The aim of the present study is to develop new magnetic polymer microspheres with functional groups available for easy protein and antibody binding. Monodisperse macroporous poly(2-hydroxyethyl methacrylate) (PHEMA-COOH) microspheres ~4 µm in diameter and containing ∼1 mmol COOH/g were synthesized by multistep swelling polymerization of 2-hydroxyethyl methacrylate (HEMA), ethylene dimethacrylate (EDMA), and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA), which was followed by MCMEMA hydrolysis. The microspheres were rendered magnetic by precipitation of iron oxide inside the pores, which made them easily separable in a magnetic field. Properties of the resulting magnetic poly(2-hydroxyethyl methacrylate) (mgt.PHEMA) particles with COOH functionality were examined by scanning and transmission electron microscopy (SEM and TEM), static volumetric adsorption of helium and nitrogen, mercury porosimetry, Fourier transform infrared (FTIR) and atomic absorption spectroscopy (AAS), and elemental analysis. Mgt.PHEMA microspheres were coupled with p46/Myo1C protein purified from blood serum of multiple sclerosis (MS) patients, which enabled easy isolation of monospecific anti-p46/Myo1C immunoglobulin G (IgG) antibodies from crude antibody preparations of mouse blood serum. High efficiency of this approach was confirmed by SDS/PAGE, Western blot, and dot blot analyses. The newly developed mgt.PHEMA microspheres conjugated with a potential disease biomarker, p46/Myo1C protein, are thus a promising tool for affinity purification of antibodies, which can improve diagnosis and treatment of MS patients.
- Klíčová slova
- affinity purification, anti-p46 kDa/Myo1C, magnetic microspheres, multiple sclerosis,
- MeSH
- imobilizované proteiny chemie imunologie MeSH
- imunoglobulin G imunologie izolace a purifikace MeSH
- lidé MeSH
- magnetismus metody MeSH
- magnety chemie MeSH
- mikrosféry MeSH
- myosin typu I chemie imunologie MeSH
- myši MeSH
- polyhydroxyethylmethakrylát chemie MeSH
- protein - isoformy chemie imunologie MeSH
- roztroušená skleróza diagnóza imunologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- imobilizované proteiny MeSH
- imunoglobulin G MeSH
- MYO1C protein, human MeSH Prohlížeč
- myosin typu I MeSH
- polyhydroxyethylmethakrylát MeSH
- protein - isoformy MeSH
A biosensor for the detection of hepatitis B antibodies in clinical saliva was developed. Compared to conventional analysis of blood serum, it offers the advantage of noninvasive collection of samples. Detection of biomarkers in saliva imposes two major challenges associated with the low analyte concentration and increased surface fouling. The detection of minute amounts of hepatitis B antibodies was performed by plasmonically amplified fluorescence sandwich immunoassay. To have access to specific detection, we prevented the nonspecific adsorption of biomolecules present in saliva by brushes of poly[(N-(2-hydroxypropyl) methacrylamide)-co-(carboxybetaine methacrylamide)] grafted from the gold sensor surface and post modified with hepatitis B surface antigen. Obtained results were validated against the response measured with ELISA at a certified laboratory using serum from the same patients.
- MeSH
- biologické markery analýza MeSH
- biosenzitivní techniky metody MeSH
- fluorescenční spektrometrie MeSH
- hepatitida B - antigeny povrchové chemie imunologie MeSH
- hepatitida B - protilátky analýza krev imunologie MeSH
- imobilizované proteiny chemie imunologie MeSH
- imunoanalýza MeSH
- lidé MeSH
- polymery chemie MeSH
- povrchová plasmonová rezonance MeSH
- sliny metabolismus MeSH
- zlato chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- biologické markery MeSH
- hepatitida B - antigeny povrchové MeSH
- hepatitida B - protilátky MeSH
- imobilizované proteiny MeSH
- polymery MeSH
- zlato MeSH
Molecular diagnostics may provide tailored and cost efficient treatment for infectious disease and cancer. Rolling circle amplification (RCA) of padlock probes guarantees high specificity to identify nucleic acid targets down to single nucleotide resolution in a multiplex fashion. This makes the assay suitable for molecular analysis of various diseases, and interesting to integrate into automated devices for point-of-care analysis. A critical prerequisite for many molecular assays is (i) target-specific isolation from complex clinical samples and (ii) removal of reagents, inhibitors and contaminants between reaction steps. Efficient solid supports are therefore essential to enable multi-step, multi-analyte protocols. Superparamagnetic micro- and nanoparticles, with large surface area and rapid liquid-phase kinetics, are attractive for multi-step protocols. Recently, streptavidin-modified magnetic monodispersed poly(2-hydroxyethyl methacrylate) (STV-mag.PHEMA) microspheres were developed by multiple swelling polymerization. They are easily separated by a magnet and exhibit low non-specific protein sorption. In this study, the performance and the binding efficiency of STV-mag.PHEMA was addressed by circle-to-circle amplification (C2CA). A lower number of RCA products were detected as compared to the gold standard Dynabeads. Nevertheless, this study was the first to successfully adapt STV-mag.PHEMA microspheres as solid support in a DNA-based protocol, which is an important finding. The STV-mag.PHEMA microspheres were larger with about 16 times less surface area as compared to the Dynabeads, which might partly explain the lower rolling circle product (RCP) count obtained. Further research is currently ongoing comparing particles of similar sizes and optimizing reaction conditions to establish their full utility in the field. Ultimately, low cost and versatile particles are a great resource to facilitate future clinical molecular diagnostics.
- Klíčová slova
- DNA, Magnetic microspheres, Poly(2-hydroxyethyl methacrylate), Rolling circle amplification,
- MeSH
- DNA chemie metabolismus MeSH
- imobilizované proteiny chemie metabolismus MeSH
- magnetismus MeSH
- mikrosféry * MeSH
- mikroskopie elektronová rastrovací MeSH
- polyhydroxyethylmethakrylát chemie MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- streptavidin chemie metabolismus MeSH
- techniky amplifikace nukleových kyselin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA MeSH
- imobilizované proteiny MeSH
- polyhydroxyethylmethakrylát MeSH
- streptavidin MeSH
Nanoparticle-protein conjugates have potential for numerous applications due to the combination of the properties of both components. In this paper we studied the conjugation of horse heart cytochrome c with ZnO nanoparticles modified by mercaptoacetic acid (MAA) which may be a material with great potential in anticancer therapy as a consequence of synergic effect of both components. Cyt c adsorption to the ZnO-MAA NPs surface was studied by UV-vis spectroscopy and by a dynamic light scattering in various pH. The results indicate that the optimal pH for the association of protein with modified nanoparticles is in range 5.8-8.5 where 90-96% of cytochrome c was assembled on ZnO-MAA nanoparticles. The interaction of proteins with nanoparticles often results in denaturation or loss of protein function. Our observations from UV-vis spectroscopy and circular dichroism performed preserved protein structure after the interaction with modified nanoparticles.
- MeSH
- adsorpce MeSH
- cirkulární dichroismus MeSH
- cytochromy c chemie MeSH
- imobilizované proteiny chemie MeSH
- koncentrace vodíkových iontů MeSH
- koně MeSH
- mikroskopie atomárních sil MeSH
- myokard chemie MeSH
- nanočástice chemie ultrastruktura MeSH
- oxid zinečnatý chemie MeSH
- spektrofotometrie MeSH
- thioglykoláty chemie MeSH
- velikost částic MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 2-mercaptoacetate MeSH Prohlížeč
- cytochromy c MeSH
- imobilizované proteiny MeSH
- oxid zinečnatý MeSH
- thioglykoláty MeSH
We report on the use of new biofunctionalized gold nanoparticles (bio-AuNPs) that enable a surface plasmon resonance (SPR) biosensor to detect low levels of carcinoembryonic antigen (CEA) in human blood plasma. Bio-AuNPs consist of gold nanoparticles functionalized both with (1) streptavidin, to provide high affinity for the biotinylated secondary antibody used in the second step of the CEA sandwich assay, and with (2) bovine serum albumin, to minimize the nonspecific interaction of the bio-AuNPs with complex samples (blood plasma). We demonstrate that this approach makes it possible for the SPR biosensor to detect CEA in blood plasma at concentrations as low as 0.1 ng/mL, well below normal physiological levels (approximately nanograms per milliliter). Moreover, the limit of detection achieved using this approach is better by a factor of more than 1,000 than limits of detection reported so far for CEA in blood plasma using SPR biosensors.
- MeSH
- biotinylace MeSH
- imobilizované proteiny chemie MeSH
- karcinoembryonální antigen krev imunologie MeSH
- lidé MeSH
- limita detekce MeSH
- nanočástice chemie MeSH
- povrchová plasmonová rezonance metody MeSH
- protilátky chemie imunologie MeSH
- sérový albumin hovězí chemie MeSH
- skot MeSH
- streptavidin chemie MeSH
- zlato chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Názvy látek
- imobilizované proteiny MeSH
- karcinoembryonální antigen MeSH
- protilátky MeSH
- sérový albumin hovězí MeSH
- streptavidin MeSH
- zlato MeSH
Magnetic macroporous PGMA and PHEMA microspheres containing carboxyl groups are synthesized by multi-step swelling and polymerization followed by precipitation of iron oxide inside the pores. The microspheres are characterized by SEM, IR spectroscopy, AAS, and zeta-potential measurements. Their functional groups enable bioactive ligands of various sizes and chemical structures to couple covalently. The applicability of these monodisperse magnetic microspheres in biospecific catalysis and bioaffinity separation is confirmed by coupling with the enzyme trypsin and huIgG. Trypsin-modified magnetic PGMA-COOH and PHEMA-COOH microspheres are investigated in terms of their enzyme activity, operational and storage stability. The presence of IgG molecules on microspheres is confirmed.
- MeSH
- biokatalýza MeSH
- chromatografie afinitní metody MeSH
- imobilizované proteiny chemie MeSH
- imunoglobulin G chemie MeSH
- kyseliny polymethakrylové chemická syntéza MeSH
- lidé MeSH
- magnety MeSH
- mikrosféry MeSH
- mikroskopie elektronová rastrovací MeSH
- polyhydroxyethylmethakrylát chemická syntéza MeSH
- polymerizace MeSH
- spektrofotometrie infračervená MeSH
- trypsin chemie MeSH
- železité sloučeniny chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- ferric oxide MeSH Prohlížeč
- imobilizované proteiny MeSH
- imunoglobulin G MeSH
- kyseliny polymethakrylové MeSH
- polyglycidyl methacrylate MeSH Prohlížeč
- polyhydroxyethylmethakrylát MeSH
- trypsin MeSH
- železité sloučeniny MeSH
One of the industrially important qualities of yeast is their ability to provide the cell-cell and cell-support interactions. This feature of yeast is responsible for technologically significant phenomena such as flocculation (brewing) and yeast biofilm formation (immobilization to supports), whereas these phenomena are time, environment, and strain dependent. Therefore, the goal of this work was to verify the possibility to predict and subsequently select yeast strains capable to colonize solid supports by using physicochemical adhesion models. Three different industrial yeast strains (Saccharomyces cerevisiae) were tested for their adhesion onto spent grain particles in the continuous gas-lift reactor. The cell adhesion energies were calculated, based on physicochemical characteristics of surfaces involved, according to three adhesion models (DLVO theory, thermodynamic approach, and extended DLVO theory). The role of physicochemical surface properties in the cell-cell and cell-support interactions was evaluated by comparing the computed predictions with experimental results. The best agreement between forecast and observation of the yeast adhesion to spent grains was achieved with the extended DLVO (XDLVO) theory, the most complex adhesion model applied in this study. Despite its relative comprehensiveness, the XDLVO theory does not take into account specific biochemical interactions. Consequently, additional understanding of the yeast adhesion mechanism was obtained by means of quantifying the expression of selected FLO genes. The presented approach provides tools to select the appropriately adhesive yeast strains and match them with solid supports of convenient surface properties in order to design immobilized biocatalysts exploitable in biotechnological processes.
- MeSH
- biotechnologie MeSH
- buněčná adheze MeSH
- flokulace MeSH
- fyzikální chemie MeSH
- imobilizované proteiny chemie MeSH
- lektiny vázající mannosu klasifikace genetika metabolismus MeSH
- molekulární sekvence - údaje MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- povrchové vlastnosti MeSH
- Saccharomyces cerevisiae - proteiny klasifikace genetika metabolismus MeSH
- Saccharomyces cerevisiae * genetika metabolismus MeSH
- sekvence nukleotidů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- FLO1 protein, S cerevisiae MeSH Prohlížeč
- imobilizované proteiny MeSH
- lektiny vázající mannosu MeSH
- Saccharomyces cerevisiae - proteiny MeSH
Liposomes represent a biocompatible platform for the construction of self-assembling proteoliposomes using nickel or zinc metallochelation. Potential applications of such structures consist in the development of new biocompatible vaccination nanoparticles and drug delivery nanoparticle systems. Here, we describe the design and construction of a flow-through ultrafiltration cell suitable for the preparation of monodisperse liposomes enabled for metallochelation and, hence, the formation of proteoliposomes. The linkage of the cell with a fast protein liquid chromatography system facilitates automation of the procedure, which fits the criteria for upscaling. Proof-of-concept experiments are performed using a mixture of egg phosphatidyl choline and nickel-chelating lipid DOGS-NTA-Ni (1,2-dioleoyl-sn-glycero-3-{[N(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl}(nickel salt)) to formulate proteoliposomes with proteins attached by metallochelation, including histidine (His)-tagged recombinant green fluorescent protein and rgp120 (derived from HIV-1 Env). These model proteoliposomes are characterized by gel permeation chromatography and by dynamic light scattering. Transmission electron microscopy and immunogold staining are used to characterize surface-bound proteins, revealing the tendency of rgp120 to form microdomains on liposome surfaces. These microdomains possess a two-dimensional crystal-like structure that is seen more precisely by atomic force microscopy.
- MeSH
- chelátory chemie MeSH
- histidin chemie genetika metabolismus MeSH
- HIV obalový protein gp120 chemie genetika metabolismus MeSH
- HIV-1 metabolismus MeSH
- imobilizované proteiny chemie genetika metabolismus MeSH
- lidé MeSH
- liposomy chemie MeSH
- micely MeSH
- mikroskopie atomárních sil MeSH
- nikl chemie MeSH
- oligopeptidy chemie genetika metabolismus MeSH
- proteolipidy chemie MeSH
- transmisní elektronová mikroskopie MeSH
- ultrafiltrace metody MeSH
- zelené fluorescenční proteiny chemie genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chelátory MeSH
- gp120 protein, Human immunodeficiency virus 1 MeSH Prohlížeč
- His-His-His-His-His-His MeSH Prohlížeč
- histidin MeSH
- HIV obalový protein gp120 MeSH
- imobilizované proteiny MeSH
- liposomy MeSH
- micely MeSH
- nikl MeSH
- oligopeptidy MeSH
- proteolipidy MeSH
- proteoliposomes MeSH Prohlížeč
- zelené fluorescenční proteiny MeSH
