Quantification of the Extracellular ROS of T24 Single Cells

Probe approach curves were recorded to help further studies for quantifications of extracellular ROS. At the beginning, the electrode was biased at 0.70 V. Depth scan images were acquired by moving the scanning stage in Y, Z plane. As shown in Figure

4.11, partial negative feedbacks were found when an electrode approached the cells. That is because when the electrode is biased at 0.70 V, hydrogen peroxide as well as Fc would get oxidized on the tip surface. The diffusion of Fc from bulk solution to the cells surface is blocked when the tip is approaching. Fc+ generated on the tip surface would not get reduced back to Fc again because cells act like insulator in this process. This leads to a decreased current. In the meanwhile, H2O2 generated by the cells caused an increased

concentration of H2O2 near cells surface and therefore an increased current. The

blockage of Fc diffusion outweighed H2O2 release from the cells, which leads to an

overall decreased current. By overlapping the experimental PACs with simulated ones, corresponding concentrations of H2O2 are determined. For cells without vitamin C

treatment, concentration was determined to be 0.036 mM. For those cells that had been incubated with 2 mM vitamin C solution for 30 minutes and 24 hours, the concentrations were 0.052 mM and 0.030 mM, respectively.

After each scanning at 0.70 V, the tip was raised to the initial position and the biased potential was changed to -0.60 V for some more depth scans. At this potential, oxygen dissolved in bulk solution and produced by cells would be reduced on the tip surface. Partial positive feedbacks were observed in Figure 4.12. The diffusion of O2 is blocked

by the cell when the tip is moving down toward the cell, resulting in a decreased current. At the meantime, the current get increased when the tip is at the vicinity of cell surface because of oxygen generation from the cell. Partial positive feedbacks were obtained as a result of the dominant role of oxygen generation. For untreated cells, the corresponding concentration of oxygen was decided to be 0.156 mM according to the simulated data. The concentrations for 30 minutes or 24 hours vitamin C treated cells were determined to be 0.166 mM and 0.152 mM, respectively.

Again, the electrode was moved up to initial position after each scan. A new potential was set at -0.80 V, at which potential both oxygen and hydrogen peroxide would get reduced on probe surface. Similar to the results obtained at -0.60 V, partial positive feedbacks were obtained in Figure 4.13. This shares the similar explanation with Figure 4.12. H2O2 and O2 production plays a dominant role.

Figure 4.11 Normalized probe approach curves (PACs) recorded over T24 single cells. The cells were treated with vitamin C for 30 mins, 24 hours and without treatment, respectively. Purple circles are experimental data obtained with a 10μm UME with a RG value of 3 at a biased potential of 0.70 V. Several simulated data are shown in solid line in different colours. Hydrogen peroxide concentrations are shown in the legend.

Figure 4.12 Normalized probe approach curves (PACs) recorded over T24 single cells. The cells were treated with vitamin C for 30 mins, 24 hours and without treatment, respectively. Purple circles are experimental data obtained with a 10μm UME with a RG value of 3 at a biased potential of -0.60 V. Several simulated data are shown in solid line in different colours. Oxygen concentrations are shown in the legend.

Figure 4.13 Normalized probe approach curves (PACs) recorded over T24 single cells. The cells were treated with vitamin C for 30 mins, 24 hours and without treatment, respectively. Purple circles are experimental data obtained with a 10μm UME with a RG value of 3 at a biased potential of -0.80 V. Several simulated data are shown in solid line in different colours. Oxygen and hydrogen peroxide concentrations are shown in the legend.

Since the concentrations of H2O2 and O2 have already been obtained in previous

simulation results at 0.70 V and -0.60 V, they were assumed to be the same at the proximities of the cell and utilized for simulation at -0.80 V. If the concentrations were correct, overlapped results of simulation and experimental ones were expected. As shown in Figure 4.13, overlapped results were observed. This confirmed the correctness of the concentrations obtained about the H2O2 and O2 concentrations in previous

simulation. It can also be concluded from the quantification results that vitamin C plays a pro-oxidant role in first 30 minutes and exerts anti-oxidant effect after several hours.

4.4

Conclusions

SECM technique was proved to be an effective tool to investigate the interaction between cancer cells and anti-cancer drugs with the extracellular ROS as the indicator. The H2O2 and O2 releases were investigated by SG/TC mode and depth scan images at

biased potential of both negative and positive sides. Qualitative and quantitative studies were conducted. The concentrations of H2O2 and O2 were determined by superimposing

experimental PACs with simulated ones.

The SECM measurements evidenced the switch-type behaviour of vitamin C on the PMA-induced ROS burst of T24 cells. When the T24 cancer cell was treated with vitamin C for 30 minutes, vitamin C was observed to be a pro-oxidant. Both H2O2 and

O2 were significantly diminished after incubating with vitamin C for 1 day, which proved

the anti-oxidant effect of vitamin C.

In Jamison's 2004 Biochemical Pharmacology paper, vitamin C induced H2O2

formation was measured fluorimetrically in cell lysates at 15-min increment for 1 hr by evaluating the oxidation of dihydrorhodamine 123 to rhodamine 123 and expressed in terms of nanomoles of hydrogen peroxide per milligram of protein. In his results, VC exposure resulted in an increase of H2O2 content during the first 15 min of incubation and

kept increasing during the second 15 min of incubation. The H2O2 content rapidly

decreased during the next 15 min and remained constant during the final 15 min of incubation17. The obtained results in my project agrees very well with their report.

4.5

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