Are reactive oxygen species generated in electrospray at low currents?

It was demonstrated that electrospraying (ES) of solvents from a glass capillary proceeds without emission of light provided that the current is kept below a certain critical level (<100 nA at positive potential and <25 nA at negative potential for 96% ethanol; < 40 nA at positive potential for water). Though the onset of corona, as detected by the appearance of light, was always accompanied by a break in the current-voltage slope, such breaks also happened before the onset of corona, so they cannot be used as an adequate indicator of corona ignition. Of four ROS studied (hydrogen peroxide, ozone, hydroxyl radicals, and superoxide anions), only H2O2 and ozone were found to be generated at a current of 150-200 nA in detectable quantities: with a yield of 0.5-1 H2O2 molecules per electron at positive potential and 1.5-3 at negative potential. Despite the low yield of the ROS, jack bean urease was shown to be inactivated when the enzyme solution with a concentration below 20 µg/mL was electrosprayed at a current of 200 nA. Addition of 0.1 mM EDTA totally protected the activity of the electrosprayed urease.

Conic electrophoretic concentrator for charged macromolecules.

A simple, rapid, and highly effective technique for concentrating charged macromolecules is described which employs electrophoresis in a conic cell made of a dialysis membrane. The cell is partly submerged in electrolyte solution, and the level of solution slowly moves down during the process. The electric field within the cell is at its maximum in the area that is level with the surface of the external solution. This maximum value increases and its location moves downward following the decreasing level of external solution carrying downward and concentrating charged macromolecules. It has been demonstrated that proteins can be concentrated within 12-15 min by a factor of ∼100,000 with the total yield of 60-80%. Concentrated proteins can be harvested from the nanoliter-sized cul-de-sac of the conic concentrator using chemically activated magnetic beads. The presence of certain protein molecules linked to the bead's surface can be further revealed by specific reaction with a microarray of antibody molecules. Such "reversed magnetic array" format was applied to a cone-concentrated exhaled breath condensate (EBC) to reveal the presence of human immunoglobulin in the EBC and to estimate its concentration. The technique may be used for concentrating and detecting trace amounts of pathogens and toxins, in protein crystallization, and in many other applications.

Force differentiation in recognition of cross-reactive antigens by magnetic beads.

Functionalized magnetic beads have been suggested recently as active labels for extremely rapid and highly sensitive immunoassay. Here we addressed the problem of specificity and cross-reactivity in such detection, which (unlike conventional immunoassay methods) cannot rely on a difference in the equilibrium binding constants to distinguish between closely related antigens. Microarrays containing spots of nine albumins from sera of different mammals (human, bovine, sheep, goat, pig, dog, rabbit, rat, and mouse) were tested for their interaction with magnetic beads functionalized with monoclonal antibodies against bovine or human serum albumin. It was demonstrated that the magnetic beads bound only those albumin spots to which antibody was reactive or cross-reactive in enzyme-linked immunosorbent assay (ELISA). The effect of cross-reactivity in the assay with magnetic beads detection could be decreased substantially by placing the array into a flow cell and subjecting the tethered beads to increasing shear flow, which removed beads first from the weakest cross-reactive antigens and then from more strong ones. Partial blocking of the antibody molecules on the bead surface was shown to reduce critical shear stress necessary to remove beads from the specific antigens, indicating that multiple antigen-antibody bonds held the beads on the array surface.