Tamara Ya. Morozova

Electrospray deposition as a method to fabricate functionally active protein films

Electrospray ionization is a routine method in MS analysis of proteins and other biopolymers. Deposition of the electrospray products onto a conductive electrode is suggested here as a means to manufacture functionally active protein films. Recovery of the specific hydrolytic activity of the electrosprayed alkaline phosphatase (AP) was used as a probe for preservation of protein intactness in the electrospray deposition (ESD). It was shown that protein inactivation upon ESD is highly dependent on voltage and current used. Humidity and the presence of protective substances in solution also affect the process. Complete preservation of the enzyme activity was observed when the ESD was performed at low current and humidity in the presence of disaccharides.

Viscoelasticity of protein crystal as a probe of the mechanical properties of a protein molecule: Hen egg-white lysozyme

Abstract Here we present a new approach to studying the anisotropy of the elastic properties of a protein globule based on an analysis of the elastic properties of a protein monocrystal. The anisotropy of the elasticity of hen egg-white lysozyme triclinic crystals, as well as its changes because of the formation of a lysozyme—N-acetyl- d -glucosamine complex, were investigated. The data can be explained on the assumption that the lysozyme molecule consists of two rigid domains connected by a flexible link. The binding of the inhibitor in the active-site cleft is accompanied by an increase of about 40% in the interdomain rigidity.

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.

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.

Mechanical detection of interaction of small specific ligands with proteins and DNA in cross-linked samples.

Cross-linked crystalline and amorphous films of different proteins and cross-linked DNA gels were found to change their mechanical properties when soaked in solutions of specific ligands at nearly physiological concentrations. This chemomechanical effect may be used to rapidly (within a few minutes) detect the ability of macromolecules to bind small (less than 1 kDa) ligand molecules, to measure concentrations of ligands (higher than 10 nM), and to estimate binding constants (lower than 10(7) M-1). Only 0.1-1 mg of protein or DNA is needed to prepare more than 10 samples sufficient for a large number of tests, provided binding is reversible. The method is recommended for rapid primary screening in search of new drugs, in biochemical studies, and as a basis for designing biosensors and other analytical instruments.

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.

A protein molecule as a bioanalytical device: Chemomechanical protein sensors

Cross‐linked protein solids are proposed as new types of chemomechanical sensors for identification of biological molecules and measurement of their concentration. New sensors make use of the ability of proteins to specifically bind its ligands and to alter their conformation upon the binding. The use of immobilized proteins enables these conformational changes to be detected through the changes in mechanical properties of protein solid samples (stress, stain or the Youngs modulus). Some advantages of the new sensors over enzyme probes are illustrated on considering the properties of papain sensor for α‐N‐benzoyl‐L‐arginine ethyl ester.