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

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.