Simulation And Experimental Study Of Single Cell Electroporation Using A Microfluidic Chip

In recent years,due to the deteriorating ecological environment in China,cancer has showed a high incidence and a sustained growth trend.With the improvement of people’s health awareness and medical means of continuous improvement,the limitations of traditional tumor treatment methods become more obvious.For instance,surgical treatment brings great trauma to patients and it exists the risk of further metastasis of radiation.Radiation therapy and chemotherapy have a negative impact on the patients’ mind and body.Pulsed electric field therapy,as a new tumor treatment technique,has come into being in recent years.The full use of pulsed electric field can preserve the integrity of tumor antigens,effectively stimulate the body’s immune system and contribute to the treatment area outside the micro-metastases of the attack.This treatment method of completely relying on pulsed electric field ablation of the tumor shows a good development prospect,and causes a large number of domestic and foreign scholars to carry out a large number of cell and animal experiments on electroporation.In the cell experiment level,the traditional electroporation device,due to much heat production,a large number of cells within the device between the interference and other shortcomings,cannot be accurately and deeply used to explore the relevant mechanism.Based on MEMS technology,microfluidic chips can solve the above problems.Also,at present most of the pulse parameters are selected according to the experience,and even the pulse parameters can be determined according to the hardware.There must be some certain blindness.In this paper,a microfluidic chip for cell electroporation experiments has been successfully developed.The experimental study on electroporation threshold curve and cell impedance measurement has been carried out in order to explore the aforementioned problems,and then to provide a certain basis for the cell,tissue and clinical experimental parameters selection later.The main results achieved are as follows:(1)The spherical single cell three-layer dielectric model and the pore radium changing mathematical model of cell membrane electroporation are established by using COMSOL Multiphysics 5.0 software.From the simulation results,it can be concluded that the larger the electric field intensity,the faster the cell membrane is charged after the pulse is applied,the faster the transmembrane potential rises,the maximum speed decreases after the maximum value,and the final stable voltage value is lower.Pore density and pore area are also greater.As the pulse duration increases,the maximum pore radium is also increasing(2)With the glass as the base material,gold as the microelectrode material,PDMS as the packaging material,a uniform electric field producing microfluidic electroporation chip is designed and fabricated.The width and depth of the channel are both 30μm,the electrode thickness is 30μm,and the width is 100μm.In front of the channel,a sheath flow structure was added,in which the angle between the sheath flow channel and the cell fluid channel was 30 ° and the length ratio was 1: 1.Cells can flow through the microchannel one by one,so that we can manipulate and analyze at single-cell level.In the meanwhile,sheath-flow can also guarantee that cells will not block at the inlet and outlet of the channel.(3)Single-cell electroporation experiments are performed on the human ovarian cancer cells on the microfluidic electroporation platform by using PI as the fluorescent indicator.The experimental results show that increasing the voltage amplitude and pulse width can enhance the intake of PI molecules,and the voltage amplitude plays a more significant role than the pulse duration.There is a threshold voltage for the cell electroporation.When the applied voltage is below the threshold voltage,the increase in pulse duration does not induce electroporation.The threshold voltage corresponding to the different pulse width is not the same,the width of the pulse width,the smaller the threshold voltage.A voltage-pulse-width electroporation threshold curve is plotted.(4)The study of human melanoma cell line Gll19 is carried out.The experimental results show that the impedance value decreases with the increase of frequency.At the same time,increasing the pulse width and voltage amplitudes can make the impedance value decrease as well.The voltage amplitude-pulse width threshold curves are plotted for varying the percentage of impedance modulus values(40%,30% and 20%)at 1 kHz and 5 kHz.The greater the degree of impedance declines,the greater the pulse parameters are required.The corresponding pulse parameters at different frequencies are also different.Impedance changes coupled with cell biopsy can provide a basis for subsequent selection of parameters for subsequent cell and clinical trials.Finally,the results of the changes of the impedance values before and after the New Zealand rabbit liver tissue ablation is showed to verify the correctness of the forementioned cell experients.