4. Fabrication of ECIS sensor arrays

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 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 7. Illustration of ECIS sensor fabrication.

sensitive changes in cell density. Therefore, ECIS sensors with smaller dimension working electrodes illustrate better detection sensitivity on changes in cell density.

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

Impedance shifts to cell density changes with sensors' Ri of 100 μm and 150 μm (n = 3). The cell density change

.

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

Another benefit is that smaller Ri requires fewer cells in cell-based assays.

the cell layer in ECIS measurements.

Figure 5.

Figure 6.

26

Ri = 150 μm).

from 90,000 cells/cm<sup>2</sup> to 100,000 or 110,000 cells/cm<sup>2</sup>

Biosensors for Environmental Monitoring

counter electrode, dio is the distance between the edges of the electrodes, and S1 and

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

The inherent impedance of the Au/Cr electrodes of fabricated ECIS sensors is measured by microwave probe station (Cascade Microtech Inc., Beaverton, OR) and impedance analyzer (Agilent 4294) as shown in Figure 9. The maximum inherent impedance was 19 Ω at 8000 Hz, which is much lower than the measured cellular impedance of thousands of ohms. Thus, the inherent impedance of the

S2 are the areas of the working and counter electrodes, respectively.

5. The application of ECIS sensors in environmental monitoring

Bovine aortic endothelial cells (BAECs, VEC Technologies, Rensselaer, NY) were used in this study. The BAECs were cultured in minimum essential medium (MEM, GIBCO, Grand Island, NY) with 10% fetal bovine serum (FBS, GIBCO, Grand Island NY) under standard mammalian cell culturing conditions (37°C and 5% CO2). Confluent BAEC were trypsinized to detach the cells from the cell culture flasks to prepare the cell suspension. Then, the cell suspension was centrifuged on the bottom of centrifuge tube followed by aspirating off the upper supernatant. Finally, certain amount of cell culture medium was added into centrifuge tube to

This study investigated the toxicant detection by using the ECIS sensors. The toxicants used in this study are phenol (RICCA, Arlington, TX), ammonia (Acros Organics, Fair Lawn, NJ), nicotine (Fisher Scientific, Hanover Park, IL), and aldi-

Dulbecco's phosphate-buffered saline (DPBS, Mediatech, Inc., Manassas, VA). The osmolarity of diluted toxicant solution was considered to be in the suitable range for cell culture because the small volume of toxicants added into DPBS will not change

Impedance analyzer Agilent 4294 and ECIS measurement system (Applied Biophysics, Troy, NY) was used to measure the cell impedance. The AC signal applied to the cells was monitored by using Tektronix oscilloscope DPO2014B. Two MAXIM DG408 Multiplexers, controlled by an NI USB-6008 multifunction data acquisition card, were used as a 16-channel multiplexer between the impedance analyzer and the sensor arrays. The NI USB-6008 and Agilent 4294 were controlled by LabVIEW programs to perform the data acquisition shown as Figure 10. The ECIS sensor arrays, covered with BAECs on the sensing electrodes, were kept in an

The cell seeding density and measurement frequency are need to be optimized to

obtain reasonable measurement results. BAECs were seeded with different cell densities of 150,000, 125,000, and 100,000 cells/cm<sup>2</sup> on ECIS sensor. The impedance values were recorded and normalized in the initial 46 hours after seeding onto

carb (SPEX CertiPrep, Metuchen, NJ). All the toxicants were diluted with

the concentration of essential ingredients of DPBS dramatically.

incubator with 37°C and 5% CO2 during the impedance measurement.

5.4 Optimization of cell seeding density and measurement frequency

sensor can be neglected.

5.1 Cell culture and preparation

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

5.2 Toxicant preparation

5.3 Experimental system setup

29

prepare specific concentration of the cell suspension.

## Figure 8. The array of eight ECIS sensors.

#### Figure 9.

Distribution of equipotential lines in the space between the ventral cell surface and electrode-electrolyte interface layer. The axisymmetric axis of the cell locates at x = 0 (red dashed line).

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

counter electrode, dio is the distance between the edges of the electrodes, and S1 and S2 are the areas of the working and counter electrodes, respectively.

The inherent impedance of the Au/Cr electrodes of fabricated ECIS sensors is measured by microwave probe station (Cascade Microtech Inc., Beaverton, OR) and impedance analyzer (Agilent 4294) as shown in Figure 9. The maximum inherent impedance was 19 Ω at 8000 Hz, which is much lower than the measured cellular impedance of thousands of ohms. Thus, the inherent impedance of the sensor can be neglected.
