Compartmentalised combinatorial chemistry by microfluidic control
Inventors
Griffiths, Andrew • Weitz, David • Link, Darren • Ahn, Keunho • Bibette, Jerome
Assignees
Medical Research Council • National Science Foundation NSF • Harvard University
Publication Number
US-9839890-B2
Publication Date
2017-12-12
Expiration Date
2024-03-31
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Abstract
The invention describes a method for the synthesis of compounds comprising the steps of: (a) compartmentalising two or more sets of primary compounds into microcapsules; such that a proportion of the microcapsules contains two or more compounds; and (b) forming secondary compounds in the microcapsules by chemical reactions between primary compounds from different sets; wherein one or both of steps (a) and (b) is performed under microfluidic control; preferably electronic microfluidic control, The invention further allows for the identification of compounds which bind to a target component of a biochemical system or modulate the activity of the target, and which is co-compartmentalised into the microcapsules.
Core Innovation
The invention provides a method for synthesising compounds by compartmentalising two or more sets of primary compounds into microcapsules such that a proportion of the microcapsules contains compounds from each set, and forming secondary compounds within these microcapsules by chemical reactions between primary compounds from different sets. The method utilizes microfluidic control, preferably electronic microfluidic control, in one or both of the compartmentalisation and reaction steps.
Additionally, the invention offers methods for identifying primary compounds that react to form secondary compounds capable of binding to or modulating the activity of a target, wherein the target is co-compartmentalised within the microcapsules. The method includes iterative steps where subsets of primary compounds identified as active can be re-compartmentalised and further reacted under microfluidic control for enhanced compound synthesis and identification.
The invention addresses limitations in current high-throughput screening (HTS) and combinatorial chemistry methods, which despite advances, remain insufficient to explore the vast chemical structure-activity space needed for drug discovery. Conventional spatially resolved parallel synthesis and split synthesis have scale, characterisation, and identification challenges, and dynamic combinatorial chemistry and virtual screening methods also have limitations in throughput and validation. The invention aims to create and screen extremely large numbers (≧10^10) of compounds quickly, in femtoliter volumes, and at low cost, overcoming prior restricted library sizes and throughput limitations.
Claims Coverage
The claims include one independent claim focusing on a method of preparing secondary compounds by attaching primary compounds to microbeads, forming microcapsules via microfluidic partitioning, merging microcapsules under an electric field, releasing primary compounds, forming secondary compounds by reaction, fusing microcapsules with a target enzyme, and determining active subsets.
Microfluidic formation of microcapsules with primary compounds attached to beads
Forming water-in-fluorocarbon oil microcapsules by partitioning aqueous fluids containing one or more sets of primary compounds attached to microbeads using microfluidic channels and fluorinated surfactants.
Merging charged microcapsules under an electric field to combine primary compounds
Merging distinct sets of water-in-fluorocarbon oil microcapsules each containing different primary compounds attached to microbeads via an applied electric field in a microfluidic channel to form merged microcapsules containing multiple sets of primary compounds.
Releasing primary compounds and synthesising secondary compounds within merged microcapsules
Releasing primary compounds from microbeads within merged microcapsules to allow chemical reactions forming secondary compounds from the released primary compounds in the aqueous phase of each microcapsule.
Fusing secondary compound-containing microcapsules with microcapsules containing a target enzyme
Fusing the merged microcapsules containing secondary compounds with further microcapsules containing a target enzyme under microfluidic control for activity assessment.
Determining and optionally sorting based on binding or modulating activity
Determining subsets of primary compounds that react to form secondary compounds capable of binding to or modulating the activity of the target enzyme, with optional sorting of active microcapsules.
The claims cover an integrated microfluidic method for combinatorial synthesis and screening of secondary compounds formed from bead-attached primary compounds compartmentalised into charged microcapsules merged under electric fields, with steps for releasing compounds, reacting, fusing with a target enzyme, and determining active compounds.
Stated Advantages
Highly monodisperse microcapsules (<1.5% polydispersity) functioning as uniform microreactors with volumes from 1 femtoliter to 1 nanoliter.
Compartmentalisation prevents diffusion and dispersion, enabling isolation of compounds for screening.
Perfluorocarbon carrier fluids prevent exchange of molecules between microcapsules.
Microcapsules can be created and analysed at rates up to 10,000 per second, enabling very high throughput (~10^9 per day).
Microcapsules can be fused and split under microfluidic and electric control allowing combinatorial mixing, reaction initiation, and multiplexed assays.
Rapid mixing of reagents within microcapsules (<2 ms) via chaotic advection enables fast kinetic measurements.
Microfluidic electronic control enables precise droplet generation, fusion, and sorting without moving parts, supporting integration of synthesis and screening.
Documented Applications
Synthesis and high-throughput screening of compound libraries for binding to or modulation of biochemical targets, including enzyme inhibitors such as PTP1B inhibitors.
Screening of combinatorial chemical libraries formed by multi-component reactions within microcapsules to identify bioactive compounds.
Use in drug discovery processes to generate vast chemical repertoires and identify lead compounds rapidly at very small reaction volumes and high throughput.
Screening enzyme activity modulation using fluorogenic substrates in fused microcapsules containing reaction products and target enzymes.
Screening with compounds attached to microbeads released inside microcapsules, with identification of active compounds via flow cytometry of beads.
Cell-based assays within microcapsules to assess compound effects on cellular targets or cellular pathways.
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