Adsorption (not to be mistaken for absorption) is the accumulation and adhesion of molecules, atoms, ions, or larger particles to a surface, but without surface penetration occurring. The adsorption of larger biomolecules such as proteins is of high physiological relevance, and as such they adsorb with different mechanisms than their molecular or atomic analogs. Some of the major driving forces behind protein adsorption include: surface energy, intermolecular forces, hydrophobicity, and ionic or electrostatic interaction. By knowing how these factors affect protein adsorption, they can then be manipulated by machining, alloying, and other engineering techniques to select for the most optimal performance in biomedical or physiological applications.
Relevance Many medical devices and products come into contact with the internal surfaces of the body, such as surgical tools and implants. When a non-native material enters the body, the first step of the immune response takes place and host extracellular matrix and plasma proteins aggregate to the material in attempts to contain, neutralize, or wall-off the injurious agent. These proteins can facilitate the attachment of various cell types such as osteoblasts and fibroblasts that can encourage tissue repair. Taking this a step further, implantable devices can be coated with a bioactive material to encourage adsorption of specific proteins, fibrous capsule formation, and wound healing. This would reduce the risk of implant rejection and accelerate recovery by selecting for the necessary proteins and cells necessary for endothelialization. After the formation of the endothelium, the body will no longer be exposed to the foreign material, and will stop the immune response.
Proteins such as collagen or fibrin often serve as scaffolds for cell adhesion and cell growth. This is an integral part to the structural integrity of cell sheets and their differentiation into more complex tissue and organ structures. The adhesion properties of proteins to non-biological surfaces greatly influences whether or not cells can indirectly attach to them via scaffolds. An implant like a hip-stem replacement necessitates integration with the host tissues, and protein adsorption facilitates this integration.
Surgical tools can be designed to be sterilized more easily so that proteins do not remain adsorbed to a surface, risking cross-contamination. Some diseases such as Creutzfeldt–Jakob disease and kuru (both related to mad cow disease) are caused by the transmission of prions, which are errant or improperly folded forms of a normally native protein. Surgical tools contaminated with prions require a special method of sterilization to completely eradicate all trace elements of the misfolded protein, as they are resistant to many of the normally used cleansing methods.
However, in some cases, protein adsorption to biomaterials can be an extremely unfavorable event. The adhesion of clotting factors may induce thrombosis, which may lead to stroke or other blockages. Some devices are intended to interact with the internal body environment such as sensors or drug-delivery vehicles, and protein adsorption would hinder their effectiveness.
Fundamentals of protein adsorption
Proteins are biomolecules that are composed of amino acid subunits. Each amino acid has a side chain that gains or loses charge depending on the pH of the surrounding environment, as well as its own individual polar/nonpolar qualities. Charged regions can greatly contribute to how that protein interacts with other molecules and surfaces, as well as its own tertiary structure (protein folding). As a result of their hydrophilicity, charged amino acids tend to be located on the outside of proteins, where they are able to interact with surfaces. It is the unique combination of amino acids that gives a protein its properties. In terms of surface chemistry, protein adsorption is a critical phenomenon that describes the aggregation of these molecules on the exterior of a material. The tendency for proteins to remain attached to a surface depends largely on the material properties such as surface energy, texture, and relative charge distribution. Larger proteins are more likely to adsorb and remain attached to a surface due to the higher number of contact sites between amino acids and the surface (Figure 1).
Energy of protein adsorption The fundamental idea behind spontaneous protein adsorption is that adsorption occurs when more energy is released than gained according to Gibbs law of free energy.
This is seen in the equation:
where: ∆ads is net change of the parameters G is Gibbs free energy T is the temperature (SI unit: kelvin) S is the entropy (SI unit: joule per kelvin) H is the enthalpy (SI unit: joule)
In order for the protein adsorption to occur spontaneously, ∆adsG must be a negative number.
Vroman effect Proteins and other molecules are constantly in competition with one another over binding sites on a surface. The Vroman effect, developed by Leo Vroman, postulates that small and abundant molecules will be the first to coat a surface. However, over time, molecules with higher affinity for that particular surface will replace them. This is often seen in materials that contact the blood where fibrinogen will bind to the surface first and over time will be replaced by kininogen.
Rate of adsorption In order for proteins to adsorb, they must first come into contact with the surface through one or more of these major transport mechanisms: diffusion, thermal convection, bulk flow, or a combination thereof. When considering the transport of proteins, it is clear how concentration gradients, temperature, protein size and flow velocity will influence the arrival of proteins to a solid surface. Under conditions of low flow and minimal temperature gradients, the adsorption rate can be modeled after the diffusion rate equation.
Diffusion rate equation
where: D is the diffusion coefficient n is the surface concentration of protein Co is the bulk concentration of proteins t is time
A higher bulk concentration and/or higher diffusion coefficient (inversely proportional to molecular size) results in a larger number of molecules arriving at the surface. The consequential protein surface interactions result in high local concentrations of adsorbed protein, reaching concentrations of up to 1000 times higher than in the bulk solution. However, the body is much more complex, containing flow and convective diffusion, and these must be considered in the rate of protein adsorption.
Flow in a thin channel
and
where: C is concentration D is the diffusion coefficient V is the velocity of flow x is the distance down the channel γ is the wall shear rate b is the height of the channel
This equation is especially applicable to analyzing protein adsorption to biomedical devices in arteries, e.g. stents.
Forces and interactions influencing protein adsorption The four fundamental classes of forces and interaction in protein adsorption are: 1) ionic or electrostatic interaction, 2) hydrogen bonding, 3) hydrophobic interaction (largely entropically driven), and 4) interactions of charge-transfer or particle electron donor/acceptor type.
Ionic or electrostatic interactions The charge of proteins is determined by the pKa of its amino acid side chains, and the terminal amino acid and carboxylic acid. Proteins with isoelectric point (pI) above physiological conditions have a positive charge and proteins with pI below physiological conditions have a negative charge. The net charge of the protein, determined by the sum charge of its constituents, results in electrophoretic migration in a physiologic electric field. These effects are short-range because of the high di-electric constant of water, however, once the protein is close to a charged surface, electrostatic coupling becomes the dominant force.
Hydrogen bonding Water has as much propensity to form hydrogen bonds as any group in a polypeptide. During a folding and association process, peptide and amino acid groups exchange hydrogen bonds with water. Thus, hydrogen bonding does not have a strong stabilizing effect on protein adsorption in an aqueous medium.
Hydrophobic interactions Hydrophobic interactions are essentially entropic interactions basically due to order/disorder phenomena in an aqueous medium. The free energy associated with minimizing interfacial areas is responsible for minimizing the surface area of water droplets and air bubbles in water. This same principle is the reason that hydrophobic amino acid side chains are oriented away from water, minimizing their interaction with water. The hydrophilic groups on the outside of the molecule result in protein water solubility. Characterizing this phenomenon can be done by treating these hydrophobic relationships with interfacial free energy concepts. Accordingly, one can think of the driving force of these interactions as the minimization of total interfacial free energy, i.e. minimization of surface area.
Charge-transfer interactions Charge-transfer interactions are also important in protein stabilization and surface interaction. In general donor-acceptor processes, one can think of excess electron density being present which can be donated to an electrophilic species. In aqueous media, these solute interactions are primarily due to pi orbital electron effects.
Other factors influencing protein adsorption
Temperature Temperature has an effect on both, the equilibrium state and kinetics of protein adsorption. The amount of protein adsorbed at high temperature is usually higher than that at room temperature. Temperature variation causes conformational changes in protein influencing adsorption. These conformational rearrangements in proteins results in an entropy gain which acts as a major driving force for protein adsorption. The temperature effect on protein adsorption can be seen in food manufacturing processes, especially liquid foods such as, milk which causes severe fouling on the wall surfaces of equipment where thermal treatment is carried out.
Ionic strength Ionic strength determines the Debye length that correlates with the damping distance of the electric potential of a fixed charge in an electrolyte. So, higher the ionic strength the shorter are electrostatic interactions between charged entities. As a result, the adsorption of charged proteins to oppositely charged substrates is hindered whereas the adsorption to like charged substrates is enhanced, thereby influencing adsorption kinetics. Also, high ionic strength increases the tendency of proteins to aggregate.
Multi-protein system When a surface is exposed to a multi-protein solution, adsorption of certain protein molecules are favored over the others. Protein molecules approaching the surface compete for binding sites. In multi-protein system attraction between molecules can occur, whereas in single-protein solutions intermolecular repulsive interactions dominate. In addition, there is a time-dependent protein spreading, where protein molecules initially make contact with minimal binding sites on the surface. With the increase in protein's residence time on the surface, the protein may unfold for interaction with additional binding sites. This results in a time-dependent increase in the contact points between protein and surface. This further makes desorption less likely.
Experimental approaches for studying protein adsorption Solution depletion technique This technique measures a concentration change of proteins in bulk solution before and after adsorption, Δcp. Any protein concentration change is attributed to the adsorbed layer, Γp.
Γp = Δcp V/Atot
where: V = total volume of protein solution Atot = Total area available for adsorption This method also requires a high surface area material such as particulate and beaded adsorbents.
Ellipsometry