**3. Materials and methods**

The MTSM/GA technique first experimentally tested with the polymer SU8-2002 layer spin coated on sensor surface. The determined properties of the layer were compared with the values obtained from literature. The technique was then applied to obtain the mechanical and geometrical properties of a protein layer adsorbed on gold layer. The methods and chemicals used in the experiments are described below.

### **a. Deposition of the thin polymer film**

The SU 8-2002 (MicroChem) polymer solution was spin coated on MTSM sensor by using the following procedure. First, the gold electrode surface of TSM sensors was cleaned using

Modeling of Biological Interfacial Processes Using Thickness–Shear Mode Sensors 249

Initially, two different thicknesses of SU8 2002 layers were spin coated on sensor surface and changes in the frequency and magnitude responses were monitored at 5, 15, 25 and 35 MHz. The thicknesses of the layers were measured by using optical profilometer (fig. 7a). The average thicknesses of the layers were 1920±25 nm and 770±50 nm respectively. Surface topography of the SU8 - 2002 layers was measured by using AFM (fig. 7b). The average

b)

SU8 layer Gold layer

Fig. 7. A) Thickness measurements from optical profilometer sample a. SU8-2000 solution sample b. 1:1 dilution of SU8-2002 and cyclopentanone B) Surface topography of SU 8 layer

First set of experiments were performed by spin coating 2 μm thick SU 8 - 2002 layer on sensor surface. The MTSM/GA determined properties are presented in table 2. The average thickness of the polymer layer determined to range from 2080 nm to 2140 nm among the harmonics. Although these values are slightly higher than the value (1920±25 nm) obtained in control experiments, they are still in less than 10% experimental errors. The variation between the frequencies for density value was also very small, ranging from 1240 to 1253 kg/m3. These numbers correlate well with the literature value of 1200 kg/m3 (Jiang et al.,

MTSM Frequency MTSM/GA Results Profilometer Jiang et al. [38] (MHz) d(nm) ρ (kg/m3) d (nm) ρ (kg/m3)

Table 2. Comparison density and thickness values of SU 8-2002 layer determined using MTSM/GA sensor at 5, 15, 25 and 35 MHz with profilometer and Jiang et al. (Jiang et al.

1920±25 1200

**a. Determination of mechanical and geometrical properties of SU8 layer of 1.92 μm** 

roughness of the layer was 20 nm and no cracks on the surface were observed.

**4. Results and discussions** 

0 0.2 0.4 0.6 0.8 Distance (mm)

Gold level

5 2120±60 1253±10

15 2140±50 1246±11 25 2080±110 1240±50 35 2080±60 1240±28

a)

Thickness (um)


**thickness** 

2003) for SU8.

2003)

Sample B

Sample A

Piranha solution (one part of 30% H2O2 in three parts H2SO4). After 2 min exposure time, the sensors were rinsed with distilled water. The surface was dried in a stream of nitrogen gas. The SU 8 – 2002 sample was dispensed on MTSM sensor surface and sensors were spin coated for 40 seconds. The sensors were then soft baked for 1 min at 95 oC. The SU 8-2002 films were exposed to UV light for 4 seconds under 25 mJ/cm2. This was followed by 1 min hard baking on hot plate at 95 oC.

### **b. Antibody adsorption on MTSM sensor surface**

The reference measurements were taken for air and phosphate buffer saline (PBS). Next, the sensors were exposed to rabit-immunoglobulin G (IgG) (50 μg/ml) suspended in diwater (Fisher Scientific, pH: 5.34, Cat No: 25—555-CM) for 50 minutes to allow IgG coating of the sensor surface by adsorption.

### **c. Characterization of geometrical properties of the thin film**

The thicknesses of the SU 8 – 2002 films were determined by using optical profilometer (Zygo Inc. Model #: NV6200). For the thickness measurements, a very small portion of MTSM sensor surface was not exposed to UV light. After the films were developed, the SU 8-2002 layer was removed from this portion. To obtained different thicknesses of film layer, 1:1 solution of SU8-2002 and cyclopentanone (Acros Organics) was prepared.

The surface topography of the film layer was measured using atomic force microscopy (AFM). The prepared samples were placed on a glass slide installed on the atomic force microscope (Bioscope; Veeco), that was mounted on the inverted fluorescence microscope (TE2000; Nikon, Melville, N.Y.). Measurements were made using contact mode with a scan rate of 2 Hz.

### **d. Measurement system and MTSM sensor data analysis technique**

A 14 mm diameter, 0.33 mm thick, 5 MHz quartz crystal with deposited 7 mm gold electrodes was placed in a custom fabricated brass sensor holder (ICM). The sensor holder was connected to a Network Analyzer (NA) (HP4395A). A LabView program on a personal computer was used to control the network analyzer and collect the data at 5, 15, 25 and 35 MHz. The experiments were done in room temperature (24oC±1oC). Magnitude and phase responses of MTSM sensor were monitored during the experiments (figure 6). The sampling rate was 30 seconds. Each experiment was repeated three times.

Fig. 6. a) Magnitude and b) phase responses of MTSM sensor
