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Functionalized magnetic micro‐ and nanoparticles: Optimization and application to μ‐chip tryptic digestion
Author(s) -
Bílková Zuzana,
Slováková Marcela,
Minc Nicolas,
Fütterer Claus,
Cecal Roxana,
Horák Daniel,
Beneš Milan,
le Potier Isabelle,
Křenková Jana,
Przybylski Michael,
Viovy JeanLouis
Publication year - 2006
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200500587
Subject(s) - glycidyl methacrylate , chemistry , chromatography , magnetic nanoparticles , alginic acid , polystyrene , methacrylate , nanoparticle , materials science , polymerization , polymer , nanotechnology , biochemistry , organic chemistry
The preparation of an easily replaceable protease microreactor for μ‐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 μ‐chip capillary (μ‐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 μ‐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 μ‐chip devices.