Combinatorial perturbation sequencing on single cells using microwell-based droplet random pairing

Xie, Run; Liu, Yang; Wang, Shiyu; Shi, Xuyang; Zhao, Zhantao; Liu, Longqi; Liu, Ya; Li, Zida

doi:10.1016/j.bios.2022.114913

Cited by 10 publications

(6 citation statements)

References 32 publications

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“…A microwell array device was used for droplet pairing and merging, as previously described. 23 Each microwell unit was composed of a large well (Φ90 μm) and a small well (Φ50 μm) that were interconnected, and the depths were 70 and 50 μm, respectively. As shown in Figure 1b,c and Video S1, primer droplets (∼Φ90 μm) were first loaded into the microwell array device and occupied the large well of each microwell unit.…”

Section: ■ Resultsmentioning

confidence: 99%

“…The primer droplets could be stored at 4 °C for at least 5 days without obvious coalescence (Figure S2). A microwell array device was used for droplet pairing and merging, as previously described . Each microwell unit was composed of a large well (Φ90 μm) and a small well (Φ50 μm) that were interconnected, and the depths were 70 and 50 μm, respectively.…”

Section: Resultsmentioning

confidence: 99%

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CoID-LAMP: Color-Encoded, Intelligent Digital LAMP for Multiplex Nucleic Acid Quantification

Fang

Zhao

et al. 2023

Anal. Chem.

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Multiplex, digital nucleic acid tests have important biomedical applications, but existing methods mostly use fluorescent probes that are target-specific and difficult to optimize, limiting their widespread applications. Here, we report colorencoded, intelligent digital loop-mediated isothermal amplification (CoID-LAMP) for the coidentification of multiple nucleic acid targets. CoID-LAMP supplements different primer solutions with different dyes, generates primer droplets and sample droplets, and collectively pairs these two types of droplets in a microwell array device to perform LAMP. After imaging, the droplet colors were analyzed to decode the primer information, and the precipitate byproducts within droplets were detected to determine the target occupancy and calculate the concentrations. We first established an image analysis pipeline based on a deep learning algorithm for reliable droplet detection and validated the analytical performance in nucleic acid quantification. We then implemented CoID-LAMP using fluorescent dyes as the coding materials and established an 8-plex digital nucleic acid assay, confirming the reliable coding performance and the capability of multiplex nucleic acid quantification. We further implemented CoID-LAMP using brightfield dyes for a 4-plex assay, suggesting that the assay could be realized solely by brightfield imaging with minimal demand on the optics. Leveraging the advantages of droplet microfluidics in multiplexing and deep learning in intelligent image analysis, CoID-LAMP offers a useful tool for multiplex nucleic acid quantification.

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Section: ■ Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

CoID-LAMP: Color-Encoded, Intelligent Digital LAMP for Multiplex Nucleic Acid Quantification

Fang

Zhao

et al. 2023

Anal. Chem.

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“…The PDMS chips were then cut into the desired shape with a cutter knife, and holes were punched at the inlet and outlet of the chip with differently sized punches. For the parameters for fabricating the droplet-generating chip, please refer to a previous paper 54 . As for the picoinjection chip, Figure S3 shows the design parameters; the diameter of the holes of all inlets and outlets was 1 mm.…”

Section: Methodsmentioning

confidence: 99%

High-throughput screening of functional neo-antigens and their specific TCRs via the Jurkat reporter system combined with droplet microfluidics

Liu

et al. 2023

Preprint

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T cell receptor-engineered T cells (TCR-T) therapy would be one of the most promising technologies in solid tumor immune therapy benefit from recognizing tumor neo-antigens both in cell surface and intracellular. However, time-consuming steps and low accuracy to screen functional antigens and their specific TCRs from thousands of candidate libraries may severely limits their practical application. Here, we developed an integrated platform which combine with Jurkat reporter system, DNA barcoding and droplet microfluidics to enable the library-on-library functional antigens and TCR screening. Next, analysis of T cell activation related genes and DNA barcode based on single-cell RNA-sequencing can determine the specific recognition between the antigens and their functional TCRs. Our proof-of principle study demonstrates that screening antigens and their functional TCRs simultaneously with high sensitivity and specificity in unbiased library-on-library screening can be achieved, which could significantly reduce the time required in operation, and accelerate progress toward cancer treatment. Moreover, it also be expected to expand the research filed of screening the cross-reactivity and off-target recognition of candidate antigens and TCRs in libraries before clinical application. Keywords: High throughput screening, functional TCR screening, jurkat reporter, droplet-based microfluidics, single-cell transcriptome

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“…Microwell arrays have also been developed for isolating single cells. [47][48][49] By designing micropores or chambers that match the size of cells, cells randomly settle into the micropores due to their own gravity, with advantages of easy microfabrication and convenient operation. Feng et al proposed a microwell array for the quantitative detection of glucose phosphate by MS in single cells (Figure 2B).…”

Section: Microfluidicsmentioning

confidence: 99%

Recent Progress in Mass Spectrometry‐Based Single‐Cell Metabolic Analysis

Sun,

Yu,

Qian

et al. 2023

Small Methods

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Single‐cell analysis enables the measurement of biomolecules at the level of individual cells, facilitating in‐depth investigations into cellular heterogeneity and precise interpretation of the related biological mechanisms. Among these biomolecules, cellular metabolites exhibit remarkable sensitivity to environmental and biochemical changes, unveiling a hidden world underlying cellular heterogeneity and allowing for the determination of cell physiological states. However, the metabolic analysis of single cells is challenging due to the extremely low concentrations, substantial content variations, and rapid turnover rates of cellular metabolites. Mass spectrometry (MS), characterized by its high sensitivity, wide dynamic range, and excellent selectivity, is employed in single‐cell metabolic analysis. This review focuses on recent advances and applications of MS‐based single‐cell metabolic analysis, encompassing three key steps of single‐cell isolation, detection, and application. It is anticipated that MS will bring profound implications in biomedical practices, serving as advanced tools to depict the single‐cell metabolic landscape.

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Combinatorial perturbation sequencing on single cells using microwell-based droplet random pairing

Cited by 10 publications

References 32 publications

CoID-LAMP: Color-Encoded, Intelligent Digital LAMP for Multiplex Nucleic Acid Quantification

CoID-LAMP: Color-Encoded, Intelligent Digital LAMP for Multiplex Nucleic Acid Quantification

High-throughput screening of functional neo-antigens and their specific TCRs via the Jurkat reporter system combined with droplet microfluidics

Recent Progress in Mass Spectrometry‐Based Single‐Cell Metabolic Analysis

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