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Photolithographic technique for controlled fabrication of metal structures
Jusková, Petra ; Hegrová, Jitka ; Foret, František
We are describing two strategies for controlled (geometry and dimension) preparation of metal micro-nano structures. Both techniques are based on photolithographic processes with selective pattern transfer. First technique utilizes agarose gel containing photosensitive silver chloride as the photosensitive layer. Silver structures are formed after irradiation of the gel through the photolithographic mask which defines shape of the resulting structures. Second approach utilizes photolithographic process to form particles from vacuum deposited thin metal layer deposited over soluble sacrificial layer.
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Iron oxide nanoparticle modified monolithic pipette tips for selective enrichment of phosphopeptides
Křenková, Jana ; Foret, František
We have developed iron oxide nanoparticle modified monolithic pipette tips for selective and efficient enrichment of phosphopeptides. Iron oxide nanoparticles were synthesized using a co-precipitation method and stabilized by citrate ions. A stable coating of nanoparticles was obtained via multivalent interactions of citrate ions on the nanoparticle surface with a quaternary amine functionalized surface of the methacrylate based monolithic tips. The performance of the developed and commercially available tips was demonstrated with the enrichment of phosphorylated peptides from tryptic digests of caseins followed by MALDI/MS detection.
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Silica-based monolithic capillary columns modified to zwitterionic stationary phase for hydrophilic interaction liquid chromatography
Moravcová, Dana ; Planeta, Josef ; Kahle, Vladislav ; Horká, Marie ; Roth, Michal
Zwitterionic monolithic capillary columns intended for isocratic gradient hydrophilic interaction chromatography (HILIC) separations are introduced. Silica-based capillary columns (150 mm x 0.1 mm) were prepared by acidic hydrolysis of tetramethoxysilane in the presence of polyethylene glycol and urea. The modification by a 3-(trimethoxysilyl)propyl methacrylate and then by a zwitterionic [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide to HILIC stationary phase bearing sulfoalkylbetaine groups on its surface followed. Prepared columns were characterized in HILIC separation mode employing mobile phase containing 10% (v/v) of 5 mM ammonium acetate pH = 4.5 in acetonitrile. Comparison with the commercially available ZIC-HILIC® column (Merck SeQuant®) under the same separation conditions using a mixture of aromatic carboxylic acids as a sample was done on the basis of separation efficiency of tested columns as well as retention factors and peak asymmetry of individual solutes.
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Micropreparative solution isoelectric focusing of peptides and proteins in nonwoven strip
Duša, Filip ; Šlais, Karel
Recently we devised a new instrument for micropreparative analysis [1]. It is based on solution phase isoelectric focusing (sIEF) performed in a narrow channel (approximately 2 – 4 mm wide) with a strip of nonwoven fabric serving as the analysis bed. The strip can be precut before an analysis to make harvesting of fractions easier. Isoelectric focusing is driven by a programmable electrophoretic power supply. Progress of analysis is monitored by addition of colored isoelectric point (pI) markers. Usually sIEF runs are performed overnight. Evaporation of water is one of crucial features of analysis and it forces free liquid to shrink into the nonwoven fabric strip. Moreover, water evaporation increases liquid viscosity by altering ethylene glycol/water ratio. Fractions can be harvested simply by collecting and washing precut segments after the sIEF run. In this paper we used sIEF device for purification of caseinomacropeptide (CMP) from crude whey. Obtained fractions were further analyzed by HPLC. From acquired data a plot with chromatograms of all fractions was constructed to show purification profile of CMP. We proved that the new instrument is capable of quantitative purification of CMP complex from a globulin fraction.
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Oriented immobilization of PNgase F on a porous polymer monolith for rapid N-glycan release
Szekrényes, A. ; Křenková, Jana ; Keresztessy, Z. ; Foret, František ; Guttman, A.
We demonstrate a simple and rapid method for the oriented immobilization of peptide-N4-(Nacetyl- glucosaminyl) asparagine amidase F (PNGase F) on a porous polymer monolith. The oriented immobilization is based on the affinity of glutathione-S-transferase (GST) tagged PNGase F towards glutathione modified monolith prepared in the capillary format. This approach allows the oriented and easily replaceable immobilization of PNGase F for rapid and efficient release of N-linked glycans. The reactor was tested by deglycosylation of several native glycoproteins such as ribonuclease B, fetuin or human immunoglobulin G. The proteins were effectively deglycosylated in several minutes and the released N-linked glycans were analyzed using off-line MALDI/MS or CE/LIF.
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Thin metal films in resistivity-based chemical sensing
Podešva, Pavel ; Foret, František
Measurement of basic electric properties (current, voltage, resistance) is the most frequent technique in practically all scientific disciplines. Direct electric measurements are simple, precise and provide very large dynamic range. Since the related equipment is simple and inexpensive sensors based on electric measurements are very attractive for many applications in chemistry, environmental sciences or medicine. In this work, we present resistivity sensor for direct measurement of thiols in liquid samples.
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Filtration microcartridge and capillary isoelectric focusing for the analysis low number of microorganisms in real samples
Kubesová, Anna ; Horká, Marie ; Šalplachta, Jiří ; Horký, J.
At present, detection and identification of microorganisms in real biological samples needs very sensitive methods for correct diagnosis and therapy in medical practice, biotechnology, agriculture, food safety and quality etc. Accordingly, it is essential to develop new cheap techniques for pathogen identification, particularly for pathogen in complex biological samples. Pre-concentration, separation, and sensitive detection of whole cells in one step are great potential and advantage for detection of pathogens in low concentration. Combination of filtration microcartridge and capillary isoelectric focusing was developed for pre-concentration and pre-separation of microorganisms from real suspensions and the possibility its application to real samples was verified.
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