Binding of Aβ-nanobodies to Aβ-peptides using SAW-biosensor

During the past years, different methods such as immunoaffinity analysis by ELISA and Western Blot, isothermal calorimetry and surface plasmon resonance (SPR) were used for the characterization of protein-ligand interaction [149]. In particular, SPR has been long established as a “gold standard” for analysis of biomolecular recognition processes at a biosensor surface and for quantification of different biopolymer interactions [150, 151]. Biosensors have several applications not only in basic research fields, but also in medical diagnostics, food quality control, detection of explosives and drugs, genetic screening and environmental monitoring [152].

The surface acoustic wave (SAW) technology was employed for bioaffinity detection.

The SAW technology, as an alternative to SPR, has recently gained increasing importance owing to its high detection sensitivity [149, 153]. SAW biosensors (e.g.

the S-sens K5 SAW sensor), on the basis of piezoelectric crystals are highly sensitive towards surface effects and are proved to be capable to detect and quantify mass and viscosity changes due to biomolecular interactions [154]. The S-sens K5 SAW biosensor (SAW instruments) consists of a read-out system into which the gold-coated quartz sensor is placed and the signals from the five sensor elements are recorded independently in real-time. The biosensor is operated in Love-wave geometry and is working at two fixed frequencies with a difference of about 0.3 MHz [153, 155]. Using two fixed frequencies, with φ(f1) – φ(f2) = 180° at a frequency range between 130 and 170 MHz, the influence of physical parameters, such as temperature, salts and viscosity on the sensor signal is significantly reduced.

Different surface materials are used for SAW biosensor, mostly involving gold surfaces. Other examples of surfaces are the ones coated with alkanethiols, carboxymethylated dextrans in combination with biotin/streptavidin interactions [156], ZnO surfaces [157], or SiO2 surfaces [158]. In the present work a gold-coated sensor chip was used. On the gold surface, a self assembled monolayer (SAM) was formed and used as a linker for the covalent immobilization of different molecules. For the formation of SAM, the gold-coated sensor chip was incubated overnight in a solution of mercaptohexanoic acid, thereby allowing for later coupling of proteins to the carboxylic groups on the chip. After activation of the carboxylic groups with a mixture of EDC/NHS, an increasing of the signal was observed. This can be explained by conversion of carboxyl groups of the SAM, present on the chip surface, into active N-hydroxysuccinimide ester. The next step was the covalent immobilization of ligand1 (e.g. peptide dissolved in phosphate buffer) on the sensor chip. Unreacted active ester groups were afterwards deactivated by ethanolamine. In order to ensure the near physiological conditions, the solvent was changed from water to phosphate buffer, causing a signal shift. The affinity binding of the analyte to the ligand was characterized by signal increase. The injection of HCl 0.1 M abolished the affinity and the signal decreased. The analyte was then eluted from the gold-chip (Figure 35).

4. Analyte

Activation Immobilization Blocking Affinity Elution

1. EDC/NHS

3. Ethanolamine 2. Ligand

5. Elution HCl, 0.1 M

∆ φ_a

∆ φ_b

Ligand

EDC/NHS

Ligand Ligand Ligand

Analyte

4. Analyte

Activation Immobilization Blocking Affinity Elution

1. EDC/NHS

3. Ethanolamine 2. Ligand

5. Elution HCl, 0.1 M

∆ φ_a

∆ φ_b

Ligand

EDC/NHS

Ligand

Ligand LigandLigand LigandLigand

Analyte

Figure 35: Schematic representation of phase shift of a single sensor element: activation of SAM using EDC and NHS (1), covalent immobilization of the ligand (2), blocking with ethanolamine (3), affinity binding of analyte (with a phase shift ∆φ_a) (4), elution of the analyte using HCl, pH 2 (∆φ_b) (5) (SAM – self assembled monolayer; NHS – N-hydroxysuccinimide;

EDC - N-(3-dimethylaminopropyl)-N-ethylcarbodiimide).

A specific binding between Aβ(1-40) and Aβ(17-28) to Nb_3 and Nb_9 was analyzed as a function of time with SAW biosensor. On the gold-chip surface, a self assembled monolayer (SAM) of 16-mercaptohexadecanoic acid was formed. The SAM was activated using N–hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC). In the first experiment, Aβ(1-40) was immobilized on the activated SAM and the free ester groups were blocked with ethanolamine. The affinity binding of 200 nM Nb_3 to Aβ(1-40) was analyzed at 25 °C in phosphate buffer. Using the sensitivity of the Love-wave sensor (515 ° cm² µg^-1), the bound mass has been calculated.

Further experiments were carried out for monitoring the affinity binding of 200 nM Aβ-nanobodies to 10 µM Aβ peptides. The affinity binding studies using SAW biosensor indicate that Nb_3 and Nb_9 bound specifically to the immobilized Aβ(1-40) and Aβ(17-28). Determination of dissociation constants was performed by increasing the concentration of Aβ-nanobodies (5 – 2000 nM) to the Aβ(1-40) and Aβ(17-28) peptides immobilized on the biosensor surface. After each injection, the unbound nanobody and the biosensor surface were regenerated with HCl 0.1 M. For further evaluation, the sensogram was exported into OriginPro 7.5 program and the

integrated FitMaster was applied. Figure 36 shows the resulting overlay plot and the individual fitting assuming a simple mono-molecular growth model of kinetic analysis of the binding events. Using FitMaster, the pseudo-first order kinetic constants kobs

were determined and plotted versus Aβ-nanobodies (Nb_3 and Nb_9). By applying the equation kobs = koff + kon * C, a linear best fit was obtained and the dissociation constant (KD) was established from KD = koff/kon. The KD values were found in nanomolar range.

0 200 400 600 800 1000 1200 1400 1600 0,00

0 200 400 600 800 1000 1200 1400 1600 0,00

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0 200 400 600 800 1000 1200 1400 1600 0,00

Figure 36: K_D determination of Nb_3 on a Aβ(1-40) and b Aβ(17-28) using SAW biosensor.

Affinity constants (KD) of the Nb_3 and Nb_9 for the Aβ(1-40) and Aβ(17-28) using SAW biosensor are summarized in Table 4. Nb_3 showed a slightly higher affinity for Aβ(1-40) (KD, 142 nM) in comparison with Aβ(17-28) (KD, 306 nM) (Figure 36). The dissociation constants of Nb_9 showed a slightly higher affinity to Aβ(1-40) (KD, 207 nM) as for Aβ(17-28) (KD,745 nM) (Figure 37).

0 500 1000

Figure 37: K_D determination of Nb_9 nanobody to immobilized a Aβ(1-40) and b Aβ(17-28).

Table 4: Kinetic rate and equilibrium dissociation constants of the Nb_3 and Nb_9 to the determination of interactions and precise evaluation of kinetic constants.