Paramagnetic separation
Dotaz
Zobrazit nápovědu
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
Sarcosine has been identified as a potential prostate cancer marker. To provide determination of this compound, a number of methods are developing. In this study, we optimized a method for its separation by hydrophilic interaction LC with electrochemical detection (ED). Due to the fact that mobile phases commonly used for this type of separation altered the LODs measured by electrochemical detectors, we applied postcolumn dosing of buffer suitable for ED. The optimized conditions were mobile phase A acetonitrile, mobile phase B water in the ratio A/B 70:30, with postcolumn addition of mobile phase C (200 mM phosphate buffer pH 9). The optimal mixing ratio was A + B/C 1:1 with a flow rate of 0.80 mL/min (0.40 + 0.40 mL/min) and detection potential of 1000 mV. Due to the optimization of the parameters for effective separation, which had to meet the optimal parameters of ED, we reached a good resolution for separation also with a good LOD (100 nM). In addition, we successfully carried out sarcosine analysis bound on our modified paramagnetic microparticles with the ability to preconcentrate sarcosine isolated from artificial urine.
- 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
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
The emergence of drug-resistant bacteria and new or changing infectious pathogens is an important public health problem as well as a serious socioeconomic concern. Immunomagnetic separation-based methods create new possibilities for rapidly recognizing many of these pathogens. Nanomaterial-based techniques including fluorescent labeling by quantum dots as well as immunoextraction by magnetic particles are excellent tools for such purposes. Moreover, the combination with capillary electrophoresis in miniaturized microchip arrangement brings numerous benefits such as fast and rapid analysis, low sample consumption, very sensitive electrochemical and fluorescent detection, portable miniaturized instrumentation, and rapid and inexpensive device fabrication. Here the use of superparamagnetic particle-based fully automated instrumentation to isolate pathogen Staphylococcus aureus and its Zn(II)-containing proteins (Zn-proteins) is reported using a robotic pipetting system speeding up the sample preparation and enabling to analyze 48 real samples within 6 h. Cell lysis and Zn-protein extractions were obtained from a minimum of 100 cells with the sufficient yield for SDS-PAGE (several tens ng of proteins).
- MeSH
- bakteriální proteiny izolace a purifikace MeSH
- elektroforéza kapilární metody MeSH
- elektroforéza mikročipová metody MeSH
- imunomagnetická separace metody MeSH
- kvantové tečky * MeSH
- Staphylococcus aureus chemie izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Currently, metallothioneins (MTs) are extensively investigated as the molecular biomarkers and the significant positive association of the MT amount was observed in tumorous versus healthy tissue of various types of malignant tumors, including head and neck cancer. Thus, we proposed a biosensor with fluorescence detection, comprising paramagnetic nanoparticles (nanomaghemite core with gold nanoparticles containing shell) for the magnetic separation of MT, based on affinity of its sulfhydryl groups toward gold. Biosensor was crafted from PDMS combined with technology of 3D printing and contained reservoir with volume of 50 μL linked to input (sample/detection components and washing/immunobuffer) and output (waste). For the immunolabeling of immobilized MT anti-MT antibodies conjugated to CdTe quantum dots through synthetic heptapeptide were employed. After optimization of fundamental conditions of the immunolabeling (120 min, 20°C, and 1250 rpm) we performed it on a surface of paramagnetic nanoparticles in the biosensor reservoir, with evaluation of fluorescence of quantum dots (λexc 400 nm, and λem 555 nm). The developed biosensor was applied for quantification of MT in cell lines derived from spinocellular carcinoma (cell line 122P-N) and fibroblasts (122P-F) and levels of the biomarker were found to be about 90 nM in tumor cells and 37 nM in fibroblasts. The proposed system is able to work with low volumes (< 100 μL), with low acquisition costs and high portability.
- MeSH
- 3D tisk * MeSH
- biosenzitivní techniky MeSH
- dimethylpolysiloxany chemie MeSH
- fluorescence MeSH
- kovové nanočástice MeSH
- kvantové tečky MeSH
- lidé MeSH
- magnetismus MeSH
- metalothionein analýza MeSH
- nádorové buněčné linie MeSH
- nádory patologie MeSH
- sloučeniny kadmia chemie MeSH
- telur chemie MeSH
- zlato chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- 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
Methanolic extract from leaves of Karwinskia humboldtiana (Schult.) Zucc. was subjected to fractionation with a pair of immiscible solvents and subsequently tested for antioxidant activity. Using DPPH the order of scavenging activities of the extracts in comparison with standards of L-ascorbic acid and quercetin was as follows: ethyl acetate fraction > L-ascorbic acid > quercetin > butan-1-ol > light petrol > chloroform > water. The capability of scavenging OH radicals determined by EPR was as follows: ethyl acetate, chloroform > light petrol > butan-1-ol > water fractions. On the basis of availability and antioxidant activity of these fractions, isolation of constituents from butanol fractions was achieved. Separation of this fraction led to the isolation of (+)-epicatechin and flavonol derivatives – quercetin, quercetin 3-O-glucoside (isoquercitrin), quercetin-3-O-galactoside (hyperosid), quercetin-3-O-arabinoside, quercetin 3-O-rutinoside (rutin), kaempferol 3-O-arabinoside and kaempferol 3-O-rutinoside. All of them might be responsible for the observed antioxidant effects.
Závěrečná zpráva o řešení grantu Interní grantové agentury MZ ČR
1 svazek : ilustrace ; 30 cm
Předmětem řešení projektu je vyhodnocení efektivity metodiky separace mrtvých (a případně i ubikvitinovaných) spermií u mužů z neplodných párů pomocí MACS na základě porovnání vyšetřovaných biomarkerů spermií v ejakulátu a následně ve spermiích separovaných technikou MACS. Porovnávány budou tyto markery: ubikvitinace (metodou SUTI); fragmentace DNA (metodou SCSA); úroveň OS (metodou EPR); viabilita (na základě funkčnosti mitochondrií). Budou-li spermie po selekci vykazovat signifikantně lepší parametrynež celkový ejakulát, bude dalším krokem zavedení metody separace spermií pomocí MACS do rutinní diagnostiky a léčby andrologického faktoru neplodnosti.; The aim of project is to evaluate effectivity of sperm separation technique based on negative selection of dead spermatozoa (and eventually subsequent selection of ubiquitinated spermatozoa using MACS technique. This technique will be applied on sperm samples of males from infertile couples. In suspensions of non-separated and separated spermatozoa, chosen biomarkers will be compared: ubiquitination (SUTI assay), DNA fragmentation (SCSA assay), free radicals production (EPR method); sperm viability (based on mitochondrial membrane potential). In the case of significant improvement of chosen biomarkers in sperm samples after separation, this technique could be implemented into routine protocol for sperm preparation for the purpose of assisted reproduction.
- MeSH
- biologické markery MeSH
- elektronová paramagnetická rezonance MeSH
- fragmentace DNA MeSH
- mužská infertilita diagnóza patofyziologie terapie MeSH
- oxidační stres MeSH
- průtoková cytometrie MeSH
- spermie MeSH
- ubikvitinace MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- andrologie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu IGA MZ ČR
Superparamagnetic particles have been attractive for molecular diagnostics and analytical chemistry applications due to their unique magnetic properties and their ability to interact with various biomolecules of interest. This paper presents a critical overview of magnetic nano- and microparticles used as a solid phase for extraction and purification of DNAs. The mechanisms of DNA binding to the surface of functionalised magnetic particles are described. The most widely used materials including silica supports, organic polymers and other materials, mostly containing magnetite or paramagnetic metallic elements are reviewed. The main application areas of magnetic particles for DNA separation are briefly described.
