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.

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.

Nanoaerosols reduce required effective dose of liposomal levofloxacin against pulmonary murine Francisella tularensis subsp. novicida infection.

BACKGROUND: The Institute of Theoretical and Experimental Biophysics in Moscow recently developed a new nanoaerosol generator. This study evaluated this novel technology, which has the potential to enhance therapeutic delivery, with the goal of using the generator to treat pulmonary Francisella tularensis subsp. novicida (F. novicida) infections in BALB/c mice. RESULTS: First, the analysis of quantum dots distribution in cryosections of murine lungs demonstrated that nanoaerosols penetrate the alveoli and spread more homogenously in the lungs than upon intranasal delivery. Second, the generator was used to aerosolize the antibiotic levofloxacin to determine the effectiveness of nanoaerosolized levofloxacin as treatment against F. novicida. The generator was capable of delivering a sufficient dose of nanoaerosolized liposome-encapsulated levofloxacin to rescue mice against 100LD50 of F. novicida. CONCLUSIONS: The nanoaerosol-delivered dosage of liposome-encapsulated levofloxacin required to rescue mice is approximately 94× lower than the oral required dose and approximately 8× lower than the intraperitoneal dose required for rescue. In addition, treatment with nanoaerosols consumes less total volume of therapeutic solutions and is gentler on sprayed material than the aerosolization by a conventional three-jet Collison nebulizer as seen by the preservation of liposomes. This could represent a significant advance for the use of expensive therapeutics and lung directed therapies.

Knockdown of Fruit Flies by Imidacloprid Nanoaerosol.

This report describes the effects of nanoaerosol particles (NAPs) from imidacloprid (IMI) on fruit flies. NAPs were produced using a newly developed generator which employs electro-hydrodynamic atomization of IMI solution in ethanol. Exposure of Drosophila melanogaster to the IMI NAPs at a concentration of C = 2.7 ± 0.1 ng/cm(3) caused knockdown in half of the flies in T50 = 88 ± 14 min at 22 °C and in T50 = 36 ± 2 min at 33 °C. A number of special experiments precluded IMI volatilization and contact or oral action of IMI upon exposure to the NAPs. It was shown that only the fraction of NAPs in the size range of 7-300 nm is responsible for the knockdown and that dependence of T50 on the NAPs' fraction mass follows Haber's rule, C × T50 = const. Comparison with the oral doses obtained when flies were fed an IMI-sucrose mixture revealed that the inhaled doses that caused knockdown were 2 orders of magnitude lower than the oral ones. This new technology may be used to quickly eliminate insects with nanoaerosols of nonvolatile insecticides in greenhouses and other closed environments.

Dry Lung as a Physical Model in Studies of Aerosol Deposition.

A new physical model was developed to evaluate the deposition of micro- and nanoaerosol particles (NAPs) into the lungs as a function of size and charges. The model was manufactured of a dry, inflated swine lung produced by Nasco company (Fort Atkinson, WI). The dry lung was cut into two lobes and a conductive tube was glued into the bronchial tube. The upper 1-2-mm-thick layer of the lung lobe was removed with a razor blade to expose the alveoli. The lobe was further enclosed into a plastic bag and placed within a metalized plastic box. The probability of aerosol deposition was calculated by comparing the size distribution of NAPs passed through the lung with that of control, where aerosol passed through a box bypassing the lung. Using this new lung model, it was demonstrated that charged NAPs are deposited inside the lung substantially more efficiently than neutral ones. It was also demonstrated that deposition of neutral NAPs well fits prediction of the Multiple-Path Particle Dosimetry (MPPD) model developed by the Applied Research Associates, Inc. (ARA).

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.

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.

Rapid simultaneous ultrasensitive immunodetection of five bacterial toxins.

Rapid ultrasensitive detection of gastrointestinal pathogens presents a great interest for medical diagnostics and epidemiologic services. Though conventional immunochemical and polymerase chain reaction (PCR)-based methods are sensitive enough for many applications, they usually require several hours for assay, whereas as sensitive but more rapid methods are needed in many practical cases. Here, we report a new microarray-based analytical technique for simultaneous detection of five bacterial toxins: the cholera toxin, the E. coli heat-labile toxin, and three S. aureus toxins (the enterotoxins A and B and the toxic shock syndrome toxin). The assay involves three major steps: electrophoretic collection of toxins on an antibody microarray, labeling of captured antigens with secondary biotinylated antibodies, and detection of biotin labels by scanning the microarray surface with streptavidin-coated magnetic beads in a shear-flow. All the stages are performed in a single flow cell allowing application of electric and magnetic fields as well as optical detection of microarray-bound beads. Replacement of diffusion with a forced transport at all the recognition steps allows one to dramatically decrease both the limit of detection (LOD) and the assay time. We demonstrate here that application of this "active" assay technique to the detection of bacterial toxins in water samples from natural sources and in food samples (milk and meat extracts) allowed one to perform the assay in less than 10 min and to decrease the LOD to 0.1-1 pg/mL for water and to 1 pg/mL for food samples.

Proteomic characterization and functional analysis of outer membrane vesicles of Francisella novicida suggests possible role in virulence and use as a vaccine.

We have isolated and characterized outer membrane vesicles (OMVs) from Francisella. Transport of effector molecules through secretion systems is a major mechanism by which Francisella tularensis alters the extracellular proteome and interacts with the host during infection. Outer membrane vesicles produced by Francisella were examined using TEM and AFM and found to be 43-125 nm in size, representing another potential mechanism for altering the extracellular environment. A proteomic analysis (LC-MS/MS) of OMVs from F. novicida and F. philomiragia identified 416 (F. novicida) and 238 (F. philomiragia) different proteins, demonstrating that OMVs are an important contributor to the extracellular proteome. Many of the identified OMV proteins have a demonstrated role in Francisella pathogenesis. Biochemical assays demonstrated that Francisella OMVs possess acid phosphatase and hemolytic activities that may affect host cells during infection, and are cytotoxic toward murine macrophages in cell culture. OMVs have been previously used as a human vaccine against Neisseria meningitidis . We hypothesized that Francisella OMVs could be useful as a novel Francisella vaccine. Vaccinated BALB/C mice challenged with up to 50 LD50 of Francisella showed statistically significant protection when compared to control mice. In the context of these new findings, we discuss the relevance of OMVs in Francisella pathogenesis as well as their potential use as a vaccine.

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.

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.

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.

Rapid active assay for the detection of antibodies to West Nile virus in chickens.

To reduce the assay time for detecting virus-specific antibodies in serum, we developed microarray-based active immunoassay techniques for detecting West Nile virus (WNV)-specific IgM molecules in chicken blood. The assay uses electrophoretic concentration of IgM molecules onto WNV antigens arrayed on a dialysis membrane followed by detection of bound IgM molecules with functionalized magnetic beads as active labels. This assay takes only 15 minutes and has the same sensitivity as a commercially available human WNV IgM antibody-capture enzyme-linked immunosorbent assay (commonly called a MAC-ELISA) modified for use with chicken sera.

Non-invasive approach to diagnosis of pulmonary tuberculosis using microdroplets collected from exhaled air.

In this report we present a proof-of-principle study aimed at developing non-invasive diagnostics for pulmonary TB that are based on analyzing TB biomarkers in exhaled microdroplets of lung fluid (MLFs). Samples were collected on electrospun filters recently developed by the authors, and then tested for the presence of Mycobacterium tuberculosis (Mtb) cells, Mtb DNA, and protein biomarkers (secreted Mtb antigens and antigen-specific antibodies). The latter were detected using rapid ultra-sensitive immunochemistry methods developed in our laboratory. Neither Mtb cells (limit of detection, LOD = 1 cell) nor Mtb DNA (LOD âˆ¼ 10 CFU) were found in the MLF samples exhaled by TB patients. However, immunoglobulin A (IgA) was found in over 90% of samples from TB patients and healthy volunteers. Antigen-specific IgA were detected at higher rates in the patient samples as compared to those from nominally healthy volunteers resulting in a modest discrimination level of 72% sensitivity and 58% specificity. As such, this novel, non-invasive and fast breath diagnostic method shows promise for further development.

A collection system for dry solid residues from exhaled breath for analysis via atomic force microscopy.

Exhaled air contains sub-micron droplets of lung liquid, which potentially bear biomarkers of lung diseases. After dehydration they form dry residue particles (DRPs). As a first step in developing techniques to characterize individual DRPs, a new electrostatic collector was designed in which DRPs are charged within a unipolar corona charger, concentrated in a cone funnel, and deposited onto a limited area of a highly oriented pyrolytic graphite surface. The collector captures 80%-90% of DRPs at an optimal flow rate of 0.15 l min-1. Atomic force microscopy (AFM) revealed flattened round particles 20-50 nm high, with notable protrusions at their surface suggestive of an inhomogeneous internal structure. Exposure to humid air resulted in the DRPs spreading over the surface, with a 50%-200% decrease in their heights and an increase in their lateral dimensions so that their volume decreased by only 10% ± 3%. Exposure to saturated chloroform vapor resulted in drainage of 10%-15% of the DRP volume (presumably lipids), forming collar-shaped rings around each particle but leaving the core size and structure unchanged. AFM measurements combined with laser counter measurements of the DRP concentrations were used to estimate that one liter of air exhaled by volunteers contained less than 100 pg of dry residue material.

Titration of trace amounts of immunoglobulins in a microarray-based assay with magnetic labels.

The existing immunoassay format that combines the electrophoretic collection of charged analytes on an antibody microarray with the detection of the bound analytes by magnetic beads coated with secondary antibodies displays extreme sensitivity and speed, but suffers from low precision because of high signal scatter and low signal-to-concentration ratio. Here we report three innovations that substantially improve the precision of this method and enable quantitative measurements of analyte concentrations as low as 10 fg/ml. The improvements were achieved by (i) employing parallel titration of analytes by measuring signal response to a series of sample dilutions with increasing analyte concentration, (ii) internally normalizing the signal (by relating signal intensity to that of positive controls on the same microarray) and (iii) taking measurements in the linear range of the calibration curve at concentrations close to the limit of detection. This improved method was used to quantitatively measure in human serum the titer of immunoglobulins specific to antigens secreted by Mycobacterium tuberculosis.

Non-invasive lung disease diagnostics from exhaled microdroplets of lung fluid: perspectives and technical challenges.

The combination of ultra-sensitive assay techniques and recent improvements in the instrumentation used to collect microdroplets of lung fluid (MLF) from exhaled breath has enabled the development of non-invasive lung disease diagnostics that are based on MLF analysis. In one example of this approach, electrospun nylon filters were used to collect MLFs from patients with pulmonary tuberculosis. The filters were washed to obtain liquid probes, which were then tested for human immunoglobulin A (h-IgA) and fractions of h-IgA specific to ESAT-6 and Psts-1, two antigens secreted by Mycobacterium tuberculosis. Probes collected for 10 min contained 100-1500 fg of h-IgA and, in patients with pulmonary tuberculosis, a portion of these h-IgA molecules showed specificity to the secreted antigens. Separate MLFs and their dry residues were successfully collected using an electrostatic collector and impactor developed especially for this purpose. Visualization of MLF dry residues by atomic force microscopy made it possible to estimate the lipid content in each MLF and revealed mucin molecules in some MLFs. This exciting new approach will likely make it possible to detect biomarkers in individual MLFs. MLFs emerging from an infection site ('hot' microdroplets) are expected to be enriched with infection biomarkers. This paper discusses possible experimental approaches to detecting biomarkers in single MLFs, as well as certain technological problems that need to be resolved in order to develop new non-invasive diagnostics based on analysing biomarkers in separate MLFs.

Mechano-chemical effects of Ca(2+) in cross-linked troponin-C films.

Changes in troponin C (TnC) conformation upon Ca(2+) binding forms the basis for regulatory and structural functions of TnC molecules. In the present study, Ca(2+)-induced conformational changes in TnC were observed by mechanical measurements. TnC films were prepared by drying or electrospraying TnC solutions, cross-linked with glutaraldehyde, and isometric tension and stiffness measured as a function of pCa. An increase in Ca(2+) from a pCa of 9 to 4 induced large-scale mechanical changes in the TnC films causing several percent shrinkage of the unloaded films. This shrinkage could be partially assigned to Ca(2+) binding to the Ca(2+)/Mg(2+) sites of TnC.

Direct detection of isotopically labeled metabolites bound to a protein microarray using a charge-coupled device.

A charge-coupled device (CCD) was used to quantitatively detect isotope-labeled ligands bound to a protein microarray. Protein microarrays with protein dots, 10-50 microm in diameter, were fabricated on an aluminized Mylar film using an electrospray deposition technique. Proteins in dots were immobilized by cross-linking in glutaraldehyde vapor. After contact with solutions of isotope-labeled metabolites, the protein microarrays were washed, dried and placed face down onto the surface of a standard B/W video CCD chip with the protective window removed. We show here that such a simple inexpensive CCD detector can be used to quantify distribution of 14C and other radioactive isotopes on microarrays.

Computer simulation of gas-phase neutralization of electrospray-generated protein macroions.

The process of neutralizing hydrated multicharged gas-phase protein ions with small counterions was simulated using a molecular dynamics (MD) technique. Hen egg white lysozyme (HEWL) molecules with different numbers of positive charges, both dry and solvated by up to 1500 water molecules, were first equilibrated. Simulations revealed that the hydration layer over a highly charged protein surface adapted a spiny structure with water protrusions composed of oriented water dipoles. MD simulations of the neutralization process showed that the impact of a small dehydrated single-charged counterion with a dehydrated HEWL ion bearing eight uncompensated charges resulted in a short local increase in temperature by 600-1000 K, which quickly (in 3-5 ps) dissipated over the whole protein molecule, increasing its average temperature by 20-25 K. When the protein ion was solvated, no drastic local increase in the temperature of the protein atoms was observed, because the impact energy was dissipated among the water molecules near the collision site.