Biomolecular Force Measurements and the Atomic Force Microscope

Current Opinion in Biotechnology. Feb, 2002  |  Pubmed ID: 11849957

The atomic force microscope (AFM) is a surface-sensitive instrument capable of imaging biological samples at nanometer resolution in all environments including liquids. The sensitivity of the AFM cantilever, to forces in the pico Newton range, has been exploited to measure breakaway forces between biomolecules and to measure folding-unfolding forces within single proteins. By attaching specific antibodies to cantilevers the simultaneous imaging of target antigens and identification of antigen-antibody interactions have been demonstrated.

Surface Patterning of Silica Nanostructures Using Bio-inspired Templates and Directed Synthesis

Langmuir : the ACS Journal of Surfaces and Colloids. Sep, 2004  |  Pubmed ID: 15379457

Natural systems excel in directing the synthesis of inorganic materials for various functional purposes. One of the best-studied systems is silica synthesis, as occurs in diatoms and marine sponges. Various biological and synthetic polymers have been shown to template and catalyze silica formation from silicic acid precursors. Here, we describe the use of poly-L-lysine to promote the synthesis of silica in neutral, aqueous solution and when immobilized onto a silicon support structure under similar conditions. Either reagent jetting or conventional photolithography techniques can be used to pattern the templating polymer. Spots created by reagent jetting led to the creation of silica structures in the shape of a ring that may be a result of the spotting process. Photolithographically defined poly-L-lysine spots led to thin laminate structures after exposure to a dilute aqueous silicic acid solution. The laminate structures were nanostructured and highly interconnected. Photolithographic patterning of (3-aminopropyl)trimethoxysilane, a reagent that mimics the lysine functional group, led to similar silica coatings even though low-molecular-weight materials do not rapidly promote silica synthesis in solution. This result highlights the importance of functional-group arrangement for templating and promoting the synthesis of inorganic materials. The described surface-patterning techniques offer a route to integrate conventional silicon-patterning technologies with biologically based material synthesis. Such combined fabrication techniques enable controlled assembly over multiple length scales and an approach to understanding interfacial silica synthesis, as occurs in natural systems.

Controlling the Dimensions of Carbon Nanofiber Structures Through the Electropolymerization of Pyrrole

Synthetic Metals. Apr, 2007  |  Pubmed ID: 18431459

Electrically conductive polymers, such as polypyrrole (PPy), show promise for modifying the dimensions and properties of micro- and nanoscale structures. Mechanisms for controlling the formation of PPy films of nanoscale thickness were evaluated by electrochemically synthesizing and examining PPy films on planar gold electrodes under a variety of growth conditions. Tunable PPy coatings were then deposited by electropolymerization on the sidewalls of individual, electrically addressable carbon nanofibers (CNFs). The ability to modify the physical size of specific nanofibers in controllable fashion is demonstrated. The biocompatibility, potential for chemical functionalization, and ability to effect volume changes of this nanocomposite can lead to advanced functionality, such as specific, nanoscale valving of materials and morphological control at the nanoscale.

Biomineralization at the Nanoscale Learning from Diatoms

Methods in Cell Biology. 2008  |  Pubmed ID: 19195546

Application of AFM in Understanding Biomineral Formation in Diatoms

Pflügers Archiv : European Journal of Physiology. Apr, 2008  |  Pubmed ID: 18058126

We review previous work and present new data on the application of atomic force microscopy (AFM) to study biomineral formation in diatoms, unicellular algae that make cell walls of silica. Previous studies examined a small subset of mostly larger diatom species, identifying a prevalence of large particulate silica on the nanoscale. We survey different structures including valves, girdle bands, and elongated spines called setae, in a variety of species, and show a diversity of nano- and meso-scale silica morphologies, even on different portions of the same structure. A general trend of highly organized mesoscale silica structure on the proximal face of cell wall components was observed, with less organized structure occurring on the distal face. The highly organized structures have features suggestive of an underlying linear template, which defines the area of initial silica polymerization. Such features have not been imaged with such clarity previously, demonstrating the advantages of AFM to image small differences in surface morphology and providing new insights and confirming evidence for models of diatom silica structure formation. In addition to its imaging capability, more developed application of AFM to map locations of organic template components on the nanoscale will greatly aid in elucidating mechanisms of diatom biosilica synthesis.

Effects of Colistin on Surface Ultrastructure and Nanomechanics of Pseudomonas Aeruginosa Cells

Langmuir : the ACS Journal of Surfaces and Colloids. Apr, 2009  |  Pubmed ID: 19227989

Chronic lung infections in cystic fibrosis patients are primarily caused by Pseudomonas aeruginosa. Though difficult to counteract effectively, colistin, an antimicrobial peptide, is proving useful. However, the exact mechanism of action of colistin is not fully understood. In this study, atomic force microscopy (AFM) was used to evaluate, in a liquid environment, the changes in P. aeruginosa morphology and nanomechanical properties due to exposure to colistin. The results of this work revealed that after 1 h of colistin exposure the ratio of individual bacteria to those found to be arrested in the process of division changed from 1.9 to 0.4 and the length of the cells decreased significantly. Morphologically, it was observed that the bacterial surface changed from a smooth to a wrinkled phenotype after 3 h exposure to colistin. Nanomechanically, in untreated bacteria, the cantilever indented the bacterial surface significantly more than it did after 1 h of colistin treatment (P-value = 0.015). Concurrently, after 2 h of exposure to colistin, a significant increase in the bacterial spring constant was also observed. These results indicate that the antimicrobial peptide colistin prevents bacterial proliferation by repressing cell division. We also found that treatment with colistin caused an increase in the rigidity of the bacterial cell wall while morphologically the cell surface changed from smooth to wrinkled, perhaps due to loss of lipopolysaccharides (LPS) or surface proteins.

Silver Nanocrystallites: Biofabrication Using Shewanella Oneidensis, and an Evaluation of Their Comparative Toxicity on Gram-negative and Gram-positive Bacteria

Environmental Science & Technology. Jul, 2010  |  Pubmed ID: 20509652

Microorganisms have long been known to develop resistance to metal ions either by sequestering metals inside the cell or by effluxing them into the extracellular media. Here we report the biosynthesis of extracellular silver-based single nanocrystallites of well-defined composition and homogeneous morphology utilizing the gamma-proteobacterium, Shewanella oneidensis MR-1, upon incubation with aqueous silver nitrate solution. Further characterization of these particles revealed that the crystals consist of small, reasonably monodispersed spheres in the 2-11 nm size range (average of 4 +/- 1.5 nm). The bactericidal effect of these nanoparticles (biogenic-Ag) is compared to chemically synthesized silver nanoparticles (colloidal-Ag and oleate capped silver nanoparticles, oleate-Ag) and assessed using Gram-negative (E. coli and S. oneidensis) and Gram-positive (B. subtilis) bacteria. Relative toxicity was based on the diameter of inhibition zone in disk diffusion tests, minimum inhibitory concentrations, live/dead assays, and atomic force microscopy. From a toxicity perspective, strain-dependent inhibition depended on the synthesis procedure and the surface coat. Biogenic-Ag was found to be of higher toxicity compared to colloidal-Ag for all three strains tested, whereas E. coli and S. oneidensis were found to be more resistant to either of these nanoparticles than B. subtilis. In contrast, oleate-Ag was not toxic to any of the bacteria. These findings have implications for the potential uses of Ag nanomaterials and for their fate in biological and environmental systems.

Atomic Force Microscopy of Biological Samples

Wiley Interdisciplinary Reviews. Nanomedicine and Nanobiotechnology. Nov-Dec, 2010  |  Pubmed ID: 20672388

The ability to evaluate structural-functional relationships in real time has allowed scanning probe microscopy (SPM) to assume a prominent role in post genomic biological research. In this mini-review, we highlight the development of imaging and ancillary techniques that have allowed SPM to permeate many key areas of contemporary research. We begin by examining the invention of the scanning tunneling microscope (STM) by Binnig and Rohrer in 1982 and discuss how it served to team biologists with physicists to integrate high-resolution microscopy into biological science. We point to the problems of imaging nonconductive biological samples with the STM and relate how this led to the evolution of the atomic force microscope (AFM) developed by Binnig, Quate, and Gerber, in 1986. Commercialization in the late 1980s established SPM as a powerful research tool in the biological research community. Contact mode AFM imaging was soon complemented by the development of non-contact imaging modes. These non-contact modes eventually became the primary focus for further new applications including the development of fast scanning methods. The extreme sensitivity of the AFM cantilever was recognized and has been developed into applications for measuring forces required for indenting biological surfaces and breaking bonds between biomolecules. Further functional augmentation to the cantilever tip allowed development of new and emerging techniques including scanning ion-conductance microscopy (SICM), scanning electrochemical microscope (SECM), Kelvin force microscopy (KFM) and scanning near field ultrasonic holography (SNFUH).

Effects of Engineered Cerium Oxide Nanoparticles on Bacterial Growth and Viability

Applied and Environmental Microbiology. Dec, 2010  |  Pubmed ID: 20952651

Interest in engineered nanostructures has risen in recent years due to their use in energy conservation strategies and biomedicine. To ensure prudent development and use of nanomaterials, the fate and effects of such engineered structures on the environment should be understood. Interactions of nanomaterials with environmental microorganisms are inevitable, but the general consequences of such interactions remain unclear, due to a lack of standard methods for assessing such interactions. Therefore, we have initiated a multianalytical approach to understand the interactions of synthesized nanoparticles with bacterial systems. These efforts are focused initially on cerium oxide nanoparticles and model bacteria in order to evaluate characterization procedures and the possible fate of such materials in the environment. The growth and viability of the Gram-negative species Escherichia coli and Shewanella oneidensis, a metal-reducing bacterium, and the Gram-positive species Bacillus subtilis were examined relative to cerium oxide particle size, growth media, pH, and dosage. A hydrothermal synthesis approach was used to prepare cerium oxide nanoparticles of defined sizes in order to eliminate complications originating from the use of organic solvents and surfactants. Bactericidal effects were determined from MIC and CFU measurements, disk diffusion tests, and live/dead assays. For E. coli and B. subtilis, clear strain- and size-dependent inhibition was observed, whereas S. oneidensis appeared to be unaffected by the particles. Transmission electron microscopy along with microarray-based transcriptional profiling was used to understand the response mechanism of the bacteria. Use of multiple analytical approaches adds confidence to toxicity assessments, while the use of different bacterial systems highlights the potential wide-ranging effects of nanomaterial interactions in the environment.

Biofabrication of Discrete Spherical Gold Nanoparticles Using the Metal-reducing Bacterium Shewanella Oneidensis

Acta Biomaterialia. May, 2011  |  Pubmed ID: 21241833

Nanocrystallites have garnered substantial interest due to their various applications, including catalysis and medical research. Consequently important aspects of synthesis related to control of shape and size through economical and non-hazardous means are desirable. Highly efficient bioreduction-based fabrication approaches that utilize microbes and/or plant extracts are poised to meet these needs. Here we show that the γ-proteobacterium Shewanella oneidensis can reduce tetrachloroaurate (III) ions to produce discrete extracellular spherical gold nanocrystallites. The particles were homogeneously shaped with multiple size distributions and produced under ambient conditions at high yield, 88% theoretical maximum. Further characterization revealed that the particles consist of spheres in the size range of ∼2-50 nm, with an average size of 12±5 nm. The nanoparticles were hydrophilic and resisted aggregation even after several months. Based on our experiments, the particles are likely fabricated by the aid of reducing agents present in the bacterial cell membrane and are capped by a detachable protein/peptide coat. Ultraviolet-visible and Fourier transform infrared spectroscopy, X-ray diffraction, energy dispersive X-ray spectra and transmission electron microscopy measurements confirmed the formation, surface characteristics and crystalline nature of the nanoparticles. The antibacterial activity of these gold nanoparticles was assessed using Gram-negative (Escherichia coli and S. oneidensis) and Gram-positive (Bacillus subtilis) bacterial species. Toxicity assessments showed that the particles were neither toxic nor inhibitory to any of these bacteria.

Effects of Sub-minimum Inhibitory Concentrations of Ciprofloxacin on Enteroaggregative Escherichia Coli and the Role of the Surface Protein Dispersin

International Journal of Antimicrobial Agents. Jul, 2011  |  Pubmed ID: 21570813

Enteroaggregative Escherichia coli (EAEC) are bacterial pathogens that cause watery diarrhoea, which is often persistent and can be inflammatory. The antibiotic ciprofloxacin is used to treat EAEC infections, but a full understanding of the antimicrobial effects of ciprofloxacin is needed for more efficient treatment of bacterial infections. In this study, it was found that sub-minimum inhibitory concentrations (sub-MICs) of ciprofloxacin had an inhibitory effect on EAEC adhesion to glass and mammalian HEp-2 cells. It was also observed that bacterial surface properties play an important role in bacterial sensitivity to ciprofloxacin. In an EAEC mutant strain where the hydrophobic positively charged surface protein dispersin was absent, sensitivity to ciprofloxacin was reduced compared with the wild-type strain. Identified here are several antimicrobial effects of ciprofloxacin at sub-MIC concentrations indicating that bacterial surface hydrophobicity affects the response to ciprofloxacin. Investigating the effects of sub-MIC doses of antibiotics on targeted bacteria could help to further our understanding of bacterial pathogenicity and elucidate future antibiotic treatment modalities.

Monodispersed Biocompatible Silver Sulfide Nanoparticles: Facile Extracellular Biosynthesis Using the γ-proteobacterium, Shewanella Oneidensis

Acta Biomaterialia. Dec, 2011  |  Pubmed ID: 21798382

Interest in engineered metal and semiconductor nanocrystallites continues to grow due to their unique size- and shape-dependent optoelectronic, physicochemical and biological properties. Therefore identifying novel non-hazardous nanoparticle synthesis routes that address hydrophilicity, size and shape control and production costs has become a priority. In the present article we report for the first time on the efficient generation of extracellular silver sulfide (Ag₂S) nanoparticles by the metal-reducing bacterium Shewanella oneidensis. The particles are reasonably monodispersed and homogeneously shaped. They are produced under ambient temperatures and pressures at high yield, 85% theoretical maximum. UV-visible and Fourier transform infrared spectroscopy, dynamic light scattering, X-ray diffraction, transmission electron microscopy and X-ray photoelectron spectroscopy measurements confirmed the formation, optical and surface properties, purity and crystallinity of the synthesized particles. Further characterization revealed that the particles consist of spheres with a mean diameter of 9±3.5 nm, and are capped by a detachable protein/peptide surface coat. Toxicity assessments of these biogenic Ag₂S nanoparticles on Gram-negative (Escherichia coli and S. oneidensis) and Gram-positive (Bacillus subtilis) bacterial systems, as well as eukaryotic cell lines including mouse lung epithelial (C 10) and macrophage (RAW-264.7) cells, showed that the particles were non-inhibitory and non-cytotoxic to any of these systems. Our results provide a facile, eco-friendly and economical route for the fabrication of technologically important semiconducting Ag₂S nanoparticles. These particles are dispersible and biocompatible, thus providing excellent potential for use in optical imaging, electronic devices and solar cell applications.