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Monitoring fibrinogen adsorption kinetics by interfacial TIRF rheometry
Author(s) -
Sanders A.,
Jennissen H. P.
Publication year - 1996
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/(sici)1099-1352(199634/12)9:5/6<503::aid-jmr292>3.0.co;2-i
Subject(s) - total internal reflection fluorescence microscope , adsorption , kinetics , chemistry , fibrinogen , analytical chemistry (journal) , shear rate , receptor–ligand kinetics , chromatography , materials science , viscosity , organic chemistry , composite material , biochemistry , receptor , physics , quantum mechanics , membrane
The adsorption kinetics of purified fibrinogen to unmodified and aminopropylsilane‐modified quartz glass surfaces were studied under pseudo‐first order (binding‐unit excess) conditions by the total internal reflection fluorescence (TIRF) method. Fluorescence in the adsorbed protein layer (350 nm) was excited by the evanescent wave at 285–290 nm. In order to reduce and possibly eliminate the influence of mass transfer on the kinetics of fibrinogen adsorption, a novel protein adsorption chamber containing a cone‐and‐plate rheometer with total internal reflection technology was employed. The aim of the study was to obtain critical shear rates, at which the adsorption rate of fibrinogen became independent of diffusion. Therefore, shear rates were varied between 0 and 7200 s −1 at initial fibrinogen concentrations of c 0 = 4.7 and 17.7 μg mL −1 . The adsorption rate of fibrinogen increased 5–17‐fold, depending on the surface, as the critical shear rate was approached. Above the critical shear rates the kinetic data of fibrinogen adsorption could be fitted at c 0 = 4.7 μg mL 1 to a single exponential function, indicating the predominance of a single binding step with a half‐life of ca 20 s. At the higher initial concentration of c 0 = 17.7 μg/mL − , however, a significant deviation from the single exponential behavior was observed in the first 10 s of the adsorption reaction, indicating a very fast initial event with a half‐life of ca 5 s in addition to a slower binding reaction with a half‐life of ca 35 s. Thus the novel TIRF rheometer can resolve kinetics down to half‐lives of 5 s and possibly even lower.