Single‐Cell Immunoblotting based on a Photoclick Hydrogel Enables High‐Throughput Screening and Accurate Profiling of Exogenous Gene Expression

Abstract

Fast and accurate profiling of exogenous gene expression in host cells is crucial for studying gene function in cellular and molecular biology, but still faces the challenge of incomplete co‐expression of reporter genes and target genes. Here, a single‐cell transfection analysis chip (scTAC) is presented, which is based on the in situ microchip immunoblotting method, for rapid and accurate analysis of exogenous gene expression in thousands of individual host cells. scTAC not only can assign information of exogenous gene activity to specific transfected cells, but enables the acquisition of continuous protein expression even in low co‐expression scenarios. It is demonstrated that scTAC can reveal the relationship of expression level between reporter genes and target genes, which is helpful for evaluating transient transfection strategy efficiency. The advantages of this method for the study of fusion protein expression and downstream protein expression in signaling pathway in rare cells are shown. Empirically, an EGFP‐TSPAN8 fusion plasmid is transfected into MCF‐7 breast cancer cells and the expressions of two cancer stemness biomarkers (ALDHA1 and SOX2) are analyzed. The scTAC method clearly reveals an interesting phenomenon that transfected adherent MCF‐7 cells exhibit some stem cell characteristics, but they do not have stem cell appearance.

Multiplex Protein Profiling by Low-Signal-Loss Single-Cell Western Blotting with Fluorescent-Quenching Aptamers

“…The production of Single-Cell Western Blot chip templates was based on our previous studies. , The film mask was first designed in CAD and then sent to Shenzhen Nano Chip Electronic Co., Ltd. for production. According to the photolithography protocol of the SU8-2050 manufacturer, the silicon wafer was coated with the SU8-2050 gel and was heated to homogenize the template surface (pre-heated at 65 °C for 1 min and then 95 °C for 8 min).…”

“…Single-cell western blotting (scWB) enables the assessment of cell heterogeneity at the protein expression level, , making it a general utility in biological and medical sciences. − Compared to other single-cell techniques such as flow cytometry, scWB is less influenced by antibody cross-reactivity due to the integration of protein electrophoresis and antibody recognition. , However, multiplex protein detection with antibody-based scWB requires repeated antibody stripping, which leads to protein loss (up to 40%) that impedes the quantification and sensitivity of scWB . Such loss compromises protein detection at single-cell resolution, especially for low-abundance proteins.…”

The Intriguing Landscape of Single‐Cell Protein Analysis

“…developed a single‐cell transfection analysis chip (scTAC) to rapidly and accurately evaluate transient transfection efficiency which is widely used in cellular and molecular biological studies. [ 76 ] scTAC can monitor exogenous gene expression in thousands of individual host cells, enabling the acquisition of continuous protein expression even in low co‐expression scenarios. Grist et al.…”

“…[73][74][75] More recently, Li et al developed a single-cell transfection analysis chip (scTAC) to rapidly and accurately evaluate transient transfection efficiency which is widely used in cellular and molecular biological studies. [76] sc-TAC can monitor exogenous gene expression in thousands of individual host cells, enabling the acquisition of continuous protein expression even in low co-expression scenarios. Grist et al introduced 3D single-cell immunoblots to detect both cytosolic and nuclear proteins from hundreds of single mammalian breast and brain tumor cells.…”

“…Despite recent developments in single-cell proteomic technologies, challenges remain in getting a deep understanding of the complex and dynamic nature of proteome in single cells. For example, there are many complex biological factors taking place including splice variants, PTMs, large concentration dy- Single-cell barcode chip (SCBC) Single-cell lysis Fluorescence detection About 40 Medium High [14][15][16][17][18][19][20][21][22][23] Miniaturized ELISA Single-cell Fluorescence detection About 30 Medium Medium [37][38][39] Droplet-based microfluidics methods Single-cell Fluorescence detection About 10 Medium Medium [40][41][42][43][44][45][46] CE-LIF Single-cell Fluorescence detection About 10 Medium Medium [ 47,48] Single-cell Western blotting (scWB) Single-cell lysis Fluorescence detection About 10 Medium Medium [72][73][74][75][76][77][78][79][80][81] Proximity ligation assay-based method Single-cell RNA sequencing Unlimited Medium High [87][88][89][90][91][92][93][94][95][96]…”