Handbook of Modern Coating Technologies

Films of biological molecules

Since SE has an excellent thickness resolution (0.01 nm or better) and could be employed in solution ambient, it is suitable for studying biological films while the resolution of SE is close to the dimensions of biological molecules and many biological reactions take place in solu­tions or at the solid/liquid interface. Analyzing the adsorption of proteins or macromolecules on planar solid surfaces is the most commonly investigation task and the classical applica­tion of SE. Based on the information extracted by SE, the properties, formation mechanisms, and surface dynamics of biological molecules films could be obtained, which is helpful to improve the performance of the films such as the accuracy and precision of biosensors.

For the adsorption of ferritin on gold [127], SE data were deconvoluted with a four-phase model. The thicknesses of the ferritin layer, the interface layer which was treated as the mixture of protein and gold based on Bruggeman EMA, as well as the optical properties of the ferritin layer were determined simultaneously. Spaeth et al. [128] studied the change of the thickness and dispersion state of the protein multilayer during the incubation period by using SE. The Cauchy model was used to fit the SE data. The authors found that a single homogeneous pro­tein layer after 5—15 incubations, and the refractive index n_D of the layer was almost a constant of 1.384 6 0.002. The thickness and mass of protein deposition per incubation step was 18.75 nm and 4.74 ng/mm², respectively. Preininger et al. [129] characterized anti-human immunoglobulin G (IgG) Langmuir—Blodgett films by using SE and measured accurately the mean thicknesses of an anti-IgG film on different substrates (glass, PVC-COOH, and aminopro- pyl sol—gel). Castilla-Casadiego et al. [130] applied IR VASE to obtain the thickness and rough­ness of the multilayers of heparin and collagen (COL), which was a bioactive surface coating and could enhance the response of human mesenchymal stromal cells to soluble interferon- gamma. The results from SE are important to optimize the preparation of the multilayers.

McArthur et al. [131] used in situ SE to dynamically monitor the adsorbed protein layers on Cu_12XAl_x (0 # x # 1) substrate. Both fibrinogen (Fib) and albumin adsorbed preferentially onto Cu-rich surfaces. When Al content is higher than 21 at.%, the thickness of the protein adsorption layer decreased significantly. In situ SE was also used to study the adsorption mechanism of yeast cytochrome c (YCC) on SiO₂/Si substrates by Toccafondi et al. [132]. Due to the variations in the Д and Ф spectra induced by the adsorption of proteins on the substrate were generally small, the difference spectra (8Д and &Ф) was introduced. After the interface effect was neglected, a simple three-layer optical model, that is, solution-film- substrate, was built to deconvolute the experimental 8Д and 8Ф spectra. Both in situ SE and the difference spectra were powerful techniques to provide the detailed insights to the pro- tein/surface interaction mechanism.

Silaghi and Zahn group [133,134] studied the optical properties of four DNA base films by using SE in the wavelength range from near IR to ultraviolet. To deconvolute the SE data, a uniaxial model was built for adenine and guanine films while an isotropic model was intro­duced for the thymine and cytosine films and the dielectric properties of the films were obtained, which were very helpful to design and manufacture DNA—based electronic and optoelectronic devices.

SE and surface plasmon resonance—enhanced ellipsometry were used to measure the thickness and to quantify the molecular orientation of biological layers which were com­posed of single-stranded DNA [135]. In the optical model to fit the SE data, a vertical gradi­ent was introduced to describe the optical index of the biolayer, and the thicknesses obtained by SE were identical with them obtained by AFM.

Nabok et al. [20,21] presented examples of applications of the TIRE method to study the DNA hybridization and the registration of low molecular weight toxins. Their results showed that the sensitivity of TIRE is almost 10 times higher than that of conventional external reflection ellipsometry. In situ TIRE with SPR was also used by Balevicius et al. [136] to investigate the binding kinetics between an immobilized glycoprotein granulocyte colony stimulating factor (GCSF) receptor and three genetically engineered ligands [GCSF monomer (mGCSF), (GCSF)₂La (GCSF-homodimer), and stem cell factor-La-GCSF]. This high sensitivity method, TIRE, provided deep insight into the mechanism of interactions between receptors and ligands.

IE has become popular in biological studies [22,137] because it could be used for quanti­fication and visualization of biomolecular interactions. Fig. 2—15 [138] shows the principle of IE schematically, in which the thickness distribution of thin layers (protein patterns) on a solid substrate was visualized by using IE. Jin et al. [137] used IE with a CCD camera to detect antigen—antibody complexes on a silicon substrate. Three types of protein layers, Fib, human serum albumin (HSA), and human IgG, adsorbed on a hydrophobic silicon substrate were studied. For the layers without incubation treatment, the results showed that the thick­ness of IgG layer was thinner than that of Fig layer and thicker than that of HSA layer. Then the samples were incubated in an anti-IgG serum [bovine serum albumin (BSA)] and were measured with IE again. It was found that thickness of the IgG dots increased significantly while the thicknesses of the Fib dots and HSA dots did not increase. It could be inferred that the antigen—antibody complexes of IgG formed. The images obtained from IE results distin­guished clearly the distributions of the proteins. IE was also used to study the competitive adsorption of COL and BSA on chemically modified silicon substrates [139]. In noncompetitive situation, the solution only contained one protein (COL or BSA), the adsorbed amounts of

Image in grayscale

Complex layer \

Ligand layer

FIGURE 2-15 Schematic model of bioprobe based on imaging ellipsometry [138].

COL or BSA on hydrophobic surfaces were two times more than these on the hydrophilic sur­faces. In the mixed solution of BSA (1 mg/mL) and COL (0.1 mg/mL), BSA and COL would be adsorbed competitively because of their different binding affinities. The results showed that, BSA was almost 100% protein adsorbed on the substrate for the hydrophobic surface, and only 6% for the hydrophilic surface. IE directly visualized and quantified the distributions of the proteins and the results agreed with these obtained using AFM well.