Counting Protein Number in a Single Cell by a Picoliter Liquid Operating Technology

Ultra-low-copy number proteins play a crucial role in exploring cellular heterogeneity and the insight of protein biomarkers in a single cell. However, counting ultra-low-copy number target proteins in a single cell remains a grand challenge. Herein, we developed a so-called single-cell picoliter liquid operating technology for counting target proteins in a single cell. An ingenious volume-controllable sampling technique was employed to capture a single cell for subsequent analysis. Remarkably, 50 pL of sample volume was employed for sample preparation, single-cell capture, in-droplet lysis, and target protein immobilization on a functionalized coverslip in a monolayer. Then, target protein antibodies coupled with quantum dots were added and incubated to label those immobilized proteins. After clean-up, a single-view image under 100× objective was taken, and the 80 × 80 μm² view image was then applied to count the precise copy number of the target proteins in the single cell. Furthermore, good linearity and repeatability were achieved for ultra-low-copy number proteins, ranging from 1 to 1500. Finally, the expression level of human epidermal growth factor receptor 2 in single cells from both MCF-7 and MDA-MB-231 cell lines was also analyzed. In a word, this work stimulated the development of capillary-based single-cell analysis and updated the connotation of counting ultra-low-copy number proteins.