Application Examples

Three examples of possible applications of TIRE in the area of protein adsorption are presented here. The first example shows a potential for microstructural analysis of protein monolayers adsorbed onto metal films. The second example addresses problems with potential industrial rele vance. It also demonstrates one of the TIRE strengths - the possibility of measurement in opaque solutions. The final example illustrates a major TIRE advantage: extremely high sensitivity under SPR conditions. The experiments were performed with spectroscopic ellipsometers (M-88 and VASE)fromJ. A. Woollam; dataanalysiswereperformedwith theWVASE32 software (J.A. Woollam Co).

In the first application example, TIRE is employed both in spectroscopic mode (in the wavelength range 400-1,200 nm) and in kinetic in situ mode for investigation of ferritin adsorption onto thin semitransparent gold films with a thickness of 43 nm. The measurements were performed at two angles of incidence: 60° and 65.5°. The second angle is optimized for the SPR effect for the gold layer thickness used. In this configuration the SPR phenomenon gives a large enhancement of the protein film thickness sensitivity. Adsorption of a monolayer of ferritin results in a change in A of more than 90° (Poksinski and Arwin 2004) compared to 3° in similar ordinary ellipsometric measurements on gold substrates (Martensson et al. 1995). This large sensitivity demonstrates a large potential for sensor applications. The 60° angle of incidence was included for comparison between SPR-enhanced TIRE and ordinary TIRE. Ellipsometric spectra before and after adsorption are presented in Fig. 8. Notice the difference in sensitivity between the two angles of incidence and the very large change in A close to the resonance (e. g., at a wavelength of 850 nm). The ferritin layer refractive index was then modeled with a Cauchy dispersion model, resulting in a layer thickness of 9.2 nm, which is in good agreement with the dimen-

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Fig. 8. Spectroscopic A data measured in TIRE mode on a thin gold film before and after the addition of ferritin to a final concentration of 1 mg/ml in a TIRE cell, recorded at the angles of incidence of 60° and 65.6°. The cell contains 2 ml of phosphate buffered saline (PBS). Reprinted with permission from Poksinski and Arwin (2004; copyright Elsevier)

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Fig. 9. Best fit to experimental TIRE data on a ferritin layer on a thin gold film. The insert shows the model used for the calculations. Reprinted with permission from Poksinski and Arwin (2004; copyright Elsevier)

sion of the ferritin molecules (Martensson et al. 1995). The fit is shown in Fig. 9 with the optical model as an insert. Further details, including the 1.2-nm interface layer, are described by Poksinski and Arwin (2004).

In the second example we demonstrate that TIRE can be used as sensor principle for measurements in a nontransparent ambient with the potential for industrial applications like in situ monitoring of growth of layers on the inside of pipelines. Adsorption from milk on gold, iron, and chromium surfaces, and subsequent cleaning with sodium hydroxide was chosen as a specific application example (Poksinski and Arwin 2003). The TIRE system used in this example was based on a rotating polarizer, multiwavelength el-lipsometer (M88, J.A. Woollam Co.) equipped with a half-cylindrical prism configuration. The experiments were performed at an angle of incidence of 64° in the wavelength range 409-762 nm. The kinetic data reported here were recorded at 762 nm. Glass slides with 43-nm-thick layers of vacuum-deposited gold were exposed to water, milk, and NaOH in the order as presented at the bottom of Fig. 10. Adsorption and desorption onto the metal layer surface can be observed as changes in the ellipsometric angle y and A, as exemplified by A versus time in Fig. 10. To obtain a more detailed view of the adsorption process, spectroscopic data were taken at certain points (marked A-E in Fig. 10) during data acquisition. Spectroscopic data for an experiment using a gold layer are presented in Fig. 11. One should notice that the SPR effect was not fully utilized in this example due to the spectral range limitations of the ellipsometer used.

To further illustrate the large sensitivity obtainable, we show in Fig. 12 the kinetics of adsorption ofhuman serum albumin (HSA) on a thin gold film.

Protein Adsorption Kinetic

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Fig. 10. Changes in the ellipsometric parameter A during adsorption from milk and subsequent cleaning on a gold surface measured at a wavelength of 762 nm and an angle of incidence of 64°. Changes in the liquid in the flow are marked with letters A-E. Reprinted with permission from Poksinski and Arwin (2003; copyright Elsevier)

Fig. 10. Changes in the ellipsometric parameter A during adsorption from milk and subsequent cleaning on a gold surface measured at a wavelength of 762 nm and an angle of incidence of 64°. Changes in the liquid in the flow are marked with letters A-E. Reprinted with permission from Poksinski and Arwin (2003; copyright Elsevier)

Wavelength (nm) Wavelength (nm)

Fig. 11. Spectral ellipsometry changes due to adsorption from milk and subsequent cleaning. Spectroscopic data were measured in the wavelength range 409-762 nm at an angle of incidence of 64° (left). A magnification of part of the spectrum is also shown (right). The letters A-E refer to spectra recorded at times as shown in Fig. 10. For clarity, only A is presented. Reprinted with permission from Poksinski and Arwin (2003; copyright Elsevier)

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Fig. 12. A versus time in TIRE mode during adsorption of human serum albumin on a 45.8-nm gold layer in phosphate-buffered saline buffer (pH 7.4). The measurements were performed at an angle of incidence of 70° and a wavelength of 650 nm

HSA is a relatively small protein, and from the evaluation of ellipsometric data we find a layer thickness of 2.7 nm. Even for such a thin layer, we measure a change of 72° in A, with a noise level corresponding to an equivalent thickness of less than 1 pm.

The application examples mentioned earlier are just a few of the various experimental possibilities offered by TIRE. We would also like to point on two interesting applications for further studies. It can be observed that with appropriate tuning of the system (i. e., the combination of the components set up for the largest possible SPR effect) one can obtain sensitivities of several orders of magnitude larger in comparison to currently available similar techniques (e.g., ellipsometry, SPR). The large sensitivity of TIRE for thin layers opens up a pathway to analyze in detail the structure of thin protein layers. With further development of the models for the protein layers, mass distribution perpendicular to the surface may be resolved. Another application of TIRE can be a parallel study on, for example, protein adsorption performed simultaneously with TIRE and traditional ellipsometry with use of two ellipsometers on the same sample under the same measurement conditions. This will increase the information obtained in an experiment, and due to the specific response of each technique should allow more ac

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