Construction Of Single-cell Precise Manipulation Platform Based On Microfluidics And Its Application In Single Cell Analysis

Cell is the basic unit of organism structure and functions.Conventional bulk methods process a large number of cells at a time.But even in the same type of cell,cell heterogeneity exists.Therefore,the average result based on conventional bulk methods ignores the intrinsic heterogeneity among single cells and single-cell analysis is in a critical need.The microfluidics has been widely applied in single-cell research,for which it offers a number of particular advantages.It can precisely manipulate single cell,due to the cellular scale dimensions.What's more,it has the ability to use very small quanties of reagents,carry out detection with high sensitivity and integrate multiple operations.In this study,microfluidic technology and single cell analysis were combined.We developed the microfluidic platforms to precisely manipulate single cell,including single cell capture and assembly,and achieved the analysis of single cell protein.The main content of this paper include three parts.1.Manipulate and pair single cell/bead based on microfluidicsThe single cell precise manipulation is an important condition for single-cell analysis.However,the developed single cell analysis platforms have their limitations.For example,the droplet microfluidics can encapsulate the single cell,whereas the single cell encapsulation efficiency is low and the long-term dynamic observation is difficult for the particular cell,for which the droplets can move.In contrast,single cell microarray can capture single cell highthrouputly and the cell can be observed in situ.However,cells are not isolated in most single cell microarray.The interference can't be avoided.To combine the advantages and overcome the limitations of droplet microfluidics and single cell microarray,we developed a microfluidic chip which can trap and paire the single cell/bead with high efficiency and throughput based on fluid dynamics.Besides,paired droplets that contain single cell and single bead could be generated,which avoided the interference during single cell analysis.The paired droplets manipulation,including droplets mixing,reagent change could be achieved by microvalve.This microfluidic platform has provided the theoretical basis for single cell analysis.2.The analysis of single cell protein based paired chipProteins are the key executors of biological process.The analysis of single cell protein can help to clarify the process of life.Two biggest challenges to analysis single cell protein are the abundance of single cell protein is low,and protein can't be directly amplified like nucleic acid.There are a few platforms available for single cell protein analysis.However,it's difficult for the platforms like mass spectrometry,flow cytometry to achieve high-sensitivity measurements of low abundant protein in single cell.And these machines are expensive.The quality and quantity of single cell isolation are limited based on passion distribution in mirowell and microvalve platform.In this paper,we described a new method to measure single cell protein based on the paired-chip.The chip could capture and pair single cell/single bead with high efficiency and high throughput based on fluid dynamics.The single cell sensitivity was improved by isolating single cell in 160 pl volume droplet.What's more,Integrated operations reduced the loss of single cell protein,like single cells lysis and measurement of single cell protein by immune-assay.At last,the quantity and heterogeneity of single cell protein were highthrouputly measured based on the fluorescence intensity of single bead.This method may have the potential to deeply understand the cancer progression,immune response and so on.3.Precisely assemle single cell based microfluidic platformThe functions of any given cell is affected by its neighboring cells in multicellular organisms.Therefore,it's important to reconstruct the cell-cell interaction in vitro.However,The current platforms mainly focuses on pairing two cells.Multiple cells can be essembled by combining microfluidics with optical,electrical,magnetic.However,these methods are time-consuming and require complex equipments.The mirowell array and droplets microfluidics are hard to get the desired number/type cell assemblages due to Poisson distribution.To overcome these limitations,we designed a microfluidic chip that could precisely assemble cell clusters.This chip had two work modes by controlling microwalve.One is single cell trap mode,the other is single cell transfer mode.The quantity and type of cell culstures could be precisely controlled by repeating the operations of single cell trap and transfer.This microfluidic platform can be used to reveal the cell-cell interaction in the future.