Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single-Molecule-Counting Immunoassays

Shim, Jung-uk; Ranasinghe, Rohan T.; Smith, Clive A.; Ibrahim, Shehu; Hollfelder, Florian; Huck, Wilhelm T. S.; Klenerman, David; Abell, Chris

doi:10.1021/nn401661d

Cited by 197 publications

(207 citation statements)

References 39 publications

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“…Theoretically, the rate-limiting step for the application of this platform is the FACS-assisted droplet-screening technology (20), which is limited to several tens of thousands of events per second. The characteristics of the MDE-FACS platform-specifically, sensitive detection combined with specific separation-allow its use in practical research on drug design, including the selection of novel desired activity, direct isolation of probiotics, antibiotic producers, and prediction of unculturable effectors.…”

Section: Discussionmentioning

confidence: 99%

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity

Terekhov

Смирнов

Stepanova

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

191

172

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Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-tocell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slowgrowing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.ultrahigh-throughput screening | microfluidic encapsulation | butyrylcholinesterase | Staphylococcus aureus | cell-cell interactions T he ultrahigh-throughput (1, 2) technique of screening (uHTS) in a double emulsion was applied in directed enzyme evolution (3, 4) to investigate the idea that a universal genotypephenotype linkage was provided by compartmentalization. Artificial compartments of double emulsions were produced with high polydispersity by shear stress (5, 6), which significantly decreased the portion of uniform droplets and thereby reduced the sensitivity and the maximal sorting rate. By contrast, sophisticated custom sorters demonstrated the screening of precise monodisperse droplets of water-in-oil emulsions generated by microfluidic technology (1, 7). However, it is not always convenient to use custom devices, and the use of oil as a continuous phase limits the sorting rate. Alternatively, compartmentalization in microfluidic double emulsion (MDE) enables uHTS of >10,000 events/s using commercially available cell sorters (8, 9). Furthermore, biocompatible oil and water phases provide viability and proliferation of Escherichia coli cells (10) inside the microenvironment of a double emulsion.Here, we propose an MDE-FACS platform that combines the benefits of previously reported systems based on compartmentalization in MDE and FACS selection together with modern omics (Fig. 1). We succeeded in assembling this platform using commercially available parts, which included straightforward microfluidics (Fig. S1) for MDE generation, multiparametric FACS for uHTS, next-generation sequencing (NGS) for bioinformatic predictions, and mass spectrometry for proteome and secretome analysis. We demonstrated this idea with several single-cell methods (Fig. S2), including the selection of di...

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Section: Discussionmentioning

confidence: 99%

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity

Terekhov

Смирнов

Stepanova

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

191

172

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“…10 Improvements in sensitivity could be possible by adapting single-molecule detection techniques [37][38][39][40] or through the implementation of amplification schemes. More generally, we have shown that our microfluidic MITOMI platform 9,41-43 is capable of measuring protein biomarkers in hundreds to thousands of samples with highsensitivity and high dynamic-range.…”

Section: Discussionmentioning

confidence: 99%

A 1024-sample serum analyzer chip for cancer diagnostics

García-Cordero

Maerkl

2014

Lab Chip

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We present a platform that combines microarrays and microfluidic techniques to measure four protein biomarkers in 1024 serum samples for a total of 4096 assays per device. Detection is based on a surface fluorescence sandwich immunoassay with a limit of detection of~1 pM for most of the proteins measured: PSA, TNF-α, IL-1β, and IL-6. To validate the utility of our platform, we measured these four biomarkers in 20 clinical human serum samples, 10 from prostate cancer patients and 10 female and male controls. We compared the results of our platform to a conventional ELISA and found a good correlation between them. However, compared to a classical ELISA, our device reduces the total cost of reagents by 4 orders of magnitude while increasing throughput by 2 orders of magnitude. Overall, we demonstrate an integrated approach to perform low-cost and rapid quantification of protein biomarkers from over one thousand serum samples. This new high-throughput technology will have a significant impact on disease diagnosis and management.

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“…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%

“…For example, very recently, Shim et al fabricated a microfluidic device capable of generating and trapping 200 000 highly uniform femtoliter droplets for the measurement of chemical reactions of single-enzyme molecules and the application to a single-molecule-counting immunoassay. 26 Takeuchi et al developed a method that utilizes an immiscible liquid phase to instantly enclose thousands of uniform femtoliter droplets within microchambers built in microchannel walls to study enzymatic activities at single-molecule levels. 27 In these two examples, droplets were stably trapped for imaging analysis, with the former relying on a complicated valve and post design and the latter using a non-scalable microchamber format.…”

Section: Introductionmentioning

confidence: 99%

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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Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single-Molecule-Counting Immunoassays

Cited by 197 publications

References 39 publications

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity

A 1024-sample serum analyzer chip for cancer diagnostics

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

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