Advanced techniques for gene heterogeneity research: Single‐cell sequencing and on‐chip gene analysis systems

Abstract

Gene heterogeneity leads to the differences in cellular behaviors in a wide range, such as tumor drug‐resistant mutation, epithelial‐mesenchymal transition, and migration, posing significant challenges to the development of biomedicine. Traditional gene analysis methods, such as polymerase chain reaction, employ a mass of cells as the gene source, resulting in that the gene properties from a specific single cell are hidden in massive gene information. Recent decades have seen the emerging single‐cell gene analysis techniques with their unprecedented opportunities to study gene heterogeneity with high precision and high throughput. In this review, we summarized the state‐of‐the‐art techniques for single‐cell sequencing and on‐chip gene analysis systems. The principles of each technique are introduced in detail, with the focus on the application scenarios in gene heterogeneity research. Looking forward, we also introduced the challenges in current technologies and point out the future direction for facilitating the technical improvement and clinical applications of single‐cell gene analysis techniques.

Construction of Exosome SORL1 Detection Platform Based on 3D Porous Microfluidic Chip and its Application in Early Diagnosis of Colorectal Cancer

“…Recently, novel analytical systems were introduced to detect biomarkers for disease diagnosis. [26][27][28][29][30][31][32] The microfluidic chip is a technology for precise control of microfluidics in submicron structures. [33,34] The use of microfluidic chips can facilitate the accurate control and detection of microfluids in the micro and nanoscale space.…”

Advances in novel strategies for isolation, characterization, and analysis of CTCs and ctDNA

“…Micromanipulation -Low cost -High accuracy -Simple process -Applicable for low cell count -Applicable for observing cells -Low throughput -Time-consuming -Manual process -Need to experienced operators -Mechanical damage to the target cell -Need to cell suspensions preparation Xu et al, 14 Tian and Li, 15 Paolillo et al, 16 Wang et al, 17 Chen et al, 18 Casasent et al 19 Microfluidic separation -Time saving -High throughput -Low reagent cost -Low contamination -Precise fluid control -Free-label separation -Low sample consumption -Low cell capture rate -Cell damage -Device clogging Xu et al, 14 Tian and Li, 15 Dong et al, 20 Wang et al, 17 Qi et al 21 DEPArray sorting system -High purity -Live imaging -Rare cells imaging -Small sample volume -Applicable for fixed or live cells -Semiautomatic separation system -Time-consuming -Low throughput size/deformability-based, hydrodynamics-based, electric-based, and acoustics-based microfluidics. Microfluidics-based CTC isolation is also done using affinity-based techniques in which cells are captured based on their surface protein expression levels involving the use of relevant antibodies or aptamers, allowing the selection of cancer-specific biomarkers (Figure 1).…”

“…Multiple studies have been conducted to assess the metastatic potential of CTCs using membranous detection of epithelial and mesenchymal 14 Aliya et al, 100 Gawad et al, 101 Tang et al, 102 Wei et al, 97 Wakai et al 20 Khan et al, 104 Slatko et al, 114 Court et al, 115 Gasch et al Castro-Giner and Aceto, 94 McDonald et al, 121 Bai et al, 122 Watanabe et al, 123 Freitas et al 124 Whole-exome sequencing Gawad et al, 101 Wakai et al, 103 McDonald et al, 121 Xie et al, 125 Lohr et al, 126 Manier et al, 127 Szczerba et al 14 Lei et al, 119 Li and Wang, 129 Mustachio and Roszik 130 (Continued)…”

Microfluidics Coupled Mass Spectrometry for Single Cell Multi‐Omics

“…Once single cells are isolated and captured, various analytical methods can be selected for single‐cell determination and characterization, including immunoassays, fluorescence techniques, imaging, flow cytometry, single‐cell sequencing, mass spectrometry (MS), etc. [ 27–31 ] For instance, proteins secreted from a single cell can be bound to a microarray by primary antibodies and then detected with secondary antibodies labeled with fluorescent markers. [ 32 ] However, antibody‐based immunoassays rely on high‐quality antibodies and are limited in throughout.…”