Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Droplet microfluidics for amplification-free genetic detection of single cells.

ABSTRACT: In this article we present a novel droplet microfluidic chip enabling amplification-free detection of single pathogenic cells. The device streamlines multiple functionalities to carry out sample digitization, cell lysis, probe-target hybridization for subsequent fluorescent detection. A peptide nucleic acid fluorescence resonance energy transfer probe (PNA beacon) is used to detect 16S rRNA present in pathogenic cells. Initially the sensitivity and quantification abilities of the platform are tested using a synthetic target mimicking the actual expression level of 16S rRNA in single cells. The capability of the device to perform "sample-to-answer" pathogen detection of single cells is demonstrated using E. coli as a model pathogen.

SUBMITTER: Rane TD

PROVIDER: S-EPMC3696383 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Droplet microfluidics for amplification-free genetic detection of single cells.

Rane Tushar D TD Zec Helena C HC Puleo Chris C Lee Abraham P AP Wang Tza-Huei TH

Lab on a chip 20120730 18

In this article we present a novel droplet microfluidic chip enabling amplification-free detection of single pathogenic cells. The device streamlines multiple functionalities to carry out sample digitization, cell lysis, probe-target hybridization for subsequent fluorescent detection. A peptide nucleic acid fluorescence resonance energy transfer probe (PNA beacon) is used to detect 16S rRNA present in pathogenic cells. Initially the sensitivity and quantification abilities of the platform are te ...[more]

PMID: 22842841

Similar Datasets

Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics.

Project description:Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

| S-EPMC5507899 | biostudies-literature

Droplet Microfluidics Enables Tracing of Target Cells at the Single-Cell Transcriptome Resolution.

Project description:The rapid promotion of single-cell omics in various fields has begun to help solve many problems encountered in research, including precision medicine, prenatal diagnosis, and embryo development. Meanwhile, single-cell techniques are also constantly updated with increasing demand. For some specific target cells, the workflow from droplet screening to single-cell sequencing is a preferred option and should reduce the impact of operation steps, such as demulsification and cell recovery. We developed an all-in-droplet method integrating cell encapsulation, target sorting, droplet picoinjection, and single-cell transcriptome profiling on chips to achieve labor-saving monitoring of TCR-T cells. As a proof of concept, in this research, TCR-T cells were encapsulated, sorted, and performed single-cell transcriptome sequencing (scRNA-seq) by injecting reagents into droplets. It avoided the tedious operation of droplet breakage and re-encapsulation between droplet sorting and scRNA-seq. Moreover, convenient device operation will accelerate the progress of chip marketization. The strategy achieved an excellent recovery performance of single-cell transcriptome with a median gene number over 4000 and a cross-contamination rate of 8.2 ± 2%. Furthermore, this strategy allows us to develop a device with high integrability to monitor infused TCR-T cells, which will promote the development of adoptive T cell immunotherapy and their clinical application.

| S-EPMC9687293 | biostudies-literature

Single-cell analysis and sorting using droplet-based microfluidics.

Project description:We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. As an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. Secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. The beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ?200 Hz as well as cell enrichment. The microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ?1 million cells, the microfluidic operations require 2-6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5-7 d.

| S-EPMC4128248 | biostudies-literature

High throughput single cell counting in droplet-based microfluidics.

Project description:Droplet-based microfluidics is extensively and increasingly used for high-throughput single-cell studies. However, the accuracy of the cell counting method directly impacts the robustness of such studies. We describe here a simple and precise method to accurately count a large number of adherent and non-adherent human cells as well as bacteria. Our microfluidic hemocytometer provides statistically relevant data on large populations of cells at a high-throughput, used to characterize cell encapsulation and cell viability during incubation in droplets.

| S-EPMC5431057 | biostudies-literature

Evolution and Single-Droplet Analysis of Fuel-Driven Compartments by Droplet-Based Microfluidics.

Project description:Active droplets are a great model for membraneless organelles. However, the analysis of these systems remains challenging and is often limited due to the short timescales of their kinetics. We used droplet-based microfluidics to encapsulate a fuel-driven cycle that drives phase separation into coacervate-based droplets to overcome this challenge. This approach enables the analysis of every coacervate-based droplet in the reaction container throughout its lifetime. We discovered that the fuel concentration dictates the formation of the coacervate-based droplets and their properties. We observed that coacervate-based droplets grow through fusion, decay simultaneously independent of their volume, and shrinkage rate scales with their initial volume. This method helps to further understand the regulation of membraneless organelles, and we believe the analysis of individual coacervate-based droplets enables future selection- or evolution-based studies.

| S-EPMC9400878 | biostudies-literature

Detection of single enzymatic events in rare or single cells using microfluidics.

Project description:In the present study we demonstrate highly sensitive detection of rare, aberrant cells in a population of wild-type human cells by combining a rolling-circle-enhanced enzyme activity single-molecule detection assay with a custom-designed microfluidic device. Besides reliable detection of low concentrations of aberrant cells, the integrated system allowed multiplexed detection of individual enzymatic events at the single cell level. The single cell sensitivity of the presented setup relies on the combination of single-molecule rolling-circle-enhanced enzyme activity detection with the fast reaction kinetics provided by a picoliter droplet reaction volume and subsequent concentration of signals in a customized drop-trap device. This setup allows the fast reliable analyses of enzyme activities in a vast number of single cells, thereby offering a valuable tool for basic research as well as theranostics.

| S-EPMC3823540 | biostudies-literature

Functional single-cell hybridoma screening using droplet-based microfluidics.

Project description:Monoclonal antibodies can specifically bind or even inhibit drug targets and have hence become the fastest growing class of human therapeutics. Although they can be screened for binding affinities at very high throughput using systems such as phage display, screening for functional properties (e.g., the inhibition of a drug target) is much more challenging. Typically these screens require the generation of immortalized hybridoma cells, as well as clonal expansion in microtiter plates over several weeks, and the number of clones that can be assayed is typically no more than a few thousand. We present here a microfluidic platform allowing the functional screening of up to 300,000 individual hybridoma cell clones within less than a day. This approach should also be applicable to nonimmortalized primary B-cells, as no cell proliferation is required: Individual cells are encapsulated into aqueous microdroplets and assayed directly for the release of antibodies inhibiting a drug target based on fluorescence. We used this system to perform a model screen for antibodies that inhibit angiotensin converting enzyme 1, a target for hypertension and congestive heart failure drugs. When cells expressing these antibodies were spiked into an unrelated hybridoma cell population in a ratio of 1:10,000 we observed a 9,400-fold enrichment after fluorescence activated droplet sorting. A wide variance in antibody expression levels at the single-cell level within a single hybridoma line was observed and high expressors could be successfully sorted and recultivated.

| S-EPMC3406880 | biostudies-other

Functional TCR T cell screening using single-cell droplet microfluidics.

Project description:Adoptive T cell transfer, in particular TCR T cell therapy, holds great promise for cancer immunotherapy with encouraging clinical results. However, finding the right TCR T cell clone is a tedious, time-consuming, and costly process. Thus, there is a critical need for single cell technologies to conduct fast and multiplexed functional analyses followed by recovery of the clone of interest. Here, we use droplet microfluidics for functional screening and real-time monitoring of single TCR T cell activation upon recognition of target tumor cells. Notably, our platform includes a tracking system for each clone as well as a sorting procedure with 100% specificity validated by downstream single cell reverse-transcription PCR and sequencing of TCR chains. Our TCR screening prototype will facilitate immunotherapeutic screening and development of T cell therapies.

| S-EPMC6279597 | biostudies-literature

Single nuclei RNA-seq using droplet microfluidics (DroNc-seq)

Project description:We report the application of DroNc-seq on archived tissue from mouse adult hippocampus and cortex barin regions

2017-10-06 | GSE104525 | GEO

Sorting by interfacial tension (SIFT): Label-free enzyme sorting using droplet microfluidics.

Project description:Droplet microfluidics has the ability to greatly increase the throughput of screening and sorting of enzymes by carrying reagents in picoliter droplets flowing in inert oils. It was found with the use of a specific surfactant, the interfacial tension of droplets can be very sensitive to droplet pH. This enables the sorting of droplets of different pH when confined droplets encounter a microfabricated trench. The device can be extended to sort enzymes, as a large number of enzymatic reactions lead to the production of an acidic or basic product and a concurrent change in solution pH. The progress of an enzymatic reaction is tracked from the position of a flowing train of droplets. We demonstrate the sorting of esterase isoenzymes based on their enzymatic activity. This label-free technology, that we dub droplet sorting by interfacial tension (SIFT), requires no active components and would have applications for enzyme sorting in high-throughput applications that include enzyme screening and directed evolution of enzymes.

| S-EPMC6850955 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data