# Surface Acoustic Wave Sensor for C-Reactive Protein Detection
## 2 Materials and Methods
### Method of Immobilization  
Figure 3 shows all the steps that were used for the immobilization of gold surface and the CRP/anti-CRP interaction process.  
1. The gold surface was cleaned by UV/ozone and ethanol and blown dry with nitrogen gas before use.
2. For the surface modification binding, 11-mercaptoethanolanoic acid (11-MUA) in absolute ethanol (4 mM) was injected into the gold sensing area and incubated for 24 h, then washed by absolute ethanol and deionized (DI) water, and dried by nitrogen (N\({}_{2}\)) gas. This was the way the self-assembled monolayer (SAM) was attached to the gold surface.
3. This reactive SAM layer was activated by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxy succinimide (NHS) mixture (1:1) for 20 min and washed by sodium acetate buffer (10 mM, pH 5.0).
4. Immobilization of anti-CRP was done in sodium acetate buffer (10 mM, pH 5.0) for 24 h.
5. Blocking was performed by incubating in ethanolamine-HCl (pH 8.5) solutions for 10 min and then washed and incubated in phosphate buffered saline (PBS) buffer. This process was done for blocking the remaining non-specific binding of antibody active sites or removing the interference.
6. For evaluating the interaction between CRP and anti-CRP, the prepared CRP with PBS buffer solution was injected over the sensing area and allowed to react with anti-CRP for 10 min. We prepared the CRP solution of 10 mL individually for each CRP concentration (0.1, 1, 10, 100, and 1000 ug/mL), which was diluted by PBS buffer. It means that we had five bottles of CRP solution with different concentrations. The proposed SAW sensor was exposed to CRP solutions of increasing concentrations (0.1, 1, 10, 100, and 1000 ug/mL). We used a pipette to suck 50 uL from the prepared CRP solutions and cover its sensing surface to interact with the CRP antibodies for each measurement. After an interaction time of 10 min, the CRP solution was carefully removed by a pipette and the surface was rinsed with PBS (500 \(\mu\)L) and DI water (1000 \(\mu\)L), separately, for the removal of non-specifically bound molecules. Next, the residue water at the surface of the SAW sensor was also carefully removed by a low nitrogen gas flow for 30 sec. After the nitrogen drying process, the frequency response of SAW sensor for detecting the CRP was recorded by a vector network analyzer (VNA). We repeated three times the abovementioned four steps (CRP interaction, removal of CRP solution, surface drying, and record spectrum response) for each CRP concentration. It should be noted that surface liquid residues should be completely removed to avoid the radiation of the acoustic energy into the liquid. For Rayleigh SAW sensors, this will cause a severe attenuation of the acoustic wave propagating at the solid/liquid interface [34]. No resonance peak was observed when the surface liquid residues had not been removed.  
Figure 4 shows the measurement system for the frequency response of SAW sensors. A VNA was used to measure the frequency response of the SAW sensor. The calibration was done by a calibration kit before each measurement. All the measurements were done at room temperature. Port 1 of the VNA outputs high-frequency AC signals to the input terminal (the first SAW interdigital transducers) of the SAW sensor. The electrical signal is converted to surface acoustic wave by the first SAW interdigital transducer. The surface acoustic waves travel across the SAW sensing area and are very sensitive to variations on the SAW sensing surface. The surface acoustic wave is converted back to an electrical signal by the second SAW interdigital transducer. Port 2 of the VNA receives the signal from the output terminal (the second SAW interdigital transducer) of the SAW sensor. The frequency or amplitude response changes at the resulting output signal, which can be correlated to the corresponding mass and mechanical variations on the sensing surface.