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Abstract
This work is dealing with the use of polystyrene (PS) nanoparticles as substrates for bioanalytical specific interactions. Different techniques were used for the accurate characterization of the PS nanoparticles of 100 nm and 196 nm before coating them with a layer of antibodies against immunoglobulins of type E (aIgE), giving to the particle a specific functionality. The formation of the aIgE adsorbed layer was monitored using centrifugal particle separation (CPS) and centrifugal field flow fractionation (CF3) experiments, which allowed to determine the size changes and the adsorbed mass. Particle sizes were also measured with DLS, used both as stand-alone instrument and coupled to CF3 (CF3-DLS). The complementary information obtained from the CPS and CF3-DLS measurements allowed the estimation of the density of the aIgE shell. The proteins immobilized at the surface fully retained their activity, as proven by the reactions between the functionalized PS-aIgE particles and immunoglobulins of type E (IgE) dispersed in suspensions prepared on purpose.
Highlights
Protein adsorption on any foreign surface exposed to a protein solution is a spontaneous, highly favorable and often irreversible process, subject of long-standing debate, principally because of the wide range of adsorption mechanisms adopted by diverse protein structures and for the wide variety of possible surfaces either in terms of shape and chemical composition
In order to correctly interpret the results of surface coating, a good characterization of the PS particle, as they are available from the supplier, is of fundamental importance. 0.1% w/v suspensions were prepared from the commercial samples using as dilution media a phosphate buffer (PB) at pH 8
The values, ζ = −62 mV and ζ = −75 mV observed in dilutions in the mobile phase (10 mM PB, pH8) respectively for the PS100 and PS196, were assumed as an indication of stable suspensions, which should preserve them from aggregation phenomena during their analysis
Summary
Protein adsorption on any foreign surface exposed to a protein solution is a spontaneous, highly favorable and often irreversible process, subject of long-standing debate, principally because of the wide range of adsorption mechanisms adopted by diverse protein structures and for the wide variety of possible surfaces either in terms of shape and chemical composition. The adsorption process is a mix of physical (electrostatic, hydrophobic, dispersion, etc.) and chemical interactions (hydrogen bonds, π–π stacking), that might lead to the formation of real covalent bonds between the protein molecules and the surface [1]. We focus on the adsorption of immunoglobulin proteins onto synthetic polymer nanoparticles because of their potential for biomedical applications [3]. Polymeric nanoparticles offer a versatile platform for developing imaging and drug delivery applications due to the possibilities of being modified with functional moieties ranging from small chemical groups to large macromolecules. Immunoglobulins (Igs), known as antibodies (Abs), have an extraordinary specificity and binding affinity toward antigens (Ags), rendering this special protein class of crucial interest for designing immunoassays, producing biosensors, chromatographic immuno-affinity columns and for biomaterials development [4]. IgE are only found in mammals and mediate allergic response that can lead to powerful immune reactions, especially in the case of allergy classified as of “type 1” (hypersensitivity) [7,8]
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