Study On Detection Of The Cell-surface Protein EpCAM Using Microfluidic Droplet Based On Enzymatic Amplification

Objective: The flow cytometry in combination with fluorophore-labeled antibodies cannot reliably detect low-abundance proteins on cell surface.With its size close to the cell,microfluidic chip-based droplet is ideally suited for single-cell analysis.This study attempted to apply microfluidic chip-based droplet,together with enzyme-based immuno-amplification,to detect the cell surface-proteins on the single cell level.Methods: This is a methodolgical research,first establishment of the method and then validation of the method.(1)Selection of appropriate labeling enzyme and fluorogenic substrate.Alkaline phosphatase(ALP)was chosen as labeling enzyme,and fluorescein diphosphate(tetraammonium salt,FDP)was chosen as substrate in this study.(2)The Ep CAM expressing A549 cells were chosen and labeled with biotin-labeled anti-Ep CAM antibody,followed by incubation with streptavidin-labeled alkaline phosphatase(ALP-SA).Then the A549 cell suspension,together with the fluorogenic substrate FDP,was encapsulated into microfluidic chip-generated water-in-oil droplets.The fluorescence of enzymatic reaction in droplets was observed using fluorescence microscope.The number of droplets containing single A549 cells and with fluorescence could be counted,so the rate of Ep CAM-expressing cells could be calculated.(3)A549 cells were sequentially labeled with biotin-labeled anti-Ep CAM antibody and FITC-labeled streptavidin(FITC-SA).The FITC-labeled cell suspension was subsequently analyzed using both droplet method and flow cytometry to calculate the rate of Ep CAM-expressing cells.(4)The rate of Ep CAM-expressing cells was further analyzed by mixing ALP-labeled A549 cells or FITC-labeled A549 cells with A549 cell without any labeling by varying quantities ratios(1:1,1:3 and 1:10).(5)Both droplet method and flow cytometry were applied for detecting Ep CAM-expressing cells and the results of the two methods were compared.Results:(1)For non-labeled A549 cell suspension,the rate of cells expressing Ep CAM was determined to be 1.05% by microfluidic-based droplet method.For ALP-labeled A549 cells,the rate was 81.32%,while for A549 cells labeled with FITC,fluorescence could not be observed using fluorescence microscope.(2)Using the microfluidic-based droplet method,the Ep CAM expressing-cell rates were 42.78%,26.44% and 8.29% for mixed suspensions of ALP-labeled cells and unlabeled cells with a mixing ratio of 1:1,1:3 and 1:10,respectively.(3)Using flow cytometry as the detection method,the rate of Ep CAM expressing cells was determined to be 0.73% for non-labeled A549 cell suspension;otherwise the rate was 50.82% for FITC-labeled A549 cells.(4)For mixed suspensions of FITC-labeled and non-labeled cells with the ratio of 1:1,1:3 and 1:10,the rates of Ep CAM expressing cells were 25.50%,15.08% and 5.58% respectively.Conclusion: In comparison with flow cytometry,microfluidic-based droplet,in combination with enzymatic amplification reaction,could significantly improve the sensitivity of detecting cell surface protein Ep CAM.This method is ideally suited for the detection of low abundance proteins expressed on the cell surface.