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Controlled proteolysis of normal and pathological prion protein in a microfluidic chip
Authors: Le Nell Anne | Minc Nicolas | Smadja Claire | Slováková Marcela | Bílková Zuzana | Peyrin Jean-Michel | Viovy Jean-Louis | Taverna Myriam
Year: 2008
Type of publication: článek v odborném periodiku
Name of source: Lab On a Chip
Publisher name: Royal Society of Chemistry
Place: Cambridge
Page from-to: 294-301
Titles:
Language Name Abstract Keywords
cze Kontrolovaná proteolýza fyziologického a patologického prionového proteinu v mikročipovém zařízení. Proteináza K byla imobilizovanána na magnetický nosič a ten byl aplikován do kanálku mikrofluidního zařízení. Mikročip byl použit ke štěpení fyziologického a patologického prionového proteinu. Další analýzou (Western blot) bylo potvrzeno, že obě formy lze snadno odlišit.
eng Controlled proteolysis of normal and pathological prion protein in a microfluidic chip A microreactor for proteinase K (PK)-mediated protein digestion was developed as a step towards the elaboration of a fully integrated microdevice for the detection of pathological prion protein (PrP). PK-grafted magnetic beads were immobilized inside a polydimethylsiloxane (PDMS) microchannel using a longitudinal magnetic field parallel to the flow direction and a magnetic field gradient, thereby forming a matrix for enzymatic digestion. This self-organization provided uniform pore sizes, a low flow resistance and a strong reaction efficiency due to a very thin diffusion layer. The microreactor?s performance was first evaluated using a model substrate, succinyl-ala-ala-ala-paranitroanilide (SAAAP). Reaction kinetics were typically accelerated a hundred-fold as compared to conventional batch reactions. Reproducibility was around 98% for on-chip experiments. This microsystem was then applied to the digestion of prion protein from brain tissues. Controlled proteolysis could be obtained by varying the on-chip flow rate, while a complete proteolysis of normal protein was achieved in only three minutes. Extracts from normal and pathological brain homogenates were finally compared and strong discrimination between normal and pathological samples was demonstrated. Proteinase K, Prion protein, microfluidic device

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