Annual epidemics of influenza cause death of hundreds of thousands people and they also have a significant economic impact. Hence, a need for fast and cheap influenza diagnostic method is arising. The conventional methods for an isolation of the viruses are time-consuming and require expensive instrumentation as well as trained personnel. In this study, we modified the surface of nanomaghemite (γ-Fe2 O3 ) paramagnetic core with tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane and the resulting particles were utilized for the isolation of H7N7 influenza virions. Consequently, we designed γ-Fe2 O3 paramagnetic core modified with calcium tripolyphosphate which was employed for the isolation of viral nucleic acid after virion's lysis. Both of these procedures can be performed rapidly in less than 10 min and, in combination with the RT-PCR, the whole influenza detection can be shortened to few hours. Moreover, the whole protocol could be easily automated and/or miniaturized, and thus can serve as a basis for use in a lab-on-a-chip device. We assume that magnetic isolation is an exceptional procedure which can significantly accelerate the diagnostic possibilities of a broad spectrum of diseases.

• MeSH
• chromatografie iontoměničová MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• kuřecí embryo MeSH
• polymerázová řetězová reakce metody   MeSH
• reverzní transkripce MeSH
• virion izolace a purifikace   MeSH
• virus chřipky A, podtyp H7N7 izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The aim of this study was to utilize paramagnetic microparticles in isolation of mRNA using electrochemical detection. In this way mRNA from maize seedlings treated with a Cd(II) salt was isolated. The interaction of paramagnetic particles with synthetic oligo- and polynucleotides of various chain lengths was investigated. The average detection limits were 0.020?0.360 nM and the quantification limits 0.080?1.200 nM.

• MeSH
• elektrochemie metody   MeSH
• financování organizované MeSH
• messenger RNA izolace a purifikace   MeSH
• nadzemní části rostlin genetika   ultrastruktura   MeSH

This study presents synthesis of paramagnetic particles formed by nanomaghemite (γ-Fe2O3) core, whose surface was modified with LaCl3 and entire composite is able to specifically bind the bacteriophage λ. In this study we described the structure of bacteriophage λ, its biochemical properties and role in the therapy, biotechnology and nanomedicine. In the next part we summarized accessible information about paramagnetic particles, their attributes and specificity of binding the bacteriophage λ. Paramagnetic particles show the ability to immobilize bacteriophage λ for subsequent analysis using ion-exchange chromatography with Vis detection. The presence of bacteriophage λ on the surface of paramagnetic beads was demonstrated by sodium dodecyl sulphate gel electrophoresis (SDS-PAGE). It can be stated that paramagnetic beads show significant binding ability towards bacteriophage λ. This phenomenon can be employed for magnetic separation of bacteriophages prior their possible modifications and subsequent application as a cytostatic nanotransporters or antimicrobial agents usable in hard-to-heal bacterial infections.

• Klíčová slova
• superparamagnetic iron oxide nanoparticles,
• MeSH
• bakteriofág lambda * chemie   MeSH
• bakteriofágy MeSH
• chromatografie iontoměničová MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• magnetické nanočástice * MeSH
• nanomedicína MeSH
• nanotechnologie MeSH
• oxidy MeSH
• železnaté sloučeniny MeSH
• železo MeSH
• Publikační typ
• práce podpořená grantem MeSH

Easy, efficient and low demanding separation of mRNA from biological material is needed to study gene expression and to use in chip technologies. It is common knowledge that each mRNA molecule contains sequence of 25 adenines. This feature can be used for binding mRNA on the surface of the particles coated by thymine chains. The present work reports on suggesting and optimizing of mRNA separation and detection from biological material via paramagnetic microparticles coupled with electrochemical detection. Primarily we optimized cyclic and square wave voltammetric conditions to detect poly(A), which was used as standard to mimic behaviour of mRNA. Under the optimized square wave voltammetric conditions (frequency 280 Hz, accumulation time 200 s, supporting electrolyte and its temperature: acetate buffer 4.6 and 35 degrees C) we estimated detection limit down to 1 ng of poly(A) per ml. To enhance effectiveness and repeatability of isolation of nucleic acid automated approach for rinsing and hybridizing was proposed. We optimized the whole procedure and experimental conditions. Using automated way of isolation and under optimized conditions the yield of poly(A) (isolated concentration of poly(A)/given concentration of poly(A)*100) was approximately 75%. The suggested and optimized method for poly(A) isolation and detection was utilized for the analysis of brain tissues of patients with traumatic brain injury. The total amount of isolated mRNA varied from 40 to 760 g of mRNA per g of brain tissue. The isolation of mRNA from six samples per run was not longer than 2.5h. Moreover, we applied the optimized procedure on fully automated pipetting instrument to isolate mRNA. The instrument was successfully tested on the analysis of extracts from roots of maize plants treated with cadmium(II) ions.

• MeSH
• adenin MeSH
• automatizace MeSH
• elektrochemické techniky metody   MeSH
• hybridizace nukleových kyselin MeSH
• kukuřice setá genetika   MeSH
• lidé MeSH
• magnetismus MeSH
• messenger RNA izolace a purifikace   MeSH
• mozek - chemie MeSH
• nukleové kyseliny izolace a purifikace   MeSH
• párování bází MeSH
• poranění mozku genetika   MeSH
• thymin MeSH
• velikost částic MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

• MeSH
• automatizace MeSH
• elektrochemie metody   MeSH
• imunomagnetická separace MeSH
• kovové nanočástice * MeSH
• kultivační techniky MeSH
• lidé MeSH
• magnetické jevy * MeSH
• nanočástice * MeSH
• sloučeniny zinku * analýza   MeSH
• Staphylococcus aureus * izolace a purifikace   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Guanosine derivatives are important for diagnosis of oxidative DNA damage including 8-hydroxy-2'-deoxyguanosine (8-OHdG) as one of the most abundant products of DNA oxidation. This compound is commonly determined in urine, which makes 8-OHdG a good non-invasive marker of oxidation stress. In this study, we optimized and tested the isolation of 8-OHdG from biological matrix by using paramagnetic particles with an antibody-modified surface. 8-OHdG was determined using 1-naphthol generated by alkaline phosphatase conjugated with the secondary antibody. 1-Naphthol was determined by stopped flow injection analysis (SFIA) with electrochemical detector using a glassy carbon working electrode and by stationary electrochemical detection using linear sweep voltammetry. A special modular electrochemical SFIA system which needs only 10  μL of sample including working buffer for one analysis was completely designed and successfully verified. The recoveries in different matrices and analyte concentration were estimated. Detection limit (3 S/N) was estimated as 5  pg/mL of 8-OHdG. This method promises to be very easily modified to microfluidic systems as "lab on valve". The optimized method had sufficient selectivity and thus could be used for determination of 8-OHDG in human urine and therefore for estimation of oxidative DNA damage as a result of oxidation stress in prostate cancer patients.

• MeSH
• alkalická fosfatasa MeSH
• deoxyguanosin analogy a deriváty   moč   MeSH
• elektrochemické techniky metody   MeSH
• ELISA metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• magnety MeSH
• mikrofluidní analytické techniky přístrojové vybavení   metody   MeSH
• mikrosféry MeSH
• nádory prostaty moč   MeSH
• naftoly MeSH
• oxidační stres MeSH
• protilátky MeSH
• průtoková injekční analýza MeSH
• robotika přístrojové vybavení   metody   MeSH
• senioři MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Determination of serum mRNA gained a lot of attention in recent years, particularly from the perspective of disease markers. Streptavidin-modified paramagnetic particles (SMPs) seem an interesting technique, mainly due to possible automated isolation and high efficiency. The aim of this study was to optimize serum isolation protocol to reduce the consumption of chemicals and sample volume. The following factors were optimized: amounts of (i) paramagnetic particles, (ii) oligo(dT)20 probe, (iii) serum, and (iv) the binding sequence (SMPs, oligo(dT)20 , serum vs. oligo(dT)20 , serum and SMPs). RNA content was measured, and the expression of metallothionein-2A as possible prostate cancer marker was analyzed to demonstrate measurable RNA content with ability for RT-PCR detection. Isolation is possible on serum volume range (10-200 μL) without altering of efficiency or purity. Amount of SMPs can be reduced up to 5 μL, with optimal results within 10-30 μL SMPs. Volume of oligo(dT)20 does not affect efficiency, when used within 0.1-0.4 μL. This optimized protocol was also modified to fit needs of automated one-step single-tube analysis with identical efficiency compared to conventional setup. One-step analysis protocol is considered a promising simplification, making RNA isolation suitable for automatable process.

• MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• magnety * MeSH
• messenger RNA krev   izolace a purifikace   MeSH
• mikrosféry MeSH
• mladý dospělý MeSH
• molekulární sondy - techniky * MeSH
• nádorové biomarkery krev   izolace a purifikace   MeSH
• nádorové buněčné linie MeSH
• nádory prostaty diagnóza   MeSH
• senioři MeSH
• studie případů a kontrol MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Zinc(II) as the only transition metal lacking redox activity is an essential part of approximately 10% proteins as a cofactor of these proteins. Considering the fact that there are numerous zinc(II) containing proteins, proteomics and metallomics studies aimed on them require accurate methods for preparation of real biological samples prior to their subsequent analysis using 2DE and MS. For this purpose, we suggested a new method based on chicken anti-zinc antibodies and magnetizable particles. Antibodies were covalently immobilized to the surface of paramagnetic beads activated with tosyl group. Binding of the antibody to the beads was confirmed by secondary anti-chicken antibody conjugated with horseradish peroxidase. The immunoextraction conditions, such as concentration of the beads (6-18 μg/mL of the sample), time of immunoextraction (6-34 min), pH and composition of the elution buffer, and time of extraction (48-300 s) were optimized. Subsequently, zinc proteins were extracted from human plasma and total concentration of zinc was monitored by electrochemical detection in the extracts. Under optimal conditions it was possible to monitor the proteins and zinc removal from the sample by chip CE, SDS-PAGE, and indirectly using electrochemistry.

• MeSH
• dospělí MeSH
• imunoglobuliny chemie   metabolismus   MeSH
• imunomagnetická separace metody   MeSH
• koncentrace vodíkových iontů MeSH
• kur domácí MeSH
• lidé MeSH
• metaloproteiny krev   chemie   izolace a purifikace   metabolismus   MeSH
• molekulová hmotnost MeSH
• protilátky chemie   metabolismus   MeSH
• sérový albumin hovězí chemie   metabolismus   MeSH
• vaječný žloutek chemie   imunologie   MeSH
• western blotting MeSH
• zinek krev   chemie   MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Lactoferrin (LF) is approximately 80 kDa iron-binding protein, which is important part of saliva and other body fluids. Due to its ability to bind metal ions, it has many biologically important functions. In this study, a method for the isolation of LF from a biological sample using robotically prepared antibody-modified paramagnetic particles was developed using robotic pipetting station. The method consisted of the following optimised steps. Protein G was bound on the paramagnetic particles, on which goat antibody (10 μg) was linked. LF was subsequently added to microtitration plate, which had affinity to goat antibody and the interaction lasted for 30 min. We found that the highest signals were obtained using the combination of goat antibody 1:3000, murine antibody 1:5000 and conjugate 1:1500. Horseradish peroxidase reducing 3,3,5,5-tetramethylbenzidine (TMB) was linked to the merged complex. The resulted product of this reaction was subsequently analysed spectrometrically with detection limit (3 S/N) as 5 ng/mL. In addition, we also determined TMB by stopped flow injection analysis with electrochemical detection. The limit of detection (3 S/N) was estimated as 0.1 μg/mL. To compare spectrometric and electrochemical approach for detection of TMB, calibration range of bead-LF-antibodies complex was prepared and was determined using a least-squares correlation with coefficient R² higher than 0.95, indicating a very good agreement of the results obtained.

• MeSH
• design vybavení MeSH
• dospělí MeSH
• elektrochemické techniky přístrojové vybavení   MeSH
• imobilizační protilátky chemie   MeSH
• laktoferrin analýza   izolace a purifikace   MeSH
• lidé MeSH
• limita detekce MeSH
• magnety chemie   MeSH
• průtoková injekční analýza přístrojové vybavení   MeSH
• sliny chemie   MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

Metallothioneins (MTs) are a group of low-molecular weight (~6 -10kDa), cysteine-rich, evolutionarily conserved proteins, found in generally all life forms. The MTs are multi-functional and play crucial roles in the transport of essential trace elements as well as in the detoxification of harmful metallic elements 1. Four major MT isoforms, MT-1, MT-2, MT-3 and MT-4, have been identified in mammals 2. Specifically, MT-3 has been reported to be secreted mainly in neural cells. Recent reports established that this isoform play an important protective role in brain injury and metal-linked neurodegenerative diseases 3. In general, MT is known to modulate three basic processes: the release of hydroxyl radical or nitric oxide 4, apoptosis 5 and the binding and exchange of heavy metals such as zinc 6, cadmium 7 or copper 8. Previous studies have shown a positive correlation between the expression of MT with invasion, metastasis and poor prognosis in various cancers, including head and neck cancers 9, where increasing levels of MT concentration in tumor (vs. healthy tissues) was observed 10. Moreover; due to high affinity towards heavy metals, MT can be employed for modification of electrodes in development of electrochemical biosensors 11. In view of these facts, immobilization of MT on paramagnetic particles (PMPs) may provide many possibilites, such as simplification of biosensing of low levels of MT, through its pre-concentration. The present study demonstrates immobilization of MT on a surface of paramagnetic particles (gold modified). For confirmation of MT presence two detection techniques were employed: matrix-assisted laser desorption/ionization - time of flight mass spectrometry and differential pulse voltammetry with adsorptive transfer technique utilizing Brdicka solution as supporting electrolyte. For binding capacity evaluation MT isolated from rabbit liver was employed and ideal particles for its immobilization were chosen, simultaneously with optimization of immobilization conditions (pH and temperature).

• Klíčová slova
• Brdičkova reakce, MALDI-TOF/TOF, MAN53,
• MeSH
• chromatografie kapalinová MeSH
• králíci MeSH
• magnetismus * MeSH
• metalothionein * chemická syntéza   chemie   izolace a purifikace   MeSH
• nádorové biomarkery MeSH
• nanočástice * MeSH
• povidon MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice statistika a číselné údaje   MeSH
• teplota MeSH
• železité sloučeniny chemická syntéza   MeSH
• zlato MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH