Publication detail

Self assembled magnetic particles: use for protein digestion in lab-on-chip.

Year: 2005

Type of publication: ostatní - přednáška nebo poster

Name of source: 2nd Czech Proteomics Conference with international participation

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Language	Name	Abstract	Keywords
cze	Samouspořádané magnetické partikule uspořádané v mikročipovém zařízení: lab-on-chip.	Enzymy trypsin a proteináza K byly imobilizovány kovalentní vazbou na nově vyvinuté magnetické partikule a tyto enzymové reaktory byly fixovány v kanálku mikročipového zařízení. Mikročipové zařízení bylo použito ke štěpení modelových proteinů a prionového proteinu.	proteináza K, trypsin; magnetické partikule, enzymový reaktor, prionový protein, mikrofluidní zařízení
eng	Self assembled magnetic particles: use for protein digestion in lab-on-chip.	We present examples of bioanalysis based on the self-organization of superparamagnetic beads under a magnetic field in microfluidic device fabricated in polydimethylsiloxane (PDMS) by soft lithography and rapid prototyping. We use here a magnet arrangement leading to field lines parallel to the flow. This reduces flow resistance and increases digestion efficiency. This arrangement retains the advantages of self assembled magnetic bead arrays: when contaminated, the plug can be easily replaced by fresh beads. Tryptic digestion of proteins. A plug of trypsin-functionalized magnetic particles was immobilized between two magnets in a microchannel for flow-through protein digestion with off-chip analysis. Kinetics studies of the hydrolysis of a model peptide BApNA show a 100 fold increase in digestion speed obtained by the microsystem when compared to a batch system. High performance and reproducible recombinant human growth hormone digestion was confirmed by analysing the digest products in both CE and MALDI-TOF-MS. Similar sequence covering (of about 35%) are obtained from MS analysis of products after 10 minutes on-chip and 4h with soluble trypsin in batch. Proteinase K degradation of prion protein. Proteinase K (PK) was covalently bound to magnetic nanoparticles functionalised with carboxylic group. The PK magnetic beads were self-assembled into the channel of the microchip by a strong magnetic gradient produced by permanent magnets. Activity and reaction kinetics of the immobilized enzyme were determined by monitoring the hydrolysis of succinyl-ala-ala-ala-p-nitroanilide as a low-molecular synthetic substrate, and showed a 100-fold increase in digestion speed. Affinity isolation of tryptic peptides on anhydrotrypsin. Anhydrotrypsin is catalytically inactive derivative of bovine trypsin and enables highly specific trapping of tryptic peptides and binds only specific peptides containing arginine and lysine at their C-termini. Magnetic affinity reactor prepared b	trypsin, proteinase K, microfluidic device, enzyme reactor, limited digestion

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