**3. Protein adsorption at the solid/liquid interface**

in a phospholipidic bilayer were widely investigated [29,30]. Well known is the sensing sys‐ tem built by the incorporation of glutamate dehydrogenase or choline oxidase into fatty acid LB films through an adsorption or an inclusion process which consists of sandwiching the protein molecules between two LB layers [31]. The molecules of glutamate dehydrogenase adsorbed on the surface of behenic acid LB films work as a protective screen against the re‐ arrangement of the multilayers induced by diffusion of the alkaline buffer inside the struc‐ ture; on the contrary, the choline oxidase molecules operate as an accelerating factor of the

The physiological activity of transmembrane proteins, however may depend on the physico‐ chemical properties of neighboring phospholipids. Such dependence has been demonstrated in the case of, among others, hydroxybutyrate dehydrogenase, Ca2+-ATPase and melibiose‐ permease. Moreover, all integral membrane proteins are surrounded by a layer of phospho‐ lipids, the annular region, which provides the adequate lateral pressure and fluidity to seal

All the amphiphilic molecules are potentially surface active agents and substantially mon‐ olayer-forming materials. It is possible to find a discussion on the range of a large variety of amphiphile compounds able to form insoluble monomolecular films [33]. Due to the synthesis of biomimetic membranes, the most important types of amphiphilic molecules are fatty acids, phospholipids and glycolipids. Cholesterol as a type of steroid extremely abundant in the cell membrane, can also form insoluble monolayers but it is generally more studied mixed with other phospholipids [34-36] in order to its implication in the for‐ mation of lipid microdomains. Figure 4 presents the examples of the principal structures of these different types of lipids. The amphiphilic nature of biological surfactants is re‐ sponsible for their aggregation at the air/water interface. Their affinity for the air/water in‐ terface is determined by the physico-chemical properties of the hydrophilic and hydrophobic parts. The monolayer forming abilities of the amphiphiles is dependent on the balance between these two opposite forces, which are determined by the size of the hydrophobic tail group (i.e. the alkyl chain) and the strength of the hydrophilic head group (i.e. size, polarity, charge, hydration capacity) [6]. If the equilibrium between hy‐ drophilic and hydrophobic part of molecule is disturbed, the material dissolve in the sub‐

Most of the lipidic cell membrane components are composed of a zwitterionic head group at pH 7.0 (phospholipids) or contain a highly hydrophilic polar group (glycolipids), and a hy‐ drophobic part which is constituted by two hydrocarbon chains per molecule and drastical‐ ly reduces the water solubility of the complete lipidic membrane molecule. Consequently, many components of cell membranes form insoluble monolayers at the air/water interface since the lipid concentration in the aqueous subphase is negligible, and some of them may

the membrane during the changes in the protein during transport events [32].

structural lipidic reorganization induced in the same conditions.

**2.1. Main membrane lipids**

42 State of the Art in Biosensors - General Aspects

phase and is not able to form a stable monolayer.

be built up into multilayer films by Langmuir-Blodgett deposition.

Protein adsorption at solid/solution interface has been a research focus for more than three decades due to its importance in the development of biocompatible materials, various bio‐ technological processes, food and pharmaceutical industries, and promising new areas such as biosensor, gene microarray, biochip, biofuel cell and so on. In order to control and manip‐ ulate protein adsorption, the mechanisms which govern the adsorption process need to be well understood.

The effect of variables like pH, temperature, the ionic strength, the properties of the protein and the surface, the nature of the solvent and other components on protein adsorption have been studied. Protein adsorption is a very complex process (Figure 5), which is driven by different protein-surface forces, including van der Waals, hydrophobic and electrostatic forces. Attention is also paid to the structural rearrangements in the protein, dehydration of the protein and parts of the surfaces, redistribution of charged groups in the interfacial layer and the role of small ions in the overall adsorption process. Protein adsorption also depends on the chemical and physical characteristic of the surface. Conformational changes in the protein can greatly contribute to the driving force for adsorption. Proteins are highly or‐ dered structures (i.e., states of low conformational entropy). Partial or complete unfolding of the protein on the sorbent surface leads to an increase in conformational entropy, which can be the driving force for protein adsorption. To assess the tendency of proteins to unfold on surfaces, it is important to have a clear picture of protein stability.

**Figure 5.** Protein adsorption; a/ adsorption in membrane, b/ classic adsorption on solid, c/ encapsulation in porous material, d/ protein with LbL assembly

Now is generally accepted that the adsorption behavior of proteins at relatively high con‐ centrations often does not follow the true equilibrium isotherm because the slow relaxation of nonequilibrium structure leads to multilayer build-up [37, 38]. Such behavior can be monitored by atomic force microscopy (AFM) measurements, neutron reflection, dual polar‐ ization interferometry, circular dichroism, and Fourier transform infrared attenuated total reflectance (FTIR/ATR) [39] as well as other techniques.

The process of macromolecular multilayer adsorption, is still too complicated to be effective‐ ly modeled by kinetic models. At low concentrations, an interfacial cavity kinetic model has been used to characterize monolayer or submonolayer protein adsorption with surface-in‐ duced structural transitions [39].

The effect of variables like pH, temperature, the ionic strength, the properties of the protein and the surface, the nature of the solvent and other components on protein adsorption have been studied. Protein adsorption is a very complex process (Figure 5), which is driven by different protein-surface forces, including van der Waals, hydrophobic and electrostatic forces. Attention is also paid to the structural rearrangements in the protein, dehydration of the protein and parts of the surfaces, redistribution of charged groups in the interfacial layer and the role of small ions in the overall adsorption process. Protein adsorption also depends on the chemical and physical characteristic of the surface. Conformational changes in the protein can greatly contribute to the driving force for adsorption. Proteins are highly or‐ dered structures (i.e., states of low conformational entropy). Partial or complete unfolding of the protein on the sorbent surface leads to an increase in conformational entropy, which can be the driving force for protein adsorption. To assess the tendency of proteins to unfold on

**Figure 5.** Protein adsorption; a/ adsorption in membrane, b/ classic adsorption on solid, c/ encapsulation in porous

Now is generally accepted that the adsorption behavior of proteins at relatively high con‐ centrations often does not follow the true equilibrium isotherm because the slow relaxation of nonequilibrium structure leads to multilayer build-up [37, 38]. Such behavior can be monitored by atomic force microscopy (AFM) measurements, neutron reflection, dual polar‐ ization interferometry, circular dichroism, and Fourier transform infrared attenuated total

material, d/ protein with LbL assembly

44 State of the Art in Biosensors - General Aspects

reflectance (FTIR/ATR) [39] as well as other techniques.

surfaces, it is important to have a clear picture of protein stability.

Many studied projects have focused on the effect of various modifications in the adsorption systems, including surface modification [40], protein modification [41], the use of saccha‐ rides [42] and surfactants [43], and adjustment of solvent conditions such as ionic strength and pH [38, 44], for the purpose of either reducing or promoting protein adsorption.

Adsorption capacity of cytochrome *c* on chelated Cu2+ bead was demonstrated to be depend‐ ent on the buffer type with the observed adsorption in the order phosphate >*N*-(2-hydrox‐ yethyl)-piperazine-*N*′-2-ethanesulfonic acid >morpholinopropane sulfonic acid >morpholinoethane sulfonic acid >tris(hydroxymethyl)-aminomethane hydrochloride (Tris-HCl) [45]. Vasina and Dejardin reported that the adsorption of α-chymotrypsin on musco‐ vite mica was depressed by increasing the concentration of Tris-HCl buffer at pH 8.6, close to α-chymotrypsin's isoelectronic point [46]. Phosphate buffered saline (PBS) is the most commonly used buffer at the pH range close to 7, since it is reported to be able to stabilize protein structure in bulk solution environment in most cases [47]. The behavior of PBS buf‐ fer is particularly complex in adsorption studies due to the various types of phosphate ions present and the tendency of these ions to adsorb competitively and/or to form complexes ei‐ ther with the proteins or with the surfaces.

It is also well known that protein size and net charge have significant effects on adsorption. Changes in protein secondary structure are frequently monitored as indications of denatur‐ ing. Denaturing upon adsorption is important in many applications such as implants, biofu‐ eling. Quantification of secondary structure is sensitive to the peak assignments.

Adsorption of a protein to a surface may induce conformational changes in the protein. The degree of conformational changes is determined by a combination of the native stability of a protein, the hydrophobicity and the charges of the protein and the sorbent surface. Protein adsorption can be driven by a conformational entropy gain especially if adsorption is endo‐ thermic. This entropy gain can arise from the release of the solvent molecules from hydro‐ phobic patches on the protein surface. Loses of translation entropy of the protein may play a minor rule [48].

Norde et al. [49] study the thermodynamics for adsorption of human serum albumin (soft) and ribonuclease (hard) on polystyrene surface (hydrophobic). That was reported that a net increase in entropy on a like-charged polystyrene surface drives the adsorption process for both proteins. The entropy increases because hydrophobic parts of the polystyrene surface gets dehydrated and structural changes in the proteins allowed the molecule feel free. Pro‐ teins with low Gibbs energy of denaturation (i.e., a protein with low native-state stability is called soft protein) are driven by entropy gains associated with the breaking down of secon‐ dary and tertiary protein structure.
