Publikace detail

Trypsin digestion in chip

Autoři: Slováková Marcela | Minc Nicolas | Viovy Jean L

Rok: 2004

Druh publikace: ostatní - přednáška nebo poster

Název zdroje: New microfluidic-Mass spectrometry technologies for high performance proteomics, Sixth scientific conference

Název nakladatele: neuveden

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Jazyk	Název	Abstrakt	Klíčová slova
cze	Tryptické štěpení v mikrofluidním čipu.	Tryptické štěpení v mikrofluidním čipu.	trypsin; mikrofluidní zařízení; enzymová kinetika
eng	Trypsin digestion in chip	We describe the use of the self-organization of grafted trypsin magnetic beads under a magnetic field in a microchip for protein digestion. As compared to our previous work [1,2], we use here a novel magnet arrangement leading to field lines parallel to the flow. This reduces flow resistance and increases digestion efficiency. We also improved the bead grafting, and performed a quantitative analysis of the flow across the plug. This arrangement retains the advantages of self assembled magnetic bead arrays as compared to other previously proposed devices for tryptic digest [3,4].. When contaminated, the plug can be easily replaced by fresh beads. Also, the grafting of trypsin on particles can be performed ex situ in large quantities in a batch process, allowing for reduced cost and better reproducibility and quality control. Formation of the plug of magnetic beads: The PDMS device uses strong magnets to create a magnetic field parallel to the flow with a strong gradient pointing through the centre of the microchannel (Fig. 1a). This allows for the formation of a low-hydrodynamic resistance plug consisting of magnetic trypsin beads that serves as a matrix for protein digestion. At the beginning of the immobilization, particles flow into the channel and self-organize in chain-like columns along the channel direction (Fig.1b, insert). When the concentration increases, the plug becomes opaque, probably due to the formation of a ?labyrinth like? structure [5] made of tortuous and ramified ?walls? with one direction collinear to the field (Fig 1b). The distance between walls is maintained by dipole-dipole repulsion, keeping the bulk of the plug channels collinear to the flow, with a thickness of a few micrometers. In those ?tubes? covered by proteases, the proteins can rapidly diffuse to the walls, thus the digestion there is not diffusion-limited. Hydrodynamic studies: Pressure versus flow characteristics of the device was studied. In constant flow rate mode, an osci	trypsin; microchip device; kinetic constants

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