A microfluidic flow cytometer enabling absolute quantification of single-cell intracellular proteins

Abstract

Quantification of single-cell proteomics provides key insights into cellular heterogeneity while conventional flow cytometry cannot provide absolute quantification of intracellular proteins of single cells due to the lack of calibration approaches. This paper presents a constriction channel (with a cross sectional area smaller than cells) based microfluidic flow cytometer, capable of collecting copy numbers of specific intracellular proteins. In this platform, single cells stained with fluorescence labelled antibodies were forced to squeeze through the constriction channel with the fluorescence intensities quantified and since cells fully filled the constriction channel during the squeezing process, solutions with fluorescence labelled antibodies were flushed into the constriction channel to obtain calibration curves. By combining raw fluorescence data and calibration curves, absolute quantification of intracellular proteins was realized. As a demonstration, copy numbers of beta-actin of single tumour cells were quantified to be 0.90 ± 0.30 μM (A549, n = 14 228), 2.34 ± 0.70 μM (MCF 10A, n = 2455), and 0.98 ± 0.65 μM (Hep G2, n = 6945). The travelling time for individual cells was quantified to be roughly 10 ms and thus a throughput of 100 cells per s can be achieved. This microfluidic system can be used to quantify the copy numbers of intracellular proteins in a high-throughput manner, which may function as an enabling technique in the field of single-cell proteomics.

A Microfluidic Fluorescent Flow Cytometry Capable of Quantifying Cell Sizes and Numbers of Specific Cytosolic Proteins

“…In addition, the numbers of single-cell β-actins of A549 cells were estimated as 9.62 ± 4.29 × 10 5 , consistent with 1) 3.57 ± 0.22 × 10 6 per cells obtained from conventional enzyme linked immunosorbent assays and 2) ~0.96 × 10 6 at the single-cell level based on previously developed microfluidic platforms 38 . Compared to the results of single-cell analysis, conventional enzyme linked immunosorbent assays located a minute higher number of β-actins per cell, indicating that some portions of β-actins are not exposed in the step of intracellular staining, although intracellular staining has been intensively used for deep phenotyping 11 , 12 and signaling state characterization 13 – 16 .…”

“…Note that since fluorescent data were measured by PMT, fluorescent signals were averaged in the detection domain. Thus, cytosolic proteins without perfect even distributions can also be characterized by the developed platform under the condition that the standard deviation to average ratios of individual pulses are kept low 38 .…”

“…1(C) ). Because the constriction microchannel is smaller than cells, cells are forced to deform through the constriction microchannel with an elongation length L c , where the middle portion of the deformed cell fully fills the constriction microchannel 38 while both the front and rear surfaces are treated as semi-spheres with diameters the same as the width of constriction microchannel ( W c ) (refer to Fig. 1(D) ).…”

“…As to the miniaturized flow cytometry, due to the lack of calibration beads, counting of specific cytosolic proteins was not reported by the majority of micro flow cytometry 21 – 23 . Recently, a modified fluorescent micro flow cytometry was reported, enabling the translation of raw fluorescent signals into specific protein concentrations, which, however, cannot be further translated to absolute numbers due to the lack of the critical information of cell sizes 38 .…”

Absolute Copy Numbers of β-Actin Proteins Collected from 10,000 Single Cells

“…In order to address this issue, in this study, absolute copy numbers of β-actin proteins were obtained, leveraging a recently reported polymeric microfluidic flow cytometry [ 11 ]. More specifically, lung, liver, and cervical tumour cell lines of A549, Hep G2 and HeLa were characterized by the microfluidic platform, yielding absolute copy numbers of β-actin proteins from ~10,000 single cells.…”