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.
Le Nel
Anne
A
Université Paris-Sud, JE2495, Protéines et Nanotechnologies en Sciences Séparatives, F-92296 Châtenay-Malabry Cedex, France.
Minc
Nicolas
N
Smadja
Claire
C
Slovakova
Marcela
M
Bilkova
Zuzana
Z
Peyrin
Jean-Michel
JM
Viovy
Jean-Louis
JL
Taverna
Myriam
M
D000818:Animals; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D019286:Endopeptidase K / Q000737:chemistry* / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008280:Magnetics; D051379:Mice; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D008970:Molecular Weight; D011328:Prions / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results; D012756:Sheep; D013499:Surface Properties
D004129:Dimethylpolysiloxanes; D004800:Enzymes, Immobilized; D011328:Prions; D019286:Endopeptidase K