Cell viability and electrical response of breast cancer cell treated in aqueous graphene oxide solution deposition on interdigitated electrode

GO characterization

The obtained GO was first characterized by using the Raman spectroscopy, XRD spectroscopy, FESEM, EDX spectroscopy and Thermogravimetric analysis (TGA) to confirm the graphitic nature, crystal structure, morphology, composition of elements and decarboxylation process respectively. Firstly, Raman spectroscopy was used in order to study the defects and the structure’s order of the GO. According to graphitic nature, the D and G peaks can be detected in the range of 1200–1500 cm⁻¹ and 1500–1800 cm⁻¹ wavenumber respectively. From the image obtained by using the Raman spectroscopy, the presence of a defect characteristic, D peak at 1377 cm⁻¹ (as shown in Fig. 1) can be seen. The peak may be produced by the disordered bands in sp² hybridized carbon materials and led to the disruption of the symmetrical hexagonal graphitic lattice as a result of edge defects, internal structural defects and dangling bonds¹⁵. Meanwhile, the G peak was centered at 1580 cm⁻¹ and was referred to as the first order of the Raman band creating a D to G ratio of 0.87. This may be caused by all the sp² hybridized carbon materials that were related to the C–C vibrational mode¹⁶. The shift in the G band from 1582 cm⁻¹ to 1599 cm⁻¹ of GO was due to the presence of isolated double bonds on the GO carbon network¹⁵. Finally, the 2D peak was also detected at 2700 cm⁻¹.

Next, the X-Ray Diffraction (XRD) was used to identify the phase identification of the crystalline structure in GO. Before conducting the test, the GO solution was first deposited onto the Silicon Dioxide (SiO₂) substrate by using a drop cast method. The XRD pattern in Fig. 2 was comparable to that obtained by Ref.¹⁶, confirming the crystalline nature of graphite and GO. In the data, graphite exhibited a sharp peak at 2Ɵ = 26.73 degrees with d-spacing at 3.33 Å. After the oxidation of graphite, the sharp reflection peak was shifted to the lower angle at 2Ɵ = 11.54 degrees with d-spacing at 7.67 Å, due to the formation of oxygen functional groups and the intercalation of water molecules into the carbon layer structure¹⁷.

For Field Emission Scanning Electron Microscopy observation, the FESEM was operated at 15 kV and at a constant working distance of 4.9 mm to produce the optimal imaging conditions. This analysis is primarily used to determine the surface morphology at high magnification. From the observation analysis of Fig. 3 showed that the GO is a very thin monolayer or few-layered structures made up of folded and wrinkled graphene films. The films were thin due to the mechanical forces produced by using an ultrasonication bath. The films were wrinkled, folded and stacked by a few layers of graphene due to the strong π–π interaction at the surfaces¹⁸.

The Energy Dispersive X-Ray (EDX) measurement was used to investigate the elemental and quantitative compositions of the materials. In this measurement, an accelerating voltage of 20 kV was used with a scan time of 100 s per sampling area. The EDX spectrum of GO is as shown in Fig. 4. The observation revealed the presence of Carbon (C), Oxygen (O), Aluminum (Al), Silicon (Si), Sulphur (S) and Potassium (K). The C content of the GO was valued at 51.64% and was obviously the highest as graphite is a carbon material. This was followed by 35.62% of O content due to the oxygen-containing functional groups produced from the oxidation process by using Hummer’s method. The mass ratio of O/C was 0.68. Lastly, the content of S, Si, Al and K were reported at 7.85%, 2.60%, 1.50% and 0.79% respectively.

Thermogravimetric analysis (TGA) was used to determine the temperature at when a material was completely decomposed. The drop in the mass shows the material was undergoing the decomposition process. Based on Fig. 5 below, a TGA comparison between graphite and GO can be seen. While graphite percentage mass maintained close to 100% TGA because of a non-volatile nature, GO showed drop throughout the process mainly once the temperature hit 150 °C. This finding can be related with a study done by Ref.¹⁹. According to them, the majority of the carbon atoms in GO have been converted from graphitic sp² to a non-graphitic sp³ hybridized carbon that contains high density of defects due to the oxidation process that occurred. The defects and weaker interaction between the exfoliated graphene layers cause the thermal degradation temperature to reach much faster. Hence the difference between the stability in mass percentage between graphite and GO can clearly be observed.

Cell viability and pH of GO

One of the components that can be taken into consideration when maintaining the solubility of GO is the pH of the prepared GO. According to Refs.^20,21, the carboxyl groups of GO at lower pH are protonated, thus making the GO less hydrophilic, while at the higher pH of GO, the carboxyl groups are deprotonated, eventually enhance the hydrophilic features and resulting in better GO solubility. The cell membranes are semipermeable and allow selected molecules to pass through the barriers and induce changes to the biological interactions. Therefore, to distinguish the interaction between the MCF7 breast cancer cells and MCF10a normal breast cells due to the presence of GO, an observation on the effect of different pH of GO towards the cell viability was done. Figure 6 shows the numbers of viable MCF7 and MCF10a cells against the pH of GO after 24 h (as shown in Fig. 6a) and 48 h (as shown in Fig. 6b) incubation. After 24 h of incubation (i.e. incubation period) for MCF10a (Fig. 6a), the percentage number of viable cells decreased to 90.5%, 92.8% and 98.4% after the GO treatments with the pH 5, 6 and 7, respectively. From the percentage obtained, only a slight reduction in the cell’s numbers can be seen. The pH value for normal cells usually ranging from pH 7.2 to pH 7.5 which is in a neutral condition²². Hence, when the MCF10a surrounding was slightly acidic due to the pH value of GO at pH 5 and pH 6, more reduction in the cell viability can be seen. Meanwhile, after 48 h of incubation, the cell viability increased significantly to 109.8%, 113.6% and 116.4%, respectively at the pH 5, 6 and 7, as compared to after 24 h incubation. A paper by Ref.²³ discussed that normal human cells undergo proliferating on average every 24 h. During this division timing, the cells were allowed to synchronize with the physiological process and the changes in their environment and hence suggested the best maximum effect to be measured at 24 h incubation period to prevent any interruption of other external factors.

As shown in Fig. 6a, the percentages of viable MCF7 cells treated with different GO pH for 24 h was relatively similar and were approximately 73.13%, 73.53% and 73.93%, respectively. This result shows greater reduce in the cell viability compared to the normal breast cells MCF10a suggesting that GO gave greater effects towards the breast cancer cells MCF7 than the normal breast cells MCF10a. Plus, when the incubation period was increased to 48 h (as shown in Fig. 6b), the percentages of viable MCF7 cells treated with different GO pH of 5, 6 and 7 dropped further to 63.07%, 58.93% and 56.98%, respectively. These continuous decreases with respect to the incubation time somehow only occurred for the MCF7 compared to the MCF10a and was supported by Ref.²⁴ that stated GO have the ability to hinders the proliferation of the cancer stem cells in wide array of cancer and is not toxic to the normal pluripotent stem cells, but stimulate their differentiation into various cell types. Moreover, the GO of pH 7 had a greater effect on the cell viability, than the GO of pH 5, and this corresponded to the hydrophilic properties. As the pH increased, the hydrophilicity of GO also increased²¹. Hence, the prepared GO resulted in better solubility for better interactions with the cells. Therefore, for the next dose-dependent cell viability study, the GO of pH 7 was selected to be tested with varying GO concentrations, as the GO of pH 7 was shown to inhibit the growth of breast cancer MCF7 cells while maintaining or increasing the viability of normal breast MCF10a cells.

For the dose-dependent cell viability study, the GO concentrations were varied into six different concentrations (2.5, 6.25, 12.5, 25, 50 and 100 µg/mL) while the pH of GO was fixed at 7. Figure 7 shows the comparison between the number of viable MCF10a and MCF7 cells against the different concentrations after 24 h incubation time. The graph in Fig. 7a shows a small percentage difference between the number of untreated and treated MCF10a cells. The average number of MCF10a cells varied between 1 to 7.89%, as compared to the untreated MCF10a cells. Here, the value larger than 100% was inferred to the activation of MCF10a. In the case of a 48 h incubation period, the results, as in Fig. 7b, showed that the percentages of viable MCF10a were much larger (ca. 43.47–60.6%), as compared to the untreated MCF10a cells. At the higher concentrations of GO (> 25 µg/mL), the percentages of viable MCF10a cells were slightly larger than that of the lower GO concentrations. This increase resembled a much higher activation of MCF10a cells. Overall, the MCF10a activation does not have a strong dependency on the GO concentrations but rather is dependent on the incubation time.

In the case of GO incubation with the MCF7 cells, the data in Fig. 7a showed the number of viable cells was slightly proportional to the GO concentrations of less than 25 µg/mL, then showed inversely proportional relation appeared for the concentrations of more than 25 µg/mL. Moreover, at 12.5 µg/mL, the number of viable cells was larger than 100%, thus indicating the activation of cells. The results were insignificant by considering the 10% error bar. However, the GO concentrations of less than 25 µg/mL are not sufficient to increase the inhibition rate, similar to the study for GO reacting with the cancer cells²⁵.

After the 48 h incubation period, a similar trend of data was observed. However, the difference between the number of viable MCF7 and MCF10a cells was much larger at the higher GO concentration. For example, at the GO concentration of 100 µg/mL, the difference was ca. 30% for the 24 h incubation period, while it was 90% for that 48 h, hence was proportional to the incubation time. GO increased MCF7 toxicity, while it has much lower toxicity to normal breast epithelial cells, MCF10a. One of the possible mechanisms that cause cytotoxicity effect is the eliciting of apoptosis such that the GO stimulated molecular and cellular apoptosis in cancer cells whereas demonstrated low apoptosis observed in treated normal breast epithelial cells²⁶. Nevertheless, the focus of current study is on the phenomenological characterization of MCF7 and MCF10a response to GO in terms of viability and electrical response, which paving the way to a more in-depth study such as the molecular dynamics studies that could explain the effect of GO on the two cell lines²⁷.

Electrical responses characterization

The MCF7 and MCF10a cells, untreated and treated with different GO pH for 24 and 48 h were characterized based on the electrical response. Gold interdigitated electrodes on glass substrate with 10 µm gaps having initial resistance of 2.0 × 10⁵ Ω were used in this measurement. A constant value of 1 V was supplied to the electrode from the LCR meter (Agilent E4980 20 Hz–2 MHz Precision LCR (Inductance, Capacitance, Resistance) meter, USA) equipped with LabVIEW 2012 software, to obtain the electrical parameters values. The measurement was done at frequency between 20 Hz to 2 MHz.

The electrical impedance and resistance responses of untreated and treated MCF7 and MCF10a cells with different pH of GO after 24 h

Figure 8a,b shows the electrical Z_cell and phase values from the untreated and treated MCF7 cells with different GO pH for 24 h. From the plot, the MCF7 cells treated with GO of pH 5 showed the highest Z_cell value, while the cells treated with GO of pH 7 showed the lowest Z_cell value. For the MCF7 cells treated with GO of pH 5, the Z_cell value measured was 315.25 Ω with phase of − 44.58° at the frequency of 5 kHz and dropped to 220.45 Ω with phase of, − 39.05° after the GO treatment at the pH of 7. The opposite was observed in the MCF10a. From the results shown in Fig. 8c,d, the MCF10a cells treated with GO at the pH of 7 produce highest Z_cell and cells treated with GO at pH 5 show the lowest impedance. The Z_cell and phase measure at 5 kHz for MCF10a treated with GO at pH 7 was 291.52 Ω with a phase of − 48.10°, while the MCF10a cells treated with GO at the pH of 5 at the same frequency showed the lower Z_cell value of 230.58 Ω with a phase of − 45.98°. The overall value of measured Z_cell of the MCF7 was slightly higher than the measured Z_cell of the MCF10a with decreasing trend as frequency increased observed in both samples. The phase for all samples was also measured in the negative values. The negative phase was due to the characteristic of the cell membranes which is insulative causing it to have capacitive effect.

As stated by Ref.¹², without the cells, the Z_cell of the system can only come from the very low electrode capacitance (C_dl) and the solution resistance (R_sol)_. For the MCF7 medium (RPMI), the R_sol value was 2.49 kΩ with the C_dl equals to 989 nF, while for the MCF10a medium (DMEM), the R_sol value was 1.63 kΩ with the C_dl equals to 1420 nF. After dropping the treated and untreated cells onto the electrode surface, the Z_cell of the attached cells is modelled as the capacitance component (C_cell) and the resistance component (R_cell)^12,28.

Table 1 shows the R_cell and C_cell of untreated and treated MCF7 and MCF10a with different pH of GO for 24 h. For the untreated cells, the MCF7 produces R_cell value (4732.9 Ω) higher than MCF10a (2467.2 Ω) with C_cell value of 775 nF and 710 nF respectively. The MCF7 shows decreased in R_cell and increased in C_cell value as the pH was increased from 5 to 7. The reverse was observed in the MCF10a where the value of R_cell increased and C_cell decreased with increasing pH value. The R_cell value can be attributed to a few factors such as cell viability, cell types and pH of the solution. For MCF7 the decreasing trend in the R_cell as the pH of GO increase was attributed mostly due to the pH of the solution as the difference in cell viability between pH was not differ by much. As for the MCF10a, the cell viability increased slightly with increasing pH of the GO. Cell membranes have insulating behavior. The increase in cell numbers will contribute to the increase of R_cell as the insulating comportment of the membranes reduces the current flow. This will also contribute to decline in the capacitance values due to the increased number of highly insulating cell membranes of viable cells that contributed to the increasing Z_cell, as the capacitance was inversely related to the impedance^12,28,29,30.

The results of Z_cell (as shown in Fig. 8) and resistance (as shown in Table 1) were compared with the number of viable cells (as shown in Fig. 6). The findings demonstrated that the MCF7 breast cancer cells had higher cell viability and lower resistance value as the pH increased, while the MCF10a also showed a similar increasing trend in cell viability and resistance value as the pH increased. It has been verified by Ref.³¹ that the smaller gaps between the cells and electrodes led to better sensitivity in the cells’ electrical impedance signals and resistances.

The electrical impedance and resistance responses of untreated and treated MCF7 and MCF10a cells with different pH of GO after 48 h

Figure 9 shows the electrical Z_cell and phase ranging over the frequency ranging 5 kHz to 1 MHz for untreated and treated MCF7 and MCF10a with different GO pH for incubation period of 48 h. The results for both cells shows that the Z_cell decreased as the frequency increased and it converged at the higher frequencies. According to Ref.¹², this condition was due to the frequency dependent characteristics, as calculated according to Eq. (1). At 5 kHz, for MCF7 the Z_cell measure decreased from 191.01 Ω with phase of − 59.97° after the treatment with GO of pH 5 to 162.30 Ω with phase of − 55.14° after the treatment with GO of pH 7. Meanwhile, for MCF10a at the same frequency, the Z_cell increased from 131.39 Ω with the phase of − 47.46° after the treatment with GO of pH 5 to 170.29 Ω with the phase of − 57.70° after the treatment with GO of pH 7. The value of measured Z_cell of the MCF7 was generally higher than the measured Z_cell of the MCF10a. It was also evident that only small Z_cell variation was observed between the ut and sample with different pH value. The phase measure for all samples was in the negative region corresponding to the capacitive behavior of the cell membranes and could be considered as inherent characteristics of cell membranes which act as dielectric layer.

The R_cell and C_cell for both the treated MCF7 and MCF10a cells were also compared, and the results are as tabulated in Table 2. The R_cell value for MCF7 cells was the lowest at 3.23 kΩ, with the C_cell equals to 673 nF, while the R_cell value MCF10a was the highest at 3.59 kΩ, with the C_cell equals to 847 nF, after the treatment with GO of pH 7. The result for the Z_cell (as shown in Fig. 9) and R_cell (as shown in Table 2) were compared with the number of viable cells (as shown in Fig. 6).

From the results it was observed that the MCF10a has higher C_cell as compared to MCF7 at respective pH of GO. This could be due to the lower capacitive behavior of the cell membrane for metastatic grade cancer, which could be caused by their low sterol and phospholipid contents³². The irregular shape of MCF7 as compared to MCF10a can cause it to become less inflexible, which contributes to lower polarization which also led to lower capacitance value³³.

It was also observed that the MCF7 has higher R_cell value decreased as pH value increased while for MCF10a the R_cell increases as pH value increased. This can be related to the viability of the cells after 48 h. In MCF7 cell viability reduced to 56.98% from 63.07% as pH of GO increased from 5 to 7. In comparison to MCF10a in which the cell viability improves from 109.8 to 116.4% cells viability. Cell membrane is made up of a highly mobile lipid molecule bilayer that is an electrically insulator³⁴. Having more cells available will further increase resistance. The more cells available will further decrease the gap between the highly insulating cell membranes of viable cells with the electrode surface and the decreased capacitance³⁵.

The electrical impedance and resistance responses of untreated and treated MCF7 and MCF10a cells with different concentrations of GO after 24 h

The dose dependent GO effects on the MCF7 and MCF10a cells are shown in Fig. 10a,b. The findings showed that the Z_cell of MCF7 cells rose after treatment with 12.5 µg/mL of GO at the pH of 7. However, the Z_cell of MCF7 cells decreased as the GO concentrations increased. At a frequency of 5 kHz, the electrical Z_cell of the MCF7 cells treated with 12.5 µg/mL of GO was 425.27 Ω with a phase of − 44.78°, while for the MCF7 cells treated with 100 µg/mL of GO, the electrical Z_cell was 220.45 Ω with a phase of − 39.05°. From Fig. 10c,d, the MCF10a cells treated with 25 µg/mL of GO showed the highest Z_cell value of 345.03 Ω with a phase of − 34.13°, while the MCF10a cells treated with 2.5 µg/mL of GO showed the lowest Z_cell of 210.27 Ω with a phase of − 62.83° at 5 kHz.

An increasing Z_cell value shows the increasing resistance of the cells. For the MCF7 treated with 12.5 µg/mL of GO, the highest R_cell value was 7.73 kΩ with the C_cell equals to 256 nF. Meanwhile, for the MCF10a treated with 25 µg/mL of GO, the highest resistance value was 6.62 kΩ with the C_cell equals to 225 nF. The resistance and capacitance results of MCF7 and MCF10a untreated and treated with different GO concentrations for 24 h were summarized in Table 3.

The Z_cell and resistance results trends were correlated to the number of viable cells trend, as shown in Fig. 7a. The highest number of viable MCF7 cells was recorded after the treatment with 12.5 µg/mL of GO, while the lowest was recorded after the treatment with 100 µg/mL of GO. Meanwhile, the highest number of viable MCF10a was recorded after the treatment with 25 µg/mL of GO, while the lowest was recorded after the treatment with 2.5 µg/mL of GO. The trend was comparable to the findings reported by Ref.³⁵. The study observed that the Z_cell and resistance increased to correspond with the decreased gap between the highly insulating cell membranes of viable cells with the electrode surface; hence it was related to the decreased capacitance. When the capacitance decreased, the Z_cell value increased, similar to the resistance value.

The electrical impedance and resistance responses of untreated and treated MCF7 and MCF10a cells with different concentrations of GO after 48 h

Figure 11 shows the electrical Z_cell and phase of MCF7 and MCF10a untreated or treated with different GO concentrations, with a longer incubation period of 48 h. At a frequency below 500 kHz, the Z_cell decreased as the frequency increased. In contrast, it intersected and became stable at frequencies higher than 500 kHz. According to Ref.¹², this condition was due to the frequency-dependent characteristics, as calculated according to Eq. (1). For the MCF7 treated with 100 µg/mL of GO, the lowest Z_cell was 162.30 Ω, with the phase of − 55.68°, while the highest Z_cell was 189.24 Ω, with the phase of − 59.87° at the frequency of 5 kHz. On the other hand, for the MCF10a, the Z_cell increased from 139.13 Ω with the phase of − 48.82° to 170.29 Ω with the phase of − 57.70° at 5 kHz after treated with a higher GO concentration of 100 µg/mL.

Table 4 shows the resistance (R_cell) and capacitance (C_cell) of MCF7 and MCF10a before and after incubation treatment with different GO concentrations for 48 h. For the MCF7, the lowest R_cell value was 3.23 kΩ, with the C_cell equals to 673 nF after treatment with 100 µg/mL of GO, while the highest R_cell value was 3.68 kΩ, with the C_cell equals to 563 nF after treatment with 12.5 µg/mL of GO.

The Z_cell and resistance trends of MCF7 were similar for 24 and 48 h, as compared to the results presented in Fig. 9 and Table 3. Thus, the results proved that the Z_cell and resistance were increased linearly with an increasing amount of highly insulating viable cell membrane on the IDEs, subsequently leading to a decrease in the capacitance. These findings were in agreement with the findings of Ref.¹². Meanwhile, the R_cell increased steadily from 2.43 to 3.59 kΩ, with the C_cell equals to 1143 nF and 847 nF for MCF10a after 100 µg/mL of GO treatment. It showed that the resistance trend was corresponding to the impedance, as shown in Fig. 11, and was comparable with the trend for the number of viable MCF7 and MCF10a cells, as shown in Fig. 7b.