SAW biosensor

3.8 SAW biosensor

The bioaffinity measurements were performed using an S-Sens K5 Biosensor instrument (SAW Instruments GmbH, Bonn, Germany). The surface acoustic wave (SAW) sensor consists of a thin layer of gold deposited on a piezoelectric substrate (quartz wafer). Two interdigitated transducers (IDT) are situated on each end of the chip. One IDT serves as input and converts an electrical signal (25-500 MHz) through the reverse piezoelectric effect into polarized transversal waves traveling parallel to the sensing surface. The other IDT serves as output, converting the wave back into an electric signal by the piezoelectric effect [222]. When the analyte binds to the ligand immobilized on the surface of the chip, the increase in mass slows the wave's velocity, which is observed as a change in the phase-shift between input and output signals.

This change is recorded versus time and constitutes the SAW phase sensorgram.

Changes in signal amplitude are also recorded and may be processed to yield additional sample information (e.g. on solvent viscosity) [222]. A best-fit of the phase-shift vs. time curve is done according to a theoretical binding model, using the data analysis software supplied with the instrument. This fit yields a pseudo-first-order rate constant called observed rate constant (kobs = koff + Canalyte * kon) for every analyte concentration. When the kobs values are plotted vs. analyte concentrations, the association rate constant (kon) is obtained from the slope of the trend line and the dissociation rate constant (koff) from the intercept. These values are used to calculate the equilibrium dissociation constant, KD = koff* kon-1.

The general workflow used for KD determinations is shown in Figure 91. A self-assembled monolayer (SAM) was obtained on the surface of the gold chip by immersing the chip overnight (12-16 hours) at 25 °C in 10 mM 16-mercaptohexadecanoic acid in chloroform. The chip was then washed with ethanol and dried under a stream of nitrogen or air. These operations were done outside the biosensor. The chip with the SAM coating was next inserted into the instrument and wetted with a 300 μL/min. flow of deionized and degassed water, to expel the air trapped in the flow cell. The flow was then switched to 30 μL/min. for all subsequent operations. All solvents employed were freshly prepared and degassed by sonication

under vacuum prior to use, to prevent introduction of air bubbles in the system, which would cause erratic signals. All sample injections were performed at 23 °C. The reaction time for each sample was determined by the sample volume and flow rate.

Activation of the carboxyl groups was carried out by injecting 150 μL (yielding a 5 min. reaction time) of a 1:1 aqueous mixture of 200 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS).

Glycoprobe immobilization was performed by injecting 150 μL from a 10 μM aqueous glycoprobe solution. This step was followed by capping of the unreacted succinate ester groups with 1 M ethanolamine (pH 8.5). The running flow was then changed from water to binding buffer (PBS, pH 7.5).

Chip cleanup a

b

g

f e

c d

Equilibrium

Regeneration

Immobilisation Association Dissociation

protein glycoprobe

Figure 91. Workflow of the SAW bioaffinity measurements described in this dissertation. The quartz chip is represented in grey and the gold coating in yellow. (a) A self-assembled monolayer (SAM) of 16-mercaptohexadecanoic acid is built on the surface of the chip and the carboxyl groups are activated with EDC/NHS. The glycoprobe is immobilized through its free amino groups and unreacted succinate ester groups are blocked with ethanolamine. (b) Injection of the analyte (carbohydrate-binding protein or peptide) is started. The analyte molecules bind non-covalently to the carbohydrate, which is observed as an increase in signal. (c) An equilibrium arises when association equals dissociation. (d) Injection is stopped and the complex is dissociated by washing continuously with buffer. A decrease in signal may be observed. (e) The regeneration step removes any remaining bound analyte, bringing the signal back to the baseline and leaving the sensor chip as it was right after glycoprobe immobilization, ready to be used for (f) the next analyte injection. (g) At the end of the experiment the chip may be completely cleaned, thus restoring the gold surface and making it suitable for a new immobilization.

For determining dissociation constants, a series of increasing analyte concentrations in PBS were injected in volumes of 150 μL at 30 μL/min., which yielded association times of 5 min. for each concentration. The dissociation time was set for 5 min. Regeneration was performed between analyte injection with an acidic, acetonitrile-based solution (usually 60 % ACN, 0.1 % TFA in water) for 2-5 min, followed by was re-equilibration for 5 min. in running buffer. At the end of the experiment the chip was removed from the instrument and stored overnight at 4 °C in 50 % aqueous ethanol. For prolonged storage the chips were washed with ethanol, dried and kept at 4 °C. After several sets of measurements, when signal degradation or loss of binding were observed, the gold surface was completely regenerated by removing all traces of organic substances. This was achieved through careful treatment with 2 mL from a 1:1 mixture of 96 % H2SO4 and 30 % H2O2 (Piranha solution) in a small clean glass container, for 40-60 min.

The recorded sensorgrams were fitted according to the theoretical 1:1 Langmuir binding model, using the program OriginPro 7.5 (OriginLab Corporation, Northampton, USA) and the FitMaster addin for Origin (SAW Instruments, Bonn, Germany). The extracted observed rate constants (kobs = koff + Canalyte * kon) were plotted versus analyte concentrations. Linear regression of the data points yielded the association (kon) and dissociation (koff) rate constants that were used to obtain the equilibrium dissociation constant KD = koff * kon-1.