Mechanisms of Fibrinogen Adsorption on Latex Particles Determined by Zeta Potential and AFM Measurements

Abstract

The adsorption of fibrinogen on polystyrene latex particles was studied using the concentration depletion method combined with the AFM detection of residual protein after adsorption. Measurements were carried out for a pH range of 3.5-11 and an ionic strength range of 10(-3)-0.15 M NaCl. First, the bulk physicochemical properties of fibrinogen and the latex particle suspension were characterized for this range of pH and ionic strength. The zeta potential and the number of uncompensated (electrokinetic) charges on the protein were determined from microelectrophoretic measurements. It was revealed that fibrinogen molecules exhibited amphoteric characteristics, being on average positively charged for pH <5.8 (isolectric point) and negative otherwise. However, the latex particles did not show any isoelectric point, remaining strongly negative for this pH range. Afterward, systematic measurements of the electrophoretic mobility of fibrinogen-covered latex were carried out as a function of the amount of adsorbed protein, expressed as the surface concentration. A monotonic increase in the electrophoretic mobility (zeta potential) of the latex was observed in all cases, indicating a significant adsorption of fibrinogen on latex for pH below 11. It was also proven that fibrinogen adsorption was irreversible, with the maximum surface concentration varying between 2.5 and 5 × 10(3) μm(-2) (weight concentration of a bare molecule was 1.4 to 2.8 mg m(-2)). These measurements revealed two main adsorption mechanisms of fibrinogen: (i) the unoriented (random) mechanism prevailing for lower ionic strength, where adsorbing molecules significantly penetrate the fuzzy polymeric layer on the latex core and (ii) the side-on adsorption mechanism prevailing for pH > 5.8 and a higher ionic strength of 0.15 M. It was also shown that in the latter case, variations in the zeta potential with the protein coverage could be adequately described in terms of the electrokinetic model, previously formulated for planar substrate adsorption. On the basis of these experimental data, an efficient procedure of preparing fibrinogen-covered latex particles of controlled monolayer structure and coverage was envisaged.

Mechanism of immunoglobulin G adsorption on polystyrene microspheres

“…3. In each case,the zeta potential increases with the IgG coverage and As previously done for fibrinogen [26,27] and rHSA [28], the experimental datapresented in Fig. 3 were interpreted in terms of the mean-field Gouy-Chapman model and the three-dimensional (3D) model.According to the GC model,the net charge density of the latex covered by IgG can be calculated from the formula [31,37,38] σ= σ 0 + q e (Av/M w ) Γ…”

“…The IgG deposition on particleswas carried out according to the procedurebefore used for recombinant albumin(rHSA) [28] and fibrinogen [26,27]: …”

“…It should be mentioned that this method has been before used to determine adsorption mechanism of fibrinogen [26,27] and albumin [28] on latex particles.…”

KfrA plasmid protein monolayers on latex particles-electrokinetic measurements

“…Knowing the zeta potential, the electrokinetic (uncompensated) surface charge of latex can be calculated using the Gouy-Chapman relationship [ 20 ]. Accordingly, for pH 3.5,…”

“…It should also be mentioned that the estimated relaxation time of KfrA monolayer formation on latex particles [ 20 ], varied between 1.5 and 1 s for the final latex concentration of 60-120 mg L −1 . Therefore, the adsorption time of 900 s ensured that all protein molecules could adsorb on latex particles.…”