SAW-biosensor

The biosensor system used (S-Sens® K5 System, Biosensor GmbH, Bonn, Germany) is based on surface acoustic waves. The waves are produced through inverse piezoelectric effect on the surface of quartz chip covered with a thin layer of gold. Viscosity changes and mass loadings on the chip’s surface affect the phase and amplitude of the acoustic waves, which are transformed back into electrical signal through direct piezoelectric effect [146, 147.]

. The biosensor employs special shear waves of Love type to achieve high sensitivity in detecting interactions that take place in solution.

Love waves, proper to a very thin layer of substance, coupled with displacement of matter parallel to the interface solid-liquid permit a high conservation of wave energy making them very sensible to surface effects (i.e. mass loading and viscosity changes) and lowers the noise level in the signal.

EXPERIMENTAL PART 149

Figure 87. Principle of the surface acoustic wave (SAW) biosensor. An electric field is converted into a mechanical wave through a piezoelectric effect. When the surface mass loading and/or liquid viscosity change, the wave will change its amplitude and phase and it is converted into electrical signal for processing. Δφ represents the phase shift and ΔA the amplitude difference.

The central element in the instrument structure is the quartz sensor chip containing five sensor elements, where the surface acoustic waves are produced, allowed to travel along the surface and transformed back into electrical signal for analysis[148-150.]. Mass loading on the chip’s surface and liquid viscosity changes in the liquid running on it will induce modifications in the amplitude and phase of the acoustic wave. In particular, mass loading will cause phase shifts, whereas viscosity changes produces modifications in both phase and amplitude [110, 112, 151-154.]

. The quartz chips are covered with a thin gold coating, used for immobilization of different compounds containing sulphur (i.e. thiol groups). In the present work 16-mercaptohexadecanoic acid was used as a linker.

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Figure 88. S-Sens® K5 Biosensor System. The instrument has 2 main parts:

autosampler and main biosensor containing the quartz- sensor system, electronics and syringe pump. The quart-sensor system is complementary with the SiO₂ chip, covered with a thin layer or gold forming 5 different channels. On gold reacts with -SH group of 16-mercaptohexadecanoic acid to form Self Assembled Monolayer (SAM).

The chip is kept overnight in a 10 μM solution of 16-mercaptohexadecanoic acid (5.77 mg acid in 2 mL chloroform) to form a so called self assembled monolayer (SAM) after the reaction of the thiol groups with the gold surface. The hydrocarbonated chains align parallel one to anther due to hydrophobic effect, and the carboxyl groups orient themselves at the free surface. Different compounds containing free amino groups can be covalently immobilized by forming a peptide bond with these groups. However, the reactivity of the carboxyl groups must be enhanced for the formation of the peptide bond. This can be achieved by modifying the carboxyl into an active ester.

EXPERIMENTAL PART 151 covalently bound protein (in this case antibody) via ammino group; e. - blocking of the remaining free sites with ethanolamine; f. - affinity bound partner (in this case peptide antigen) to be quantified by Biosensor instrument. [110, 112, 151-154.]

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After the chip is placed in the instrument under air free liquid, first injection will contain a mixture of 200 mM EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and 50 mM NHS (N-hydroxysuccinimide) solutions, in a volumetric ratio of 1:1. The resulting hydroxysuccinimide ester reacts with

EXPERIMENTAL PART 152 the compound to be immobilized (peptide, protein or antibody), injections

of different concentrations and volumes being used. The free remaining active ester groups are blocked with 1M solution of ethanolamine, pH 8.5.

By injecting solutions of different compounds, their affinity to the immobilized partner can be observed. To elute the affinity bound compounds from the chip the pH of the buffer is changed to 2,5 from 7.

This was possible by injecting acidic solutions as 0,1 % trifluoroacetic acid, 2 % formic acid, glycine 50 mM (adjusted to pH 2.0) or HCl 0.1 M (pH 1.0).

The gold coated chips can be reused after a thorough cleaning step with Piranha-solution (H2O2 30 % / H2SO4 98 % 1:1) for 45 min, that would each all organic compounds on the surface. The quantity of bound compound to the chip can be evaluated from the measured phase shift (recorded with the K15 software) using the following sensitivity calibration factor:

Figure 90. Biosensor sensogram used for the determination of KD [110, 112, 151-154.]

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EXPERIMENTAL PART 153 For KD determinations, Aβ (1-40) was immobilized on the chip and the

affinity was investigated at different concentrations of Aβ-autoantibody found in solution, in a sequential series of injections, each of them followed by an acidic elution step (regeneration of the antibody surface).The measurements were recorded and the instrument was controlled by the Biosense K12 software. The series of injections from autosampler was programmed using the SequenceMaster 6.0 software.

The binding curves were analyzed with Origin Pro 7.5 and its engine was also employed by FitMaster for fitting the binding curves according to a mathematical model that considered a remaining residue affinity bound to the surface even after the acidic elution step, as well as for determining the dissociation and association reaction rates (koff and kon) from the shape of the fitted curves. A linear best fit was applied using the equation Kobs = koff + kon * C. The average koff [Unit in sec-1] equals the intersection with the y-axis. The slope of the fitted straight line is a measure of the kon rate [Unit in Conc-1 sec-1]. The KD value was calculated with KD = koff /kon[110, 112, 151-154.]

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