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A fluorescence spectroscopy assay for real-time monitoring of enzyme immobilization into mesoporous silica particles

Pegah S. Nabavi Zadeh (Institutionen för kemi och kemiteknik, Fysikalisk kemi) ; Kassam Abdel Mallak (Institutionen för kemi och kemiteknik, Fysikalisk kemi) ; Nils Carlsson (Institutionen för kemi och kemiteknik, Fysikalisk kemi) ; Björn Åkerman (Institutionen för kemi och kemiteknik, Fysikalisk kemi)
Analytical Biochemistry (0003-2697). Vol. 476 (2015), p. 51-58.
[Artikel, refereegranskad vetenskaplig]

Mesoporous silica particles are used as support material for immobilization of enzymes. Here we investigated a fluorescence-based assay for real-time monitoring of the immobilization of lipase, bovine serum albumin, and glucose oxidase into micrometer-sized mesoporous silica particles. The proteins are labeled with the dye epicocconone, and the interaction with the particles is observed as an increase in emission intensity of the protein–dye conjugates that can be quantified if correcting for a comparatively slow photobleaching. The immobilization occurs in tens of minutes to hours depending on particle concentration and type of protein. In the limit of excess particles over proteins, the formation of the particle–protein complexes can be described by a single exponential growth for all three investigated proteins, and the fitted pseudo-first-order rate constant increases linearly with particle concentration for each protein type. The derived second-order rate constant k varies with the protein hydrodynamic radius according to k ∼ RH−4.70±0.01, indicating that the rate-limiting step at high particle concentrations is not the diffusional encounter between proteins and particles but rather the entry into the pores, consistent with the hydrodynamic radii of the three proteins being smaller but comparable to the pore radius of the particles.

Nyckelord: Enzyme; Immobilization; Mesoporous silica; Kinetics; Fluorescence; Epicocconone

Denna post skapades 2015-03-25. Senast ändrad 2015-05-21.
CPL Pubid: 214301


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Institutionen för kemi och kemiteknik, Fysikalisk kemi



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