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Background Microplate-based immunoassays are widely used in clinical and research settings to measure a broad range of biomarkers present in complex matrices. Assay variability within and between microplates can give rise to false-negative and false-positive results leading to incorrect conclusions. To date, the contribution of microplates to this variability remains poorly characterized and described. This study provides new insights into variability in immunoassays attributable to surface characteristics of commercial microplates. Methods Well-to-well assay variation in γ-treated and nontreated 96-well opaque microplates suitable for chemiluminescence assays was determined by use of a validated sandwich ELISA. Microplate surface characteristics were assessed by sessile drop contact angle measurements, scanning electron microscopy, energy dispersive x-ray spectroscopy, and atomic force microscopy. Results All microplate types tested exhibited vendor-specific assay response profiles; and “rogue” plates with very high intraassay variation and deviant mean assay responses were found. Within-plate, location-dependent bias in assay responses and variability in well contact angle were also observed. We demonstrate substantial differences in well-surface properties with putative effects on protein-coating reproducibility and hence consistency in immunoassay responses. A surface “cleaning” effect on manufacturing residues was attributed to γ-irradiation, and treated microplates manifest increased polar functionalities, surface roughness, and assay responses. Conclusions Our data suggest that tighter control of variability in surface roughness, wettability, chemistry, and level of residual contaminants during microplate preparation is warranted to improve consistency of ELISA assay read out.

An ionic KBr(001) crystal surface covered with a thin water layer was observed with a frequency modulation atomic force microscope (FM-AFM) with atomic resolution. By immersing only the tip apex of the AFM cantilever in the thin water layer, the Q-factor of the cantilever in probing the solid-liquid interface can be maintained as high as that of FM-AFM operation in air, leading to improvement of the minimum detection of a differential force determined by the noise. Two types of images with atom-resolved contrast were observed, possibly owing to the different types of ions (K(+) or Br(-)) adsorbed on the tip apex that incorporated into the hydration layers on the tip and on the sample surface. The force-distance characteristics at the solid-water interface were analyzed by taking spatial variation maps of the resonant frequency shift of the AFM cantilever with the high Q-factor. The oscillatory frequency shift-distance curves exhibited atomic site dependence. The roles of hydration and the ions on the tip and on the sample surface in the measurements were discussed.

The two-dimensional supramolecular structures of a series of N,N'-bis(n-alkyl)naphthalenediimides (NDIs), whose chain lengths span from C3 to C18, at a liquid-HOPG surface interface, studied by STM and FM-AFM, are assigned with the help of molecular dynamics/molecular mechanics calculations to demonstrate that the C3- and C4-NDIs show lamellar structures, the C4- to C12-NDIs show honeycomb (KAGOME) structures, and the C14- to C18-NDIs show lamellar structures again. The change in supramolecular structure depending on chain length can be explained semiquantitatively by the balance of entropy and enthalpy terms to show the importance of "self-avoiding walk" of the alkyl chain in entropy terms.

An α-Al2O3(0112) wafer was immersed in an aqueous KCl solution of 1 mol L−1 and observed with a frequency-modulation atomic force microscope. The tip−surface force was precisely determined as a fu...

Effects of l -aspartic acid ( l -Asp) on dissolution of calcite were investigated. The step retreat velocity and dissolution rate of calcite were measured simultaneously using an AFM flow-through system. The etch-pit morphology of calcite was observed using confocal laser scanning microscopy. Results show that the etch-pit morphologies changed drastically depending on the l -Asp concentration ([ l -Asp]) in the order of rhomboidal, pentagonal, and triangular (not perfectly, but retaining an extra step). The change in obtuse step directions and appearance of the [0 1 0] step triggered these morphological changes. Addition of l -Asp accelerated all step retreats at [ l -Asp] l -Asp on the diffusive barrier. In contrast, at [ l -Asp]>0.01 M, l -Asp inhibited the retreats of obtuse steps and [0 1 0] step, although the retreat velocities of acute steps were constant irrespective of [ l -Asp]. These results suggest that the directional changes and the inhibition of retreat velocities of obtuse steps were attributed to the generation of [ 4 1 1] and [4 5 1 ] steps caused by l -Asp. Moreover, we confirmed the preferential effects of l -Asp on the [4 8 1 ]+ to [ 4 4 1]± step edge, and proposed the preferential effects of l -Asp on the [ 4 1 1] to [4 5 1 ] step edge.

An intercomparison of nanometric lateral scales, which are special one-dimensional (1D) grating standards with sub-hundred-nanometre pitches, among a deep-ultraviolet (DUV) laser diffractometer, a critical dimension scanning electron microscope (CD-SEM) and different types of atomic force microscope (AFM) was performed. The reference value and its expanded uncertainty were provided by the National Metrology Institute of Japan (NMIJ) using an atomic force microscope with differential laser interferometers (DLI-AFM). The consistency of the measurement results obtained using the DUV laser diffractometer, CD-SEM and some AFMs was satisfactory; however, that in the measurement results obtained using other AFMs was unsatisfactory. An improvement in AFM calibration technology using nanometrological standards is required for both AFM manufacturers and AFM users, including metrology institutes.

We constructed a scanning near-field optical microscope (SNOM) on a commercially available atomic force microscopy (AFM) apparatus (SPM-9500J2; Shimadzu Corp.) to measure the stress distribution in ceramic composite materials. Features of our SNOM system are: (1) a compact SNOM head substituted for the original AFM head; (2) a wide scanning range (125 × 125 μm2) inherited from the original scanner; (3) use of conventional shear-force regulation; (4) an optical system for the illumination-collection (I-C) mode; (5) excitation by a 488 nm line of an Ar-ion laser, and (6) light detection by photon counting or a polychromator equipped with an electronically cooled charge coupled device (CCD). This SNOM system was used to measure the surface structure and stress distribution of an Al2O3/ZrO2 eutectic composite. We simultaneously measured topographic images and fluorescence spectra of an Al2O3/ZrO2 eutectic composite. We estimated its peak intensity, peak position, and peak width from the fluorescence spectrum during scanning, which respectively correspond to the abundance of Al2O3, stress in the grain, and the anisotropy of that stress. Mapping images showed that the stress and its anisotropy were weaker in the center of the Al2O3 grain than its boundary between Al2O3 and ZrO2. That observation suggests that Al2O3 underwent intense anisotropic stress induced by volume expansion in the phase transition of ZrO2 from the cubic phase to the monoclinic phase during preparation.

The crystal growth of isotactic polystyrene (it-PS) is investigated in very thin, 11 nm thick films. The it-PS crystals grown in the thin films show quite different morphology from that in the bulk. With decreasing crystallization temperature, the branching morphology in a diffusion field appears: dendrite and compact seaweed. The branching morphology is formed through a morphological instability caused by the gradient of film thickness around a crystal; the thicker the film thickness, the larger the lateral growth rate of crystals. Regardless of the morphological change, the growth rate as well as the lamellar thickness depends on crystallization temperature as predicted by the surface kinetics., 7 pages, 8 figures (EPS), LaTeX, accepted for publiaction in Polymer

We propose a van der Pol type self-excited micro-cantilever probe for AFM (atomic force microscope). Because the response frequency of self-excited oscillators is its natural frequency, the equivalent natural frequency depending on the atomic force is easily measured from detecting the response frequency of the self-excited micro-cantilever probe. While the amplitude of the linear self-excited oscillator grows with time, it can be kept very small by the nonlinear effect as van ver Pol oscillator. Therefore, if the micro-cantilever probe has the same dynamics as that of van Pol oscillator, the contact of the micro-cantilever to the material surface is prohibited and non-contact mode AFM is realized. In the present study, we design a van der Pol type micro-cantilever for the application to the practical AFM system.Copyright © 2009 by ASME

The film thickness dependence of crystal growth is investigated for isotactic polystyrene (it-PS) in thin films, the thickness of which is from 20nm down to 4nm. The single crystals of it-PS grown at 180 in the ultrathin films show the morphology typical in the diffusion-controlled growth: dense branching morphology (DBM), fractal seeweed (FS). The characteristic length of the morphology, i.e. the width of the branch, increases with decreasing film thickness. The thickness dependence of the growth rate of crystals shows a crossover around the lamellar thickness of the crystal, 8 nm. The thickness dependences of the growth rate and morphology are discussed in terms of the diffusion of chain molecules in thin films., Comment: Latex2e files; 7 pages, 6 figures; submitted to J. Macro. Sci. Phys

In order to determine the molecular structure by x-ray diffraction analysis, it is very important to grow high quality protein crystals. The molecular resolution imaging of soluble protein crystals such as the tetragonal lysozyme (110) face in saturated solution is demonstrated using frequency-modulation atomic force microscopy (FM-AFM). The surface structure of the (110) face and the crystallographic position of individual molecules were determined from molecular resolution images. For observation of protein crystals, FM-AFM is a favorable technique as an alternative to contact mode or amplitude-modulation AFM.

The topography and solvation structure of a solution–TiO2 interface were observed in the dark using highly sensitive, frequency-modulated atomic force microscopy (FM-AFM). The nucleation and growth of an ionic solute, KCl, in this study, were observed in constant frequency-shift topography. The force applied to the tip was determined as a function of tip–surface distance. Modulations were identified on some force curves and were found to be related to the site-specific density of water molecules.

We observed local electrical properties of polycrystalline silicon films by conductive atomic force microscopy. Moreover, we investigated the effects of hydrogen termination on the polycrystalline silicon films. Before hydrogen termination, conductive regions in grain disappeared with the repeated scanning of the cantilever, while conductive regions in grain boundary almost unchanged. It is considered that hopping conduction is a major electrical conduction mechanism at grain boundaries. After 5 min hydrogen termination, locally nonterminated regions were observed near grain boundaries. This suggests that hydrogen termination of the polycrystalline silicon does not randomly progress, and there are regions that cannot be easily inactivated near grain boundaries.

We investigate the thickness dependence of the lattice constants of a chloroiron(III) derivative of tetraphenylporphine (ClTPPFe) epitaxial crystals grown on alkali–halide substrates, KCl, KBr and KI, in order to study the accommodation of the misfit strain, and do not observe any obvious dependence. Moreover, the orientational disturbance of the epitaxial crystals on the alkali halides and the formation of amorphous islands on an amorphous carbon film are observed. On the basis of these results, we propose the mechanism that the orientation of surface-diffusing molecules contributes significantly to the epitaxial growth of ClTPPFe on the alkali halides.

Optical methods have been proposed for the profile measurement of aspheric lens, but further investigation is needed to improve the accuracy of the machining. A method for the measurement of small-diameter lenses was developed using a scanning electron microscope. Photo-semiconducdors were used to detect backscattered electrons which were reflected on the surface of the lens. A new simple algorithm which makes it possible to derive direction angle and slant angle of the normal at the respective observed points was proposed. The slope of a plastic aspheric lens was measured in terms of the normal. Contour circles of equi-slope were illustrated. The difference in the radii of the circles for that the slope was varied with constant step was deformed due to the aspherity. A profile of the aspherity along the radial direction could be obtained by integrating the slope. The error from the designed profile could be evaluated based on the result.