Ramanome technology platform for label-free screening and sorting of microbial cell factories at single-cell resolution

He, Yuehui; Wang, Xixian; Ma, Bo; Xu, Jian

doi:10.1016/j.biotechadv.2019.04.010

Cited by 68 publications

(90 citation statements)

References 131 publications

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“…Screening of enzyme libraries for in vivo activities is frequently a rate-limiting step in the design of enzymes as well as microbial cell factories ( 20 , 21 ). Being single-cell-level, label-free, noninvasive, and richly informative, RACS technologies address the limitations of GC/LC-MS–based methods ( 14 , 19 ) and FACS ( 22 – 26 ), thus are finding expanding applications in microbiome research and synthetic biology ( 1 ). However, existing RACS systems are either of low throughput ( 4 – 9 ) or not applicable for sorting phenotypes associated with nonresonance Raman peaks (which represent the majority of peaks in an SCRS) ( 10 – 12 ).…”

Section: Discussionmentioning

confidence: 99%

“…For example, as many high-value compounds in the cell carry nonresonance (e.g., starch, protein, and nucleic acids) or resonance Raman signal (e.g., pigments) ( 1 , 28 ), pDEP-RADS can be extended to mining or de novo design of the numerous synthetic enzymes or cells. As a generally applicable single-cell “phenome” ( 1 ), SCRS can also distinguish or model bacterial species ( 6 ), general or substrate-specific metabolic activity ( 5 ), intracellular levels of starch and protein ( 15 ), drug sensitivity ( 29 , 30 ), cross-species metabolite exchange ( 31 ), etc. ; most of these phenotypes are associated with nonresonance Raman peaks and thus can take advantage of pDEP-RADS.…”

Section: Discussionmentioning

confidence: 99%

“…Consequently, a RACS device featuring both generality in tackleable phenotypes and high-throughput sorting is in urgent need ( Fig. 1A ) ( 1 ).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Positive dielectrophoresis–based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo

Wang

Ren

et al. 2020

Sci. Adv.

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The potential of Raman-activated cell sorting (RACS) is inherently limited by conflicting demands for signal quality and sorting throughput. Here, we present positive dielectrophoresis–based Raman-activated droplet sorting (pDEP-RADS), where a periodical pDEP force was exerted to trap fast-moving cells, followed by simultaneous microdroplet encapsulation and sorting. Screening of yeasts for triacylglycerol (TAG) content demonstrated near-theoretical-limit accuracy, ~120 cells min−1 throughput and full-vitality preservation, while sorting fatty acid degree of unsaturation (FA-DU) featured ~82% accuracy at ~40 cells min−1. From a yeast library expressing algal diacylglycerol acyltransferases (DGATs), a pDEP-RADS run revealed all reported TAG-synthetic variants and distinguished FA-DUs of enzyme products. Furthermore, two previously unknown DGATs producing low levels of monounsaturated fatty acid–rich TAG were discovered. This first demonstration of RACS for enzyme discovery represents hundred-fold saving in time consumables and labor versus culture-based approaches. The ability to automatically flow-sort resonance Raman–independent phenotypes greatly expands RACS’ application.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Positive dielectrophoresis–based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo

Wang

Ren

et al. 2020

Sci. Adv.

Self Cite

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“…However, such an approach is limited to a narrow range of enzyme reactions and often requires the use of expensive, surrogate substrates. To overcome these limitations, highthroughput Fourier transform infrared (FTIR) spectroscopy [7] and Raman spectroscopy [8] have been utilized in label-free optical screening, which relies on characteristic spectral features or "fingerprints" so that very limited structural selectivity can be achieved. Moreover, transcription factor-based biosensors that correlate product formation with the expression level of a fluorescence protein have been created to facilitate protein engineering [9][10][11].…”

Section: Introductionmentioning

confidence: 99%

Recent advances in high-throughput mass spectrometry that accelerates enzyme engineering for biofuel research

Zhang

2020

BMC Energy

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Enzymes play indispensable roles in producing biofuels, a sustainable and renewable source of transportation fuels. Lacking rational design rules, the development of industrially relevant enzyme catalysts relies heavily on highthroughput screening. However, few universal methods exist to rapidly characterize large-scale enzyme libraries. Therefore, assay development is necessary on an ad hoc basis to link enzyme properties to spectrophotometric signals and often requires the use of surrogate, optically active substrates. On the other hand, mass spectrometry (MS) performs label-free enzyme assays that utilize native substrates and is therefore generally applicable. But the analytical speed of MS is considered rate limiting, mainly due to the use of time-consuming chromatographic separation in traditional MS analysis. Thanks to new instrumentation and sample preparation methods, direct analyte introduction into a mass spectrometer without a prior chromatographic step can be achieved by laser, microfluidics, and acoustics, so that each sample can be analyzed within seconds. Here we review recent advances in MS platforms that improve the throughput of enzyme library screening and discuss how these advances can potentially facilitate biofuel research by providing high sensitivity, selectivity and quantitation that are difficult to obtain using traditional assays. We also highlight the limitations of current MS assays in studying biofuel-related enzymes and propose possible solutions.

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“…[5][6][7] In recent years, SERS has also shown great promise in thē eld of detecting microorganisms, and di®erent SERS methods have been reported for studying the detection of low-concentration viruses. 8,9 For example, Guo et al developed a tape-like SERS substrate for the point-of-care test of wound infectious pathogens (Pseudomonas aeruginosa and S. aureus), which allowed the whole detection completed within 8 h. 10 Huang et al integrated SERS technique with micro°uidic microwell device, resulting to a rapid antibiotic susceptibility test on E. coli and S. aureus. 11 The electromagnetic and chemical mechanisms are the two generally accepted mechanisms for the Raman enhancement of SERS substrate.…”

Section: Introductionmentioning

confidence: 99%

Rapid label-free SERS detection of foodborne pathogenic bacteria based on hafnium ditelluride-Au nanocomposites

Guo

et al. 2020

J. Innov. Opt. Health Sci.

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Two-dimensional (2D) nanomaterials have captured an increasing attention in biophotonics owing to their excellent optical features. Herein, 2D hafnium ditelluride (HfTe[Formula: see text], a new member of transition metal tellurides, is exploited to support gold nanoparticles fabricating HfTe2-Au nanocomposites. The nanohybrids can serve as novel 2D surface-enhanced Raman scattering (SERS) substrate for the label-free detection of analyte with high sensitivity and reproducibility. Chemical mechanism originated from HfTe2 nanosheets and the electromagnetic enhancement induced by the hot spots on the nanohybrids may largely contribute to the superior SERS effect of HfTe2-Au nanocomposites. Finally, HfTe2-Au nanocomposites are utilized for the label-free SERS analysis of foodborne pathogenic bacteria, which realize the rapid and ultrasensitive Raman test of Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and Salmonella with the limit of detection of 10 CFU/mL and the maximum Raman enhancement factor up to [Formula: see text]. Combined with principal component analysis, HfTe2-Au-based SERS analysis also completes the bacterial classification without extra treatment.

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Ramanome technology platform for label-free screening and sorting of microbial cell factories at single-cell resolution

Cited by 68 publications

References 131 publications

Positive dielectrophoresis–based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo

Positive dielectrophoresis–based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo

Recent advances in high-throughput mass spectrometry that accelerates enzyme engineering for biofuel research

Rapid label-free SERS detection of foodborne pathogenic bacteria based on hafnium ditelluride-Au nanocomposites

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