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

Abstract

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.

New Method for Counting and Picking Out Single Circulating Tumor Cells from Microliter-Volume Samples for Tumor Progression Surveillance and Single-Cell Heterogeneity Analysis

“…The cell-sampling apparatus was designed and established according to our previous work. 35 Briefly, it consists of a onesided sharpened capillary combined with a 1 μL syringe through a zero-dead-volume union, and the suction and discharge of the sample were realized and controlled by a highaccuracy step-size adjustable, two-way syringe pump. The whole capillary-based cell sampling apparatus was fixed on a 3-D platform for precise control.…”

“…Sampling Single CTCs from Real Peripheral Blood Samples. Ten microliters of tip blood was collected in tubes pretreated with EDTA-K2 on day 0 (before tumor-cell transplantation), day 4, day 7, day 10, and day 12 35 In brief, anti-CK19-functionalized coverslips covered with perfluoropolyether (PFPE) oil and quantum-dot-anti-CK19 fluorescent labels were prepared first. Then, the single-CTC sampling and cell lysis in situ processes were implemented.…”

“…Our method possesses great advantages in single-CTC sampling and CTC number counting from microliter-volume samples, and certain limitations still should be acknowledged. First, the antibody- Furthermore, the number of CTC-specific biomarker CK19 in single CTCs was counted as reported, 35 and significant heterogeneities were disclosed. Twenty single CTCs were numbered and picked out from real blood samples on day 12 (Figure 4b), and then, the isolated single CTCs were analyzed by number.…”

“…Capitalizing on the special characteristic that cells in solution tend to adhere to the substance of a different phase, the air bubble is employed as the 'glue' for target SiCS. 35 Based on the one-sided sharpened capillary and homemade bubble-glue SiCS apparatus, target single cells can be directly sampled from the single-cell suspension under visualization. The novel SiCS method possesses several advantages, including (1) both the volume of the solvent containing a target single cell and the bubble size in one SiCS operation are controllable (down to about 20 pL) by applying a high-precision, two-way syringe pump, which enabled the separation of single cells from microliter-level samples, (2) considerable improvements of SiCS efficiency in a targeted, two-dimensional way (picking out what you need in situ) and spatial context preserved, compared to the onedimensional methods (such as FACS), (3) easy fabrication and imitation, (4) non-invasive to both the morphology and function of the cells being sampled, and (5) sampling cells with any manipulated number according to different experimental purposes after simple method adjustments.…”

“…To meet all the expectations of live single-CTC sampling, herein we introduced a bubble-glue single-cell sampling (bubble-glue SiCS) method. Capitalizing on the special characteristic that cells in solution tend to adhere to the substance of a different phase, the air bubble is employed as the ‘glue’ for target SiCS . Based on the one-sided sharpened capillary and homemade bubble-glue SiCS apparatus, target single cells can be directly sampled from the single-cell suspension under visualization.…”