Single-Cell Phenotypic Analysis and Digital Molecular Detection Linkable by a Hydrogel Bead-Based Platform
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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.
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Single-cell assays, including flow cytometry, scRNA-seq, and microfluidics-based assays, allow analysis of gene expression, cell viability, and drug tolerance in individual fungal cells.
“These include techniques such as flow cytometry, single-cell RNA sequencing (scRNA-seq), and microfluidics-based assays, allowing for the analysis of gene expression, cell viability, and drug tolerance in individual fungal cells [170,171].”
From Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance (2025) · cites this paper as [170]
The Introduction (page unavailable) says microfluidic single-cell techniques can reveal viability phenotypes and genotypes, and the Results and Discussion describe the paper’s Gelbead-based viability assay and molecular detection. The source studies bacterial cells, not fungal cells; it does not establish the cited capabilities for flow cytometry or scRNA-seq, nor demonstrate analysis of gene expression and drug tolerance in individual fungal cells. (LLM, conf=0.88)
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Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance
Su, Y., Li, Y., Yi, Q., Xu, Y., Sun, T., & Li, Y. (2025) · Journal of Fungi · doi:10.3390/jof11020143 · Cited by 18 · see citing works
“These include techniques such as flow cytometry, single-cell RNA sequencing (scRNA-seq), and microfluidics-based assays, allowing for the analysis of gene expression, cell viability, and drug tolerance in individual fungal cells [170,171].”
- Authors
- Zhu, Y., Li, J., Lin, X., Huang, X., & Hoffmann, M. R.
- Affiliations
- California Institute of Technology (US)
- Corresponding author
- Hoffmann, Michael R.
- Venue
- ACS Applied Bio Materials
- Publisher
- American Chemical Society (ACS)
- Published
- 2021-02-12
- Volume
- 4
- Issue
- 3
- Pages
- 2664-2674
- ISSN
- 2576-6422 (ISSN-L)
- PMC
- PMC7976597
- OpenAlex
- W3130257407
- Cited by
- 18
- References
- 52
- Citations by year
- 2025: 4 · 2024: 4 · 2023: 4 · 2022: 4 · 2021: 2
- FWCI
- 1.06
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Locations
- ACS Applied Bio Materials (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- CaltechAUTHORS (California Institute of Technology) (submittedVersion, cc-by)
- Funders
- Bill and Melinda Gates Foundation (OPP1111252, OPP1192379)
- Topics
- Physical Sciences › Engineering › Biomedical Engineering › Microfluidic and Capillary Electrophoresis Applications
- Physical Sciences › Engineering › Biomedical Engineering › Innovative Microfluidic and Catalytic Techniques Innovation
- Physical Sciences › Engineering › Biomedical Engineering › Biosensors and Analytical Detection
- Keywords
- hydrogel beads, poly(ethylene glycol) hydrogels, thiol-Michael addition, digital PCR, loop-mediated isothermal amplification, bacterial heterogeneity, genotype-phenotype correlation, disposable microfluidic devices
- MeSH terms
- Biocompatible Materials, Cells, Cultured, Materials Testing, Particle Size, Salmonella typhi, Hydrogels, Single-Cell Analysis
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