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A droplet microfluidic approach to single-stream nucleic acid isolation and mutation detection.

ABSTRACT: In this work, a droplet microfluidic platform for genetic mutation detection from crude biosample is described. Single-stream integration of nucleic acid isolation and amplification is realized on a simple fluidic cartridge. Subsequent DNA melting curve is employed with signal normalizing algorithm to differentiate heterozygous K-ras codon 12 c.25G>A mutant from the wildtype. This technique showcases an alternative to modular bench-top approaches for genetic mutation screening, which is of interest to decentralized diagnostic platforms.

SUBMITTER: Shin DJ

PROVIDER: S-EPMC4223799 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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A droplet microfluidic approach to single-stream nucleic acid isolation and mutation detection.

Shin Dong Jin DJ Zhang Yi Y Wang Tza-Huei TH

Microfluidics and nanofluidics 20140801 2

In this work, a droplet microfluidic platform for genetic mutation detection from crude biosample is described. Single-stream integration of nucleic acid isolation and amplification is realized on a simple fluidic cartridge. Subsequent DNA melting curve is employed with signal normalizing algorithm to differentiate heterozygous K-ras codon 12 c.25G>A mutant from the wildtype. This technique showcases an alternative to modular bench-top approaches for genetic mutation screening, which is of inter ...[more]

PMID: 25386112

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Project description:Although digital detection of nucleic acids has been achieved by amplification of single templates in uniform microfluidic droplets and widely used for genetic analysis, droplet-based digital detection of proteins has rarely been reported, largely due to the lack of an efficient target amplification method for protein in droplets. Here, we report a key step towards digital detection of proteins using a highly parallel microfluidic droplet approach for single enzyme molecule detection in picoliter droplets via enzyme catalyzed signal amplification. An integrated microfluidic chip was designed for high throughput uniform droplet generation, monolayer droplet collection, incubation, detection, and release. Single β-galatosidase (β-Gal) molecules and the fluorogenic substrate fluorescein di-β-D-galactopyranoside were injected from two separated inlets to form uniform 20 μm droplets in fluorinated oil at a frequency of 6.6 kHz. About 200 000 droplets were captured as a monolayer in a capture well on-chip for subsequent imaging detection. A series of β-Gal solutions at different concentrations were analyzed at the single-molecule level. With no enzyme present, no droplets were found to fluoresce, while brightly fluorescent droplets were observed under single-enzyme molecule conditions. Droplet fluorescence intensity distribution analysis showed that the distribution of enzyme molecules under single-molecule conditions matched well with theoretical prediction, further proving the feasibility of detecting single enzyme molecules in emulsion droplets. Moreover, the population of fluorescent droplets increased as the β-Gal concentration increased. Based on a digital counting method, the measured concentrations of the enzyme were found to match well with input enzyme concentration, establishing the accuracy of the digital detection method for the quantification of β-Gal enzyme molecules. The capability of highly parallel detection of single enzyme molecules in uniform picoliter droplets paves the way to microdroplet based digital detection of proteins.

Dataset Information

A droplet microfluidic approach to single-stream nucleic acid isolation and mutation detection.

Publications

A droplet microfluidic approach to single-stream nucleic acid isolation and mutation detection.

Similar Datasets

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