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Leonardo da Vinci

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Analysis of H7N7 Equine Influenza Virus by Spectrometric and Electrochemical Methods

Influenza viruses belong to the family Orthomyxoviridae with a segmented genome made of eight single-stranded negative RNA segments, most of which encode only one viral protein (e.g. hemagglutinin, neuraminidase, matrix protein, and/or nucleoprotein). Fifteen HA subtypes (H1–H15) and nine NA subtypes (N1–N9) have been identified. H1N1, H1N2 and H3N2 subtypes are only currently circulating in the human population [1,2]. Influenza A viruses are characterized by subtype by the two major surface glycoproteins, hemagglutinin and neuraminidase. Influenza A viruses infect poultry, aquatic birds, pigs, horses and sea mammals. The influenza virus is highly contagious and infects as much as 15% of the human population every year and it has a significant impact on today's society with over 500,000 deaths worldwide and 5 million annual hospitalizations attributed to influenza every year. Due to the fact that there is a threat of future pandemics, the development of methods for early influenza detection is very important.

The influenza virus, vaccine and antiviral drugs that target the virus can be characterized by different genetic and proteomic approaches. One of the most important and advantageous is mass spectrometry (MS), especially high resolution mass spectrometry or mass mapping. By this manner primary structure of the surface protein antigens was investigated and described as it was described by Downard et al., whose mass spectrometric immunoassay was developed and applied to assess the structure and antigenicity of the virus in terms of the hemagglutinin antigen. The characterization and quantitation of antiviral drugs against the virus, and their metabolites, were monitored in blood, serum, and urine [3]. A matrix-assisted laser desorption ionization (MALDI) mass spectrometry was used for theidentification of inhibitor binding to active site residues of influenza neuraminidase [4]. It enables us to screen new inhibitor drug candidates and also to identify strains resistant to antiviral inhibitors. MALDI and tandem mass spectrometry (MS/MS) were used to the antigenic characterization of three H3N2 type A influenza strains [5]. Parainfluenza viruses (PIVs) are one of the most common causes of respiratory tract infections in children and can be life-threatening when the airway becomes obstructed. Matrix-assisted laser desorption ionization fourier transform-ion cyclotron resonance (maldi ft-icr) Mass Spectrometry was shown as a new proteotyping approach to be able to distinguish common human serotypes of the PIV from the perspective of all surface and internal viral proteins [6]. The principle of MALDI is based on the dispersion of the sample in a large excess of matrix. Matrix contains chromophore and heavily absorbs UV laser light and in the presence in a large molar excess it absorbs essentially all of the laser radiation. Matrix material isolates sample molecules in a chemical environment, which enhances the probability of ionization without fragmentation. MALDI as a soft ionization technique used in mass spectrometry is the one of the most frequently applied for both, characterization the structure and antigenicity of the influenza virus in terms of its component antigens. In developing this MALDI-based approach, it is possible to both preserve and ionize solution-specific antigen–antibody complexes on MALDI targets without the immobilization, pretreatment or affinity capture of either component [7-9]. Another technique enables large, highly polar biopolymers to be introduced directly into a mass spectrometer is electrospray ionization (ESI) [9].

In some cases of detection it is necessary to connect mass spectrometry with various separation methods. Chromatographic methods, especially capillary electrophoresis (CE), are applied. Capillary electrophoresis mass spectrometry was used for the characterization of glycans from fish serum [10]. In this work highly sialylated negatively charged O-acetylated N-glycans are CE-separated and MS-detected in negative electrospray ionization (ESI) mode without the need for derivatization. The method of capillary isoelectric focusing (CIEF) with UV detection for influenza viruses was used to test the purification of the virus from the biological samples of equine and swine influenza viruses present in infected allantoic fluid of specific pathogen free embryonated chicken eggs [11]. CIEF was found to be a suitable method for the rapid testing of the efficiency of the virus purification. Influenza detection is often realized by fluorescence measurement, especially fluorescence immunoassay. Indirect fluorescence immunoassay provides a novel method for the detection of avian influenza virus (AIV) [12]. The method was based on label-free quantum dot probe and lateral flow test strip. By the sandwich immunoreaction performed on lateral flow test strip, the gold nanoparticle labels were captured in the test zone and further dissolved to release a large number of gold ions as a signal transduction bridge that was detected by the QDs-based fluorescence quenching method. The limit of detection was estimated to be 0.09 ng mL-1 AIV, which was 100-fold greater than enzyme-linked immunosorbent assay (ELISA). Different kinds of dynamic fluorescence microscopy methods, such as fluorescence photoactivation localization microscopy (FPALM), Förster’s (or fluorescence) resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP) were used for lipid domain association of in?uenza virus proteins detection [13]. Fluorescence imaging was applied for detection of whole viruses and viral proteins with a new label-free platform based on spectral reflectance imaging. The interferometric reflectance imaging sensor (IRIS) was shown to be capable of sensitive protein and DNA detection in a real time and high-throughput format [14]. For the fluorescence measurement it is necessary to prepare molecules exhibiting fluorescence properties. For this purpose organic dyes and nanoparticles including quantum dots can be used. Quantum dots (QDs) are nanometer-scale semiconductor crystals, typically of size range 2–6 nm [15]. They compose of groups II–VI or III–V elements and they have unique optical and electronic properties in comparison with organic dyes and fluorescent proteins. They belong to new class of fluorescent probes for biomolecular and cellular imaging, especially in vivo imaging [16]. CdSe or CdTe QDs can be also applied as chemiluminescent probes for the detection of trace metal ions. Metal ions including Sb, Se, Ag and Cu were investigated due to the inhibition of the chemiluminescence when QDs are oxidized by H2O2. For better selectivity and sensitivity of the chemiluminescent assay, liquid–liquid–liquid microextraction was implemented [17,18]. Biomolecules labeled with nanoparticles or quantum dots including proteins, nucleic acids, lipids and biologically active small molecules. Thus formed conjugates can be observed by different analytical procedures. One of the advantageous techniques is metal detection or determination by application of various spectroscopic techniques. Metal included in quantum dot can be advantageously determined by laser-induced breakdown spectroscopy (LIBS). This sensitive optical technique is capable of fast multi-elemental analysis of solid, gaseous and liquid samples [19], and is based on spectroscopic analysis of radiation, which is emitted by a micro-plasma induced on the analyte surface by a laser pulse. Different kinds of samples have been analyzed by this method as minerals, bricks, soil pellets, biominerals, bones, dried or fried plant leaves or algal biomass [19-24]. Besides to spectrometric methods electrochemical methods can be used advantageously to detect influenza viruses or proteins. Differential pulse voltammetry at hanging mercury drop electrode (HMDE) and/or glassy carbon electrode (GCE) was applied for detection of cadmium sul?de quantum dots – influenza viral protein complexes isolated using paramagnetic microparticles [25]. Hybridization design probes consisting of paramagnetic particles and quantum dots with targeted DNA and their application for detection of avian influenza virus (H5N1) were investigated by Krejcova et al. [26]. Differential pulse voltammetry was also used for detection of cadmium(II) ions and square wave voltammetry for detection of cytosine-adenine peak in ODN-SH-Cd complex. Multi target bar code assay based on the electrochemical analysis of isolated target molecules labeled with quantum dots was introduced also in our previous work [27]. The aim of this study is the studying of quantum dots containing cadmium detection. For QDs detection, fluorescence based and LIBS were used as well as above mentioned electrochemical ones.

Práce je spojená s projektem NanoBioMetalNet CZ.1.07/2.4.00/31.0023

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