Rational Optimization of Raman-Activated Cell Ejection and Sequencing for Bacteria.

In Raman-activated cell ejection and sequencing (RACE-Seq), success rate and sequence coverage have generally been low for shotgun sequencing of individual post-RACE cells. Here we quantitatively evaluated the influence of cell lysis condition, nucleic acid amplification condition, and parameters of Raman measurement on RACE-Seq performance. Variations in laser energy input during Raman signal acquisition, but not duration of alkaline lysate lysis, temperature, or measurement under dry or aqueous conditions, are vital to the success of multiple displacement amplification (MDA). In fact, laser irradiation is reversely linked to MDA product quality. However, introduction of oils prior to MDA, by mitigating such negative effects of Raman irradiation, elevates genome coverage of post-RACE Escherichia coli cells from <20% to ∼50%, while greatly improving the success rate of RACE-Seq for soil microbiota. Our findings provide a practical solution for enhancing RACE-Seq performance and pinpoint protection of cells from laser irradiation as a priority in method development.

Raman-activated cell sorting and metagenomic sequencing revealing carbon-fixing bacteria in the ocean.

It is of great significance to understand CO2 fixation in the oceans. Using single cell Raman spectra (SCRS) as biochemical profiles, Raman activated cell ejection (RACE) was able to link phenotypes and genotypes of cells. Here, we show that mini-metagenomic sequences from RACE can be used as a reference to reconstruct nearly complete genomes of key functional bacteria by binning shotgun metagenomic sequencing data. By applying this approach to 13 C bicarbonate spiked seawater from euphotic zone of the Yellow Sea of China, the dominant bacteria Synechococcus spp. and Pelagibacter spp. were revealed and both of them contain carotenoid and were able to incorporate 13 C into the cells at the same time. Genetic analysis of the reconstructed genomes suggests that both Synechococcus spp. and Pelagibacter spp. contained all genes necessary for carotenoid synthesis, light energy harvesting and CO2 fixation. Interestingly, the reconstructed genome indicates that Pelagibacter spp. harbored intact sets of genes for ß-carotene (precursor of retional), proteorhodopsin synthesis and anaplerotic CO2 fixation. This novel approach shines light on the role of marine 'microbial dark matter' in global carbon cycling, by linking yet-to-be-cultured Synechococcus spp. and Pelagibacter spp. to carbon fixation and flow activities in situ.

Correction: Towards high-throughput microfluidic Raman-activated cell sorting.

Correction for 'Towards high-throughput microfluidic Raman-activated cell sorting' by Qiang Zhang et al., Analyst, 2015, DOI: 10.1039/c5an01074h.

Towards high-throughput microfluidic Raman-activated cell sorting.

Raman-activated cell sorting (RACS) is a promising single-cell analysis technology that is able to identify and isolate individual cells of targeted type, state or environment from an isogenic population or complex consortium of cells, in a label-free and non-invasive manner. However, compared with those widely used yet labeling-required or staining-dependent cell sorting technologies such as FACS and MACS, the weak Raman signal greatly limits the further development of the existing RACS systems to achieve higher throughput. Strategies that can tackle this bottleneck include, first, improvement of Raman-acquisition efficiency and quality based on advanced Raman spectrometers and enhanced Raman techniques; second, development of novel microfluidic devices for cell sorting followed by integration into a complete RACS system. Exploiting these strategies, prototypes for a new generation of RACS have been demonstrated, such as flow-based OT-RACS, DEP-RACS, and SERS/CARS flow cytometry. Such high-throughput microfluidic RACS can provide biologists with a powerful single-cell analysis tool to explore the scientific questions or applications that have been beyond the reach of FACS and MACS.

Effect of fiber length on carbon nanotube-induced fibrogenesis.

Given their extremely small size and light weight, carbon nanotubes (CNTs) can be readily inhaled by human lungs resulting in increased rates of pulmonary disorders, particularly fibrosis. Although the fibrogenic potential of CNTs is well established, there is a lack of consensus regarding the contribution of physicochemical attributes of CNTs on the underlying fibrotic outcome. We designed an experimentally validated in vitro fibroblast culture model aimed at investigating the effect of fiber length on single-walled CNT (SWCNT)-induced pulmonary fibrosis. The fibrogenic response to short and long SWCNTs was assessed via oxidative stress generation, collagen expression and transforming growth factor-beta (TGF-ß) production as potential fibrosis biomarkers. Long SWCNTs were significantly more potent than short SWCNTs in terms of reactive oxygen species (ROS) response, collagen production and TGF-ß release. Furthermore, our finding on the length-dependent in vitro fibrogenic response was validated by the in vivo lung fibrosis outcome, thus supporting the predictive value of the in vitro model. Our results also demonstrated the key role of ROS in SWCNT-induced collagen expression and TGF-ß activation, indicating the potential mechanisms of length-dependent SWCNT-induced fibrosis. Together, our study provides new evidence for the role of fiber length in SWCNT-induced lung fibrosis and offers a rapid cell-based assay for fibrogenicity testing of nanomaterials with the ability to predict pulmonary fibrogenic response in vivo.

Dual detection of cancer biomarker CA125 using absorbance and electrochemical methods.

An enzyme-linked immunoassay based on dual signal transduction mechanisms has been developed for detection of ovarian cancer biomarker CA125. The immunoassay uses a nanoelectrode array (NEA) chip and absorbance methods for the dual detection. The NEA is used to confirm the optical detection of CA125 that is carried out in a high-binding 96-well plate. An alkaline phosphatase (AP) enzyme was used to label the detection antibody to allow for both the optical and electrochemical detection of CA125. Two kinds of substrates were catalyzed by the AP enzyme. para-Nitrophenylphosphate (PNPP) produces chromogenic para-nitrophenol (PNP), which can be optically detected at 405 nm. para-Aminophenylphosphate (PAPP) produces electroactive para-aminophenol (PAP), which can be detected amperometrically between -0.1 and 0.3 V. The linear ranges have been determined to be 5-1000 U mL(-1) and 5-1000 U mL(-1) for the optical and electrochemical immunoassays, respectively. The limit of detection of the optical immunoassay is 1.3 U mL(-1) and 40 U mL(-1) for the optical and electrochemical methods, respectively.

Molding a silver nanoparticle template on polydimethylsiloxane to efficiently capture mammalian cells.

Herein, a functional template made up of in situ synthesized silver nanoparticles (AgNPs) is prepared on polydimethylsiloxane (PDMS) for the spatial control of cell capture, where the residual Si-H groups in the PDMS matrix are used as reductants to reduce AgNO(3) for forming AgNPs. In virtue of microfluidic system, a one-dimensional array pattern of AgNPs is obtained easily. Further combining with plasma treatment, a two-dimensional array pattern of AgNPs could be achieved. The obtained PDMS-AgNPs composite is characterized in detail. The PDMS-AgNPs composite shows good antibacterial property in E. coli adhesion tests. The patterns possess hifi and high resolution (ca. 8 microm). Cell patterns with high efficiency and spatial selectivity are further formed with the aid of H-Arg-Gly-Asp-Cys-OH (RGDC) tetrapeptide which is grafted on the AgNPs template. Cells immobilized on the template show a good ability for adhesion, spreading, migration, and growth.

Seasonal dynamics of the coastal bacterioplankton at intensive fish-farming areas of the Yellow Sea, China revealed by high-throughput sequencing.

Marine aquaculture areas are facing stressed environmental challenges, especially the degradation of coastal ecosystems. Here a coordinated time-series study was used to investigate the coastal bacterioplankton biodiversity dynamics of the Yellow Sea, China. Bacterial 16S rRNA gene sequencing revealed a temporal pattern of decreasing of diversity in summer. Functional prediction indicated that metabolic pathways related to the adenosine triphosphate (ATP)-binding cassette transporters and other membrane transporters were significantly enriched in May, while the genetic information processing category was most abundant in March. The May microbiomes showed most significant positive correlation with phosphate concentration, while the August and November microbiomes correlated with temperature and chemical oxygen demand (COD) most, and the March microbiomes showed significant correlation with Cu2+ level, pH and salinity. The correlations between representative bacteria and environmental parameters revealed in this study may provide insights into the potential influences of human aquaculture activities, on the biodiversity of coastal bacterioplankton.

Development of a facile droplet-based single-cell isolation platform for cultivation and genomic analysis in microorganisms.

Wider application of single-cell analysis has been limited by the lack of an easy-to-use and low-cost strategy for single-cell isolation that can be directly coupled to single-cell sequencing and single-cell cultivation, especially for small-size microbes. Herein, a facile droplet microfluidic platform was developed to dispense individual microbial cells into conventional standard containers for downstream analysis. Functional parts for cell encapsulation, droplet inspection and sorting, as well as a chip-to-tube capillary interface were integrated on one single chip with simple architecture, and control of the droplet sorting was achieved by a low-cost solenoid microvalve. Using microalgal and yeast cells as models, single-cell isolation success rate of over 90% and single-cell cultivation success rate of 80% were demonstrated. We further showed that the individual cells isolated can be used in high-quality DNA and RNA analyses at both gene-specific and whole-genome levels (i.e. real-time quantitative PCR and genome sequencing). The simplicity and reliability of the method should improve accessibility of single-cell analysis and facilitate its wider application in microbiology researches.

Label-free, rapid and quantitative phenotyping of stress response in E. coli via ramanome.

Rapid profiling of stress-response at single-cell resolution yet in a label-free, non-disruptive and mechanism-specific manner can lead to many new applications. We propose a single-cell-level biochemical fingerprinting approach named "ramanome", which is the collection of Single-cell Raman Spectra (SCRS) from a number of cells randomly selected from an isogenic population at a given time and condition, to rapidly and quantitatively detect and characterize stress responses of cellular population. SCRS of Escherichia coli cells are sensitive to both exposure time (eight time points) and dosage (six doses) of ethanol, with detection time as early as 5 min and discrimination rate of either factor over 80%. Moreover, the ramanomes upon six chemical compounds from three categories, including antibiotics of ampicillin and kanamycin, alcohols of ethanol and n-butanol and heavy metals of Cu2+ and Cr6+, were analyzed and 31 marker Raman bands were revealed which distinguish stress-responses via cytotoxicity mechanism and variation of inter-cellular heterogeneity. Furthermore, specificity, reproducibility and mechanistic basis of ramanome were validated by tracking stress-induced dynamics of metabolites and by contrasting between cells with and without genes that convey stress resistance. Thus ramanome enables rapid prediction and mechanism-based screening of cytotoxicity and stress-response programs at single-cell resolution.