Single Cell Sonoporation Microfluidic Chip Based On Microcapillary Arrays

Cells are basic units to maintain biological structures and functions.The targets of traditional cell analysis are populations of cells,ignoring the heterogeneity among various cells and concealing the significant microscopic heterogeneity among individual cells,thus making it difficult to reflect the biological rules at the single-cell level.Therefore,single-cell analysis techniques can reveal the nature and rules of life activities at a deeper level and provide fundamental scientific basis for the cause,development and treatment of diseases.On the other hand,exogenous gene transfection at the single-cell level poses great potential to cure some of the rare genetic diseases.However,the current physical membrane opening techniques（photoelectric perforation）for intracellular delivery still have many drawbacks.For example,the optical piercing method requires expensive and sophisticated equipment as well as trained persons and high costs.The electroporation method may cause irreversible damage to cells.Most vector-mediated techniques are interfered by antibodies that can specifically bind to the cell and cannot deliver the target gene to the specified location.Therefore,simple,non-invasive and high-resolution transfection of exogenous genes at the single-cell level are urgently required.In this paper,we designed and fabricated a microfluidic chip for single cell transfection,which is capable of selectively capturing specific cells,such as tumor cells,and transfecting the target cells with the aid of acoustic waves.The tumor cell A549with a diameter of about 15μm was selectively captured and separated from the leukocyte with a diameter of about 7.5μm by using dielectrophoretic force through a microcapillary arrays.The positive and negative dielectrophoretic transover frequencies of both cells were measured:1)₀=29.23 k Hz for A549 and1)₁=127.58 k Hz for leukocyte.The density of tumor cells A549 used in the experiment was1×10⁶ cells/ml,and the density of leukocytes was 3×10⁶ cells/ml,which corresponds to a mixing ratio of 1:1.Moreover,plasmid transfection experiments were conducted.A plasmid modified with red fluorescent protein gene expression（m RFP-C3）was transfected by acoustic surface wave.After 48 h of cell culture,the expression of fluorescent plasmid was observed and counted,and the effects of acoustic wave power and treatment time on GFP expression as well as cell activity were investigated.It was found that under the power of 28 d Bm and 10 s treatment duration time,the transfection efficiency of fluorescent plasmid reached up to 87%and the cell survival rate was higher than 85%,which is 15%higher than the efficiency of the traditional electroporation method and about 7%higher than the current better acoustic transfection.The cell viability after treatment was maintained at a high level.The proposed chip with integrated microcapillaries can achieve cell sorting through dielectrophoresis and perform high-efficient single cell transcription,which may have broad application prospects in the field of stem cell gene therapy.