Electrochemical Characterization of Bovine Serum Albumin Adsorption

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Blood-material interactions are critical for the performance and biocompatibility of biomedical devices implanted in thousands of patients every day. When an implant is introduced into the body, protein adsorption and activation of complement proteins occur on the foreign surface [1-3] and a series of interactions happen [4], firstly, water molecules reach the surface of the implant and build a water shell around it on a time scale that is of the order of nanoseconds. The interaction of the water molecules with the surface of the implant is dependent on the surface properties of the kind of material the implant is made of.

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This property also determines which proteins and other molecules will adhere following the formation of the hydration shell. Secondly, from a few seconds to hours after implantation, the implant becomes covered in an adsorbed layer of proteins primarily present in the extracellular matrix. Thirdly, cells eventually reach the ‘surface’ interacting through the protein covering; thus cell-surface interactions can be described as the interaction between cells and surface-bound proteins. This stage occurs from as early as minutes or up to days after implantation [4]. As the time after material implantation increases from a few hours to several days, adhesion and migration followed by differentiation of cells occur. This third stage is influenced by biological molecules (extracellular matrix proteins, cell membrane proteins and cytoskeleton proteins); the biophysical environment and the evolving material physicochemical characteristics at the surface (chemistry, nano and micro-topographies); and the released soluble products from the material and its micro-structure (porosity) [5, 6]. The fourth and final stage in the useful life of the implant, which can last from a few days (biodegradable suture) up to several decades (total hip replacement), is the continuing development of the early implant stages. Adverse responses (clots or fibrous capsule formation, for example) and implant failure can occur “ processes that can result from material degradation or mineralization [4]. Thus, the initial protein adsorption onto a biomaterial surface plays a key role in how the body responds to an implanted biomaterial.

Surface modifications are often intentionally made to the surface of implants in order to improve their functionality and biocompatibility. The future development of improved biomaterials and novel coating techniques looks towards promoting fast healing and integration to prevent implant failure.

Protein adsorption is crucial in cell adhesion for tissue regeneration, even though frequent biofouling is seen as an undesirable process in some applications. Protein adsorption on biomaterial surface is therefore of interest for bioengineering research

Human serum albumin (HSA), a globular protein, is the most abundant component of many biofluids, serving as the transport of various metabolites and the regulation of the osmotic pressure [7, 8]. Bovine serum albumin (BSA) is used for protein adsorption studies since its structure is similar to the HSA structure [9], with nearly the same isoelectric point [10, 11] and molecular weight [10] and also because of its low-cost. Albumin is important in biomedical applications as it has been identified on implants ex vivo [12, 13].

There are a few studies available in the literature on protein adsorption on nickel-titanium materials (nitinol) [14]. One of these, by Shabalovskaya on polished, chemically-etched, heat treated and electropolished nickel-titanium surfaces [15], suggested that the amount of albumin adsorbed should be proportional to the nickel surface content of the alloy. Michiardi et al. have established a correlation between protein adsorption and surface energy on oxidized nickel-titanium surfaces [16]. Moreover, Clarke et al. found that the surface roughness and hydrophobicity appeared to have no effect on albumin adsorption [17].

Herein, we coated nickel-titanium with two phenolic compounds: pyrogallol and tannic acid, a coating procedure previously developed by Messersmith et al. on different surfaces such as gold, titanium dioxide, stainless steel, and polytetrafluoroethylene [18]. Nickel-titanium has been coated here with phenolic compounds because these bio-inspired compounds have anti-inflammatory properties “ they are anti-proliferative agents for normal re-endothelialisation, nontoxic to mammalian cells [18, 19]. Polyphenols are also able to scavenge reactive oxygen species likely to oxidatively damage endothelial cells. In the presence of a metal, phenolic compounds are capable of forming metal ion complexes producing chelate ions; and on a surface, they build thin adherent films that induce minor changes in surface roughness [18, 19].

In this study, BSA adsorption on phenolic coated nickel-titanium has been investigated by different electrochemical tests. Firstly, the open circuit potential analysis (OCP), this type of measurement provides information about the ‘natural’ corrosion behavior of the system undisturbed by any external voltage or current source and, therefore, in the absence of induced corrosion effects [20]. The corrosion potentials (EOCP) reflect the composite results of electrochemical reactions taking place at the surface/solution interface. Therefore, the variation in the corrosion potential, with immersion time, can be employed to study the electrochemical processes. Secondly, electrochemical impedance spectroscopy (EIS) “ EIS is a non-destructive sensitive technique enabling the detection of any changes occurring at the electrode/electrolyte interface by the study of the impedance of an electrochemical system considered as a function of the frequency of an applied AC wave [20]. The Nyquist plots and Bode diagrams from spectra analyses allow understanding the processes that occur at the metal/electrolyte boundary before, during and after protein adsorption on an electrode surface by applying the equivalent electrical circuit approach. Additionally, the electron transfer-initiated chemical reactions at the interface surface / protein were assessed by cyclic voltammetry.

Nickel ion release has also been studied to investigate the possible diffusion of ions on the adsorbed protein layer. The objective of this paper was to study the protein interaction on phenolic coated nickel-titanium surface and simulate its adsorption in a physiological environment. This information is of clinical importance for future implantable biomedical devices to minimize ailing and failing implants.

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