Abstract
Proteins play a significant role in the key activities of cells. Single-cell protein analysis provides crucial insights in studying cellular heterogeneities. However, the low abundance and enormous complexity of the proteome posit challenges in analyzing protein expressions at the single-cell level. This review summarizes recent advances of various approaches to single-cell protein analysis. We begin by discussing conventional characterization approaches, including fluorescence flow cytometry, mass cytometry, enzyme-linked immunospot assay, and capillary electrophoresis. We then detail the landmark advances of microfluidic approaches for analyzing single-cell protein expressions, including microfluidic fluorescent flow cytometry, droplet-based microfluidics, microwell-based assay (microengraving), microchamber-based assay (barcoding microchips), and single-cell Western blotting, among which the advantages and limitations are compared. Looking forward, we discuss future research opportunities and challenges for multiplexity, analyte, throughput, and sensitivity of the microfluidic approaches, which we believe will prompt the research of single-cell proteins such as the molecular mechanism of cell biology, as well as the clinical applications for tumor treatment and drug development.
Highlights
As the physical basis for all life and the main component of living organisms, proteins dominate or participate in almost all biological activities and biological functions like providing structural supports, molecule transportations, cell growth and adhesion, signal transductions, catalytic biochemical processes, etc. [1,2]
Protein expression analysis is critical for the studies of cellular molecular mechanisms, clinical diagnosis and treatments, and drug developments [5]
As a technology well-suited for analysis of small heterogeneous samples, capillary electrophoresis was reported by Phillips et al to measure protein tyrosine phosphatases in single cells of human epidermoid carcinoma, which provided a powerful tool for the analysis of human biopsy specimens [62]
Summary
As the physical basis for all life and the main component of living organisms, proteins dominate or participate in almost all biological activities and biological functions like providing structural supports, molecule transportations, cell growth and adhesion, signal transductions, catalytic biochemical processes, etc. [1,2]. In the past few decades, various methods have been developed for protein analysis, such as gel electrophoresis [6], immunoassay [7], chromatography and mass spectrometry [8], and Raman imaging [9] These methods provide a comprehensive understanding of the biological functions of different proteins, which facilitate the developments of molecular biology and medicine [10]. Most of these conventional approaches are limited to protein analysis at tissue levels and only able to measure population-averaged protein expressions from large amounts of cells [11], masking the single-cell heterogeneity within a population [12,13]. We discuss the performance of each system in terms of multiplexity, analyte (e.g., membrane, intracellular, and secreted proteins), throughput and sensitivity, comparing advantages and limitations, and providing our perspectives on the potential development directions of future studies
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