, vesicle size and the size distribution are important factors in the determination of the dosage, cell specificity, and rate of clearance from the body. Current size measurement techniques for vesicles are electron microscopy and dynamic light scattering, but their results are not equal. Therefore atomic force microscopy was attempted as another size measurement technique. After adsorption of the vesicles from a low-concentration solution of vesicles on mica substrate, each vesicle is generally found as a flattened structure. The diameters of vesicles in these solutions and their distribution have been successfully estimated from the surface area of the flattened structure of each vesicle. At higher concentrations, we have found a monolayer crammed with dome-shaped vesicles on the substrate. The diameters of vesicles in these solutions have also been successfully estimated from the surface area of the dome-shaped structure of each vesicle. Diameters of vesicles in solution estimated from two different vesicle concentrations are not close to those reported by electron microscope studies but are close to those reported by dynamic light scattering studies.

Adsorption↗
PubMed2003

Molecular arrangements of self-assembled surfactant films: characterization from atomic force microscopy data.

The adsorbed surfactant film molecular arrangement with thickness of approximately 5 nm is determined by measurements of the film dielectric permittivity. Before the advent of atomic force microscopy the dielectric permittivity was a macroscopic parameter, appropriate only for describing uniform environments since its profile was difficult to measure for local intermolecular interactions and its spatial distribution was frequently settled without experimental justification. Here, we show that atomic force microscopy made it possible to measure the dielectric permittivity profile in a scale below 5 nm for adsorbed layers of self-assembled surfactant films in water. The measured values of the film's dielectric permittivity and the film's thickness determine the compactness of the adsorbed film and consequently the presence of water molecules in the film and the conformal structure of the adsorbed molecules.

Journal Article↗
PubMed2002

Direct visualization of ligand-protein interactions using atomic force microscopy.

1. Streptavidin is a 60-kDa tetramer which binds four molecules of biotin with extremely high affinity (K(A) approximately 10(14) M(-1)). We have used atomic force microscopy (AFM) to visualize this ligand-protein interaction directly. 2. Biotin was tagged with a short (152-basepair; 50-nm) DNA rod and incubated with streptavidin. The resulting complexes were then imaged by AFM. The molecular volume of streptavidin calculated from the dimensions of the protein particles (105+/-3 nm(3)) was in close agreement with the value calculated from its molecular mass (114 nm(3)). Biotinylation increased the apparent size of streptavidin (to 133+/-2 nm(3)), concomitant with an increase in the thermal stability of the tetramer. 3. Images of streptavidin with one to four molecules of DNA-biotin bound were obtained. When two ligands were bound, the angle between the DNA rods was either acute or obtuse, as expected from the relative orientations of the biotin binding sites. The ratio of acute : obtuse angles (1 : 3) was lower than the expected value (1 : 2), indicating a degree of steric hindrance in the binding of the DNA-biotin. The slight under-representation of higher occupancy states supported this idea. 4. Streptavidin with a single molecule of DNA-biotin bound was used to tag biotinylated beta-galactosidase, a model multimeric enzyme. 5. The ability to image directly the binding of a ligand to its protein target by AFM provides useful information about the nature of the interaction, and about the effect of complex formation on the structure of the protein. Furthermore, the use of DNA-biotin/streptavidin tags could potentially shed light on the architecture of multi-subunit proteins.

Binding Sites↗
PubMed2000

The use of atomic force microscopy for the observation of corneal epithelium surface.

PURPOSE: To evaluate the feasibility of imaging normal corneal epithelium by means of atomic force microscopy (AFM). METHODS: Twelve normal corneas from six albino rabbits were examined using a commercial atomic force microscope. Six corneas were examined in balanced salt solution after fixation in glutaraldehyde 2.5% and six without any fixation. Rectangular silicon nitride cantilevers with a spring constant of 10 to 20 mN/m were used. The measured forces after imaging were less than 100 pN. All reported images were made with 512x512-pixel definition with typical scan rates ranging from 1 to 5 Hz. RESULTS: High-quality images of corneal epithelium surface were obtained from fixed and unfixed specimens in magnifications ranging from x2000 to x2,000,000. Imaging of fixed specimens was always easier. In unfixed specimens fuzzy images were very common, probably because of the presence of the cell glycocalyx. AFM revealed the typical polygonal corneal epithelial cells. The cell surface was covered by microprojections; at cell borders the microprojections were arranged in two characteristic parallel rows. Craterlike formations were revealed in several specimens. The microprojections' morphology and their surface details were revealed using magnifications up to x2,000,000. Three-dimensional representation of the images facilitated better understanding of the surface topography. Measurements in horizontal and vertical plane were made using the section analysis tool. CONCLUSIONS: In this work the AFM parameters appropriate for corneal epithelium imaging in physiological medium were defined. AFM represents a new powerful tool for corneal epithelium imaging, and its application in this field warrants further investigation.

Animals↗
PubMed2001

Dynamic interactions of p53 with DNA in solution by time-lapse atomic force microscopy.

Dynamic interactions of the tumor suppressor protein p53 with a DNA fragment containing a p53-specific recognition sequence were directly observed by time-lapse tapping mode atomic force microscopy (AFM) in liquid. The divalent cation Mg(2+) was used to loosely attach both DNA and p53 to a mica surface so they could be imaged by the AFM while interacting with each other. Various interactions of p53 with DNA were observed, including dissociation/re-association, sliding and possibly direct binding to the specific sequence. Two modes of target recognition of p53 were detected: (a) direct binding, and (b) initial non-specific binding with subsequent translocation by one-dimensional diffusion of the protein along the DNA to the specific site.

Aluminum Silicates↗
PubMedDate not supplied

Chitosan-induced restructuration of a mica-supported phospholipid bilayer: an atomic force microscopy study.

Chitosan has emerged as a promising material for biomedical applications. However, the effect of chitosan adsorption on the structure of model biomembrane is not known. In this study, atomic force microscopy (AFM) is employed to investigate the interaction between chitosan and mica-supported dipalmitoylphosphocholine (DPPC) bilayer. First, in situ AFM measurement indicates that nucleation of chitosan occurs around the membrane defects at the initial stage of chitosan incubation. Eventually, DPPC-chitosan binding and chitosan intermolecular association lead to chitosan aggregation on the membrane surface which is quantified by average height measurement and RMS roughness analysis. Lateral force microscopy (LFM) confirms that the adsorbed chitosan has distinct material properties. Furthermore, the trend of surface pressure-area isotherms supports the condensation of DPPC monolayer induced by chitosan in the aqueous subphase. Surface coverage and surface roughness analysis show that the extent of chitosan aggregation on the supported membrane is affected by the incubation time during long-term chitosan incubation.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2006

Atomic force microscopy study of the interaction of DNA and nanostructured beta-Gallia rutile.

The ability to attach DNA molecules to solid planar substrates is desired for imaging the molecule and for building DNA-mediated nanostructures. The deposition of DNA on [001] rutile and beta-gallia rutile (BGR) substrates from buffer solutions containing various divalent cations was studied using tapping mode atomic force microscopy (AFM). beta-Gallia rutile intergrowths were prepared by spin-coating gallium isopropoxide onto [001]-oriented TiO2 single-crystal slabs and heating above 1350 degrees C for >24 h, resulting in the formation of intergrowth lines along the {210} planes in the parent rutile structure. Rutile and BGR intergrowth substrates were exposed to various buffered solutions containing DNA and the following divalent cations: Ca(II), Co(II), Cu(II), Fe(II), Mg(II), Mn(II), Ni(II), and Zn(II). Among all the cations examined, only Ni(II) resulted in the attachment of DNA on the rutile surfaces. DNA attachment to BGR surfaces was strong enough to allow AFM imaging when the deposition buffer contained one of the following cations: Co(II), Mg(II), Mn(II), Ni(II), and Zn(II). For all of these cations, DNA attachment occurred preferentially, but not exclusively, along BGR intergrowth lines. When buffers without cation additions and those containing Ca(II), Cu(II), and Fe(II) were used, DNA failed to bind the BGR surfaces strongly enough to allow AFM imaging. The mechanism(s) by which DNA attaches to the BGR surface is (are) not well understood but may involve the incorporation of divalent cations at the tunnel sites of the BGR intergrowths.

DNA↗
PubMed2001

Cleanability of soiled stainless steel as studied by atomic force microscopy and time of flight secondary ion mass spectrometry.

The hygienic status of food contact surfaces can deteriorate with wear. Effective cleaning regimes must remove any adsorbed organic material as well as microorganisms. Previous work has determined the extent of surface wear occurring on a stainless steel surface within the food industry, and we have reproduced representative samples in vitro. Two surface analytical techniques, atomic force microscopy and time of flight secondary ion mass spectrometry were combined with fluorescence microscopy to give detailed analysis of stainless steel surfaces fouled with starch and milk powder, then cleaned with water either by a spray or brushing method. It was found that the surface cleanability is affected by the cleaning regime and the surface roughness, not only the average vertical roughness but also by the shape of the surface defects, with sharp scratches more difficult to clean than wider surface defects. Spray cleaning with distilled water was found to be a selective method by preferentially removing proteinaceous material more easily than fatty acid ester material. The analytical techniques employed provided information on selective cleanability and surface topography at a hitherto unexplored level, and the information gained may be of value in the design and investigation of novel cleaning regimes and hygienic surfaces.

Detergents↗
PubMed2002

In situ single-molecule detection of antibody-antigen binding by tapping-mode atomic force microscopy.

We performed in situ detection of specific and nonspecific binding during immunoreaction on surfaces at the same location before and after analyte was injected using tapping-mode atomic force microscopy (TM-AFM) in liquid and demonstrated the ability of TM-AFM to monitor the occurrence of single-molecule binding events and to distinguish nonspecific from specific binding by examining topographical change. Two antigen/antibody pairs were investigated: chorionic gonadotropin (hCG)/mouse monoclonal anti-hCG and goat IgG (anti-intact hCG)/ mouse monoclonal anti-goat IgG. Antibody (or antigen) molecules were covalently immobilized on uniform mixed self-assembled monolayers (SAMs) terminated with carboxylic acid and hydroxyl groups. Mixed SAMs allow the control of the density of immobilized antibody (or antigen) on surfaces to achieve the detection of individual antigens, antibodies, and antigen/antibody complexes. This in situ TM-AFM-based detection method allows the single-molecule detection of antigen/antibody binding under near-physiological environment and the distinction of nonspecific from specific binding. It could be extended into a microarray.

Adsorption↗
PubMed2003

Nanodissection and high-resolution imaging of the Rhodopseudomonas viridis photosynthetic core complex in native membranes by AFM. Atomic force microscopy.

In photosynthesis, highly organized multiprotein assemblies convert sunlight into biochemical energy with high efficiency. A challenge in structural biology is to analyze such supramolecular complexes in native membranes. Atomic force microscopy (AFM) with high lateral resolution, high signal-to-noise ratio, and the possibility to nanodissect biological samples is a unique tool to investigate multiprotein complexes at molecular resolution in situ. Here we present high-resolution AFM of the photosynthetic core complex in native Rhodopseudomonas viridis membranes. Topographs at 10-A lateral and approximately 1-A vertical resolution reveal a single reaction center (RC) surrounded by a closed ellipsoid of 16 light-harvesting (LH1) subunits. Nanodissection of the tetraheme cytochrome (4Hcyt) subunit from the RC allows demonstration that the L and M subunits exhibit an asymmetric topography intimately associated to the LH1 subunits located at the short ellipsis axis. This architecture implies a distance distribution between the antenna and the RC compared with a centered location of the RC within a circular LH1, which may influence the energy transfer within the core complex. The LH1 subunits rearrange into a circle after removal of the RC from the core complex.

Image Processing, Computer-Assisted↗
PubMed2004

Inactivated enzymes as probes of the structure of arabinoxylans as observed by atomic force microscopy.

The complex structures of water-soluble wheat arabinoxylans have been mapped along individual molecules, and within populations, using the visualisation of the binding of inactivated enzymes by atomic force microscopy (AFM). It was demonstrated that site-directed mutagenesis (SDM) can be used to produce inactive enzymes as structural probes. For the SDM mutants AFM has been used to compare the binding of different xylanases to arabinoxylans. Xylanase mutant E386A, derived from the Xyn11A enzyme (Neocallimastrix patriciarium), was shown to bind randomly along arabinoxylan molecules. The xylanase binding was also monitored following Aspergillus niger arabinofuranosidase pre-treatment of samples. It was demonstrated that removal of arabinose side chains significantly altered the binding pattern of the inactivated enzyme. Xylanase mutant E246A, derived from the Xyn10A enzyme (Cellvibrio japonicus), was found to show deviations from random binding to the arabinoxylan chains. It is believed that this is due to the effect of a small residual catalytic activity of the enzyme that alters the binding pattern of the probe. Control procedures were developed and assessed to establish that the interactions between the modified xylanases and the arabinoxylans were specific interactions. The experimental data demonstrates the potential for using inactivated enzymes and AFM to probe the structural heterogeneity of individual polysaccharide molecules.

Carbohydrate Sequence↗
PubMed1996

Viscoelasticity in wild-type and vinculin-deficient (5.51) mouse F9 embryonic carcinoma cells examined by atomic force microscopy and rheology.

We have been studying mouse F9 embryonic carcinoma cells which contain no detectable vinculin protein (5.51 cells), and compared them with F9 wild-type cells. Employing atomic force microscopy, we probed the elastic properties of individual F9 wild-type and 5.51 cells by measuring the dynamic response of controlled loads of the cantilever tip. An elastic modulus (Young) of approximately 3.8 and approximately 2.5 kPa was calculated for wild-type and 5.51 cells, respectively. Using disc rheometry, we detected a marked change in shear of a 1000g pellet of approximately 55 x 10(6) cells between wild-type and 5.51 mutants. These differences are attributed to the loss of vinculin and altered cytoskeletal organization in these cells.

Animals↗
PubMed2005

Immobilization of DNA on 11-mercaptoundecanoic acid-modified gold (111) surface for atomic force microscopy imaging.

Immobilized DNA on preformed 11-mercaptoundecanoic acids (MUDA) self-assembled monolayers (SAMs) on a gold (111) surface was bound by a divalent cation bridges was imaged by atomic force microscopy (AFM). The DNA immobilization was attributed to the formation of ionic bridges between the carboxylate groups of MUDA and the phosphate groups of DNA. AFM images revealed that DNA molecules could be immobilized strongly enough to permit stable and reproducible imaging. The effect of different bridge cations, such as Mg(2+), Zn(2+) and Cu(2+), and the pH of DNA assembled solution on immobilization and conformation of DNA was studied. Plasmid DNA pBR 322/Pst I molecules were straightened by using a molecular combing technique on the MUDA surface.

DNA↗
PubMed1995

Characterization of lacrymal component accumulation on worn soft contact lens surfaces by atomic force microscopy.

The purpose of this study was to investigate lacrymal component accumulation on a soft contact lens (SCL) surface after various periods of continuous wear, using the recently developed atomic force microscopy (AFM). AFM allowed high resolution images of unworn and worn SCL, and presented two main advantages. 1. The SCL are analysed under nearly physiological conditions without being dried or destroyed. So the same SCL was analysed at various times during a long wearing period. To identify the deposited tear proteins, a qualitative analysis of solubilized deposit by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on 4-15% gradient minigels was performed as well. We present typical images which emphasize the importance of the coating by lacrymal components. AFM analysis of worn SCL showed the deposition on the surface of a uniform lacrymal component coating (named deposit type I) with a progressive accumulation of numerous discrete granules (named deposit type II). SDS-PAGE of extracted deposits revealed the main tear proteins as: IgA, lactoferrin, tear lipocalin and lysozyme and the unknown protein of molecular weight 30,000. There is no clear difference in the protein patterns of the two types of deposits. Furthermore, a particular mode of use of AFM is described to illustrate the potential of this technique as a local tool for measuring protein coating thickness. Thus, for analysis of protein deposits on SCL surfaces, SDS-PAGE on minigels and AFM were easy and rapid to perform. When associated, these two techniques could find use in a wide range of worn SCL evaluation and most generally in biocompatibility evaluation studies.

Adult↗
PubMed2004

Examination of surface-bound Ku-DNA complexes in an aqueous environment using MAC mode atomic force microscopy.

In the development of biosensors, it is essential to understand how the signal-transducing element may perturb surface-bound proteins and nucleic acids. The tip of the atomic force microscope is such an element in atomic force microscopy. In this paper, we describe the influence of tip-sample interactions on the measured height of the DNA repair protein, Ku, that has been adsorbed onto a mica surface which was submerged in aqueous solution. We find that the measured height of the Ku molecule depends critically on whether or not it is associated with DNA. Additionally, we observed that the conditions (time and concentration) under which Ku is incubated with DNA, affect the appearance (number and type) of the DNA-Ku complexes observed.

Biosensing Techniques↗