Microfluidic Analyzer Enabling Quantitative Measurements of Specific Intracellular Proteins at the Single-Cell Level.

Lixing Liu,Xiufeng Li,Jian Chen,Diancan Wang,Junbo Wang,Deyong Chen,Ting Zhang,Beiyuan Fan,Yeqing Song,Yixiang Wang

doi:10.3390/mi9110588

Abstract

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), ncell = 13,689) vs. 0.90 ± 0.58 × 105/cell (salivary adenoid cystic carcinoma lung metastasis cell line (SACC-LM), ncell = 15,341); 0.89 ± 0.69 × 106/cell (oral carcinoma cell line (CAL 27), ncell = 7357) vs. 0.93 ± 0.69 × 106/cell (oral carcinoma lymphatic metastasis cell line (CAL 27-LN2), ncell = 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.

Highlights

Single-cell protein expressions provide key insights in studying cellular heterogeneities such as tumour heterogeneities and immune response variations [1,2,3]
Microfluidics is an approach to processing fluids based on microfabricated channels (1–100 μm) [10,11], and due to their dimensional comparisons with biological cells, microfluidic instruments have been developed for single-cell protein analysis [12,13]
Barcoding microchips with a commercial brand of “Isoplexis” were developed where individual cells are confined within microchambers, and the absolute quantification of specific intracellular proteins is realized by cell lysis and the captures of target cellular proteins by preprinted antibodies on the bottom surfaces of the microchips [14,15,16,17]

Summary

Introduction

Single-cell protein expressions provide key insights in studying cellular heterogeneities such as tumour heterogeneities and immune response variations [1,2,3]. Flow cytometry is the golden instrument for single-cell protein analysis where cells stained with fluorescence-labelled antibodies rapidly travel through a capillary while the fluorescent intensities are measured [4,5].

Results

Conclusion