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

Breast cancer is one of the most reported cancers that can lead to death. Despite the advances in diagnosis and treatment procedures, the possibility of cancer recurrences is still high in many cases. With that in consideration, researchers from all over the world are showing interest in the unique features of Graphene oxide (GO), such as its excellent and versatile physicochemical properties, to explore further its potential and benefits towards breast cancer cell treatment. In this study, the cell viability and electrical response of GO, in terms of resistivity and impedance towards the breast cancer cells (MCF7) and normal breast cells (MCF10a), were investigated by varying the pH and concentration of GO. Firstly, the numbers of MCF7 and MCF10a were measured after being treated with GO for 24 and 48 h. Next, the electrical responses of these cells were evaluated by using interdigitated gold electrodes (IDEs) that are connected to an LCR meter. Based on the results obtained, as the pH of GO increased from pH 5 to pH 7, the number of viable MCF7 cells decreased while the number of viable MCF10a slightly increased after the incubation period of 48 h. Similarly, the MCF7 also experienced higher cytotoxicity effects when treated with GO concentrations of more than 25 µg/mL. The findings from the electrical characterization of the cells observed that the number of viable cells has corresponded to the impedance of the cells. The electrical impedance of MCF7 decreased as the number of highly insulating viable cell membranes decreased. But in contrast, the electrical impedance of MCF10a increased as the number of highly insulating viable cell membranes increased. Hence, it can be deduced that the GO with higher pH and concentration influence the MCF7 cancer cell line and MCF10a normal breast cell.

The new potential of GO in affecting cancerous cells is explored, as it has good surface functionality due to the oxidation process produced by using Hummer's method. Hence, GO was chosen to observe the treatment effects on MCF7 breast cancer cells and MCF10a normal breast based on the cell viability and after the incubation period of 24 h and 48 h. Even though the studies elucidating the effects of GO on normal cells are very limited in comparing studies on GO impact on cancer cells, the precise mechanism that cause difference effect GO on normal and cancer cells is still unknown 13 . Therefore, in contrast to the previous research, the electrical responses and effects of MCF7 and MCF10a without using any redox solutions or biomarkers were studied to observe the direct interactions between the cells and GO. This is very important to fully understand the electrical responses between the cells, as it may be useful for developing an early breast cancer detection device.

Experimental details
Pre-oxidized graphite. The first step of the study was to prepare the pre-oxidized graphite by following the protocol reported by Ref. 14 . Firstly, a mixture was prepared by dissolving 3 g of graphite powder (NE Scientific, Malaysia), 2.5 g of potassium persulfate (P 2 S 2 O 8 ) (Sigma-Aldrich, USA) and 2.5 g of phosphorus pentoxide (P 2 O 5 ) (Sigma-Aldrich, USA) in 12 mL of sulfuric acid (H 2 SO 4 ) (Sigma-Aldrich, USA). Then, the solution mixture was kept in an oil bath for four and a half hours under the temperature of 80 °C. Next, the mixture was cooled to the ambient temperature before it was diluted further with 500 mL of deionized (DI) water and left overnight. Finally, the mixture was filtered by using a 0.22 μm PTFE membrane and was washed with DI water to achieve neutral conditions through removing the residual acid.
Modified Hummer's method. This section described Hummer's method to produce the aqueous GO with some modification. First, the pre-oxidized graphite was added into 120 mL of concentrated sulfuric acid pre-chilled to 0 °C and was kept incubated inside an ice bath. Then, 15 g of potassium permanganate (KMnO 4 ) (Sigma-Aldrich, USA) was added gradually into the mixture to prevent extravagant heat. The mixture was stirred at 800 rpm and must be kept under 20 °C. Then, the mixture was stirred for 2 h under 35 °C. After that, Cell culture. The  . Briefly, the cell's medium was firstly removed, then MCF7 and MCF10a with and without the GO treatment were trypsinized and the reaction was terminated by adding newly fresh medium. The cells in suspension were then centrifuged, in order to get the cell's pellets. Then, 1.5 mL of medium was added and the cells were manually counted using hemocytometer. Lastly, 5 µL of the MCF7 and MCF10a cells were deposited onto the IDE by using the drop cast method. The Z cell and resistance changes before and after deposition were then monitored at a frequency between 20 Hz to 2 MHz using the LCR meter.
(5) Average media = summation of three well plates for media only ÷ 3, Average untreated cells = summation of three well plates for untreated cells ÷ 3,

Results and discussion
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 sp 2 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 bonds 15 . 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 sp 2 hybridized carbon materials that were related to the C-C vibrational mode 16 . 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 network 15 . 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 (SiO 2 ) 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 structure 17 .
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 surfaces 18 .
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 sp 2 to a non-graphitic sp 3 hybridized carbon that contains high density of defects due to the oxidation process that occurred. The defects and weaker www.nature.com/scientificreports/ 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 condition 22 . Hence, when the MCF10a surrounding was slightly acidic due to the pH  www.nature.com/scientificreports/ 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 increased 21 . 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  www.nature.com/scientificreports/ 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 cells 25 .
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  www.nature.com/scientificreports/ 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 12,28-30 .
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. 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 electrical impedance and resistance responses of untreated and treated MCF7 and MCF10a cells with different pH of GO after 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 contents 32 . 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 value 33 .
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 insulator 34 . 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 capacitance 35 .  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 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.   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    www.nature.com/scientificreports/ 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.

Conclusion
For this research, a few layers of GO, with an average size of 0.56-0.96 µm and an average thickness of 1.24-1.32 nm, were successfully produced by using the Hummer's method. Then, the GO was further synthesized and characterized by using the Raman spectroscopy, XRD spectroscopy, FESEM, EDX spectroscopy and TGA analysis. The cell viability for both the MCF7 and MCF10a before and after treatment with GO was successfully determined by using the PrestoBlue cell viability assay. After treating both the MCF7 and MCF10a cells with three different pH (e.g., pH 5, 6 and 7), it was that when the GO pH increased to 7, the number of MCF7 viable cells was decreased, while the number of MCF10a viable cells was maintained at 24 h incubation time. Also, the number of MCF7 viable cells was further decreased with the increasing number of MCF10a viable cells at 48 h incubation time. Hence, it can be deduced that the GO of pH 7 was the most suitable pH to inhibit the MCF7 proliferation compared to pH 5 and pH 6, with the optimum incubation period at 24 h. In terms of the dosedependent interaction between cells and GO, the concentration of GO was varied into six different concentrations (e.g., 2.5, 6.25, 12.5, 25, 50 and 100 µg/mL). For the electrical characterization part, the electrical properties of MCF7 and MCF10a cells before and after exposures to GO were successfully demonstrated by using 10 µm-gaps gold interdigitated electrodes connected to the LCR meter. In conclusion, the trend showing the number of viable cells was comparable with the capacitance, impedance, and resistance results. Thus, it was proven that the capacitance reduction was due to the increase in highly insulating MCF7 and MCF10a cells membrane. When dropped on the electrode surface, the highly insulated cells membrane caused an increase in electrical Z cell and resistance. In conclusion, the electrical characterization and cell viability analyses of breast cancer cells using the GO deposited on IDE were successful.