Synergistic Antimicrobial Effect of Cold Atmospheric Plasma and Redox-Active Nanoparticles.

Cold argon plasma (CAP) and metal oxide nanoparticles are well known antimicrobial agents. In the current study, on an example of Escherichia coli, a series of analyses was performed to assess the antibacterial action of the combination of these agents and to evaluate the possibility of using cerium oxide and cerium fluoride nanoparticles for a combined treatment of bacterial diseases. The joint effect of the combination of cold argon plasma and several metal oxide and fluoride nanoparticles (CeO2, CeF3, WO3) was investigated on a model of E. coli colony growth on agar plates. The mutagenic effect of different CAP and nanoparticle combinations on bacterial DNA was investigated, by means of a blue-white colony assay and RAPD-PCR. The effect on cell wall damage, using atomic force microscopy, was also studied. The results obtained demonstrate that the combination of CAP and redox-active metal oxide nanoparticles (RAMON) effectively inhibits bacterial growth, providing a synergistic antimicrobial effect exceeding that of any of the agents alone. The combination of CAP and CeF3 was shown to be the most effective mutagen against plasmid DNA, and the combination of CAP and WO3 was the most effective against bacterial genomic DNA. The analysis of direct cell wall damage by atomic force microscopy showed the combination of CAP and CeF3 to be the most effective antimicrobial agent. The combination of CAP and redox-active metal oxide or metal fluoride nanoparticles has a strong synergistic antimicrobial effect on bacterial growth, resulting in plasmid and genomic DNA damage and cell wall damage. For the first time, a strong antimicrobial and DNA-damaging effect of CeF3 nanoparticles has been demonstrated.

Antitumor Effect of Bleomycin Nanoaerosol in Murine Carcinoma Model.

Bleomycin, which is widely used as an antitumor agent, possesses serious adverse effects such as pulmonary toxicity. Local nanoaerosol deposition for lung cancer treatment is a promising alternative to drug delivery to lung lesions. The aim of this work is to test the hypothesis that bleomycin nanoaerosol can be effectively used to treat multiple lung metastases. To obtain bleomycin nanoaerosol, an aerosol generator based on electrospray of a solution of a nonvolatile substance with gas-phase neutralization of charged aerosol particles was used. Lung metastases in murine Lewis lung carcinoma and B16 melanoma animal models were counted. The effect of inhaled bleomycin nanoparticles on the number and volume of metastases, as well as pulmonary side effects, was investigated. Using a mouse exposure chamber, the dose-dependent effect of inhaled bleomycin on tumor volume was evaluated in comparison with intraperitoneal administration. Bleomycin nanoaerosol reduced the volume of metastases and produced a higher antitumor effect at much lower doses. It has been established that long-term exposure to nanoaerosol with a low dose of bleomycin is capable of suppressing cancer cell growth. The treatment was well tolerated. In the lungs, minor changes were found in the form of focal-diffuse infiltration of the lung parenchyma.

Cancer-Retina Antigens in the Urine of Bladder and Prostate Cancer Patients.

It has recently been shown that combination of arrestin and recoverin can serve as an effective urinary biomarker for renal cell carcinoma with sensitivity and specificity of over 92%. In this work, we studied the possibility of detecting these antigens in the urine in other urological oncological diseases - bladder cancer (BC) and prostate cancer (PCa). Urine samples from 40 BC patients and 40 PCa patients were analyzed using an ultrasensitive microarray immunoassay with a detection limit of 0.1 pg/ml. It was shown that in BC the sensitivity of determining combination of arrestin with recoverin is 58% (AUC 0.76, 95% CI 0.66-0.86), while in PCa it is 60% (AUC 0.7, 95% CI 0.68-0.88). It has been established that in patients with bladder and prostate cancer who had a positive test, these antigens are not detected in 90% of cases after removal of the tumor. In the future, the obtained results could become the basis for developing new approaches for timely detection of relapses of such diseases and treatment control, as well as for the development of new diagnostic methods.

Non-Invasive Diagnostics of Renal Cell Carcinoma Using Ultrasensitive Immunodetection of Cancer-Retina Antigens.

Renal cell carcinoma (RCC) is the most common urological malignancy with a high mortality and low detection rate. One of the approaches to improving its diagnostics may be the search for new non-invasive biomarkers in liquid biopsy and development of more sensitive methods for their detection. Cancer-retina antigens, which are known to be aberrantly expressed in malignant tumors, are present in liquid biopsy at extremely low concentrations. Using the developed multiplex immunoassay with a detection limit of 0.1 pg/ml, urine and serum samples of 89 patients with RCC and 50 non-cancer patients were examined for the presence of cancer-retina antigens (arrestin, recoverin, rhodopsin kinase, and transducin); the difference between the RCC and control groups was evaluated with the χ2 test. The results showed high diagnostic efficiency of a combination of arrestin and recoverin: at a threshold of 0.1 pg/ml, the sensitivity was 96%, specificity 92%, and AUC = 0.96 (95% confidence interval, 0.93-0.99). Seven days after nephrectomy, the concentration of the antigens returned to the level characteristic of the control group. Therefore, arrestin in a combination with recoverin can serve as a diagnostic non-invasive urinary biomarker of RCC.

Rapid Generation of Neurospheres from Hippocampal Neurons Using Extracellular-Matrix-Mimetic Scaffolds.

3D models of brain organoids represent an innovative and promising tool in neuroscience studies. However, the process of neurosphere formation in vitro remains complicated and is not always very effective. This is largely due to the lack of growth factors, guidance cues, and scaffold structures commonly found in tissues. Here we present a new, simple, and efficient method for generating neurospheres using scaffolds composed of electrospun nylon fibers with a diameter of 40-180 nm, which makes them similar to the brain extracellular matrix (ECM) components. Several main advantages of the proposed method should be highlighted. The method is fast, and the biomaterial consumption is low. Also, the resulting neurospheres are attached to the scaffold nanofibers. This not only provides the experimental convenience but also suggests that the resulting organoid models can potentially demonstrate fundamentally new properties, being closer to the nervous tissue in vivo. We demonstrate the influence of the fibrous scaffold structure on the formation, morphology, and composition of neurospheres and confirm adequate functional activity of the cellular components of these spheroids. The proposed approach can be further used for drug screening, modeling of neurodevelopmental, neurodegenerative disorders, and, potentially, therapeutic tissue engineering.

Amyloid Aggregates of Smooth-Muscle Titin Impair Cell Adhesion.

Various amyloid aggregates, in particular, aggregates of amyloid ß-proteins, demonstrate in vitro and in vivo cytotoxic effects associated with impairment of cell adhesion. We investigated the effect of amyloid aggregates of smooth-muscle titin on smooth-muscle-cell cultures. The aggregates were shown to impair cell adhesion, which was accompanied by disorganization of the actin cytoskeleton, formation of filopodia, lamellipodia, and stress fibers. Cells died after a 72-h contact with the amyloid aggregates. To understand the causes of impairment, we studied the effect of the microtopology of a titin-amyloid-aggregate-coated surface on fibroblast adhesion by atomic force microscopy. The calculated surface roughness values varied from 2.7 to 4.9 nm, which can be a cause of highly antiadhesive properties of this surface. As all amyloids have the similar structure and properties, it is quite likely that the antiadhesive effect is also intrinsic to amyloid aggregates of other proteins. These results are important for understanding the mechanisms of the negative effect of amyloids on cell adhesion.

ECM-Mimetic Nylon Nanofiber Scaffolds for Neurite Growth Guidance.

Numerous nanostructured synthetic scaffolds mimicking the architecture of the natural extracellular matrix (ECM) have been described, but the polymeric nanofibers comprising the scaffold were substantially thicker than the natural collagen nanofibers of neural ECM. Here, we report neuron growth on electrospun scaffolds of nylon-4,6 fibers with an average diameter of 60 nm, which closely matches the diameter of collagen nanofibers of neural ECM, and compare their properties with the scaffolds of thicker 300 nm nanofibers. Previously unmodified nylon was not regarded as an independent nanostructured matrix for guided growth of neural cells; however, it is particularly useful for ultrathin nanofiber production. We demonstrate that, while both types of fibers stimulate directed growth of neuronal processes, ultrathin fibers are more efficient in promoting and accelerating neurite elongation. Both types of scaffolds also improved synaptogenesis and the formation of connections between hippocampal neurons; however, the mechanisms of interaction of neurites with the scaffolds were substantially different. While ultrathin fibers formed numerous weak immature ß1-integrin-positive focal contacts localized over the entire cell surface, scaffolds of submicron fibers formed ß1-integrin focal adhesions only on the cell soma. This indicates that the scaffold nanotopology can influence focal adhesion assembly involving various integrin subunits. The fabricated nanostructured scaffolds demonstrated high stability and resistance to biodegradation, as well as absence of toxic compound release after 1 month of incubation with live cells in vitro. Our results demonstrate the high potential of this novel type of nanofibers for clinical application as substrates facilitating regeneration of nervous tissue.

Improving Immunoassay Performance with Cleavable Blocking of Microarrays.

Among the key issues that are commonly associated with the development of microarray-based assays are nonspecific binding and diffusion constraints. Here we present a novel strategy addressing both of these challenges simultaneously. The essence of the method consists in blocking the microarray surface with a blocking agent containing a perfluoroalkyl chain and a disulfide linker. The resulting surface is hydrophobic, and no immiscible liquid layer remains on it upon cyclically draining and replenishing the sample solution, ensuring an efficient mass transfer of an analyte onto a microarray. Prior to the signal detection procedure, disulfide bonds are chemically cleaved, and the perfluoroalkyl chains are removed from the microarray surface along with nonspecifically adsorbed proteins, resulting in extremely low background. Using conventional fluorescent detection, we show a 30-fold increase in signal/background ratio compared to a common epoxy-modified glass substrate. The combination of this technique with magnetic beads detection results in a simple and ultrasensitive cholera toxin (CT) immunoassay. The limit of detection (LOD) is 1 fM, which is achieved with an analyte binding time of 1 h. Efficient mass transfer provides highly sensitive detection of whole virus particles despite their low diffusion coefficient. The achieved LOD for vaccinia virus is 104 particles in 1 mL of sample. Finally, we have performed for the first time the simultaneous detection of whole virus and CT protein biomarker in a single assay. The developed technique can be used for multiplex detection of trace amounts of pathogens of various natures.

Rapid Ultrasensitive Gel-Free Immunoblotting with Magnetic Labels.

Immunoblotting is widely used for the detection of proteins using specific antibodies. We present here a new immunoblotting method, which is characterized by exceptional sensitivity, rapidness, and low consumption of antibodies. A thin conductive layer between touching hydrophilic cellulose membranes instead of polyacrylamide gel is used for the electrophoretic separation of proteins. Contrary to common Western blotting, the separation occurs in nondenaturing conditions. The membrane surface is smoothed by deposition of the cellulose layer and modified with azidophenyl groups, allowing for the photochemical in situ immobilization of proteins, which are carried out after the electrophoresis. Thus, the additional step of transferring the protein from the gel onto the membrane is eliminated. Specific protein bands are then visualized by decoration with magnetic beads. The limit of detection of interleukin IL-1ß reaches 0.3 fg or ∼104 molecules, whereas the total blotting time is about 5 min. The application of the technique is demonstrated by the detection of IL-1ß, total IgA, and IgA specific to Mycobacterium tuberculosis antigen in the exhaled breath samples, obtained from healthy subjects and tuberculosis patients.

Can new immunoassay techniques improve bladder cancer diagnostics With protein biomarkers?

The search for new diagnostic tests for cancer or ways to improve existing tests is primarily driven by the desire to identify the disease as early as possible. In this report, we summarize the current knowledge of the most promising diagnostic protein bladder cancer (BC) markers reported over the last decade. Unfortunately, analysis of published data suggests that a reliable, highly sensitive biomarker test-system based on ELISA for detecting BC has not yet been developed. The use of more sensitive assays to detect ultra-low concentrations of biomarkers not available for ELISA, could be very beneficial. Based on the literature and pilot experimental data, we conclude that a highly sensitive immunoassay using microarrays and magnetic labels, could be an effective and cheap technique suitable for the detection of diagnostically relevant BC biomarkers.

Rapid Amplification-Free Microarray-Based Ultrasensitive Detection of DNA.

We present a multiplex microarray-based assay of DNA fragments, which allows the detection of less than 10000 DNA fragments in a sample of 100 µL (corresponding to ∼0.1 fM analyte concentration) in less than 5 min. High speed and sensitivity are due to three main features of the assay. First, biotinylated adapter oligonucleotides are hybridized to the DNA fragment. Second, it is electrophoretically concentrated from the sample onto the microarray. Third, biotin labels are detected by scanning the microarray surface with streptavidin-coated magnetic beads. Prior to analysis, dsDNA fragments and genomic DNA samples were first denatured and then annealed in the presence of blocking oligonucleotides, generating ssDNA fragments capable of hybridizing with oligonucleotide probes on the microarray. The multiplexity of the assay system was demonstrated by the simultaneous detection of the genomic DNAs of three microorganisms: E. coli, B. cereus, and M. neoaurum.

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.

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.

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.

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.

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.

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.

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.

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.

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.