Atomic force microscopy for the characterization of immobilized enzyme molecules on biosensor surfaces

The development of biosensors has been one of the key areas in biotechnology and biomedical studies. Often it is difficult to investigate the immobilized biomolecules on the surfaces for biosensor optimization. Atomic force microscopy (AFM) should provide an ideal means for the visualization of biosensor surface and for the investigation of biomolecule activities. Therefore, AFM has been employed to study the surface topography of immobilized glutamate dehydrogenase (GDH) on two-dimensional glutamate biosensor surfaces. Correlation between the surface topography and the activity of the biosensor was investigated. Surface analysis has revealed that the enzymatic activity of the immobilized GDH molecules on the biosensor surface is linked to surface roughness, as measured by the peak-to-valley distance. Fractal dimension of the immobilization sensor surface was found to be a good parameter for judging the quality of the immobilized biosensors. As enzyme immobilization time increases, the biosensor has its maximum activity with around 18 h of immobilization in 10^–6 M GDH solution. Various biosensors prepared under different experimental conditions have been studied by AFM. This technique is shown to be an effective tool to characterize biosensor surfaces.     

Atomic force microscopy for the characterization of immobilized enzyme molecules on biosensor surfaces

The development of biosensors has been one of the key areas in biotechnology and biomedical studies. Often it is difficult to investigate the immobilized biomolecules on the surfaces for biosensor optimization. Atomic force microscopy (AFM) should provide an ideal means for the visualization of biosensor surface and for the investigation of biomolecule activities. Therefore, AFM has been employed to study the surface topography of immobilized glutamate dehydrogenase (GDH) on two-dimensional glutamate biosensor surfaces. Correlation between the surface topography and the activity of the biosensor was investigated. Surface analysis has revealed that the enzymatic activity of the immobilized GDH molecules on the biosensor surface is linked to surface roughness, as measured by the peak-to-valley distance. Fractal dimension of the immobilization sensor surface was found to be a good parameter for judging the quality of the immobilized biosensors. As enzyme immobilization time increases, the biosensor has its maximum activity with around 18 h of immobilization in 10(-6) M GDH solution. Various biosensors prepared under different experimental conditions have been studied by AFM. This technique is shown to be an effective tool to characterize biosensor surfaces.     

Atomic force microscopy of microvillous cell surface dynamics at fixed and living alveolar type II cells

Scanning probe techniques enable direct imaging of morphology changes associated with cellular processes at life specimen. Here, glutaraldehyde-fixed and living alveolar type II (ATII) cells were investigated by atomic force microscopy (AFM), and the obtained topographical data were correlated with results obtained by scanning electron microscopy (SEM) and confocal microscopy (CM). We show that low-force contact mode AFM at glutaraldehyde-fixed cells provides complementary results to SEM and CM. Both AFM and SEM images reveal fine structures at the surface of fixed cells, which indicate microvilli protrusions. If ATII cells were treated with Ca²⁺ channel modulators known to induce massive endocytosis, changes of the cell surface topography became evident by the depletion of microvilli. Low force contact mode AFM imaging at fixed ATII cells revealed a significant reduction of the surface roughness for capsazepine and 2-aminoethoxydiphenyl-borate (CPZ/2-APB)-treated cells compared to untreated control cells (Rc of 99.7 ± 6.8 nm vs. Rc of 71.9 ± 4.6 nm for N = 22), which was confirmed via SEM studies. CM of microvilli marker protein Ezrin revealed a cytoplasmic localization of Ezrin in CPZ/2-APB-treated cells, whereas a submembranous Ezrin localization was observed in control cells. Furthermore, in situ AFM investigations at living ATII cells using low force contact mode imaging revealed an apparent decrease in cell height of 17% during stimulation experiments. We conclude that a dynamic reorganization of the microvillous cell surface occurs in ATII cells at conditions of stimulated endocytosis.     

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.     

Stretched polymer surfaces: Atomic force microscopy measurement of the surface deformation and surface elastic properties of stretched polyethylene

The surface structure and surface mechanical properties of low‐ and high‐density polyethylene were characterized by atomic force microscopy (AFM) as the polymers were stretched. The surfaces of both materials roughened as they were stretched. The roughening effect is attributed to deformation of nodular structures, related to bulk spherulites, at the surface. The surface‐roughening effect is completely reversible at tensile strains in the elastic regime and partially reversible at tensile strains in the plastic regime until the polymers are irreversibly drawn into fibers. AFM force versus distance interaction curves, used to measure changes in the stiffness of the surface and the surface elastic modulus as a function of elongation, show that the surfaces become softer as the polymers are drawn into fibers at high strains. At low elastic strains, however, the surface elastic modulus of HDPE increases—attributed to elastic energy stored by the amorphous regions. © 2001 John Wiley & Sons, Inc. J Polym Sci Part B: Polym Phys 39: 2263–2274, 2001     

Atomic force microscopy (AFM)-based nanografting for the study of self-assembled monolayer formation of organophosphonic acids on Al2O3 single-crystal surfaces

Adsorption, stability, and organization kinetics of organophosphonic acids on single-crystalline alumina surfaces were investigated by means of atomic force microscopy (AFM)-based imaging, nanoshaving, and nanografting. AFM friction and phase imaging have shown that chemical etching and subsequent annealing led to heterogeneities on single-crystalline surfaces with (0001) orientation. Self-assembly and stability of octadecylphosphonic acid (ODPA) were shown to be strictly dependent upon the observed heterogeneities of the surface termination, where it was locally shown that ODPA can loosely or strongly bind on different terminations of the crystal surface. Organization kinetics of ODPA was monitored with nanografting on (0001) surfaces. Supported by measurements of surface wettability and diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), it was demonstrated that the lack of organization within the protective adsorbed hexylphosphonic acid (HPA) monolayer on alumina surfaces facilitated the reduced confinement effect during nanografting, such that kinetics information on the organization process of ODPA could be obtained.     

Acoustics and atomic force microscopy for the mechanical characterization of thin films

The science and technology of thin films require the development of nondestructive methods for their quantitative mechanical characterization with nanometric spatial resolution. High-frequency ultrasonic techniques—especially acoustic microscopy—and atomic force microscopy (AFM) have been demonstrated to represent versatile tools for developing such methods. In particular, in the last 15 years, the combination of AFM, which can probe the surface of a sample by applying ultralow loads (from micronewtons down to piconewtons) with a micromachined tip having an apex radius of a few nanometers, and ultrasonics techniques led researchers to develop some unique tools which allow one to perform not only spot measurements of the sample elastic modulus, but also to obtain both the qualitative imaging of mechanical properties and the quantitative mapping of the elastic modulus of the sample surface with nanometric lateral resolution. In the present review, firstly a brief overview of the main ultrasound-based techniques for thin film characterization is reported. Then, some of the ultrasonic AFM techniques are described, emphasizing their capability of retrieving maps of both the tip–sample contact stiffness and the sample elastic modulus. Although these techniques are less affected by the mechanical properties of the substrates than standard indentation tests, a method for the correction of the substrate effect in ultrathin films is reported in detail. Finally, by probing the mechanical properties of a small portion of the sample volume underneath the tip, we illustrate the techniques as tools for the qualitative and quantitative characterization of variations in the adhesion between a thin film and a buried interface, as well as for detecting subsurface defects, voids, cracks, and dislocations.     

The use of atomic force microscopy for imaging the surfaces of polyamide, 6

The surface morphologies of PA 6 resulting from the use of various processing methods were studied by tapping mode atomic force microscopy. Three PA 6 samples: (1) a thin film, spin coated on a silicon wafer, (2) a freestanding film, i.e. a foil and (3) a monofilament, show definite morphological differences revealing typical supramolecular structures. The thin film having thickness of app. 35 nm is a good example of the initial step of spherulite formation where the sheaf development is still prominent. In an area of 100 μm² 1-4 spherulites can be detected which are typical of crystallization from the solution. The annealing (vacuum, 195°C, 3.5h) causes additional crystallization, which leads to a radial coordination and enlargement of spherulites to app. 50% in diameter and up to 40% in height. The morphology of foil (thickness of 100 μm) can be interpreted as a system of spherulites formed from the melt, and a typical fibrillar structure is observed on the surface of monofilament.     

Recent progress in the application of in situ atomic force microscopy for rechargeable batteries

High energy density batteries are urgently required for sustainable life. The intrinsic understanding of the reaction mechanism at the interfaces is essential for the progress. In this short overview, recent advances in rechargeable batteries by in situ atomic force microscopy are summarized, providing nanoscale information on the solid product evolution and metal plating/stripping inside working batteries. Besides, the multifunctional imaging of the morphology along with mechanical and electrical properties can be achieved to assist further interfacial design. Extensive applications of in situ atomic force microscopy are encouraged to explore the electrochemical mechanism and advanced engineering.     

Surface characterization using chemical force microscopy and the flow performance of modified polydimethylsiloxane for microfluidic device applications

The widespread interest in micro total analysis systems has resulted in efforts to develop devices in cheaper polymer materials such as polydimethylsiloxane (PDMS) as an alternative to expensive glass and silicon devices. We describe the oxidation of the PDMS surface to form ionizable groups using a discharge from a Tesla coil and subsequent chemical modification to augment electroosmotic flow (EOF) within the microfluidic devices. The flow performance of oxidized, amine-modified and unmodified PDMS materials has been determined and directly compared to conventional glass devices. Exact PDMS replicas of glass substrates were prepared using a novel two step micromolding protocol. Chemical force microscopy has been utilized to monitor and measure the efficacy of surface modification yielding information about the acid/base properties of the modified and unmodified surfaces. Results with different substrate materials correlates well with expected flow modifications as a result of surface modification. Oxidized PDMS devices were found to support faster EOF (twice that of native PDMS) similar to glass while those derivatized with 3-aminopropyl triethoxysilane (APTES) showed slower flow rates compared to native PDMS substrates as a result of masking surface charge. Results demonstrate that the surface of PDMS microdevices can be manipulated to control EOF characteristics using a facile surface derivatization methodology allowing surfaces to be tailored for specific microfluidic applications and characterized with chemical force microscopy.     

A proteomic approach for the study of Saccharomyces cerevisiae cell wall biogenesis

In fungi, cell shape is determined by the presence of a rigid cell wall which separates the cell from the extracellular medium. This highly dynamic structure is essential for the maintenance of cell integrity and is involved in several phenomena such as flocculation, adherence and pathogenicity. The composition of the fungal cell wall is well known, but issues such as the assembly and remodeling of its components remain poorly understood. In an attempt to study the de novo construction of the yeast cell wall, we have undertaken a large-scale proteomic approach to analyze the proteins secreted by regenerating protoplasts. Upon incubation of protoplasts in regenerating conditions, numerous proteins are secreted into the culture medium. These presumably include proteins destined for the cell wall, comprising both structural proteins as well as enzymes involved in cell wall biogenesis. This work reports the establishment of a reference map of proteins secreted by regenerating protoplasts by means of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and their identification by mass spectrometry. Thirty-two different proteins have been identified, including known cell wall proteins, glycolytic enzymes, heat shock proteins, and proteins involved in several other processes. Using this approach, novel proteins possibly involved in cell wall construction have also been identified. This reference map will allow comparative analyses to be carried out on a selected collection of mutants affected in the cell wall.     

Synthesis of the hexasaccharide repeating unit corresponding to the cell wall lipopolysaccharide of Azospirillum irakense KBC1

A convenient chemical synthesis of the hexasaccharide repeating unit of the cell wall lipopolysaccharide of Azospirillum irakense KBC1 has been successfully achieved. A stereo- and regioselective [4 + 2] block glycosylation strategy has been used to obtain the target hexasaccharide as its octyl glycoside. All synthetic intermediates have been prepared in high yields from commercially available reducing sugars following a series of protection–deprotection reactions. An oxidation–reduction methodology has been applied to convert β-d-glucosidic unit to a β-d-mannosidic moiety.     

A cell for in situ incident-light microscopy for the study of electrochromism of solid state electrochemical reactions

A cell is described which allows in situ incident-light microscopy to be used for the analysis of solid state electrochemical reactions studied by abrasive stripping voltammetry. The cell provides the possibility to screen solid compounds with respect to their electrochromic properties without requiring the preparation of special electrodes on transparent and conducting materials. Silver octacyanomolybdate(IV) and silver octacyanotungstate(IV) have been used to study the performance of the cell because both compounds exhibit a stable electrochromic and voltammetric behaviour.     

A force field for guanidinium-based ionic liquids that utilizes the electron charge distribution of the actual liquid: a molecular simulation study

We propose a new all-atom force field for guanidinium-based ionic liquids (GILs) which is based on the charge distribution in the actual liquid. It comprises all cations that can be built by attaching alkyl chains of variable length to an acyclic or cyclic guanidinium compound and that are paired with nitrate or perchlorate anions. We based the parametrization of the force field on liquid-phase charge distributions to improve the prediction of energetic and dynamic properties of GILs. The impact of electron charge transfer and polarization on various properties of GILs is systematically assessed. A significant average electron charge transfer between -0.12 and -0.06 e from anions to the central guanidinium group of the cations and a strong polarization of acyclic cations are observed by applying a combined quantum mechanical/molecular mechanical (QM/MM) approach. Molecular dynamics simulations of GILs are performed, utilizing the proposed force field. Derived structures approach the accuracy of QM/MM structures, and a previously reported crystal structure remains stable throughout the simulations. Mass densities are reproduced with a deviation of only 1.4% from experimental data. The calculated melting point of a GIL crystal deviates only 8% from the measured value. Self-diffusion coefficients of various GILs are reported, and a comparison with a diffusion coefficient derived from experimental data indicates that the values are within a reasonable range. We observe that the melting point of a GIL crystal was lowered up to 60 K and that diffusion coefficients are substantially increased by a factor of up to 3.5 upon consideration of charge transfer and polarization. The results demonstrate that liquid-phase partial charges are capable of improving the quality of ionic liquid force fields substantially and that their utilization led to a model that can be applied to predict structural, energetic, and dynamic properties of GILs.     

Evidence that gas-phase ion-molecule reactions are responsible for the behaviour of crown ethers under fast atom bombardment conditions

The fast atom bombardment mass spectrometry of some crown ethers shows the formation of both [M + H]⁺ and [M ? H]⁺ ions, paralleling behaviour already observed using electron impact ionization. The study of these oily samples with and without a glycerol matrix, trifluoroacetic acid or alkali metal salts, suggests that the ionization process does not occur in the condensed phase, but in the selvedge region by gas-phase ion-molecule reactions in accordance with the ‘gas-phase explosion model’. Positive-ion chemical ionization experiments support this proposal.