An automated two-phase microfluidic system for kinetic analyses and the screening of compound libraries

Clausell-Tormos, Jenifer; Griffiths, Andrew D.; Merten, Christoph A.

doi:10.1039/b921754a

Cited by 99 publications

(97 citation statements)

References 22 publications

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“…However, it is difficult to maintain stable coflows that exceed a 1∶9 ratio, and hence to achieve dilutions of greater than 10-fold, which makes this method poorly adapted for studying phenomena such as inhibition where concentrations must be varied logarithmically. Prediluting compounds in microplates (6,14), composing droplets one at a time in a glass capillary (35), or employing on-chip diffusive mixing (36, 37) permits a wider concentration range to be explored, but the sampling of this range becomes sparse, as with microplate-based methods (≤10 concentrations). Better sampling (23 concentrations) can be achieved by manipulating a single nanoliter-volume droplet trapped in a microfluidic channel by splitting and merging buffer droplets (38), but this method has not been applied to screening.…”

Section: Discussionmentioning

confidence: 99%

“…As a result of the miniaturization inherent in this approach, our system ( Fig. 1) is capable of generating doseresponse curves at materially higher resolutions than existing microplate-based (3) and microfluidics-based approaches (6)(7)(8)(9)(10)(11)(12)(13)(14). Each dose-response curve contains approximately 10,000 data points, 1,000 times more than in conventional systems, resulting in extremely precise measurement of dose-response relationships using minimal quantities of reagents.…”

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confidence: 99%

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High-resolution dose–response screening using droplet-based microfluidics

Miller

Harrak²,

Mangeat³

et al. 2011

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

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257

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A critical early step in drug discovery is the screening of a chemical library. Typically, promising compounds are identified in a primary screen and then more fully characterized in a dose-response analysis with 7-10 data points per compound. Here, we describe a robust microfluidic approach that increases the number of data points to approximately 10,000 per compound. The system exploits Taylor-Aris dispersion to create concentration gradients, which are then segmented into picoliter microreactors by droplet-based microfluidics. The large number of data points results in IC 50 values that are highly precise ( 2.40% at 95% confidence) and highly reproducible (CV 2.45%, n 16). In addition, the high resolution of the data reveals complex dose-response relationships unambiguously. We used this system to screen a chemical library of 704 compounds against protein tyrosine phosphatase 1B, a diabetes, obesity, and cancer target. We identified a number of novel inhibitors, the most potent being sodium cefsulodine, which has an IC 50 of 27 0.83 μM.high-throughput screening | HTS | small molecule library I n the early 16th century the Swiss chemist Paracelsus declared "all substances are poisons, there is none which is not a poison; only the right dose makes a substance non-poisonous." This idea that the biological effects of a chemical compound are dependent upon its concentration was quantified by A. V. Hill in 1910 (1). However, despite the fact that compounds can display complex concentration-dependent relationships, varying in potency, efficacy, and steepness of response, usually just a single measurement at a single concentration (approximately 10 μM) is obtained for each compound in the chemical library during a primary drug screen, even when using state-of-the-art robotic microplate-based screening systems. This results in high numbers of false positives and false negatives (2), as well as the inability to identify subtle, complex pharmacology, such as partial agonism or antagonism. Even when dose-response curves are generated during a quantitative primary screen (3) or, more typically, during the follow-up of a single-point screen, the time and cost limitations mean that the curves typically contain only 7-10 data points each. With ≤10 nonduplicated data points, and as many as four adjustable nonlinear parameters (e.g., in the four-parameter Hill function), the results are highly sensitive to the data quality: For example, the presence of a single outlying data point can substantially alter the fit of the data, unless the outliers are identified and removed (4).We have developed a system that uses droplet-based microfluidics to generate high-quality dose-response data during drug screening. Droplet-based microfluidics is itself a new technology for creating and manipulating picoliter-volume aqueous droplets that function as independent microreactors (for a review see ref. 5). As a result of the miniaturization inherent in this approach, our system (Fig. 1) is capable of generating doseresponse curves at materiall...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

High-resolution dose–response screening using droplet-based microfluidics

Miller

Harrak²,

Mangeat³

et al. 2011

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

Self Cite

257

217

View full text Add to dashboard Cite

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“…In the present work, each concentration of chemicals was prepared and tested independently, which somehow diminished throughput and user convenience. Several clever methods have been demonstrated by other group to generate concentration gradients inside droplets, including by varying flow rates of constituting streams of liquid, 30 by on chip-dilution, 31 by using aspiration technique, 32 by droplet-on-demand technology, 33 or by use of droplet libraries. 34 Single cell screen coupled with generation of concentration gradient on the same chip was demonstrated recently.…”

Section: Discussionmentioning

confidence: 99%

Microfluidic droplet encapsulation of highly motile single zoospores for phenotypic screening of an antioomycete chemical

et al. 2011

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Highly motile Phytophthora sojae (P. sojae) zoospores of an oomycete plant pathogen and antioomycete candidate chemicals were encapsulated into microdroplets. Random fast self-motion of P. sojae zoospores was overcome by choosing an appropriate flow rate for a zoospore suspension. To influence stochastic loading of zoospores into a microfluidic channel, a zoospore suspension was directly preloaded into a microtubing with a largely reduced inner diameter. A relatively high single zoospore encapsulation rate of 60.5% was achieved on a most trivial T-junction droplet generator platform, without involving any specially designed channel geometry. We speculated that spatial reduction in the diameter direction of microtubing added a degree of zoospore ordering in the longitudinal direction of microtubing and thus influenced positively to change the inherent limitation of stochastic encapsulation of zoospores. Comparative phenotypic study of a plant oomycete pathogen at a single zoospore level had not been achieved earlier.Phenotypic changes of zoospores responding to various chemical concentration conditions were measured in multiple droplets in parallel, providing a reliable data set and thus an improved statistic at a low chemical consumption. Since each droplet compartment contained a single zoospore, we were able to track the germinating history of individual zoospores without being interfered by other germinating zoospores, achieving a high spatial resolution. By adapting some existing droplet immobilization and concentration gradient generation techniques, the droplet approach could potentially lead to a medium-to-high throughput, reliable screening assay for chemicals against many other highly motile zoospores of pathogens.

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“…In addition, a robust method for sample tracking is required. In previous studies [25][26][27] plugs were simply stored in a sequential fashion (within a microfluidic channel or a piece of tubing), wherein the common fusion or splitting of plugs causes "frameshifts" resulting in loss of information on sample composition.…”

Section: Introductionmentioning

confidence: 99%

Rapid identification of optimal drug combinations for personalized cancer therapy using microfluidics

Eduati

Utharala

Madhavan

et al. 2016

Preprint

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Functional screening of live patient cancer cells holds great potential for personalized medicine and allows to overcome the limited translatability of results from existing in--vitro and ex--vivo screening models. Here we present a plug--based microfluidics approach enabling the testing of drug combinations directly on cancer cells from patient biopsies. The entire procedure takes less than 48 hours after surgery and does not require ex vivo cultivation. We screened more than 1100 samples for different primary human tumors (each with 56 conditions and at least 20 replicates), and obtained highly specific sensitivity profiles. This approach allowed us to derive optimal treatment options which we further validated in two different pancreatic cancer cell lines. This workflow should pave the way for rapid determination of optimal personalized cancer therapies at assay costs of less than US$ 150 per patient.

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An automated two-phase microfluidic system for kinetic analyses and the screening of compound libraries

Cited by 99 publications

References 22 publications

High-resolution dose–response screening using droplet-based microfluidics

High-resolution dose–response screening using droplet-based microfluidics

Microfluidic droplet encapsulation of highly motile single zoospores for phenotypic screening of an antioomycete chemical

Rapid identification of optimal drug combinations for personalized cancer therapy using microfluidics

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