Evaluation of the anticancer effects induced by cold atmospheric plasma on leukemia stem cells

Hydrogen peroxide detection kit [35] and total nitric oxide detection kit produced by Beyotime Biotechnology Co., Ltd were used to detect the liquid-phase active particles of the cell culture solution treated by the jet. The number of the hydrogen peroxide detection kit is S0038, and the number of the nitric oxide detection kit is S0023. In the experiment, the type of power supply and the gas passed in the jet device were used as variables, and the discharge power and processing time of the jet device were controlled to be the same. The hydrogen peroxide and nitric oxide contents in the cell culture fluid after the treatment were measured. According to different control conditions, the experiment is divided into four groups: sinusoidal power source + He and 0.5% ${{\rm{N}}}_{2},$ sinusoidal power source + He and 0.5% ${{\rm{O}}}_{2},$ pulse power source + He and 0.5% ${{\rm{N}}}_{2},$ pulse power source + He and 0.5% ${{\rm{O}}}_{2}.$

During the experiment, the jet generating device was fixed on an iron stand, and the cell culture solution was placed directly under the jet device. Then we connect the power supply and the oscilloscope and control the oscilloscope power display to 1 watt. It's necessary to adjust the height of the iron stand so that the plasma plume could just touch the culture solution page. The discharge processing time is 30 s. The processed culture medium sample is added to the kit reagent and tested in a microplate reader to obtain a measurement result.

Since each atom has its own characteristic spectral line, it is possible to identify the substance and determine its chemical composition based on the spectrum. This method is called spectral analysis [36] For spectral analysis, either emission spectrum or absorption spectrum can be used. The advantages of this method are very sensitive and fast. If the content of a certain element in the substance reaches ${10}^{-10}$ grams, and its characteristic spectral line can be found from the spectrum, so it can be checked out.

The measurement of the absorption spectrum is based on the characteristic spectrum of the element to be measured. The content is calculated from the intensity of the measured element's spectrum after being absorbed by the ground state atoms in the sample vapor. It complies with Beer–Lambert Law [37]:

$$\begin{eqnarray*}&&{\rm{A}}=\,{\rm{lg}}\,\displaystyle \frac{{I}_{0}}{I}=\,\mathrm{lg}\,\displaystyle \frac{1}{T}=Kbc\end{eqnarray*}$$

K is the absorbance and T is the transmittance. I represents the intensity of the outgoing light and ${I}_{0}$ represents the intensity of the incident light. K is the molar absorption coefficient, c is the concentration of the light absorbing substance, and b is the thickness of the absorption layer.

Therefore, during the discharge of the device, we used an optical lens-assisted spectrometer to measure the absorption spectrum of the plasma plume in the discharge part, so as to explore the material composition and relative content of the discharge area during the discharge process.

When the output voltage of the power supply is adjusted so that the discharge power of the jet device is 1 watt, the voltage and current waveforms are shown in figure 2. Figure 2(a) is a voltage and current waveform diagram of a pulse power supply under constant discharge power, and figure 2(b) is a voltage and current waveform diagram of a sinusoidal power supply under constant discharge power.

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When the output voltage of the power supply is adjusted so that the output voltage is 8 kV, the voltage and current waveforms are shown in figure 3. Figure 3(a) is a voltage and current waveform diagram of a pulse power supply at a constant output voltage, and figure 3(b) is a voltage and current waveform diagram of a pulse power supply at a constant output voltage.

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The power and processing time are consistent during the control of the discharge process, and the discharge power and background gas components are used as variables. The contents of hydrogen peroxide and nitrite ions in the cell culture fluid of the four groups were measured respectively: pulsed power + Helium and 0.5% nitrogen, pulsed power + Helium and 0.5% oxygen, sinusoidal power + Helium and 0.5% nitrogen, sinusoidal power + Helium and 0.5% oxygen. The content of hydrogen peroxide in the four groups of the cell culture fluids is shown in figure 4(a), and the content of nitrite ions is shown in figure 4(b).

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Considering only the driving power and the same background gas, we found that under the same gas composition, the amount of hydrogen peroxide produced by the pulse power driving device was slightly higher than that of the sinusoidal power. When the background gas is only taken into consideration, the amount of hydrogen peroxide generated when helium is doped with oxygen is higher than when helium is doped with nitrogen. Overall, when the device is driven by a pulsed power source and the background gas is Helium and 0.5% nitrogen, the hydrogen peroxide content in the cell culture fluid is the highest, which is about 4–6 times that of the other three groups.

Considering only the driving power and the same background gas, we found that under the same gas composition, the amount of nitrite ion produced by the pulse power driving device was slightly higher than that of the sinusoidal power. When the background gas is only taken into consideration, the amount of nitrite ion generated when helium is doped with oxygen is higher than when helium is doped with nitrogen. Overall, when the device is driven by a pulsed power source and the background gas is Helium and 0.5% nitrogen, the hydrogen peroxide content in the cell culture fluid is the highest, which is about 8–9 times that of the other three groups.

For the above experimental results, we think that the reason why the pulsed power supply generates more active particles is that during the pulse discharge, the peak voltage is higher and the sustaining time is longer, which results in more energy concentration and more active particles. For the phenomenon that oxygen-doped helium produces more active particles than nitrogen-doped helium, we think that the main reason is that the main long-life active particles such as hydrogen peroxide and nitrite ions contain oxygen element. Therefore, doping an appropriate amount of oxygen in the easily ionized background gas helium helps the production of these oxygen-containing active particles. Aiming at the problem that the nitrogen doping in helium has little effect on the content of nitrate ions, we suspect that the nitrogen content in the air at the jet exit is enough to combine with the reactive oxygen generated. So doping nitrogen in the background gas has little effect on the content of nitrate ions.

Using the discharge power source and the background gas components as variables, the power during the discharge process is controlled, and the absorption spectra of the plasma plumes of the four groups of jet devices are detected respectively: pulsed power + Helium and 0.5% nitrogen, pulsed power + Helium and 0.5% oxygen, sinusoidal power + Helium and 0.5% nitrogen, sinusoidal power + Helium and 0.5% oxygen.

In the case of 1 watt power, figure 5 compares the relative intensity of the plasma absorption spectrum under different conditions. Comparing figures 5(a) and (c), when the same gas is inserted in, the absorption line spectrum of the pulse power source and the sinusoidal power source is about the same. It indicates that different power sources do not change the material composition of the plasma plume. Comparing figure 5(b) with figure 5(d), as well as combining the spectral line identification in figure 6, it is found that when the gas is helium and 0.5% oxygen, the relative intensity of O (3p−3s) with a line length around 780 nm is obviously different. When driven by pulse power, the relative intensity of O (3p−3s) is 1350 a.u. When driven by sinusoidal power, the relative intensity of O (3p−3s) is 1200 au. Under the same conditions, the intensity of the pulse power is greater than that of sinusoidal power 11%. It also confirms that in the example of liquid-phase active particles, the amount of active particles generated by the pulse power source is more than that of the sinusoidal power source under the same conditions.

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Then, under the same power supply and gas conditions, when the discharge power is 0.5 watt and 2.5 watt, we measure the absorption spectrum of the plasma plume. The measurement results are shown in figure 7. Figure 7 shows that in the same background gas and power source, changing the discharge power only affects the intensity of the absorption spectrum without changing the spectral line distribution and shape of the absorption spectrum. The larger the discharge power, the stronger the relative intensity of the absorption spectrum.

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It is easy to find the characteristic spectral lines representing the oxygen and nitrogen atoms in figure 6. We set the spectral line near the wavelength of 337 nm as the characteristic line of the nitrogen atom, and set the spectral line near the wavelength of 777 nm as the characteristic line of the oxygen atom. Five discharge power gradients of 0.5 watt, 1 watt, 1.5 watt, 2 watt, and 2.5 watt are set to compare the relative intensities of the spectral lines. They are all driven by pulse power. Figure 8(a) shows that when the background gas is different, N-337 nm is more sensitive to power changes. With the increase of power, the absorption spectrum intensity of N-337 nm in the experimental group using helium + 0.5% nitrogen increased much faster than the experimental group using helium +0.5% oxygen. Figure 8(b) shows that O-777 nm is not sensitive to power changes when the background gas is different. When the power is low, the difference between different background gases is greater than that of high power. Accordingly, we conclude that different atoms have different sensitivities to power growth. Based on this characteristic, the proportion of each component in the discharge product can be affected by controlling the discharge power.

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