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_cell = 14 228), 2.34 ± 0.70 μM (MCF 10A, n_cell = 2455), and 0.98 ± 0.65 μM (Hep G2, n_cell = 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.

This article reviews recent developments in microfluidic systems enabling high-throughput characterization of single-cell proteins. Four key perspectives of microfluidic platforms are included in this review: (1) microfluidic fluorescent flow cytometry; (2) droplet based microfluidic flow cytometry; (3) large-array micro wells (microengraving); and (4) large-array micro chambers (barcode microchips). We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on three key performance parameters (absolute quantification, sensitivity, and throughput).

This study presents a microfluidics based cytometry capable of characterizing cell sizes and counting numbers of specific cytosolic proteins where cells were first bound by antibodies labelled with fluorescence and then aspirated into a constriction microchannel in which fluorescent levels were measured. These raw fluorescent pulses were further divided into a rising domain, a stable domain and a declining domain. In addition, antibody solutions with labelled fluorescence were aspirated through the constriction microchannel, yielding curves to translate raw fluorescent levels to protein concentrations. By using key parameters of three domains as well as the calibration curves, cell diameters and the absolute number of β-actins at the single-cell level were quantified as 14.2 ± 1.7 μm and 9.62 ± 4.29 × 10⁵ (A549, n_cell = 14 242), 13.0 ± 2.0 μm and 6.46 ± 3.34 × 10⁵ (Hep G2, n_cell = 35 932), 13.8 ± 1.9 μm and 1.58 ± 0.90 × 10⁶ (MCF 10 A, n_cell = 16 650), and 12.7 ± 1.5 μm and 1.09 ± 0.49 × 10⁶ (HeLa, n_cell = 26 246). This platform could be further adopted to measure numbers of various cytosolic proteins, providing key insights in proteomics at the single-cell level.

Semi-quantitative studies have located varied expressions of β-actin proteins at the population level, questioning their roles as internal controls in western blots, while the absolute copy numbers of β-actins at the single-cell level are missing. In this study, a polymeric microfluidic flow cytometry was used for single-cell analysis, and the absolute copy numbers of single-cell β-actin proteins were quantified as 9.9 ± 4.6 × 10⁵, 6.8 ± 4.0 × 10⁵ and 11.0 ± 5.5 × 10⁵ per cell for A549 (n_cell = 14,754), Hep G2 (n_cell = 36,949), and HeLa (n_cell = 24,383), respectively. High coefficients of variation (~50%) and high quartile coefficients of dispersion (~30%) were located, indicating significant variations of β-actin proteins within the same cell type. Low p values (≪0.01) and high classification rates based on neural network (~70%) were quantified among A549, Hep G2 and HeLa cells, suggesting expression differences of β-actin proteins among three cell types. In summary, the results reported here indicate significant variations of β-actin proteins within the same cell type from cell to cell, and significant expression differences of β-actin proteins among different cell types, strongly questioning the properties of using β-actin proteins as internal controls in western blots.

This paper presents a microfluidic instrument capable of quantifying single-cell specific intracellular proteins, which are composed of three functioning modules and two software platforms. Under the control of a LabVIEW platform, a pressure module flushed cells stained with fluorescent antibodies through a microfluidic module with fluorescent intensities quantified by a fluorescent module and translated into the numbers of specific intracellular proteins at the single-cell level using a MATLAB platform. Detection ranges and resolutions of the analyzer were characterized as 896.78–6.78 × 10⁵ and 334.60 nM for Alexa 488, 314.60–2.11 × 10⁵ and 153.98 nM for FITC, and 77.03–5.24 × 10⁴ and 37.17 nM for FITC-labelled anti-beta-actin antibodies. As a demonstration, the numbers of single-cell beta-actins of two paired oral tumor cell types and two oral patient samples were quantified as: 1.12 ± 0.77 × 10⁶/cell (salivary adenoid cystic carcinoma parental cell line (SACC-83), n_cell = 13,689) vs. 0.90 ± 0.58 × 10⁵/cell (salivary adenoid cystic carcinoma lung metastasis cell line (SACC-LM), n_cell = 15,341); 0.89 ± 0.69 × 10⁶/cell (oral carcinoma cell line (CAL 27), n_cell = 7357) vs. 0.93 ± 0.69 × 10⁶/cell (oral carcinoma lymphatic metastasis cell line (CAL 27-LN2), n_cell = 6276); and 0.86 ± 0.52 × 10⁶/cell (patient I) vs. 0.85 ± 0.58 × 10⁶/cell (patient II). These results (1) validated the developed analyzer with a throughput of 10 cells/s and a processing capability of ~10,000 cells for each cell type, and (2) revealed that as an internal control in cell analysis, the expressions of beta-actins remained stable in oral tumors with different malignant levels.