Una aproximación psicolingüística

The primary aim of this thesis is to develop and apply RAS to monitor, in real time, electron transfer induced conformational change in Cytochrome P450 Reductase (CPR) adsorbed onto an Au(110) surface. In order to understand conformational

events using RAS the protein must first be adsorbed onto the Au(110) substrate surface, therefore understanding the adsorption process is vital to the success of the research.

The interaction of biological molecules with solid surfaces is an area of huge interest. Understanding this interaction has application in biology, medicine, food processing and the area which has had rapid growth, the development of biomedical materials [27]. It is clear that monitoring of the adsorption of biological materials on solid surfaces is crucial, creating opportunities for the development of new adsorption monitoring techniques.

A Quartz Crystal Microbalance (QCM) is a nanogram sensitive device used to measure adsorbed mass per unit area. It utilises the resonant behaviour of the piezoelectric quartz crystal and the fact that the resonant frequency of the crystal changes when the mass of the crystal changes, therefore as mass is adsorbed onto the crystal accurate measurement of the frequency changes allows very small masses to be measured.

This was only possible after 1959, when Sauerbrey demonstrated the linear relationship of frequency changes of an oscillating material are related to the mass adsorbed on the material [28].

Δm=(C/n)Δf

where Δf is the frequency change, Δm is the mass adsorbed, n=1,3… is the overtone number and C is the mass sensitivity constant given by:

C=(t

ρ

)/f

where tq is the thickness of quartz, ρq is the density of quartz, f0 is the resonant frequency of the crystal in Hz and C= 17.7 Hz ngcm-2 for a 5 MHz crystal.

The Sauerbrey relationship is accurate as long as three assumptions are held, i) the adsorbed mass is small relative to the mass of the quartz crystal, ii) the mass adsorbed is rigidly adsorbed and iii) the adsorbed mass is evenly distributed over the

active area of the crystal [29]. Initial studies using QCM’s were carried out in gaseous or vacuum environments where metal oxidation [30], gas adsorption [31], dry etching and catalytic reactions [32] were the major areas of research.

The technique expanded into the liquid phase after Nomura and Okuhara [33] demonstrated that, if one face of the sensor is exposed to the liquid and the other to the gas phase it is possible to operate a QCM in the liquid phase. This expansion of the QCM capabilities created not only new experimental opportunities, but also introduced difficulties in the interpretation of the frequency changes. QCMs operating in the liquid phase introduced viscous and elastic contributions to the frequency change [29], which violates the assumption in the Sauerbrey relationship that the adsorbed mass is rigidly adsorbed, questioning the validity of the Sauerbrey relationship in the liquid phase.

The sensitivity of the QCM to the viscoelasticity of adsorbed layers can be the reason for experimentally observed deviations from the Sauerbrey linear relationship between frequency change and adsorbed mass [34]. The viscoelastic adsorbed layer brings the adjacent liquid layer into motion as a result of the crystal oscillating, which causes inaccuracies in the Sauerbrey relation. Voinova et al. [35] used continuum mechanics to show that the mechanical properties of viscoelastic materials are usually related to energy storage and dissipation processes resulting from the balance between applied stress and the ensuing relaxation process in the material. To use a QCM in the liquid phase, applying it to non-rigid overlayers, it is necessary to monitor the damping of the crystal oscillation or its dissipation factor along with the frequency change. The dissipation factor is defined as:

D=E

/(2πE

)

where D is the dissipation, EDissipated is the energy dissipated during one oscillating cycle and EStored is the energy stored in the oscillating system. Dissipation is the sum of various energy dissipating subsystems in the composite oscillator which can reveal the dissipative properties of the viscoelastic overlayers.

The Sauerbrey relation is not obeyed by non-rigid adsorbed layers. This causes a dissipation shift which has been shown to violate the Sauerbrey relation. Consequently just using this relation and measuring frequency alone would greatly underestimate the adsorbed mass of sufficiently non-rigid adsorbed layers [29]. It has been suggested that the failure of the Sauerbrey relationship derives from two sources. The first source is related to the propagation of the shear acoustic wave in the viscoelastic film. A thin and rigid film, adsorbed on the crystal acts as ‘dead’ mass, whereas viscoelastic or thicker films constitute a coupled oscillation for which the change in frequency is not proportional to the change in mass. The second source is related to the definition of mass. At solid liquid interfaces, mass is often defined as molar mass or ‘dry’ mass. In QCM measurements liquid may couple as additional mass through direct hydration or entrapment in cavities of the adsorbed film [36].

Voinova et al. [35] explained that the simplest way to account for dissipative losses in viscoelastic layers was to introduce a shear viscosity coefficient η and a shear elasticity modulus μ within one of the two basic models “Maxwell” or “Voight”. The Voight model, unlike the Maxwell model, does not describe flow at a steady rate, instead the viscoelastic element is described by a complex shear modulus of which the real part (the storage modulus) is independent of frequency and the imaginary part (the loss modulus) increases linearly with frequency [35]. The two models are applicable when using different solutions. The Maxwell model is usually applied to polymer solutions with low shear rate that demonstrate purely liquid like behaviour, whereas the Voight model is applied for polymers that conserve their shape and do not flow [35].

The analysis of viscoelastic layers adsorbed on resonant crystal surfaces has lead to the development of commercial Quartz Crystal Microbalance with Dissipation monitoring (QCM-D). The first QCM-D devices were developed by Q- sense in 1996. These QCM-D devices have been used to monitor protein adsorption, and it has been shown that when a monolayer or a multilayer of biomolecules are adsorbed in the liquid phase the energy dissipated increases. The monitoring of the change in dissipation as well as frequency, at multiple harmonics of resonant frequencies in the millisecond timescale, can be modelled with theory to extract

meaningful data from the experimental data, such as mass, adsorbed layer thickness, viscosity or storage modulus [29]. This allows a much broader characterisation for viscoelastic systems that do not obey the linear Sauerbrey relation, than QCMs which monitor frequency changes alone. Biomedical material development is an area that has benefitted greatly from the development of QCM-D. An important factor in biomaterial development is the biocompatibility of the material. The ability of QCM-D to measure accurately the amount of protein adsorbed onto different surfaces provides insight into whether the material inhibits or promotes protein adsorption, which plays a crucial role in the development of biomedical material development and selection. Dissipation losses during protein adsorption have been shown to originate from several sources: interactions at the protein/substrate interface, interactions at the protein/liquid interface and/or processes within the protein layer (proteins plus trapped liquid) [37].

Figure 2.7: Q sense E4 QCM-D module. (Reproduced from www.q-sense.com).

In the research reported in this thesis, protein adsorption has been monitored uses a Q-Sense E4 Module QCM-D (figure 2.7). This is a 4 chamber module capable of monitoring the frequency and dissipation changes on four quartz crystals

at once. The frequency and dissipation changes are monitored using Q-Soft 401 software and analysis of layer thickness, viscosity and mass can be modelled and fitted to experimental data using Q-Tools 3.0 software. The frequency and dissipation changes can be monitored to an accuracy of 1 Hz and 1x10-6 units of dissipation respectively, for the protein solutions used in this research [38].

The quartz crystals are ‘AT’ cut which provides favourable stability properties and the crystal oscillates in a purely shear mode [39]. The crystals comprise a thin AT cut quartz disc sandwiched between two electrodes, which are used to establish an electric field across the crystal. The electrodes can be made of any metal material and in the case of this research gold is used, which is commonly used due to its inert nature. A polycrystalline Au coated sensor is shown in figure 2.8.

Figure 2.8: Q-Sense Au coated Sensor. (Reproduced from www.q-sense.com).

The E4 modules (figure 2.9) are temperature controlled and for the experimental research in this thesis the temperature was kept at 20˚C. All crystals were cleaned and UV-Ozone treated before use. Once placed inside the flow module ultra pure water was first pumped over the sensor until a steady trace for both frequency and dissipation was reached, which was then repeated with buffer solution to produce a baseline before a known concentration of protein was added to the solution, which was then pumped through the cell whilst the frequency and dissipation changes were monitored. It is important to rinse the cell thoroughly with buffer solution before the Δf and ΔD data are analysed, to ensure excess protein is rinsed off the crystal surface.

Figure 2.9: Q-Sense flow module with gold coated quartz sensor crystal in position. (Reproduced from www.q-sense.com).