Selection by compartmentalised screening
First Claim
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1. A method for screening compounds, the method comprising:
- introducing a first aqueous fluid comprising a target enzyme and a second aqueous fluid comprising a repertoire of small molecule compounds by separately injecting the first aqueous fluid and the second aqueous fluid into an immiscible fluorocarbon oil continuously flowing through a first microfluidic channel to form a plurality of first water-in-fluorocarbon oil microcapsules, wherein the first immiscible fluorocarbon oil comprises a fluorosurfactant, and wherein each aqueous portion of each of the plurality of the first water-in-fluorocarbon oil microcapsule comprises the target enzyme and a subset of the repertoire of small molecule compounds;
continuously flowing the plurality of the first water-in-fluorocarbon oil microcapsules through the first microfluidic channel and conducting a first target enzyme catalyzed reaction between the target enzyme and the subset of the repertoire of small molecule compounds within the aqueous portion of each of the plurality of the first water-in-fluorocarbon oil microcapsules to produce a first reaction product;
introducing a third aqueous fluid comprising a fluorogenic substrate from a second microfluidic channel, wherein the fluorogenic substrate is specific for the target enzyme;
merging a plurality of second water-in-fluorocarbon oil microcapsules flowing through the second microfluidic channel with the plurality of the first water-in-fluorocarbon oil microcapsules flowing through the first microfluidic channel to form a plurality of merged water-in-fluorocarbon oil microcapsules, wherein the aqueous portion of each of the plurality of second water-in-fluorocarbon oil microcapsules comprises the fluorogenic substrate, to form a plurality of merged water-in-fluorocarbon oil microcapsules, wherein the aqueous portion of the plurality of merged water-in-fluorocarbon oil microcapsules comprises the first reaction product and the fluorogenic substrate;
conducting a second enzyme catalyzed reaction between the first reaction product and the fluorogenic substrate within the aqueous portion of each of the plurality of the merged water-in-fluorocarbon oil microcapsules to produce a second reaction product; and
optically detecting the second reaction product.
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Abstract
The invention describes a method for the identification of compounds which bind to a target component of a biochemical system or modulate the activity of the target, comprising the steps of: a.) compartmentalizing the compounds into microcapsules together with the target, such that only a subset of the repertoire is represented in multiple copies in any one microcapsules; and b.) identifying the compound which binds to or modulates the activity of the target. The invention enables the screening of large repertoires of molecules which can serve as leads for drug development.
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Citations
17 Claims
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1. A method for screening compounds, the method comprising:
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introducing a first aqueous fluid comprising a target enzyme and a second aqueous fluid comprising a repertoire of small molecule compounds by separately injecting the first aqueous fluid and the second aqueous fluid into an immiscible fluorocarbon oil continuously flowing through a first microfluidic channel to form a plurality of first water-in-fluorocarbon oil microcapsules, wherein the first immiscible fluorocarbon oil comprises a fluorosurfactant, and wherein each aqueous portion of each of the plurality of the first water-in-fluorocarbon oil microcapsule comprises the target enzyme and a subset of the repertoire of small molecule compounds; continuously flowing the plurality of the first water-in-fluorocarbon oil microcapsules through the first microfluidic channel and conducting a first target enzyme catalyzed reaction between the target enzyme and the subset of the repertoire of small molecule compounds within the aqueous portion of each of the plurality of the first water-in-fluorocarbon oil microcapsules to produce a first reaction product; introducing a third aqueous fluid comprising a fluorogenic substrate from a second microfluidic channel, wherein the fluorogenic substrate is specific for the target enzyme; merging a plurality of second water-in-fluorocarbon oil microcapsules flowing through the second microfluidic channel with the plurality of the first water-in-fluorocarbon oil microcapsules flowing through the first microfluidic channel to form a plurality of merged water-in-fluorocarbon oil microcapsules, wherein the aqueous portion of each of the plurality of second water-in-fluorocarbon oil microcapsules comprises the fluorogenic substrate, to form a plurality of merged water-in-fluorocarbon oil microcapsules, wherein the aqueous portion of the plurality of merged water-in-fluorocarbon oil microcapsules comprises the first reaction product and the fluorogenic substrate; conducting a second enzyme catalyzed reaction between the first reaction product and the fluorogenic substrate within the aqueous portion of each of the plurality of the merged water-in-fluorocarbon oil microcapsules to produce a second reaction product; and optically detecting the second reaction product. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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Specification