title: Investigating the kinetics of paramagnetic-beads linked alkaline phosphatase enzyme through microchannel resistance measurement in dielectric microchip.
abstract: Real time monitoring of electrolyte resistance changes during hydrolysis of 4-nitrophenylphosphate (pNPP) by alkaline phosphatase (ALP) bound on paramagnetic-beads was performed into a small dielectric channel. The reaction kinetic fit with a non-competitive substrate-inhibition equation. Michaelis-Menten apparent constant, KM(app), was determined as 0.33±0.06mM and the maximum apparent rate, Vmax(app) as 98±5pMs(-1). The detection limits were 15fM for ALP and 0.75mM for pNPP. This miniaturized device constitutes a powerful tool for analysis of interaction between ligands.
authors.affiliation: CNRS, UMR 8235, LISE, F-75005 Paris, France; Sorbonne Universités, UPMC Univ Paris 06, UMR 8235, Laboratoire Interfaces et Systèmes Electrochimiques, F-75005 Paris, France.
authors.forename: Mathilde
authors.initials: M
authors.lastname: Faure
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, UMR 7622, Laboratoire Biologie des Semences, 4 Place Jussieu, F-75005 Paris, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Sotta
authors.affiliation: CNRS, UMR 8235, LISE, F-75005 Paris, France; Sorbonne Universités, UPMC Univ Paris 06, UMR 8235, Laboratoire Interfaces et Systèmes Electrochimiques, F-75005 Paris, France. Electronic address: jean.gamby@upmc.fr.
authors.forename: Jean
authors.initials: J
authors.lastname: Gamby
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis / Q000737:chemistry*; D000814:Aniline Compounds / Q000032:analysis / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation; D003217:Conductometry / Q000295:instrumentation*; D017097:Electric Impedance; D004789:Enzyme Activation; D004867:Equipment Design; D019544:Equipment Failure Analysis; D018189:Immunomagnetic Separation / Q000295:instrumentation*; D007700:Kinetics; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D008904:Miniaturization; D009943:Organophosphorus Compounds / Q000032:analysis / Q000737:chemistry*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000295:instrumentation*
chemical_list: D000814:Aniline Compounds; D009943:Organophosphorus Compounds; C068798:4-aminophenylphosphate; D000469:Alkaline Phosphatase
keywords: 4-Nitrophenylphosphate; Alkaline phosphatase; Dielectric microchannel; Electric impedance; Enzyme kinetics; Paramagnetic-beads