scholarly journals Sorting droplets into many outlets

2021 ◽  
Author(s):  
Saurabh Vyawahare ◽  
Michael Brundage ◽  
Aleksandra Kijac ◽  
Michael Gutierrez ◽  
Martina de Geus ◽  
...  
Keyword(s):  
Droplet Microfluidics ◽  
Aqueous Samples ◽  
Single Droplet ◽  
Single Cell Sequencing ◽  
Droplet Pcr ◽  
Injection Molded ◽  
Droplet Sorting ◽  
Sorting System ◽  
Downstream Analysis ◽  
Successful Technology

Droplet microfluidics is a commercially successful technology, widely used in single cell sequencing and droplet PCR. Combining droplet making with droplet sorting has also been demonstrated, but so far found limited use, partly due to difficulties in scaling manufacture with injection molded plastics. We introduce a droplet sorting system with several new elements, including: 1) an electrode design combining metallic and ionic liquid parts, 2) a modular, multi-sorting fluidic design with features for keeping inter-droplet distances constant, 3) using timing parameters calculated from fluorescence or scatter signal triggers to precisely actuate dozens of sorting electrodes, 4) droplet collection techniques, including ability to collect a single droplet, and 5) a new emulsion breaking method to collect aqueous samples for downstream analysis. We use these technologies to build a fluorescence based cell sorter that sorts with high purity. We also show that these microfluidic designs can be translated into injection molded thermoplastic, suitable for industrial production. Finally, we tally the advantages and limitations of these devices.

scholarly journals Optimized double emulsion flow cytometry with high-throughput single droplet isolation

2019 ◽  
Author(s):  
Kara K. Brower ◽  
Catherine Carswell-Crumpton ◽  
Sandy Klemm ◽  
Bianca Cruz ◽  
Gaeun Kim ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Single Cell ◽  
High Throughput ◽  
Single Cell Analysis ◽  
Double Emulsion ◽  
Droplet Microfluidics ◽  
Cell Analysis ◽  
Single Droplet ◽  
Emulsion Droplets ◽  
Droplet Sorting

Droplet microfluidics has made large impacts in diverse areas such as enzyme evolution, chemical product screening, polymer engineering, and single-cell analysis. However, while droplet reactions have become increasingly sophisticated, phenotyping droplets by a fluorescent signal and sorting them to isolate variants-of-interest remains a field-wide bottleneck. Here, we present an optimized double emulsion workflow, sdDE-FACS, that enables high-throughput phenotyping, selection, and sorting of droplets using standard flow cytometers. Using a 130 μm nozzle, we demonstrate robust post-sort recovery of intact droplets, with little to no shear-induced droplet breakage, at high sort frequency (12-14 kHz) across two industry-standard FACS instruments. We report the first quantitative plate statistics for double emulsion droplet isolation and demonstrate single droplet recovery with >70% efficiency. In addition, we establish complete downstream recovery of nucleic acids from single, sorted double emulsion droplets, an advance in droplet sorting comparable with the capabilities of single-cell FACS. This work resolves several hurdles in the field of high-throughput droplet analysis and paves the way for a variety of new droplet assays, including rare variant isolation and multiparameter single-cell analysis, marrying the full power of flow cytometry with droplet microfluidics.

scholarly journals An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Mathias Girault ◽  
Hyonchol Kim ◽  
Hisayuki Arakawa ◽  
Kenji Matsuura ◽  
Masao Odaka ◽  
...  
Keyword(s):  
Single Cell ◽  
Real Time ◽  
Shape Recognition ◽  
Target Cells ◽  
Recognition Method ◽  
System A ◽  
Time Shape ◽  
Droplet Sorting ◽  
On Chip ◽  
Sorting System

scholarly journals Dual sequentially addressable dielectrophoretic array for high-throughput, scalable, multiplexed droplet sorting

2021 ◽  
Vol 25 (4) ◽  
Author(s):  
Akihiro Isozaki ◽  
Dunhou Huang ◽  
Yuta Nakagawa ◽  
Keisuke Goda
Keyword(s):  
High Throughput ◽  
Cell Biology ◽  
Digital Pcr ◽  
Industrial Applications ◽  
Droplet Microfluidics ◽  
Diverse Range ◽  
Droplet Sorting ◽  
On Chip ◽  
Single Droplets

AbstractDroplet microfluidics is a powerful tool for a diverse range of biomedical and industrial applications such as single-cell biology, synthetic biology, digital PCR, biosafety monitoring, drug screening, and food, feed, and cosmetic industries. As an integral part of droplet microfluidics, on-chip multiplexed droplet sorting has recently gained enthusiasm, since it enables real-time sorting of single droplets containing cells with different phenotypes into multiple bins. However, conventional sorting methods are limited in throughput and scalability. Here, we present high-throughput, scalable, multiplexed droplet sorting by employing a pair of sequentially addressable dielectrophoretic arrays (SADAs) across a microchannel on a microfluidic chip. A SADA is an on-chip array of electrodes, each of which is sequentially activated and deactivated in synchronization to the position and speed of a flowing droplet of interest. The dual-SADA (dSADA) structure enables high-throughput deflection of droplets in multiple directions in a well-controlled manner. For proof-of-concept demonstration and characterization of the dSADA, we performed fluorescence-activated droplet sorting (FADS) with a 3-way dSADA at a high throughput of 2450 droplets/s. Furthermore, to show the scalability of the dSADA, we also performed FADS with a 5-way dSADA at a high throughput of 473 droplets/s.

scholarly journals High-throughput single-cell DNA sequencing of AML tumors with droplet microfluidics

2017 ◽  
Author(s):  
Maurizio Pellegrino ◽  
Adam Sciambi ◽  
Sebastian Treusch ◽  
Robert Durruthy-Durruthy ◽  
Kaustubh Gokhale ◽  
...  
Keyword(s):  
Single Cell ◽  
Clonal Evolution ◽  
Droplet Microfluidics ◽  
Next Generation Sequencing Data ◽  
Sequencing Data ◽  
Single Cell Sequencing ◽  
Selection For ◽  
Genetic Profiles ◽  
Generation Sequencing

ABSTRACTTo enable the characterization of genetic heterogeneity in tumor cell populations, we developed a novel microfluidic approach that barcodes amplified genomic DNA from thousands of individual cancer cells confined to droplets. The barcodes are then used to reassemble the genetic profiles of cells from next generation sequencing data. Using this approach, we sequenced longitudinally collected AML tumor populations from two patients and genotyped up to 62 disease relevant loci across more than 16,000 individual cells. Targeted single-cell sequencing was able to sensitively identify tumor cells during complete remission and uncovered complex clonal evolution within AML tumors that was not observable with bulk sequencing. We anticipate that this approach will make feasible the routine analysis of heterogeneity in AML leading to improved stratification and therapy selection for the disease.

scholarly journals Integration of Droplet Microfluidic Tools for Single-cell Functional Metagenomics: An Engineering Head Start

2021 ◽  
Author(s):  
David Conchouso ◽  
Amani Al-Ma’abadi ◽  
Hayedeh Behzad ◽  
Mohammed Alarawi ◽  
Masahito Hosokawa ◽  
...  
Keyword(s):  
Head Start ◽  
Single Cell ◽  
High Throughput ◽  
Single Cells ◽  
Droplet Microfluidics ◽  
Metagenomic Data ◽  
Functional Screening ◽  
Functional Metagenomics ◽  
Technical Details ◽  
Droplet Sorting

<p>Droplet microfluidics techniques have shown promising results to study single-cells at high throughput. However, their adoption in laboratories studying “-omics” sciences is still irrelevant because of the field’s complex and multidisciplinary nature. To facilitate their use, here we provide engineering details and organized protocols for integrating three droplet-based microfluidic technologies into the metagenomic pipeline to enable functional screening of bioproducts at high throughput. First, a device encapsulating single-cells in droplets at a rate of ~ 250 Hz is described considering droplet size and cell growth. Then, we expand on previously reported fluorescent activated droplet sorting (FADS) systems to integrate the use of 4 independent fluorescence-exciting lasers (e.g., 405, 488, 561, 637 nm) in a single platform to make it compatible with different fluorescence-emitting biosensors. For this sorter, both hardware and software are provided and optimized for effortlessly sorting droplets at 60 Hz. Then, a passive droplet merger was also integrated into our method to enable adding new reagents to already made droplets at a rate of 200 Hz. Finally, we provide an optimized recipe for manufacturing these chips using silicon dry-etching tools. Because of the overall integration and the technical details presented here, our approach allows biologists to quickly use microfluidic technologies and achieve both single-cell resolution and high-throughput (> 50,000 cells/day) capabilities to mining and bioprospecting metagenomic data.</p>

scholarly journals Sample demultiplexing, multiplet detection, experiment planning and novel cell type verification in single cell sequencing

2019 ◽  
Author(s):  
Hongyi Xin ◽  
Qi Yan ◽  
Yale Jiang ◽  
Qiuyu Lian ◽  
Jiadi Luo ◽  
...  
Keyword(s):  
Single Cell ◽  
State Of The Art ◽  
Cell Types ◽  
Gaussian Mixture ◽  
Experiment Planning ◽  
Cell Type ◽  
Single Cell Sequencing ◽  
Artificial Cell ◽  
Real Cell ◽  
Downstream Analysis

AbstractIdentifying and removing multiplets from downstream analysis is essential to improve the scalability and reliability of single cell RNA sequencing (scRNA-seq). High multiplet rates create artificial cell types in the dataset. Sample barcoding, including the cell hashing technology and the MULTI-seq technology, enables analytical identification of a fraction of multiplets in a scRNA-seq dataset.We propose a Gaussian-mixture-model-based multiplet identification method, GMM-Demux. GMM-Demux accurately identifies and removes the sample-barcoding-detectable multiplets and estimates the percentage of sample-barcoding-undetectable multiplets in the remaining dataset. GMM-Demux describes the droplet formation process with an augmented binomial probabilistic model, and uses the model to authenticate cell types discovered from a scRNA-seq dataset.We conducted two cell-hashing experiments, collected a public cell-hashing dataset, and generated a simulated cellhashing dataset. We compared the classification result of GMM-Demux against a state-of-the-art heuristic-based classifier. We show that GMM-Demux is more accurate, more stable, reduces the error rate by up to 69×, and is capable of reliably recognizing 9 multiplet-induced fake cell types and 8 real cell types in a PBMC scRNA-seq dataset.

scholarly journals Ultrahigh-throughput–directed enzyme evolution by absorbance-activated droplet sorting (AADS)

2016 ◽  
Vol 113 (47) ◽  
pp. E7383-E7389 ◽  
Author(s):  
Fabrice Gielen ◽  
Raphaelle Hours ◽  
Stephane Emond ◽  
Martin Fischlechner ◽  
Ursula Schell ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Droplet Microfluidics ◽  
Enzyme Molecule ◽  
Functional Metagenomics ◽  
Oil Emulsion ◽  
Emulsion Droplets ◽  
Droplet Sorting ◽  
Water In Oil Emulsion ◽  
Increased Activity ◽  
Coupled Assay

Ultrahigh-throughput screening, in which members of enzyme libraries compartmentalized in water-in-oil emulsion droplets are assayed, has emerged as a powerful format for directed evolution and functional metagenomics but is currently limited to fluorescence readouts. Here we describe a highly efficient microfluidic absorbance-activated droplet sorter (AADS) that extends the range of assays amenable to this approach. Using this module, microdroplets can be sorted based on absorbance readout at rates of up to 300 droplets per second (i.e., >1 million droplets per hour). To validate this device, we implemented a miniaturized coupled assay for NAD+-dependent amino acid dehydrogenases. The detection limit (10 μM in a coupled assay producing a formazan dye) enables accurate kinetic readouts sensitive enough to detect a minimum of 1,300 turnovers per enzyme molecule, expressed in a single cell, and released by lysis within a droplet. Sorting experiments showed that the AADS successfully enriched active variants up to 2,800-fold from an overwhelming majority of inactive ones at ∼100 Hz. To demonstrate the utility of this module for protein engineering, two rounds of directed evolution were performed to improve the activity of phenylalanine dehydrogenase toward its native substrate. Fourteen hits showed increased activity (improved >4.5-fold in lysate; kcat increased >2.7-fold), soluble protein expression levels (up 60%), and thermostability (Tm, 12 °C higher). The AADS module makes the most widely used optical detection format amenable to screens of unprecedented size, paving the way for the implementation of chromogenic assays in droplet microfluidics workflows.

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