5. The application of ECIS sensors in environmental monitoring

#### 5.1 Cell culture and preparation

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 prepare specific concentration of the cell suspension.

## 5.2 Toxicant preparation

Figure 8.

Figure 9.

28

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 array of eight ECIS sensors.

Biosensors for Environmental Monitoring

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 aldicarb (SPEX CertiPrep, Metuchen, NJ). All the toxicants were diluted with 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 the concentration of essential ingredients of DPBS dramatically.

#### 5.3 Experimental system setup

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

#### 5.4 Optimization of cell seeding density and measurement frequency

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 the ECIS sensor array, as shown in Figure 11. The morphology of cells with seeding density 125,000 cells/cm<sup>2</sup> at different time points was also shown in Figure 11. The cells gradually spread on the surface of ECIS sensors after seeding and eventually form a monolayer with stable impedance. The cell impedance gradually increased in the initial 8–20 hours, which indicates the initial formation of a loose monolayer and stable up to the end of the impedance measurements. The cell morphology was

checked under microscope frequently. The corresponding impedance readings were used to represent the impedance of the cell monolayer for cell-based assays. In Figure 11, the impedance of cell monolayer with higher seeding densities increases more rapidly than cells with lower seeding densities because higher seeding densities allow the cells to have tighter and stronger intercellular junctions and the corresponding ion insulating abilities are better. The impedance of cells with the

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

(around 8 hours) of cell monolayer due to the cell movement on the surface of ECIS sensors. Also, the impedance of cells with 150,000 cells/cm<sup>2</sup> seeding density is not stable as those with 125,000 and 100,000 cells/cm<sup>2</sup> seeding densities. The cells with a seeding density of 100,000 cells/cm<sup>2</sup> need 20 hours to be confluent and have low impedance compared with those with higher seeding densities. Hence, the

The optimal measurement frequency allows the sensors to obtain the largest difference in measured impedance between a sample with and without cells [19]. In this study, the impedance of cell monolayer was measured with different frequencies from 500 Hz to 64 kHz. The optimal measurement frequency was optimized to

The ECIS sensors need to be prepared before the toxicity testing. ECIS sensors were cleaned by oxygen plasma to provide a sterilized surface for cell seeding. Then phosphate-buffered saline (PBS, GIBCO, Grand Island, NY) was used to clean the sensor surface again. Before cell seeding, 30 μg/ml fibronectin (GIBCO, Grand Island, NY) was coated on the surface of the sensors to improve cell attachment. BAECs were seeded onto each sensor with a seeding density of 125,000 cells/cm<sup>2</sup>

The cell morphology was checked under microscope. The selected toxicants were introduced to each well to perform toxicity testing after monolayer formation. Figure 12 shows normalized impedance response after introducing 0.1 and 0.2 mM aldicarb and the cell morphology after introducing 0.2 mM aldicarb. Some of the cells detached from the substrate. The normalized impedance decreases to 0.84 and

, decreased after initial formation

, was chosen as the preferred seeding density

.

highest seeding density, 150,000 cells/cm<sup>2</sup>

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

cell seeding density, 125,000 cells/cm<sup>2</sup>

be 8000 Hz in experimental measurements.

in the toxicity testing.

5.5 Toxicity testing

Figure 12.

31

The normalized impedance of BAEC exposed to aldicarb.

Figure 10.

Experimental setup of cell impedance measurement.

Figure 11. Impedance response of BAECs measured by an ECIS sensor array at 8000 Hz and the image of cell morphology.

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

checked under microscope frequently. The corresponding impedance readings were used to represent the impedance of the cell monolayer for cell-based assays. In Figure 11, the impedance of cell monolayer with higher seeding densities increases more rapidly than cells with lower seeding densities because higher seeding densities allow the cells to have tighter and stronger intercellular junctions and the corresponding ion insulating abilities are better. The impedance of cells with the highest seeding density, 150,000 cells/cm<sup>2</sup> , decreased after initial formation (around 8 hours) of cell monolayer due to the cell movement on the surface of ECIS sensors. Also, the impedance of cells with 150,000 cells/cm<sup>2</sup> seeding density is not stable as those with 125,000 and 100,000 cells/cm<sup>2</sup> seeding densities. The cells with a seeding density of 100,000 cells/cm<sup>2</sup> need 20 hours to be confluent and have low impedance compared with those with higher seeding densities. Hence, the cell seeding density, 125,000 cells/cm<sup>2</sup> , was chosen as the preferred seeding density in the toxicity testing.

The optimal measurement frequency allows the sensors to obtain the largest difference in measured impedance between a sample with and without cells [19]. In this study, the impedance of cell monolayer was measured with different frequencies from 500 Hz to 64 kHz. The optimal measurement frequency was optimized to be 8000 Hz in experimental measurements.

#### 5.5 Toxicity testing

the ECIS sensor array, as shown in Figure 11. The morphology of cells with seeding density 125,000 cells/cm<sup>2</sup> at different time points was also shown in Figure 11. The cells gradually spread on the surface of ECIS sensors after seeding and eventually form a monolayer with stable impedance. The cell impedance gradually increased in the initial 8–20 hours, which indicates the initial formation of a loose monolayer and stable up to the end of the impedance measurements. The cell morphology was

Impedance response of BAECs measured by an ECIS sensor array at 8000 Hz and the image of cell morphology.

Figure 10.

Figure 11.

30

Experimental setup of cell impedance measurement.

Biosensors for Environmental Monitoring

The ECIS sensors need to be prepared before the toxicity testing. ECIS sensors were cleaned by oxygen plasma to provide a sterilized surface for cell seeding. Then phosphate-buffered saline (PBS, GIBCO, Grand Island, NY) was used to clean the sensor surface again. Before cell seeding, 30 μg/ml fibronectin (GIBCO, Grand Island, NY) was coated on the surface of the sensors to improve cell attachment. BAECs were seeded onto each sensor with a seeding density of 125,000 cells/cm<sup>2</sup> . The cell morphology was checked under microscope. The selected toxicants were introduced to each well to perform toxicity testing after monolayer formation. Figure 12 shows normalized impedance response after introducing 0.1 and 0.2 mM aldicarb and the cell morphology after introducing 0.2 mM aldicarb. Some of the cells detached from the substrate. The normalized impedance decreases to 0.84 and

Figure 12. The normalized impedance of BAEC exposed to aldicarb.

0.76 times its original impedance value within 3 hours when treated with 0.1 and 0.2 mM aldicarb, respectively. The cell morphology changed and even detached from the sensors. Figure 13 shows the normalized impedance response after introducing 0.1 and 0.2 mM phenol as toxicant. The BAEC detached from substrate after introducing 0.2 mM phenol. The decreasing impedance curves indicate the toxic effect on BAECs. The normalized impedance values rapidly decreased to 0.80 and 0.74 times its original impedance value within 2 hours when treated with 0.1 and 0.2 mM phenol, respectively. The image shows the cells obviously detached from the sensor. Figure 14 shows the normalized impedance response after introducing 2 and 5 mM ammonia as toxicant. Those lines shows that the normalized impedance values rapidly decreased to 0.78 and 0.68 times its original impedance value within 1 hour when treated with 2 and 5 mM ammonia, respectively. The image shows the cell morphology after 1 hour after introducing ammonia. The cells morphology changed and very easily detached from the sensor substrate. Figure 15 shows the

normalized impedance of BAEC after exposing to 0.8 and 1.3 mM nicotine as toxicant. The normalized impedance rapidly decreased to 0.92 and 0.75 times its original value within 2 hours when treated with 1.3 and 0.8 mM nicotine, respectively. The image shows the BAEC morphology after exposed to 1.3 mM nicotine. Most of the cell detached from the sensor due to the toxic effect of nicotine.

The normalized impedance of BAEC exposed to 0.8 mM and 1.3 mM nicotine. The image shows the cell

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

approaches.

Figure 15.

morphology after exposure to 1.3 mM nicotine.

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

6. Conclusions

Acknowledgements

33

The cell morphology and decreasing impedance curves indicate the toxic effect and the effectiveness of ECIS sensing on environmental monitoring within short period of time. Different concentrations of toxicants are qualified according to the gradients of normalized impedance. ECIS sensing technique is able to perform environmental monitoring effectively and efficiently compared with other

In this study, the biosensors based on ECIS sensing technique were used to monitor and measure the environmental toxicants, including the phenol, ammonia, nicotine, and aldicarb. A model, validated by experimental results, was created to analyze the electric potential distribution of ECIS sensing and guide the designing, especially the sensing area of sensor electrodes. The detection sensitivity of ECIS sensors was optimized. The experimental results show that ECIS sensors are capable to detect and qualify the environmental toxicants rapidly. The concentration of toxicants can be indicated from the gradients of normalized cell impedance.

We appreciate Dr. Ioana Voiculescu's and Andres Rivera's support in this study.

Figure 13. The normalized impedance of BAEC exposed to phenol.

#### Figure 14.

The normalized impedance of BAEC exposed to 2 mM and 5 mM ammonia, and the image shows the BAEC morphology after exposure to 5 mM of ammonia.

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

Figure 15.

0.76 times its original impedance value within 3 hours when treated with 0.1 and 0.2 mM aldicarb, respectively. The cell morphology changed and even detached from the sensors. Figure 13 shows the normalized impedance response after introducing 0.1 and 0.2 mM phenol as toxicant. The BAEC detached from substrate after introducing 0.2 mM phenol. The decreasing impedance curves indicate the toxic effect on BAECs. The normalized impedance values rapidly decreased to 0.80 and 0.74 times its original impedance value within 2 hours when treated with 0.1 and 0.2 mM phenol, respectively. The image shows the cells obviously detached from the sensor. Figure 14 shows the normalized impedance response after introducing 2 and 5 mM ammonia as toxicant. Those lines shows that the normalized impedance values rapidly decreased to 0.78 and 0.68 times its original impedance value within 1 hour when treated with 2 and 5 mM ammonia, respectively. The image shows the cell morphology after 1 hour after introducing ammonia. The cells morphology changed and very easily detached from the sensor substrate. Figure 15 shows the

The normalized impedance of BAEC exposed to 2 mM and 5 mM ammonia, and the image shows the BAEC

Figure 13.

Figure 14.

32

The normalized impedance of BAEC exposed to phenol.

Biosensors for Environmental Monitoring

morphology after exposure to 5 mM of ammonia.

The normalized impedance of BAEC exposed to 0.8 mM and 1.3 mM nicotine. The image shows the cell morphology after exposure to 1.3 mM nicotine.

normalized impedance of BAEC after exposing to 0.8 and 1.3 mM nicotine as toxicant. The normalized impedance rapidly decreased to 0.92 and 0.75 times its original value within 2 hours when treated with 1.3 and 0.8 mM nicotine, respectively. The image shows the BAEC morphology after exposed to 1.3 mM nicotine. Most of the cell detached from the sensor due to the toxic effect of nicotine.

The cell morphology and decreasing impedance curves indicate the toxic effect and the effectiveness of ECIS sensing on environmental monitoring within short period of time. Different concentrations of toxicants are qualified according to the gradients of normalized impedance. ECIS sensing technique is able to perform environmental monitoring effectively and efficiently compared with other approaches.

### 6. Conclusions

In this study, the biosensors based on ECIS sensing technique were used to monitor and measure the environmental toxicants, including the phenol, ammonia, nicotine, and aldicarb. A model, validated by experimental results, was created to analyze the electric potential distribution of ECIS sensing and guide the designing, especially the sensing area of sensor electrodes. The detection sensitivity of ECIS sensors was optimized. The experimental results show that ECIS sensors are capable to detect and qualify the environmental toxicants rapidly. The concentration of toxicants can be indicated from the gradients of normalized cell impedance.

### Acknowledgements

We appreciate Dr. Ioana Voiculescu's and Andres Rivera's support in this study.

Biosensors for Environmental Monitoring

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