Single-Cell Phenotypic Analysis and Digital Molecular Detection Linkable by a Hydrogel Bead-Based Platform

Abstract

Cell heterogeneity, such as antibiotic heteroresistance and cancer cell heterogeneity, has been increasingly observed. To probe the underlying molecular mechanisms in the dynamically changing heterogeneous cells, a high throughput platform is urgently needed to establish single cell genotype-phenotype correlations. Herein, we report a platform combining single-cell viability phenotypic analysis with digital molecular detection for bacterial cells. The platform utilizes polyethylene glycol hydrogel that cross-links through a thiol-Michael addition, which is biocompatible, fast, and spontaneous. To generate uniform nanoliter-sized hydrogel beads (Gelbeads), we developed a convenient and disposable device made of needles and microcentrifuge tubes. Gelbead-based single cell viability and molecular detection assays were established. Enhanced thermal stability and uncompromised efficiency were achieved for digital polymerase chain reaction (PCR) and digital loop-mediated isothermal amplification (LAMP) within the Gelbeads. Reagent exchange for in situ PCR following viability phenotypic analyses was demonstrated. The combined analyses may address the genotypic differences between cellular subpopulations exhibiting distinct phenotypes. The platform promises unique perspectives in mechanism elucidation of environment-evolution interaction that may be extended to other cell types for medical research.

Membrane-Based In-Gel Loop-Mediated Isothermal Amplification (mgLAMP) System for SARS-CoV-2 Quantification in Environmental Waters

“…As the RT-LAMP reaction vessel, the PEG hydrogel has high tolerance to environmental inhibitors by limiting their diffusion, 24 , 45 while a large amount of amplification products are generated. 46 The target-specific QUASR probe facilitates fluorescence amplicon dot formation in the hydrogel. (v) With the fluorescence illumination in the mgLAMP prototype, fluorescent signals can be imaged and counted in a cloud server to deliver absolute quantification.…”

“…Other than expanding the dot size, reducing the gel concentration from 10 to 5% also boosted the dot count from 80 ± 26 to 201 ± 21, which could be explained by higher amplification efficiency with a loosened hydrogel network leading to increased diffusivity of amplification intermediate structures and amplification products. 46 Therefore, a gel concentration of 5% was used further in this study. At this gel concentration, the incubation time was optimized to be 30 min ( Supporting Information Text S3 and Figure S7).…”

“…The reagents contain Triton X-100 for in-assay viral lysis. As the RT-LAMP reaction vessel, the PEG hydrogel has high tolerance to environmental inhibitors by limiting their diffusion, , while a large amount of amplification products are generated . The target-specific QUASR probe facilitates fluorescence amplicon dot formation in the hydrogel.…”

Near‐Infrared Responsive Droplet for Digital PCR

“…[12,13] In particular, it can form and process hydrogel droplets to alleviate the evaporating shortcomings, enhance thermal stability, and prevent external contaminations of dPCR. [14,15] Hydrogel materials can also assist dPCR by immobilizing microgel array on a chip, [16] counting through PCR amplicons-ligated beads after demulsification, [17] etc. Although with many successes, the droplet-based dPCR (ddPCR) still faces challenges in practice.…”

Heterogeneous Signal Fusion Method in Driving Fatigue Detection Signals

“…It is a generalized single hidden layer feedforward neural network used for classification, regression, clustering, sparse approximation, compression, and single or multilayer hidden nodes. Feature learning [14,15] is where the parameters of the hidden nodes (not just the weights connected to the input to the hidden nodes) do not need to be adjusted [16]. ese hidden nodes can be randomly assigned and never updated (i.e., they are random projections, but have nonlinear transformations), or they can be inherited from their parents without being changed [17,18].…”