Elena A. Shlyapnikova

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.

Ionic conductivity, transference numbers, composition and mobility of ions in cross-linked lysozyme crystals

Micromethods for measurements of electric conductivity, transference numbers and concentrations of inorganic ions within immobilized protein crystals have been developed and applied to study tetragonal lysozyme crystals cross-linked with glutaraldehyde. Donnan equilibria and mobilities of ions in this crystal were calculated using the data of these methods and the data of crystal pH titration. Taken together these results characterize the lysozyme crystal as an ion exchanger whose electrical properties and ion composition differ greatly from those of the external solution. Although anions transfer most of the current in the crystals, anion mobility is considerably lower than that of cations. Mobility of all ions in the crystal is considerably lower than in solution (3.5-50 times for cations and 120-330 times for anions) and depends on steric restrictions and charges of both ions and lysozyme molecules. Similar features in behavior of crystalline and biological channels are discussed.

Detection of microarray-hybridized oligonucleotides with magnetic beads.

The efficiency of hybridization analysis with oligonucleotide microarrays depends heavily on the method of detection. Conventional methods based on labeling nucleic acids with fluorescent, chemiluminescent, enzyme, or radioactive reporters suffer from a number of serious drawbacks which demand development of new detection techniques. Here, we report two new approaches for detection of hybridization with oligonucleotide microarrays employing magnetic beads as active labels. In the first method streptavidin-coated magnetic beads are used to discover biotin-labeled DNA molecules hybridized with arrayed oligonucleotide probes. In the second method biotin-labeled DNA molecules are bound first to the surface of magnetic beads and then hybridized with arrayed complementary strands on bead-array contacts. Using a simple low-power microscope with a dark-field illumination and a pair of complementary primers as a model hybridization system we evaluated sensitivity, speed, and cost of the new detection method and compared its performance with the detection techniques employing enzyme and fluorescent labels. It was shown that the detection of microarray-hybridized DNA with magnetic beads combines low cost with high speed and enhanced assay sensitivity, opening a new way to routine hybridization assays which do not require precise measurements of DNA concentration.

Carboxymethyl cellulose film as a substrate for microarray fabrication.

Magnetic beads (MB) are widely used for quick and highly sensitive signal detection in microarray-based assays. However, this technique imposes stringent requirements for smoothness and adhesive properties of the surface, which most common substrates do not satisfy. We report here a new type of substrate for microarrays with a low adhesion to MB-thermally cross-linked carboxymethyl cellulose (CMC) film. This substrate can be readily fabricated on a conventional glass slide. A highly cross-linked CMC film (∼1 cross-link per monomer unit) possesses a surface smooth on a nanometer scale and a low adhesion to protein-coated MB, which partly originates from electrostatic repulsion of MB from negatively charged CMC surface. The efficiency of the CMC substrate is demonstrated hereby in fabrication of microarrays for the detection of three bacterial toxins: cholera toxin, staphylococcal enterotoxin A, and toxic shock syndrome toxin. The assay employing a primary antibodies arrayed on a CMC surface and detection of the bound bacterial toxins with a biotinylated secondary antibodies and streptavidin-coated MB resulted in a limits of detection as low as 0.1 ng/mL. The CMC-based microarrays demonstrated very high storage stability; their activity did not change after one year storage at room temperature.

Reversible and Irreversible Mechanical Damaging of Large Double-Stranded DNA upon Electrospraying

Electrohydrodynamic spraying (or electrospaying, ES) of DNA solutions is an attractive technique for applications in mass spectrometry, in microarray fabrication, and in generation of DNA nanoaerosols. Here we report how ES affects DNA structure and evaluate possible ways to reduce DNA damage upon ES. It is shown that under any ES conditions, linear λ-phage DNA is subjected to intensive rupture producing a mixture of fragments. In addition to such fragmentation, notable reversible changes in the DNA structure were revealed by a slight increase in DNA electrophoretic mobility. The degree of fragmentation was shown to decrease with decreased DNA length and with increased flow rate through the ES capillary. Fragments shorter than 5 kbp did not show any notable damage upon ES. Both experimental data and theoretical estimations of the forces acting on DNA during ES indicate that DNA is damaged by mechanical forces, and the damage takes place in the vicinity of the Taylor cone tip, presumably due to the high shear stress or/and viscous drag forces operating there. Condensation of λ-DNA with hexamminecobalt(III) ions completely protected it from any damage upon ES.

Ballistic Penetration of Highly Charged Nanoaerosol Particles through a Lipid Monolayer

To be used as a drug, inhaled nanoaerosol particles (NAPs) must first penetrate the lipid layer on top of the lung fluid before they will be able to reach the lung epithelium. We investigated how the penetration of NAPs through a model lipid monolayer (LM) depends upon their charging level and size. It was shown that deposition of NAPs 20-200 nm in diameter and charged to the Rayleigh limit gradually increased the surface tension of a dipalmitoylphosphatidylcholine monolayer (DPPC), indicating a loss of lipid molecules from the monolayer. This phenomenon was reproduced with a variety of NAPs produced from glucose, proteins, and polymers. Transfer of the lipid material into the subphase was documented by direct visualization of lipid nanoparticles in the subphase with atomic force microscopy after deposition of glucose NAPs on a DPPC monolayer, followed by collection of the lipid nanoparticles on a mica surface. Partial restoration of tension upon storage indicates that some of the lipid may return to the monolayer. Experiments with the deposition of highly charged calibrated polystyrene nanoparticles showed that the amount of lipid removed from the surface was roughly proportional to the overall surface area of the deposited NAPs. When the number of charges on the NAPs was reduced from their Rayleigh level of 103-104 units to 1-10 units, no notable changes in monolayer surface tension were observed even with prolonged deposition of such NAPs. It was therefore concluded that only highly charged NAPs of a certain size acquire sufficient speed from their attraction by mirror charges to enable ballistic penetration through a lipid monolayer.

Exposure to bleomycin nanoaerosol does not induce fibrosis in mice

Abstract Bleomycin (BLM), a cytostatic drug widely used in the treatment of cancer, often induces lung fibrosis as a side effect. This phenomenon has been exploited in biomedical studies to create a fibrosis model. Inhalation of BLM nanoaerosol particles (NAPs) was expected to induce severe fibrosis in mice. Instead, inhalation of BLM NAPs for 13 days with a total inhaled dose of ~1 mg/kg did not cause pneumofibrosis in CD-1 mice; only marked pneumonitis with infiltration of macrophages in all parts of the lungs was observed in all eight mice exposed. These changes were notably reversible within 2 weeks of cessation of exposure. Despite the limited number of animals it was confidently concluded that slow administration of BLM in the form of NAPs did not cause fibrosis as the bolus instillation of similar doses does.