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The integration of several controlled parameters within a single test system is experiencing increased demand. However, multiplexed test systems typically require complex manufacturing. Here, we describe a multiplexed immunochromatographic assay that incorporates a conventional nitrocellulose membrane, which is used together with microspot printing, to construct adjacent microfluidic "tracks" for multiplexed detection. The 1 mm distance between tracks allows for the detection of up to four different analytes. The following reagents are applied in separate zones: (a) gold nanoparticle conjugates with antibodies against each analyte, (b) other antibodies against each analyte, and (c) antispecies antibodies. The immersion of the test strip in the sample initiates the lateral flow, during which reagents of different specificities move along their tracks without track erosion or reagent mixing. An essential advantage of the proposed assay is its extreme rapidity (1-1.5 min compared with 10 min for common test strips). This assay format was applied to the detection of cardiac and inflammatory markers (myoglobin, D-dimer, and C-reactive protein) in human blood, and was characterized by high reproducibility (8%-15% coefficient of variation) with stored working ranges of conventional tests. The universal character of the proposed approach will facilitate its use for various analytes., Competing Interests: The authors declare that they have no known competition for financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Object: To study the main causes of severe course and high mortality in patients with nervous form of listeriosis., Materials and Methods: The analysis of the course of Listeria meningoencephalitis (LM) in 36 patients aged from 9 to 85 years, who were treated in the Infectious clinical hospital No. 2 DZM (IKB No. 2 DZM). Along with standard examination methods, blood and cerebrospinal fluid (CSF) polymerase chain reaction tests were performed to identify Listeria monocytogenes. The sensitivity of the pathogen to antibiotics was determined by serial dilutions on the WalkAway 96 Plus device of Siemens, USA., Results: LM in 84% of cases developed in patients with disorders in the immune system, in particular, with infection with the human immunodeficiency virus - in 25% of cases. The clinical picture of the disease, changes in CSF were not typical for bacterial purulent meningitis of another etiology. It is noted that LM is characterized by early involvement of the substance and ventricles of the brain in the process., Conclusion: Severe course and high mortality are due to atypical picture of the disease, late diagnosis, low bioavailability of the pathogen for antibiotics (intracellular persistence of the pathogen) and frequent resistance to them. The mortality from the nervous form of listeriosis was 33.3%.

Multiarray on a test strip (MATS) was developed for the detection of eight important potato pathogens. The proposed assay combines the rapidity of immunochromatography with the high throughput of array techniques. The test zone of the immunochromatographic strip comprises ordered rows of spots containing antibodies specific for different potato pathogens. The assay benefits from the simplicity of immunochromatography; colored immune complexes form at the corresponding spots within the test zone. The presence and intensity of the coloration are used for identification of the target pathogens. The MATS was applied to the simultaneous detection of eight priority potato pathogens, characterized by the following limits of detection: 1 ng/mL for potato virus X and the ordinary type of potato virus Y, 10 ng/mL for potato virus M, 20 ng/mL for potato leaf roll virus, 40 ng/mL for necrotic-type potato virus Y, 100 ng/mL for potato virus S, 300 ng/mL for potato virus A, and 10(4) cells/mL for Clavibacter michiganensis subsp. sepedonicus. Analysis time was 15 min. The observed sensitivity of the MATS was comparable to the traditional enzyme-linked immunosorbent assay. The developed technique was tested on potato leaf extracts, and its efficiency for on-site control of the pathogens was confirmed in 100 % by commercial LFIA test strips. Graphical abstract Location of binding zones in the developed multiarray on a test strip (MATS) for simultaneous detection of eight pathogens.

The development of sector of "fast-testing" i.e. test-systems permitting carrying out analysis during home visit of doctor or at primary examination of patient without any additional devices and reagents is predominant tendency in international practice. The immunochromatography is an effective technical solution in out-laboratory diagnostic, which nowadays is actively applied in controlling hundreds of diagnostically significant markers of infectious diseases, metabolic and functional disorders. However, common immunochromatography is focused on qualitative visual evaluation of results of study i.e. conclusion on presence or absence of coloration of particular zones of test-band. Therefore, the technical solutions retaining such merits of immunochromatography as expressness and technical simplicity and at the same time providing objectivity of diagnostic and increasing its informativeness are extremely in demand. The review considers main methodical solutions and tendencies of their practical implementation targeted at device documentation, processing and interpretation of results of immunochromatography analysis. The optical systems of registration in visible area of spectrum dominating in assortment of modern detectors are presented. The new solutions oriented on working with fluorescent, magnetic and electroconductive markers are presented too. The perspectives of further development of this direction are characterized including application as detectors of domestic communication devices and formation of cloud data bases for storage and processing of information concerning results of examinations.

A novel rapid (20 min) fluorescent lateral flow test for chloramphenicol (CAP) detection in milk was developed. The chosen format is a binding-inhibition assay. Water-soluble quantum dots with an emission peak at 625 nm were applied as a label. Milk samples were diluted by 20 % with phosphate buffer to eliminate the matrix effect. The result of the assay could be seen by eye under UV light excitation or registered by a portable power-dependent photometer. The limit of CAP detection by the second approach is 0.2 ng/mL, and the limit of quantitation is 0.3 ng/mL.

A microarray analytical system for performing tests of latex agglutination reaction in microformat with digital image registration was developed. The system allows the application of latex microdrops to the surface of the carrier in the form of a regular microarray and mixing of the latex droplets with the individual samples in each droplet of the microarray. The reaction is performed in a total mixture volume of about 1 microl for each of 30 samples simultaneously with video registration and interpretation of the results using a scanning device and specially developed software. The results of the semi-quantitative determination of C-Reactive Protein, Rheumatoid Factor and Anti-Streptolysin O concentrations by traditional macro- and proposed micro-arrayagglutination method were compared with the immunoturbidimetric measurements used as reference method. It was concluded that the suggested method for performing latex agglutination reactions on the basis of a microarray approach with digital image evaluation of results can provide a high throughput and reliable results and also offers significant advantages to the traditional latex agglutination tests with visual interpretation. Comprehensive documentation and objectification of readouts show a siginificant improvement to the present methodology.

From July 25 to October 1, 1999, 826 patients were admitted to Volgograd Region, Russia, hospitals with acute aseptic meningoencephalitis, meningitis, or fever consistent with arboviral infection. Of 84 cases of meningoencephalitis, 40 were fatal. Fourteen brain specimens were positive in reverse transcriptase-polymerase chain reaction assays, confirming the presence of West Nile/Kunjin virus.

Intramolecular compact structures formed by high molecular weight circular superhelical DNA molecules due to interaction with synthetic oligopeptide trivaline (1) were studied by atomic force and electron microscopy. Three DNA preparations were used: plasmids pTbol, pRX10 and cosmid 27,877, with sizes 6,120 bp, 10,500 bp and 44,890 bp respectively. Plasmid pTbo1 and pRX10 preparations along with monomers contained significant amount of dimers and trimers. Main structures in all preparations observed were compact particles, which coincide in their appearance and compaction coefficient (3,5-3,7) with triple rings described earlier. The size and structure characteristics of triple rings and other compact particles on atomic force images in general coincide with those obtained by EM (2). AFM (3) images allow to get additional information about the ultrastructural organization and arrangement of DNA fibers within the compact structures. Along with triple rings in pTbol and pRX10-TVP complexes significant amount of compact structures were observed having the shape of two or three compact rings attached to each other by a region of compact fibre. Basing on the data of contour length measurements and the shape of the particles it was concluded that these structures were formed due to compaction of dimeric and trimeric circular DNA molecules. Structures consisting of several attached to each other triple rings were not found for pTbol, pRX10 monomers or cosmid preparations--TVP complexes where only single triple rings were observed. The conclusion is made that initiation of compact fibre formation within the circular molecules depends on the primary structure and for dimeric or trimeric circular molecules two or three compaction initiation points are present, located in each monomer unit within one circular DNA molecule. The nucleotide sequence dependent compaction mechanism providing independent compaction of portions of one circular molecule can be of interest for understanding of DNA compaction processes in vivo.

Electron microscopical data is presented on the varying morphology of the complexes formed between DNA and a number of synthetic beta-oligopeptides. In general, these peptides produce DNA compaction or condensation, resulting in two main types of complexes: toroids and rods. By controlling the ratio of peptide to DNA, interpretations of the possible packing of DNA within the various compact particles are advanced. Some understanding of the mechanism of peptide-induced DNA compaction in vitro may be of significance in relation to DNA condensation and the discrimination of gene domains in vivo.

The levels of serum IgG, IgA and IgM were examined in 191 adults including 103 patients with various forms of meningococcal infection, 32 meningitis convalescents and 56 carriers, in order to elucidate the causes of different susceptibility to the meningococcal infection. The IgD level was determined in 54 meningitis patients as well as in convalescents and carriers. The amount of immunoglobulins was determined by radial immunodiffusion. The level of IgG at the beginning of the disease in patients with the generalized forms of meningococcal infection (meningitis, meningitis combined with meningococcaemia, meningococcaemia) was found to be considerably lower than in healthy subjects. The levels of all immunoglobulins, particularly of IgA and IgM, increased in the course of the disease. The levels of IgG, IgA and IgM in meningitis convalescents a year after recovery were found to be the same as in the controls. The levels of IgG, IgA and IgM in patients with meningococcal nasopharyngitis were significantly lower than in healthy subjects. The carriers showed a decreasd level of IgA and a considerably increased level of IgG while the levels of IgM and IgD did not differ from the control.

The organization of chromatin in macronuclei of Bursaria truncatella cells that completed their growth and differentiation was electron microscopically studied. The data obtained showed that (1) inactive macronuclear chromatin was organized in compact chromatin clumps 120 to 180 nm in diameter linked by one or several chromatin fibres, and (2) in low salt buffer the chromatin clumps gradually unraveled, radial loops of supranucleosomal or, more often, nucleosomal structure appearing around chromatin clumps. Upon prolonged incubation in low salt buffer chromatin clumps were completely transformed into nucleosomal fibres. The data obtained evidenced in favour of a loop-packed structure of chromatin clumps.

The morphology of complexes between relaxed circular plasmid pBR322 DNA and tripeptide L-Val-L-Val-L-Val-NH-NH-Dns (TVP) at different peptide/DNA ratios was studied by electron microscopy. The results show that interaction of TVP with circular DNA leads to the formation of perfect torus-shaped particles. The torus parameter measurements offer the possibility to conclude that DNA condensation observed is of intermolecular nature. On the basis of the analysis of the structures corresponding to the early stages of DNA compaction the model for intermolecular condensation of circular DNA into torus-shaped particles is proposed.

Complexes of circular superhelical pBR322 DNA with a synthetic tripeptide capable of beta-structure formation (dansylhydrazide trivaline) were studied at different peptide/DNA ratios by electron microscopy. It was shown on rotary-shadowed preparations that peptide binding induces intramolecular DNA condensation and compact ring-shaped particles are formed from fibres 120 A thick. The analysis of the morphology of the ring structures observed at various peptide/DNA ratios as well as contour length measurements enabled us to draw conclusions about the organization of the double-stranded DNA filaments in these structures. It was established that the fibres forming compact rings contain three double-stranded DNA segments closely associated due to DNA-peptide and peptide-peptide interactions. The mechanisms leading to the formation of the triple rings may be important in DNA condensation in vivo.

Chromatin from calf thymus isolated under hypotonic conditions in the presence of various agents was investigated by methods of electron microscopy prior to and after EDTA treatment. It is shown that the presence of chelating agents and, especially, the application of considerable mechanical forces in the course of isolation may cause damage to the nucleosomal structure of the chromatin. Moreover, sufficiently great mechanical forces are liable to destroy the structure of the chromatin nucleosomal fibres even when they are packed in structures of a higher order of organization of the chromatin.

Structural changes of chromatin induced by interaction of netropsin (Nt) with DNA has been examined by analysis of CD and electromicroscopic measurements. The results demonstrate the existence of a transition from the condensed globular state of chromatin into nucleosomal fibres generated by extremely low Nt concentration up to 1 mole Nt per 200 nucleotides. A second transition occurs at high Nt ratio per DNA phosphate (v' = 0.3). involving disorganization of nucleosomal particles. The interference of the Nt binding with chromatin proteins maintaining the sub- and superstructure will be discussed.

Binding to DNA of two synthetic peptides, Val-Thr-Thr-Val-Val-NH-NH-Dns and Thr-Val-Thr-Lys-Val-Gly-Thr-Lsy-Val-Gly-Thr-Val-Val-NH-NH-Dns (where Dns is a residue of 5-dimethylaminonaphthalene-1-sulfonic acid), has been studied by circular dichroism, electron microscopy and fluorescence methods. It has been found that these two peptides can self-associate in aqueous solution as follows from the fact that concentration-dependent changes are observed in the UV absorbance and fluorescence spectra. The two peptides can bind to DNA both in self-associated and monomeric forms. The pentapeptide in the beta-associated form binds more strongly to poly(dG).poly(dC) than to poly[d(A-C)].poly[d(G-T)] and poly(dA).poly(dT) whereas the tridecapeptide exhibits an opposite order of preferences binding more strongly to poly[d(A-C)].poly[d(G-T)] and poly(dA).poly(dT) than to poly(dG).poly(dC). Binding is a cooperative process which is accompanied by the DNA compaction at peptide/DNA base pair ratios greater than 1. At the initial stage of the compaction process, the coalescence of DNA segments covered by bound peptide molecules leads to the formation of DNA loops stabilized by the interaction between peptide molecules bound to different DNA segments. Further increase in the peptide/DNA ratio leads to the formation of rod-like structures each consisting of two or more double-stranded DNA segments. The final stage of the compaction process involves folding of fibrillar macromolecular complexes into a globular structure containing only one DNA molecule.

Electron microscopy shows that EDTA treatment or partial removal of histone HI converts 200-250 A chromatin fibres characteristic for native chromatin, isolated in low ionic strength conditions into fibres consisting of nucleosomes connected by segments of DNA. This structural transition is accompanied by an increase in the amplitude of positive band of CD spectra at 280 nm. Comparison of electron microscopic, thermal denaturation and electrophoretic data suggests that multiphasic character of melting curves, observed for chromatin, lacking histone HI is due to the removal of histone HI and destabilisation of the DNA segments, connecting nucleosomes. It is also shown that bivalent cations play an important part both in the stabilisation of 200 A globules and of nucleosomes.