A comparative study of protein adsorption on titanium oxide surfaces using in situ ellipsometry, optical waveguide lightmode spectroscopy, and quartz crystal microbalance/dissipation
Introduction
Adsorption of proteins at solid–liquid interfaces is a process of central importance for biomedical technologies, such as biosensors and biochips [1], biomaterials for medical implants [2], [3] and non-fouling surfaces in industry and marine environments. In spite of vast number of studies, it is a process that still is only rudimentarily understood at the molecular level [4], [5]. Further progress in the understanding of protein adsorption is highly dependant on reliable measurement techniques that can produce rich and accurate information about protein adsorption. A large number of techniques, based on different principles such as optical absorption, refractive index changes, radiolabeling, electromechanical microbalances, fluorescence markers and others (see [6] and references therein), have been and are used for this purpose. To obtain a broad and reliable data set for progress in understanding and modeling it is important it is necessary to measure results using common protocols with the different techniques. Few such studies are reported [4], [5], [7], [8], [9], [10].
The main objective of the present work is to perform such a multi-technique study, using three different techniques, two of them based on similar but not identical optical principles, and one based on a piezoelectric, i.e. electro-mechanical, oscillator principle. The measurements were performed with a common protocol, including identically prepared surfaces that were characterized by state-of-the-art surface spectroscopies (X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS)), and with several proteins with different sizes and molecular weights, known to have quite different biological functions. In addition to the primary protein adsorption experiments, additional data were obtained by exposure of the adsorbed proteins to specific and non-specific polyclonal antibodies.
As model systems in this study we used sputter-deposited TiO2 films and three different proteins: albumin, fibrinogen and hemoglobin, all known to interact strongly with most solid surfaces [4], [5], [11], [12]. Albumin is a globular, extracellular protein (dimension: 8×8.7×6 nm [13], Mw: 65 kD) and a main constituent of blood. Fibrinogen is an extracellular protein with an elongated shape (dimension: 45×9×6 nm [14], Mw: 340 kD) important in the context of both blood/surface interactions and cell adhesion. Hemoglobin is a globular, intracellular protein, built up from four myoglobin-like subunits (dimension: 5×5.5×6.5 nm [15], Mw: 64.5 kD) responsible for oxygen transport. For the antibody reaction, polyclonal IgG antibodies were used (dimension: 5.9×13.1×14.3 nm, Mw: ∼150 kD).
The three measurement techniques, briefly described below, that were compared in this study are ellipsometry (ELM), optical waveguide lightmode spectroscopy (OWLS) and the dissipative quartz crystal microbalance (QCM-D) techniques.
Ellipsometry (ELM) is an optical method that has been used extensively for protein adsorption studies by many research laboratories [16], [17], [18], [19], [20]. The method is based on the change upon protein adsorption of the state of polarization of elliptically polarized light reflected at a planar surface. From the changes in the ellipsometric angles (Δ, ψ), the refractive index and the thickness of the film can be deduced [18]. Since the refractive index of adsorbed protein films is always close to n=1.5, which the film thickness can be calculated with quite good accuracy. The main advantage of this method (and the additional techniques discussed below) is that the proteins under investigation require no chemical treatments with markers etc. before use. Also, the measurement procedure is quite fast (on the order of a few seconds) and the results achieved have been shown to agree fairly well with quantitative radioimmunoassay (RIA) methods [8], [10]. The disadvantages with the method include the requirement of reflecting surfaces, and the rather complex theory in cases when extremely detailed information is required or if systems with unknown optical properties are investigated.
Optical waveguide lightmode spectroscopy (OWLS) is based on grating-assisted coupling of light into and guidance within an optical wave-guide layer [21]. By varying the angle of the incident light beam, different guided transverse electric (TE) and transverse magnetic (TM) modes can be excited. The evanescent wave typically extends to a distance of about 200 nm beyond the surface into the adjacent air or solution. If the waveguide is coupled to a liquid cell designed for adsorption studies (e.g. a flow-through cell), the in-coupling angle of the incident light is sensitive to the refractive index and the thickness of the adsorbed film. Provided that the refractive index of the protein film is known, as for ellipsometry, the film thickness can be calculated. Alternatively, if one measures both the TE and TM modes, the refractive index of the protein film can also be calculated [22]. The method is fast and well suited for in situ studies of protein adsorption kinetics [23] with the major advantage of a very high sensitivity of approximately 0.5 ng/cm2 (in the order of 0.5% of an average protein monolayer). The main disadvantage is the fact that only highly transparent surfaces can be investigated.
The quartz crystal microbalance technique (QCM) is a well-established technique for monitoring mass and film thickness in coating equipments in vacuum and for investigations of gas adsorption and surface reactions in the monolayer range [24], [25], via changes in the resonant frequency, f. More recently, the technique has also been proven valuable for studying surface-related processes in liquids [24], [25], including protein adsorption [7], [9], [12], [26], [27]. In contrast to the optical techniques, which are not sensitive to water associated with adsorbed proteins, the f-shift of the QCM is due to the change in total coupled mass, including hydrodynamically coupled water, water associated with the hydration layer of e.g. proteins and/or water trapped in cavities in the film [9], [27]. A recent extension of the technique, called QCM-D, to simultaneously measure changes in the frequency, Δf, and in the energy dissipation, ΔD, of the QCM [28], [29], [30] provides new insight into e.g. protein adsorption processes [12], [26], [27] as well as other surface-related processes [31], [32], [33]. In comparison with OWLS and ELM, one advantage of the QCM technique is that the surface material can be fairly freely chosen without having to consider special properties such as optical transparency or reflectivity, as long as the preferred material can be deposited as a thin film onto the sensor crystals. An apparent complication is that the method measures coupled water. However, this can actually constitute an advantage, especially when combined with water-insensitive techniques.
Section snippets
Substrates
The different substrates used for the three measurement techniques were:
- 1.
Flat glass slides cut out of glass stripes of dimensions 12×250 mm covered by a 100 nm thick, optically reflecting titanium metal film for the ellipsometry study.
- 2.
Planar optical waveguides of dimension 8×12 mm consisting of a Schott AF45 glass substrate coated with a SiO2·TiO2 waveguiding layer of approximately 160 nm thickness, deposited by the sol–gel technique. The optical grating consist of 2400 lines per mm with a
Surface analysis of the clean substrate
The surface-analytical results of the substrates used in the present study have been published previously [34]. Therefore, only a brief summary is given here.
XPS and ToF-SIMS measurements have shown that the outermost few nanometers of the surface are indeed composed of titanium oxide and that there are—apart from adventitious hydrocarbons—no other contaminants present at appreciable concentrations. Most of the surface-analytical studies were carried out on coated waveguides; the results are
Protein adsorption
In an attempt to obtain additional information about how the studied proteins are arranged on the surface, the experimentally determined mass uptakes are compared with theoretical values calculated according to two different models: (i) assuming a close-packed monolayer; and (ii) using an adsorption model that has been described and is generally accepted in the literature: the random sequential adsorption (RSA) model. Table 5 summarizes the relevant properties of the proteins, the theoretical
Conclusions
The three measurement techniques compared in this work, optical waveguide lightmode spectroscopy, ellipsometry and quartz crystal microbalance dissipation are all suitable for the in situ real time monitoring of protein adsorption and protein–antibody interaction kinetics. The combination of the optical techniques with the quartz crystal microbalance proved to be particularly revealing by providing data about both the molar (‘dry’) mass of proteins and water coupled to the adlayers. With the
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