Mechanism of fibrinogen /microparticle complex deposition on solid substrates: Role of pH
Graphical abstract
Introduction
Protein immobilization on carrier particles of various size is essential for their efficient separation and purification by chromatography and filtration, for biosensing, enzymatic catalysis, bioreactors, immunological assays, etc. [1]. In the case of metal particles, a controlled protein attachment leads to the ‘corona’ formation [[2], [3], [4], [5]] that creates conjugates, which can be applied for drug delivery, hyperthermia therapy and contrast imaging.
Analogously, a physical (non-specific) adsorption of protein molecules on polymer microparticles (often referred to as latex) is advantageous because such complexes show much larger stability than the protein solution themselves. This allows to study their electrokinetic properties, for example to determine the isoelectric point in a reliable way if trace amounts of proteins are available [[6], [7], [8], [9]]. Polymer particles conjugated with various antibodies (immunoglobulins), referred to as immunolatex [1] are currently used in plethora of sensitive agglutination immunoassays for various infections and disease, for example Salmonella, E.Coli, etc.
Because of its significance, antibody molecule adsorption on polymer particles was extensively studied in the literature [[10], [11], [12], [13], [14], [15], [16]] mainly using the electrophoretic mobility measurements and the concentration depletion methods. In Refs [17,18]. adsorption of albumins on polymer microparticles was studied and in Ref [[6], [7], [8], [9]]. adsorption of bovine and human serum fibrinogen on negatively charged polystyrene microparticles. In the latter references, a quantitative interpretation of the experimental results was performed in terms of an electrokinetic model representing the extension of the Smoluchowski approach for heterogeneous (particle covered) surfaces. It was shown that using this model, the protein coverage on polymer carrier particles can be precisely determined in situ, which is not feasible by other methods. It was also confirmed [[6], [7], [8], [9]] that the orientation of adsorbed molecules can be regulated by pH, ionic strength of the adsorption processes and by the surface charge of the microparticles.
However, despite the significance of the protein/microparticle conjugates for biosensing purposes, no experimental investigations were reported in the literature focused on studying the kinetics of their deposition on solid substrates. Therefore, in view of the deficit of experimental data, the main goal of this work is to unravel the mechanisms of protein/microparticle complex interactions with mica used as a model substrate and oxidized silicon wafers that can mimic the behavior of sensors used in QCM [19] or in reflectometric studies [20,21].
Attention in this work is focused on fibrinogen, which can be considered as model antigen. It is one of the most abundant blood plasma glycoprotein [22] playing an essential role in the clotting cascade, platelet adhesion, leucocyte binding, thrombosis, angiogenesis, inflammatory response, tumor growth, fouling in artificial organs (stents, valves, pacemakers, catheters) [[23], [24], [25]] and implant failure.
As far as the primary structure is concerned, the fibrinogen molecule is composed of two symmetric parts each consisting of three different polypeptide chains named Aα, Bβ, and γ, that are joined together by 29 disulfide bonds [[26], [27], [28]] and major parts of the Aα chains extend from the core of the molecule, forms two appendages each having a molar mass equal to 42.3 kg mol−1 whereas the entire molecule molar mass is equal to 338 kg mol−1.
The fibrinogen molecule dimensions were determined in Refs. [[29], [30], [31]] where it is established that it possesses a largely elongated tri-nodular shape with the total length of 47.5 nm. Veklich et al. [32] confirmed that at pH 7.4 the fibrinogen molecules assume a collapsed conformation with the Aα chains attached to the central nodule. In contrast, at lower pHs, the molecule assumed a more expanded conformation with the flexible Aα chains directed under a right angle against the core part of the molecule. These results were theoretically confirmed in Refs [33,34]. where a bead model of the fibrinogen molecule was developed by explicitly considering the presence of the flexible side arms, which were positively charged at pH range 3–10 that facilitates fibrinogen molecule adsorption at various substrates comprising negatively charged polymer microparticles. In the expanded state appearing at pH < 4 the effective molecule length is ca. 80 nm and at pH 7.4 the length is equal to 48 nm.
The net charge of the molecule derived from electrophoretic mobility measurements is positive for pH below 5.8 that is considered as its isoelectric point, and negative otherwise [35].
In this work, complete characteristics of fibrinogen, the microparticles and the substrates are acquired that facilitate a quantitative interpretation of the deposition kinetics of the fibrinogen/microparticle complexes measured using direct in situ methods and interpreted in terms of a hybrid mass transfer model. This is of a considerable interest to basic sciences, enabling to acquire new knowledge about the colloid interactions in heterogeneous systems at various pHs, and in consequence to determine the validity of the mean-field theoretical approaches.
Additionally, the results can be exploited for establishing optimum conditions for an efficient performance of biosensing assays of fibrinogen serving as antigen.
Section snippets
Materials and methods
Fibrinogen from human blood plasma, in the form of crystalline powder containing 65% protein, 25% sodium chloride and 15% sodium citrate, was supplied by Sigma and used without further purification.
Fibrinogen solutions were prepared by dissolving an appropriate amount of the powder under gentle stirring at appropriate pH and 298 K in a sodium chloride solution. Afterward, the suspension was passed through the 0.45 μm filter and the bulk concentration of fibrinogen was spectrophotometrically
Fibrinogen, particle and substrate characteristics in the bulk
It was established performing the DLS diffusion coefficient measurements that fibrinogen solutions were stable for pH < 5 and pH > 7, which enabled precise measurements of its electrophoretic mobility μe as a function of pH. At pH 3.5 and a NaCl concentration equal to 10-3 M, the fibrinogen molecule electrophoretic mobility was equal to 2.0 ± 0.2 × 10-8 m2 (Vs−1) = 2.0 ± 0.2 μm cm (Vs−1) (the latter unit is used hereafter in this work) and a NaCl concentration of 10-2 M, μe = 1.6 ± 0.1 μm cm s−1
Conclusions
An efficient method for the preparation of fibrinogen/polymer microparticle complexes was developed. Using the in situ electrophoretic mobility LDV and the DLS measurements, the coverage and molecule orientation in the monolayers adsorbed on the microparticles were determined.
It was demonstrated that the complexes are stable over prolonged time period at pH below 4.5 and above 5.5 for large suspension concentration of 2.000 mg L−1 and NaCl concentration up to 0.01 M. For lower concentrations
Acknowledgements
This work was supported by the Research Project for Young Employee of ICSC PAS and the NCN Preludium Grant 2013/09/N/ST4/00320.
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