Imaging of biological macromolecules on mica in humid air by scanning electrochemical microscopy

Imaging of DNA, keyhole limpet hemocyanin, mouse monoclonal IgG, and glucose oxidase on a mica substrate has been accomplished by scanning electrochemical microscopy with a tungsten tip. The technique requires the use of a high relative humidity to form a thin film of water on the mica surface that allows electrochemical reactions to take place at the tip and produce a faradaic current (≈1 pA) that can be used to control tip position. The effect of relative humidity and surface pretreatment with buffer solutions on the ionic conductivity of a mica surface was investigated to find appropriate conditions for imaging. Resolution of the order of 1 nm was obtained.

Manufacturing DNA microarrays of high spot homogeneity and reduced background signal

Analyses on DNA microarrays depend considerably on spot quality and a low background signal of the glass support. By using betaine as an additive to a spotting solution made of saline sodium citrate, both the binding efficiency of spotted PCR products and the homogeneity of the DNA spots is improved significantly on aminated surfaces such as glass slides coated with the widely used poly-l-lysine or aminosilane. In addition, non-specific background signal is markedly diminished. Concomitantly, during the arraying procedure, the betaine reduces evaporation from the microtitre dish wells, which hold the PCR products. Subsequent blocking of the chip surface with succinic anhydride was improved considerably in the presence of the non-polar, non-aqueous solvent 1,2-dichloroethane and the acylating catalyst N-methylimidazole. This procedure prevents the overall background signal that occurs with the frequently applied aqueous solvent 1-methyl-2-pyrrolidone in borate buffer because of DNA that re-dissolves from spots during the blocking process, only to bind again across the entire glass surface.

Rapid Regulation by Acid pH of Cell Wall Adjustment and Leaf Growth in Maize Plants Responding to Reversal of Water Stress1

The role of acid secretion in regulating short-term changes in growth rate and wall extensibility was investigated in emerging first leaves of intact, water-stressed maize (Zea mays L.) seedlings. A novel approach was used to measure leaf responses to injection of water or solutions containing potential regulators of growth. Both leaf elongation and wall extensibility, as measured with a whole-plant creep extensiometer, increased dramatically within minutes of injecting water, 0.5 mm phosphate, or strong (50 mm) buffer solutions with pH ≤ 5.0 into the cell-elongation zone of water-stressed leaves. In contrast, injecting buffer solutions at pH ≥ 5.5 inhibited these fast responses. Solutions containing 0.5 mm orthovanadate or erythrosin B to inhibit wall acidification by plasma membrane H+-ATPases were also inhibitory. Thus, cell wall extensibility and leaf growth in water-stressed plants remained inhibited, despite the increased availability of (injected) water when accompanying increases in acid-induced wall loosening were prevented. However, growth was stimulated when pH 4.5 buffers were included with the vanadate injections. These findings suggest that increasing the availability of water to expanding cells in water-stressed leaves signals rapid increases in outward proton pumping by plasma membrane H+-ATPases. Resultant increases in cell wall extensibility participate in the regulation of water uptake, cell expansion, and leaf growth.

Electrostatic force microscope for probing surface charges in aqueous solutions.

A scanning force microscope was converted to an electrostatic force microscope by charging the usually neutral cantilever with phospholipids. The electrostatic force microscope was used to study surface electrostatic charges of samples in aqueous solutions. Lysozymes, DEAE-Sephadex beads, 3-propyltriethoxysilane-treated glass and mica were imaged in water or phosphate buffer with electrostatic force microscopy. The adhesion force measured when a charged probe and oppositely charged specimen interacted was up to 500 times greater than when a bare probe was used. This dramatic increase in measured adhesion force can be attributed to the energy required to break the salt bridges formed between the charged probe and the specimen. The use of phospholipids to functionalize the cantilever tip allows the incorporation of other biomolecules and ligands that can be used as biologically specific tips (e.g., receptors, drugs) for the study of intermolecular interactions.