3.1.2 The relationship between the distance between the edges of the sensing electrodes (dio) and cell impedance

The distance between the edges of the sensing electrodes (dio) is another factor that should be considered in designing ECIS sensors. Figure 4 shows the experimental impedance and the simulated impedance with different dio. The average experimental impedance slightly changed from 12.50 to 12.52 KΩ, when dio changed from 1000 to 3500 μm. The simulated impedance was calculated by using Eq. (7).

The Modeling, Design, Fabrication, and Application of Biosensor Based on Electric Cell… DOI: http://dx.doi.org/10.5772/intechopen.81178

#### Figure 4.

Relationships between dio and experimental impedance, and between dio and simulated impedance, measured at 8000 Hz (n = 6 � 7, Ri = 100 μm). The three images show the cell morphology of BAECs on the ECIS sensors with dio of 1 mm, 2 mm, and 3.5 mm, respectively.

dio only slightly influences the simulated impedance because the natural logarithm of the quotient of ð Þ Ri þ dio and Ri makes the influence of dio on simulated impedance more slightly in Eq. (7). The simulated impedance is consistent with the experimental data with maximum difference 0.63%, which validates the model. The experimental and simulated impedance indicates that dio in the range of 1000–3500 μm has only a little influence on the impedance because dio influenced the impedance of medium, which is only a small portion of measured impedance. Thus, dio cannot dramatically influence the measured impedance. However, dio should be large enough to avoid the current bypassing the cell monolayer between sensing electrodes.

#### 3.2 The influence of electrode dimensions on the detection sensitivity of ECIS

Detection sensitivity reflects the fineness of impedance response to the changes of cell behavior in cell-based assay environmental monitoring. The detection sensitivity of ECIS sensors is influenced by Ri. According to the previous experimental results, ECIS sensors with Ri larger than 200 μm do not respond sensitively and quickly on cell morphology changes. So, ECIS sensors with Ri of 100 and 150 μm were fabricated to study the influence of Ri on the detection sensitivity. Cell densities, 90,000, 100,000, and 110,000 cells/cm<sup>2</sup> , were used to study the relationship between cell density and impedance. Figure 5 shows the impedance shifts versus the cell density changes with Ri of 100 and 150 μm. Figure 6 shows the corresponding cell morphology on different ECIS sensors. When the cell density changes are 10,000 cells/cm<sup>2</sup> (from 90,000 to 100,000 cells/cm<sup>2</sup> ), the impedance increased 597 and 350 Ω for the sensors with Ri of 100 and 150 μm, respectively. When the cell density changes are 20,000 cells/cm<sup>2</sup> (from 90,000 to 110,000 cells/cm<sup>2</sup> ), the impedance increased 1336 and 880 Ω for the sensors with Ri of 100 and 150 μm, respectively. The experimental results indicate that the sensors with larger Ri illustrate less impedance changes with the same amount of cell density changes. Therefore, the sensors with smaller Ri are able to detect more

simulated impedance curve matches the experimental data closely with maximum difference 13.29%, which is acceptable when considering the fluctuation of measured impedance. The consistency of the simulated impedance with the experimental impedance validates this model's ability to optimize the Ri according to the range of measured cell number and expected output impedance level during sensor

Relationships between Ri and experimental impedance, and between Ri and simulated impedance at 8000 Hz

3.1.2 The relationship between the distance between the edges of the sensing electrodes

The distance between the edges of the sensing electrodes (dio) is another factor that should be considered in designing ECIS sensors. Figure 4 shows the experimental impedance and the simulated impedance with different dio. The average experimental impedance slightly changed from 12.50 to 12.52 KΩ, when dio changed from 1000 to 3500 μm. The simulated impedance was calculated by using Eq. (7).

designing.

24

Figure 3.

(n = 4 � 6, dio = 3.5 mm).

Figure 2.

BAEC monolayer on ECIS sensors with different Ri (dio = 3.5 mm).

Biosensors for Environmental Monitoring

(dio) and cell impedance

4. Fabrication of ECIS sensor arrays

DOI: http://dx.doi.org/10.5772/intechopen.81178

Figure 7.

27

Illustration of ECIS sensor fabrication.

The fabrication of ECIS sensors can follow different photolithography techniques. The substrates are usually nonconductive materials, includes glass, printed circuit board (PCB) [1–4, 19, 23], and polymer including polydimethylsiloxane (PDMS) [9] and polycarbonate [1–3, 18, 19]. The ECIS arrays were fabricated on glass by thin film deposition and lift-off photolithography technique, as shown in Figure 7. Initially, the photoresist AZ5214E (MicroChemicals, Somerville, NJ) was coated on glass slides with spinning coater at 2000 rpm. After baking on hotplate at 110°C for 50 seconds, the coated photoresist was exposure to ultraviolet (UV) light. Then, a reversal bake is carried out at 120°C for 2 minutes. Finally, UV light with intensity larger than 200 mJ/cm<sup>2</sup> was exposure on the photoresist pattern. The electrode pattern was created after immersing the slides with photoresist in the AZ 100 Remover (MicroChemicals, Somerville, NJ). The remover is able to dissolve the photoresist without the first exposure (image reverse). A 20-nm-thick chromium (Cr) followed by a 150-nm-thick gold (Au) was coated on the substrate to form the sensor's electrodes by thermal evaporation. The sensing electrodes were formed after the lift-off process. Then, the photoresist SU-8 (MicroChem, Westborough, MA) was used to cover the substrate except the sensing areas. The sensor arrays were treated with 95% sulfuric acid at 60°C for 15 seconds [48] followed by washing

The Modeling, Design, Fabrication, and Application of Biosensor Based on Electric Cell…

with deionized water (DI) and then treated with 8% (3-aminopropyl) triethoxysilane (APTES) at 50°C for 2 hours to improve the surface

biofunctionality. Finally, cell culture wells (Lab-Tek 8-well culture wares) were glued onto the sensor array. Figure 8 shows the fabricated ECIS sensor array and its configuration. Ri is the radius of the working electrode, Rco is the outer radius of the

#### Figure 5.

Impedance shifts to cell density changes with sensors' Ri of 100 μm and 150 μm (n = 3). The cell density change from 90,000 cells/cm<sup>2</sup> to 100,000 or 110,000 cells/cm<sup>2</sup> .

#### Figure 6.

Cell morphology with 90,000, 100,000, and 110,000 cells/cm<sup>2</sup> cell densities on ECIS sensors (Ri = 100 μm and Ri = 150 μm).

sensitive changes in cell density. Therefore, ECIS sensors with smaller dimension working electrodes illustrate better detection sensitivity on changes in cell density. Another benefit is that smaller Ri requires fewer cells in cell-based assays.

Based on the analysis above, the ECIS sensors with Ri of 100–125 μm and dio of 3.5 mm are preferred in environmental monitoring because Ri of 100–125 μm will allow the ECIS sensors to be sensitive to sense the cell morphology changes due to environment influence and own good anti-interference ability. The area of counter electrodes should be as large as possible to guarantee sufficient contact area between electrode and cells. dio of 3.5 mm is enough to avoid the current bypassing the cell layer in ECIS measurements.

The Modeling, Design, Fabrication, and Application of Biosensor Based on Electric Cell… DOI: http://dx.doi.org/10.5772/intechopen.81178
