On-a-chip tryptic digestion of transthyretin: a step toward an integrated microfluidic system for the follow-up of familial transthyretin amyloidosis

Autor: jeanne bataille, Antoine Pallandre, Jörg Peter Kutter, Arthur Viode, Stéphanie Descroix, Josiane P. Lafleur, François Becher, Isabelle Le Potier, Fanny Varenne, Myriam Taverna, Christian Poüs, Iago Pereiro, camille roesch, Claire Smadja
Přispěvatelé: Université Paris Saclay (COmUE), Faculté de Pharmacie, Université Paris-Sud - Paris 11 (UP11), Institut Curie [Paris], University of Copenhagen = Københavns Universitet (KU), IZON Sciences Ldt, Partenaires INRAE, Hôpital Antoine Béclère, Assistance Publique - Hôpitaux de Paris (AP-HP), Laboratoire de Chimie Physique D'Orsay (LCPO), Université Paris-Sud - Paris 11 (UP11)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre de Nanosciences et Nanotechnologies (C2N (UMR_9001)), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), fellowship programme of DIM Analytics, DIGIDIAG projects - French National Research Agency (ANR) as part of the Investissements d'Avenir programme [ANR 10-NANO 0207]
Jazyk: angličtina
Rok vydání: 2018
Předmět:
0301 basic medicine
proteolysis
Hydrochloride
diagnosis
monoliths
[SDV]Life Sciences [q-bio]
Peptide
protein digestion
01 natural sciences
Biochemistry
magnetic beads
Analytical Chemistry
03 medical and health sciences
Hydrolysis
chemistry.chemical_compound
Electrochemistry
medicine
solvent systems
Humans
Prealbumin
Environmental Chemistry
Monolith
Spectroscopy
chemistry.chemical_classification
enzyme reactors
Amyloid Neuropathies
Familial
geography
geography.geographical_feature_category
Chromatography
biology
Chemistry
010401 analytical chemistry
high-efficiency
Reproducibility of Results
Substrate (chemistry)
nutritional and metabolic diseases
Microfluidic Analytical Techniques
mass-spectrometry
Trypsin
0104 chemical sciences
Transthyretin
trypsin
030104 developmental biology
biology.protein
Microreactor
medicine.drug
Zdroj: Analyst
Analyst, Royal Society of Chemistry, 2018, 143 (5), pp.1077-1086. ⟨10.1039/c7an01737e⟩
ISSN: 0003-2654
1364-5528
Popis: International audience; A microfluidic microreactor for trypsin mediated transthyretin (TTR) digestion has been developed as a step towards the elaboration of a fully integrated microdevice for the detection of a rare and disabling disease, the familial transthyretin amyloidosis (ATTR) which is related to specific TTR mutations. Therefore, an enzymatic microreactor coupled to an analytical step able to monitor the mutation of TTR on specific peptide fragments would allow an accurate monitoring of the treatment efficiency of ATTR. In this study, two types of immobilized trypsin microreactors have been investigated: a new miniaturized, microfluidic fluidized bed packed with trypsin functionalized magnetic particles (MPs), and a thiol-ene (TE) monolithbased chip. Their performances were first demonstrated with N-benzoyl-DL-arginine-4-nitroanilide hydrochloride BApNA, a low molecular weight substrate. High reaction yields (75.2%) have been reached within 0.6 min for the TE-based trypsin microreactor, while a lower yield (12.4%) was obtained for the micro-fluidized bed within a similar residence time. Transposition of the optimized conditions, developed with BApNA, to TTR digestion in the TE-based trypsin microreactor was successfully performed. We demonstrated that the TE-chip can achieve an efficient and reproducible digestion of TTR. This has been assessed by MS detection. In addition, TTR hydrolysis led to the production of a fragment of interest allowing the therapeutic follow-up of more than twenty possible ATTR mutations. High sequence coverage (90%), similar to those obtained with free trypsin, was achieved in a short time (2.4 min). Repeated experiments showed good reproducibility (RSD = 6.8%). These promising results open up the route for an innovative treatment follow-up dedicated to ATTR.
Databáze: OpenAIRE
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