Single-enzyme analysis in a droplet-based micro- and nanofluidic system

Arayanarakool, Rerngchai; Shui, Lingling; Kengen, Servé W. M.; Berg, Albert van den; Eijkel, Jan C.T.

doi:10.1039/c3lc41100a

Cited by 50 publications

(44 citation statements)

References 39 publications

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“…Arayanarakool et al fabricated a nano-and micro-fluidic device capable of generating a large number of droplets with diameter around 3 lm for parallel single-enzyme kinetic analysis. 28 However, no digital detection was achieved in this work. More importantly, in their system, the enzyme reaction time was reported to be more than 20 h, which precludes the use for practical digital enzyme detection.…”

Section: Introductionmentioning

confidence: 83%

“…2(c-f)-2(c-h)) and the chip can be reused. Compared with other droplet trapping methods, 27,28 our design offers the advantages of simple device fabrication, scalable droplet generation, facile droplet trapping, and rapid device regeneration.…”

Section: The Choice Of Fluorinated Oil Gh-135 and Surfactant E2k0660mentioning

confidence: 99%

“…Over the years, great effort has been spent on creating uniform reactors, including fabricated microarrays [20][21][22][23][24][25] and microfluidic droplets, [26][27][28] in a high throughput manner for single enzyme analysis. Compared with a microarray, microdroplets have the advantages of ultrahigh throughput, low cost, flexible manipulation, and low non-specific adsorption to channel/ chamber wall by oil phase isolation, offering attractive alternative reactors for protein quantification.…”

Section: Introductionmentioning

confidence: 99%

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A highly parallel microfluidic droplet method enabling single-molecule counting for digital enzyme detection

et al. 2014

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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 b-galatosidase (b-Gal) molecules and the fluorogenic substrate fluorescein di-b-D-galactopyranoside were injected from two separated inlets to form uniform 20 lm 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 b-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 b-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 b-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. V C 2014 AIP Publishing LLC. [http://dx

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Section: Introductionmentioning

confidence: 83%

Section: The Choice Of Fluorinated Oil Gh-135 and Surfactant E2k0660mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A highly parallel microfluidic droplet method enabling single-molecule counting for digital enzyme detection

et al. 2014

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“…Femtoliter to picoliter-sized droplets encapsulated in continuous oil phase, have been found to be compatible with enzymatic fluorescent signal amplification. For instance, single enzyme activity analysis [16], single molecule immunoassays on magnetic beads [15], and cellsurface biomarker detection [17] were recently investigated in droplets on micro/nanofluidic chips. These advancements have made enzymatic reaction in droplet microfluidics a versatile tool for various biotechnological applications.…”

Section: Introductionmentioning

confidence: 99%

Digital droplet ELOHA for nucleic acid molecule counting and analysis

Guan

Chen

Rane

et al. 2015

2015 Transducers - 2015 18th International Conference on Solid-State Sensors, Actuators and Microsystems (TRANSDUCERS)

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We present a droplet digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for reference-free absolute quantification of nucleic acids. Droplet digital ELOHA is able to accurately quantify as few as several hundred copies of nucleic acids in a large, mL-sized sample volume (equivalent to 1 aM or lower) without molecular replication. Droplet digital ELOHA provides a linear response to the number of copies present in a sample to allow for detecting a small fold change unattainable by real time quantitative PCR. Its capability to cope with a large sample volume benefits from the use of magnetic beads to streamline capturing of nucleic acids from the sample and subsequent surface hybridization reactions in droplets. Droplet digital ELOHA is particularly attractive for RNA quantification since it does not require reverse transcription. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria Gonorrhoeae 16S rRNA to show its potential diagnostic value.

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“…Miniaturization and automation are key features in technology developments. Over the past ten years, droplet-based microfluidics has emerged as a groundbreaking technology for the miniaturization and automation of biochemical assays: [1][2][3][4][5][6] it provides essential elements to both miniaturize assays down to the scale of single microorganisms or even single molecules [7][8][9][11][12][13][14][15][16] and automatize sample processing. 17,18 In many biochemical applications, high-throughput droplet generation with a precise control of the size is required.…”

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confidence: 99%

Parallelized ultra-high throughput microfluidic emulsifier for multiplex kinetic assays

et al. 2015

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Droplet-based microfluidic technologies are powerful tools for applications requiring high-throughput, for example, in biochemistry or material sciences. Several systems have been proposed for the high-throughput production of monodisperse emulsions by parallelizing multiple droplet makers. However, these systems have two main limitations: (1) they allow the use of only a single disperse phase; (2) they are based on multiple layer microfabrication techniques. We present here a pipette-and-play solution offering the possibility of manipulating simultaneously 10 different disperse phases on a single layer device. This system allows high-throughput emulsion production using aqueous flow rates of up to 26 ml/h (>110 000 drops/s) leading to emulsions with user-defined complex chemical composition. We demonstrate the multiplex capabilities of our system by measuring the kinetics of β-galactosidase in droplets using nine different concentrations of a fluorogenic substrate.

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Single-enzyme analysis in a droplet-based micro- and nanofluidic system

Cited by 50 publications

References 39 publications

A highly parallel microfluidic droplet method enabling single-molecule counting for digital enzyme detection

A highly parallel microfluidic droplet method enabling single-molecule counting for digital enzyme detection

Digital droplet ELOHA for nucleic acid molecule counting and analysis

Parallelized ultra-high throughput microfluidic emulsifier for multiplex kinetic assays

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