Biomedical subjects

Jana Krenková

Publications and source records attributed to Jana Krenková.

3 recordsLinked to original sources

PubMed2006

Functionalized magnetic micro- and nanoparticles: optimization and application to micro-chip tryptic digestion.

The preparation of an easily replaceable protease microreactor for micro-chip application is described. Magnetic particles coated with poly(N-isopropylacrylamide), polystyrene, poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate), poly(glycidyl methacrylate), [(2-amino-ethyl)hydroxymethylen]biphosphonic acid, or alginic acid with immobilized trypsin were utilized for heterogeneous digestion. The properties were optimized, with the constraint of allowing immobilization in a microchannel by a magnetic field gradient. To obtain the highest digestion efficiency, sub-micrometer spheres were organized by an inhomogeneous external magnetic field perpendicularly to the direction of the channel. Kinetic parameters of the enzyme reactor immobilized in micro-chip capillary (micro-chip immobilized magnetic enzyme reactor (IMER)) were determined. The capability of the proteolytic reactor was demonstrated by five model (glyco)proteins ranging in molecular mass from 4.3 to 150 kDa. Digestion efficiency of proteins in various conformations was investigated using SDS-PAGE, HPCE, RP-HPLC, and MS. The compatibility of the micro-chip IMER system with total and limited proteolysis of high-molecular-weight (glyco)proteins was confirmed. It opens the route to automated, high-throughput proteomic micro-chip devices.

Amyloid beta-Peptides↗

PubMed2005

Characterization of a monolithic immobilized trypsin microreactor with on-line coupling to ESI-MS.

The preparation and characterization of a miniaturized trypsin reactor using on-line coupling with an ESI-TOF mass spectrometer are described. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-trypsin was covalently immobilized on poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith prepared in a 75 microm ID fused silica capillary resulting in a bioreactor with high local concentration of the proteolytic enzyme. Covalent immobilization of trypsin on this support was performed using the epoxide functional groups in either a one- or a multistep reaction. For on-line protein digestion-MS analysis the bioreactor was coupled with the mass spectrometer using a liquid junction microelectrospray interface. The performance of the reactor was tested using an on-line flow through the system with flow rates of 50-300 nL/min. The resulting protein consumption was in the atto- to low femtomole range. Proteolytic activity was characterized in a wide range of conditions with respect to the flow rate, pH, and temperature. Complete protein digestion was achieved in less than 30 s at 25 degrees C with the sequence coverage of 80% (cytochrome c), which is comparable to 3 h digestion in solution at 37 degrees C. Besides the good performance at laboratory temperature, the immobilized trypsin in the bioreactor also performed well at lower pH compared to the standard in-solution protocols.

Capillary Action↗

PubMed2004

Immobilized microfluidic enzymatic reactors.

The use of enzymes for cleavage, synthesis or chemical modification represents one of the most common processes used in biochemical and molecular biology laboratories. The continuing progress in medical research, genomics, proteomics, and related emerging biotechnology fields leads to exponential growth of the applications of enzymes and the development of modified or new enzymes with specific activities. Concurrently, new technologies are being developed to improve reaction rates and specificity or perform the reaction in a specific environment. Besides large-scale industrial applications, where typically a large processing capacity is required, there are other, much lower-scale applications, benefiting form the new developments in enzymology. One such technology is microfluidics with the potential to revolutionize analytical instrumentation for the analyses of very small sample amounts, single cells or even subcellular assemblies. This article aims at reviewing the current status of the development of the immobilized microfluidic enzymatic reactors (IMERs) technology.

Animals↗