Robust Spontaneous Raman Flow Cytometry for Single‐Cell Metabolic Phenome Profiling via pDEP‐DLD‐RFC

Abstract

A full-spectrum spontaneous single-cell Raman spectrum (fs-SCRS) captures the metabolic phenome for a given cellular state of the cell in a label-free, landscape-like manner. Herein a positive dielectrophoresis induced deterministic lateral displacement-based Raman flow cytometry (pDEP-DLD-RFC) is established. This robust flow cytometry platform utilizes a periodical positive dielectrophoresis induced deterministic lateral displacement (pDEP-DLD) force that is exerted to focus and trap fast-moving single cells in a wide channel, which enables efficient fs-SCRS acquisition and extended stable running time. It automatically produces deeply sampled, heterogeneity-resolved, and highly reproducible ramanomes for isogenic cell populations of yeast, microalgae, bacteria, and human cancers, which support biosynthetic process dissection, antimicrobial susceptibility profiling, and cell-type classification. Moreover, when coupled with intra-ramanome correlation analysis, it reveals state-and cell-type-specific metabolic heterogeneity and metabolite-conversion networks. The throughput of ≈30-2700 events min −1 for profiling both nonresonance and resonance marker bands in a fs-SCRS, plus the >5 h stable running time, represent the highest performance among reported spontaneous Raman flow cytometry (RFC) systems. Therefore, pDEP-DLD-RFC is a valuable new tool for label-free, noninvasive, and high-throughput profiling of single-cell metabolic phenomes.

Chemical Nose Strategy with Metabolic Labeling and “Antibiotic-Responsive Spectrum” Enables Accurate and Rapid Pathogen Identification

“…The overuse and misuse of antibiotics are among the leading causes of the evolution of antibiotic-resistant bacteria. − Thus, monitoring the evolution of pathogens from antibiotic-sensitive to antibiotic-resistant ones could provide valuable information for adjusting the treatment in time and thereby preventing the worsening of AMR. To evaluate the capability of the present sensor array system to monitor the evolution of antibiotic-resistant bacteria, we prepared mixtures of antibiotic-resistant and -sensitive strains with different ratios to simulate the evolution process at various stages by using S.…”

Single cell metabolic phenome and genome via the ramanome technology platform: Precision medicine of infectious diseases at the ultimate precision?

“…Notably, when pathogen isolation is required, the cell sorting module in both FlowRACS and RACS-seq systems can be coupled to a DNA/RNA sequencer or mass spectrometer for proteome or metabolome profiling and cultivation of the sorted cells with the target SCRS (Figure 2). In fact, designs of the positive dielectrophoresis induced deterministic lateral displacement-based Raman Flow Cytometry chip [44] in the Flow RACS instrument and the RAGE chip in the RACS-seq instrument [40] have carefully ensured the preservation of cellular vitality along the complete operation that includes sample preparation, SCRS acquisition, cell sorting, and cell exportation. This RACS-culture procedure, which has been demonstrated for both humans [40] and environmental microbiomes [39], selects a target cell directly from a clinical specimen and then deposits the cell into a well of a 96/384-well plate or into a microdroplet for liquid medium-based cultivation.…”

“…(a) The clinical antimicrobial susceptibility test ramanometry (CAST-R) instrument [38,41], which includes hardware devices for automated clinical sample pretreatment and intelligent SCRS acquisition and analysis in a parallel, 60-well platebased format, reports AST results for dozens of antimicrobials in each patient sample and processes dozens of patient samples in each run. (b) The flow-mode Ramanactivated cell sorter (FlowRACS) instrument [43,44], which is a microfluidics-based system, profiles the full spectrum spontaneous SCRS in a cell population with high speed (30 per min for Escherichia coli and 270 per min for budding yeasts) via its Raman flow cytometry mode, thereby providing a novel solution for automated, high-throughput SCRS-based ID and AST of pathogens [44]. (c) The RACS-seq instrument sorts individual microbial cells by their SCRS (i.e., based on identity, antimicrobial susceptibility, or other phenotypes as depicted by SCRS) and corresponding morphological phenome as depicted by brightfield microscopic images, and then exports target cells to a 96-well plate in an index-based, what-you-see-is-what-you-get and one-cell-one-well manner.…”

“…By adopting the Raman flow cytometry mode in FlowRACS [43,44], new clinical or scientific applications such as high-throughput, label-free metabolic profiling of live microbial cells will emerge, which are highly valuable to resolve the long-standing challenge of detecting and quantifying hetero-resistance [49]. At the one-cell RACS-seq or RACS-culture step, new designs of the RAGE chip that improve the precision of cell capture, such as optical tweezer-assisted pool screening [50] or the use of artificial intelligence for automated sorting of cells [50,51], can greatly enhance the throughput, while the automation of SCRS acquisition, sorting, and one cell-one well disposition would ensure downstream single cell genome extraction, amplification and sequencing or cultivation can proceed without human intervention.…”

Multi‐omics analysis of disulfidptosis regulators and therapeutic potential reveals glycogen synthase 1 as a disulfidptosis triggering target for triple‐negative breast cancer

“…Liu et al. further conducted a CRISPR/Cas9 loss‐of‐function screening and reported candidate agents participating in disulfidptosis 11,12 . We defined them as disulfidptosis regulators based on their roles in regulating disulfidptosis activity.…”