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1.
Huiling Yuan Ran Tu Xinwei Tong Yuping Lin Yuanyuan Zhang Qinhong Wang 《Journal of industrial microbiology & biotechnology》2022,49(3)
Droplet-based microfluidics has emerged as a powerful tool for single-cell screening with ultrahigh throughput, but its widespread application remains limited by the accessibility of a droplet microfluidic high-throughput screening (HTS) platform, especially to common laboratories having no background in microfluidics. Here, we first developed a microfluidic HTS platform based on fluorescence-activated droplet sorting technology. This platform allowed (i) encapsulation of single cells in monodisperse water-in-oil droplets; (ii) cell growth and protein production in droplets; and (iii) sorting of droplets based on their fluorescence intensities. To validate the platform, a model selection experiment of a binary mixture of Bacillus strains was performed, and a 45.6-fold enrichment was achieved at a sorting rate of 300 droplets per second. Furthermore, we used the platform for the selection of higher α-amylase-producing Bacillus licheniformis strains from a mutant library generated by atmospheric and room temperature plasma mutagenesis, and clones displaying over 50% improvement in α-amylase productivity were isolated. This droplet screening system could be applied to the engineering of other industrially valuable strains. 相似文献
2.
Parametric studies on droplet generation reproducibility for applications with biological relevant fluids
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Stefan Wiedemeier Marko Eichler Robert Römer Andreas Grodrian Karen Lemke Krees Nagel Claus‐Peter Klages Gunter Gastrock 《Engineering in Life Science》2017,17(12):1271-1280
Although the great potential of droplet based microfluidic technologies for routine applications in industry and academia has been successfully demonstrated over the past years, its inherent potential is not fully exploited till now. Especially regarding to the droplet generation reproducibility and stability, two pivotally important parameters for successful applications, there is still a need for improvement. This is even more considerable when droplets are created to investigate tissue fragments or cell cultures (e.g. suspended cells or 3D cell cultures) over days or even weeks. In this study we present microfluidic chips composed of a plasma coated polymer, which allow surfactants‐free, highly reproducible and stable droplet generation from fluids like cell culture media. We demonstrate how different microfluidic designs and different flow rates (and flow rate ratios) affect the reproducibility of the droplet generation process and display the applicability for a wide variety of bio(techno)logically relevant media. 相似文献
3.
Xavier Casadevall i Solvas Xize Niu Katherine Leeper Soongwon Cho Soo-Ik Chang Joshua B. Edel Andrew J. deMello 《Journal of visualized experiments : JoVE》2011,(58)
The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection. 相似文献
4.
Anming Xu Jiawei Liu Shixiang Cao Bin Xu Chengzhi Guo Ziyi Yu Xiaoqiang Chen Jie Zhou Weiliang Dong Min Jiang 《Microbial biotechnology》2023,16(2):474-480
Application of polyester-degrading microorganisms or enzymes should be considered as an eco-friendly alternative to chemical recycling due to the huge plastic waste disposal nowadays. However, current impranil DLN-based screening of polyester-degrading microorganisms is time-consuming, labour-intensive and unable to distinguish polyesterases from other protease- or amidase-like enzymes. Herein, we present an approach that combined a novel synthetic fluorescent polyurethane analogue probe (FPAP), along with the droplet-based microfluidics to screen polyurethane-degrading microorganisms through fluorescence-activated droplet sorting (FADS) pipeline. The fluorescent probe FPAP exhibited a fluorescence enhancement effect once hydrolysed by polyesterases, along with a strong specificity in discriminating polyesterases from other non-active enzymes. Application of FPAP in a microfluidic droplet system demonstrated that this probe exhibited high sensitivity and efficiency in selecting positive droplets containing leaf-branch compost cutinase (LCC) enzymes. This novel fluorogenic probe, FPAP, combined with the droplet microfluidic system has the potential to be used in the exploitation of novel PUR-biocatalysts for biotechnological and environmental applications. 相似文献
5.
Fuqiang Ma Tianjie Guo Yifan Zhang Xue Bai Changlong Li Zelin Lu Xi Deng Daixi Li Katsuo Kurabayashi Guang-yu Yang 《Environmental microbiology》2021,23(2):996-1008
Uncultivable microbial communities provide enormous reservoirs of enzymes, but their experimental identification by functional metagenomics is challenging, mainly due to the difficulty of screening enormous metagenomic libraries. Here, we propose a reliable and convenient ultrahigh-throughput screening platform based on flow cytometric droplet sorting (FCDS). The FCDS platform employs water-in-oil-in-water double emulsion droplets serving as single-cell enzymatic micro-reactors and a commercially available flow cytometer, and it can efficiently isolate novel biocatalysts from metagenomic libraries by processing single cells as many as 108 per day. We demonstrated the power of this platform by screening a metagenomic library constructed from domestic running water samples. The FCDS assay screened 30 million micro-reactors in only 1 h, yielding a collection of esterase genes. Among these positive hits, Est WY was identified as a novel esterase with high catalytic efficiency and distinct evolutionary origin from other lipolytic enzymes. Our study manifests that the FCDS platform is a robust tool for functional metagenomics, with the potential to significantly improve the efficiency of exploring novel enzymes from nature. 相似文献
6.
Manpreet Kaur Aura Ferreiro Cheng-Yu Hung Gautam Dantas Anand K. Ramasubramanian 《Biotechnology progress》2023,39(2):e3317
Functional metagenomics is an attractive culture-independent approach for functional screening of diverse microbiomes to identify known and novel genes. Since functional screening can involve sifting through tens of thousands of metagenomic library clones, an easy high-throughput screening approach is desirable. Here, we demonstrate a proof-of-concept application of a low-cost, high-throughput droplet based microfluidic assay to the selection of antibiotic resistance genes from a soil metagenomic library. Metagenomic library members encapsulated in nanoliter volume water-in-oil droplets were printed on glass slides robotically, and cell growth in individual drops in the presence of ampicillin was imaged and quantified to identify ampicillin-resistant clones. From the hits, true positives were confirmed by sequencing and functional validation. The ease of liquid handling, ease of set-up, low cost, and robust workflow makes the droplet-based nano-culture platform a promising candidate for screening and selection assays for functional metagenomic libraries. 相似文献
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Anming Xu Jiawei Liu Shixiang Cao Bin Xu Chengzhi Guo Ziyi Yu Xiaoqiang Chen Jie Zhou Weiliang Dong Min Jiang 《Microbial biotechnology》2023,16(2):474
Application of polyester‐degrading microorganisms or enzymes should be considered as an eco‐friendly alternative to chemical recycling due to the huge plastic waste disposal nowadays. However, current impranil DLN‐based screening of polyester‐degrading microorganisms is time‐consuming, labour‐intensive and unable to distinguish polyesterases from other protease‐ or amidase‐like enzymes. Herein, we present an approach that combined a novel synthetic fluorescent polyurethane analogue probe (FPAP), along with the droplet‐based microfluidics to screen polyurethane‐degrading microorganisms through fluorescence‐activated droplet sorting (FADS) pipeline. The fluorescent probe FPAP exhibited a fluorescence enhancement effect once hydrolysed by polyesterases, along with a strong specificity in discriminating polyesterases from other non‐active enzymes. Application of FPAP in a microfluidic droplet system demonstrated that this probe exhibited high sensitivity and efficiency in selecting positive droplets containing leaf‐branch compost cutinase (LCC) enzymes. This novel fluorogenic probe, FPAP, combined with the droplet microfluidic system has the potential to be used in the exploitation of novel PUR‐biocatalysts for biotechnological and environmental applications.A complete FADS pipeline using FPAP as the fluorogenic probe for screening polyester‐degrading microorganisms was developed. 相似文献
9.
Microbial systems often exhibit staggering diversity, making the study of rare, interesting species challenging. For example, metagenomic analyses of mixed-cell populations are often dominated by the sequences of the most abundant organisms, while those of rare microbes are detected only at low levels, if at all. To overcome this, selective cultivation or fluorescence-activated cell sorting (FACS) can be used to enrich for the target species prior to sequence analysis; however, since most microbes cannot be grown in the lab, cultivation strategies often fail, while cell sorting requires techniques to uniquely label the cell type of interest, which is often not possible with uncultivable microbes. Here, we introduce a culture-independent strategy for sorting microbial cells based on genomic content, which we term PCR-activated cell sorting (PACS). This technology, which utilizes the power of droplet-based microfluidics, is similar to FACS in that it uses a fluorescent signal to uniquely identify and sort target species. However, PACS differs importantly from FACS in that the signal is generated by performing PCR assays on the cells in microfluidic droplets, allowing target cells to be identified with high specificity with suitable design of PCR primers and TaqMan probes. The PACS assay is general, requires minimal optimization and, unlike antibody methods, can be developed without access to microbial antigens. Compared to non-specific methods in which cells are sorted based on size, granularity, or the ability to take up dye, PACS enables genetic sequence-specific sorting and recovery of the cell genomes. In addition to sorting microbes, PACS can be applied to eukaryotic cells, viruses, and naked nucleic acids. 相似文献
10.
The natural separation maintained by microfluidic flows is employed as the basis of a particle/cell sorting device. This method of separating particulate suspensions exploits the inherent laminar nature of microscale fluid dynamics and incorporates applied fields and image cytometry to enable sorting based upon any visually identifiable difference between colloid-sized cells or particles. This technique may be used to easily isolate, separate, sort, or enrich virtually any suspension of microscale biological or colloidal particles within a microfluidic system. The entire footprint of the device described here is less than 0.01 mm(2), allowing it to be readily incorporated within highly integrated micro total analysis systems (microTAS). 相似文献
11.
Dennis J. Eastburn Yong Huang Maurizio Pellegrino Adam Sciambi Louis J. Ptá?ek Adam R. Abate 《Nucleic acids research》2015,43(13):e86
Targeted sequence enrichment enables better identification of genetic variation by providing increased sequencing coverage for genomic regions of interest. Here, we report the development of a new target enrichment technology that is highly differentiated from other approaches currently in use. Our method, MESA (Microfluidic droplet Enrichment for Sequence Analysis), isolates genomic DNA fragments in microfluidic droplets and performs TaqMan PCR reactions to identify droplets containing a desired target sequence. The TaqMan positive droplets are subsequently recovered via dielectrophoretic sorting, and the TaqMan amplicons are removed enzymatically prior to sequencing. We demonstrated the utility of this approach by generating an average 31.6-fold sequence enrichment across 250 kb of targeted genomic DNA from five unique genomic loci. Significantly, this enrichment enabled a more comprehensive identification of genetic polymorphisms within the targeted loci. MESA requires low amounts of input DNA, minimal prior locus sequence information and enriches the target region without PCR bias or artifacts. These features make it well suited for the study of genetic variation in a number of research and diagnostic applications. 相似文献
12.
Yohei Nishikawa Masahito Hosokawa Toru Maruyama Keisuke Yamagishi Tetsushi Mori Haruko Takeyama 《PloS one》2015,10(9)
Whole genome amplification (WGA) is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA), using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 105 droplets (67 pL) within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidic-generated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples. 相似文献
13.
Phenotypic profiling of natural, engineered or synthetic cells has increasingly become a bottleneck in the mining and engineering of cell factories. Single-cell phenotyping technologies are highly promising for tackling this hurdle, yet ideally they should allow non-invasive live-cell probing, be label-free, provide landscape-like phenotyping capability, distinguish complex functions, operate with high speed, sufficient throughput and low cost, and finally, couple with cell sorting so as to enable downstream omics analysis. This review focuses on recent progress in Ramanome Technology Platform (RTP), which consists of Raman spectroscopy based phenotyping, sorting and sequencing of single cells, and discuss the key challenges and emerging trends. In addition, we propose ramanome, a collection of single-cell Raman spectra (SCRS) acquired from individual cells within a cellular population or consortium, as a new type of biological phenome datatype at the single-cell resolution. By establishing the phenome-genome links in a label-free, single-cell manner, RTP should find wide applications in functional screening and strain development of live microbial, plant and animal cell factories. 相似文献
14.
The article reviews applications of flow cytometry sorting in manufacturing of pharmaceuticals. Flow cytometry sorting is an extremely powerful tool for monitoring, screening and separating single cells based on any property that can be measured by flow cytometry. Different applications of flow cytometry sorting are classified into groups and discussed in separate sections as follows: (a) isolation of cell types, (b) high throughput screening, (c) cell surface display, (d) droplet fluorescent-activated cell sorting (FACS). Future opportunities are identified including: (a) sorting of particular fractions of the cell population based on a property of interest for generating inoculum that will result in improved outcomes of cell cultures and (b) the use of population balance models in combination with FACS to design and optimize cell cultures. 相似文献
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细胞机械性能与细胞的生理状态与功能存在密切联系。早期对于细胞机械性能的研究受制于技术条件,只能获得细胞群的弹性或剪切模量,使得少量异质细胞的机械表型被淹没。近年来,单细胞机械性能检测技术得到了蓬勃发展。原子力显微镜、微吸管技术、光镊与光学拉伸、磁扭转流变仪与磁镊等单细胞机械性能检测技术展现出非常高的检测精度,但检测通量相对较低。新型微流控高通量检测方法的出现使检测通量呈几何式增长,有望解决大样本快速检测的需求。本文首先综述原子力显微镜、微吸管、光镊与光学拉伸和磁扭转流变仪与磁镊等单细胞机械性能检测技术。在此基础上,重点介绍细胞过孔、剪切诱导细胞变形和拉伸诱导细胞变形3种新兴微流控高通量检测技术的工作原理及最新研究进展,探讨各类方法的优缺点。最后,本文展望单细胞机械性能检测技术的未来发展方向。 相似文献
17.
Richard J. Thomas Daniel Webber William Sellors Aaron Collinge Andrew Frost Anthony J. Stagg Stephen C. Bailey Pramukh N. Jayasekera Rosa R. Taylor Steve Eley Richard W. Titball 《Aerobiologia》2009,25(2):75-84
Flow-focussing technology was harnessed to enable generation of large droplet aerosols within high-level microbiological containment.
The Collison nebuliser and flow-focussing aerosol generator (FFAG) produced aerosols from distilled water with average mass
median aerodynamic diameters (MMAD) of 4.19 and 11.93 μm, respectively. The medium type [water, phosphate buffered saline
(PBS) or microbiological broth] had minimal effect on the droplet size generated by the FFAG. The FFAG can be modulated to
generate reproducible aerosols with a wide range of MMADs (9–105 μm). The number of particulates (i.e. fluospheres) contained
within the droplets increased as the MMAD increased from 12 to 50 μm. The technology described can be used for the exposure
of small-animal models to large droplet aerosols (>10 μm) and has applications in healthcare, pharmaceutical, agricultural
and biodefence environments. 相似文献
18.
Small volume fluid handling in single and multiphase microfluidics provides a promising strategy for efficient bio-chemical assays, low-cost point-of-care diagnostics and new approaches to scientific discoveries. However multiple barriers exist towards low-cost field deployment of programmable microfluidics. Incorporating multiple pumps, mixers and discrete valve based control of nanoliter fluids and droplets in an integrated, programmable manner without additional required external components has remained elusive. Combining the idea of punch card programming with arbitrary fluid control, here we describe a self-contained, hand-crank powered, multiplex and robust programmable microfluidic platform. A paper tape encodes information as a series of punched holes. A mechanical reader/actuator reads these paper tapes and correspondingly executes operations onto a microfluidic chip coupled to the platform in a plug-and-play fashion. Enabled by the complexity of codes that can be represented by a series of holes in punched paper tapes, we demonstrate independent control of 15 on-chip pumps with enhanced mixing, normally-closed valves and a novel on-demand impact-based droplet generator. We demonstrate robustness of operation by encoding a string of characters representing the word “PUNCHCARD MICROFLUIDICS” using the droplet generator. Multiplexing is demonstrated by implementing an example colorimetric water quality assays for pH, ammonia, nitrite and nitrate content in different water samples. With its portable and robust design, low cost and ease-of-use, we envision punch card programmable microfluidics will bring complex control of microfluidic chips into field-based applications in low-resource settings and in the hands of children around the world. 相似文献
19.
We have investigated the accuracy with which droplets containing cells can be sorted individually onto known and thus relocatable positions. The presence and random arrival of cells and particles in the sorter jet disturbs the orderly production and deflection of droplets, causing a dispersion of sorted droplet trajectories. The magnitude of this dispersion is a function of the phase relationship between the arrival of a cell at the end of the jet and the droplet formation. Using a modified Becton Dickinson Fluorescence-Activated Cell Sorter, we selected for sorting only those droplets that formed with a cell near the center of the droplet. We used this technique to sort Lewis lung tumor cells. The dispersion of droplet positions was reduced from over 3% to about 1% of an average deflection of typically 15 mm for a nozzle with a 50-micron diameter orifice. Sorting onto a surface such as magnetic tape or a microscope slide introduces another uncertainty in position because the cell may be located anywhere within the wetted radius of the droplet on the slide. Sorting onto less-wettable surfaces reduces the wetted radius and thus the variation in cell position. 相似文献
20.
巴斯德毕赤酵母是当前应用最为方便和广泛的外源蛋白表达系统之一,为了进一步提高其表达外源蛋白的能力,文中建立了基于液滴微流控的毕赤酵母高通量筛选方法,并以木聚糖酶融合荧光蛋白为例,筛选获得木聚糖酶表达和分泌能力提高的突变株。通过PCR扩增得到木聚糖酶xyn5基因和绿色荧光蛋白gfp基因融合片段,并克隆到毕赤酵母表达载体pPIC9K中构建出木聚糖酶融合绿色荧光蛋白的质粒pPIC9K-xyn5-gfp,电转化至毕赤酵母GS115中得到表达木聚糖酶和绿色荧光蛋白的毕赤酵母SG菌株。该菌株经过常压室温等离子体诱变后进行单细胞液滴包埋,液滴培养24h后进行微流控筛选,获得高表达木聚糖酶的突变菌株,进而用于下一轮的诱变突变库构建和筛选。以此类推,经过5轮液滴微流控筛选,获得一株高产菌株SG-m5,其木聚糖酶活为149.17U/mg,较出发菌株提升300%,分泌外源蛋白的能力较出发菌株提高160%。文中建立的毕赤酵母单细胞液滴微流控高通量筛选方法能达到每小时10万菌株的筛选通量,筛选百万级别的菌株库仅需10h,消耗荧光试剂体积100μL,对比传统的微孔板筛选方法降低试剂成本近百万倍,为高效、低成本筛选获得表达和分泌外源蛋白能力提高的毕赤酵母提供了一条新途径。 相似文献