PDMS Microwell Stencil Based Multiplexed Single Cell Secretomic Analysis

In recent years,multiple single-cell protein secretion assays have provided in-depth exploration of secreted protein-mediated intercellular communication and cellular heterogeneity.However,increasing the proteomic parameters analyzed from each single cell has been a challenge.Therefore,we constructed a PDMS-based microfluorescence speckle analysis platform consisting of two antibody slides and a PDMS microwell array perforated membrane.Two different antibody sets can be immobilized by a static coating,eliminating the need for complex fluid handling or batch equipment,enabling multiple proteomic parameter detection,and analysing cellular differences in secretory omics characteristics in populations.Qualitative,can be a powerful tool for the analysis of high-throughput multi-single cell cytokine secretion,the main contents are as follows:(1)The secreted proteins of U937 population cells were detected by antibody barcode chip and sandwich immunofluorescence method.The effects of different stimulation conditions and secreted proteins on U937 population cells were investigated.We found that:(a)at the same stimulation time,the amount of secreted protein in the group with LPS and PMA stimulating a was significantly higher than that in the group containing only with LPS stimulating;(b)when the same stimulant was added,the amount of secreted protein increased with the stimulation time and peaked at a certain time point.And stable existence.The antibody slides were well-adsorbed and the antibody protein was specifically recognized,and a standard curve of protein content and fluorescence intensity was determined.(2)The PDMS microporous array perforated membrane was designed and fabricated,it can capture more than 1500 single cells through the analysis platform,,which ensures the high single cell throughout of the experiment.The IL-8 secreted protein was detected by the upper and lower slides,and the secretion data of the cells were analyzed.It was confirmed that there was no significant difference in the secretion of secreted proteins between the top and bottom antibody slides(P=0.188,paired t-test)and their performance With high correlation(R=0.98),these results demonstrate the successful implementation of a micro-fluorescence speckle analysis platform based on PDMS chips.Finally,U937 single-cell multiple secreted protein was detected,and the signal intensity of the secreted protein in the detection region corresponding to the same single-cell was different,indicating that there is a difference in secretion function between the same single cells,among which IL-10,IL-lb secreted is highest,IL-8 is in the middle,secreting TNF-?,MCP-1 and MIP-lb are less.