Compartmentalised combinatorial chemistry by microfluidic control

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.

1 . A method for preparing a repertoire 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 multiple copies of one or more compounds representative of each of said sets, and wherein said one or more compounds form a subset of the set of primary compounds;    (b) forming secondary compounds in the microcapsules by chemical reactions between primary compounds from different sets; wherein one or more of steps (a) and (b) are performed under microfluidic control.    
     
     
         2 . A method according to  claim 1 , wherein the subset of primary compounds constitutes 10% or less of the set of primary compounds.  
     
     
         3 . A method according to  claim 2 , wherein the subset of primary compounds constitutes 1% or less of the set of primary compounds.  
     
     
         4 . A method according to  claim 3 , wherein the subset of primary compounds is a single compound.  
     
     
         5 . A method according to  claim 1 , wherein, in step (a), the modal number of different primary compounds per compartment will be equivalent to the number of primary compounds forming the secondary compound in step (b).  
     
     
         6 . A method according to  claim 1 , wherein step (a) comprises forming separate emulsion compartments comprising primary compounds and mixing the emulsion compartments to form an emulsified set of primary compounds wherein a subset of the set of primary compounds is represented in multiple copies in any one microcapsule.  
     
     
         7 . A method according to  claim 1 , wherein step (a) comprises 
 (a) attaching the set of primary compounds onto microbeads, such that only a subset of the set of primary compounds is represented on any one microbead; and    (b) compartmentalising the microbeads into microcapsules; such that a subset of the set of primary compounds is represented in multiple copies in any one microcapsule;wherein one or more of steps (a) and (b) are performed under microfluidic control.    
     
     
         8 . A method according to  claim 1 , wherein at least one set of primary compounds comprises a repertoire of different compounds.  
     
     
         9 . A method according to  claim 1 , wherein one of the sets of primary compounds consists of a single compound.  
     
     
         10 . A method according to  claim 7 , wherein the compounds are attached to the beads non-covalently.  
     
     
         11 . A method according to  claim 7 , wherein the compounds are attached to the beads covalently.  
     
     
         12 . A method according to  claim 7 , wherein the compounds are attached to the microbeads by reversible physical or chemical mechanisms.  
     
     
         13 . A method according to  claim 11 , wherein the compounds are attached to the microbeads by photochemically cleavable linkers.  
     
     
         14 . A method for identifying primary compounds which react together to form secondary compounds capable of binding to or modulating the activity of a target, 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;    (b) forming secondary compounds in the microcapsules by chemical reactions between primary compounds from different sets; and    (c) identifying subsets of primary compounds which react to form secondary compounds which bind to or modulate the activity of the target; wherein one or more of steps (a) and (b) and (c) are performed under microfluidic control.    
     
     
         15 . A method for synthesising compounds with enhanced ability to bind to or modulate the activity of the target, comprising the steps of:: 
 (a) compartmentalising into microcapsules subsets of primary compounds identified in step (c) of the second aspect of the invention and, optionally, compartmentalising additional sets of primary compounds;    (b) forming secondary compounds in the microcapsules by chemical reactions between primary compounds from different sets; and    (c) identifying subsets of primary compounds which react to form secondary compounds which bind to or modulate the activity of the target;wherein one or more of steps (a) and (b) and (c) are performed under microfluidic control.    
     
     
         16 . A method according to  claim 15 , wherein steps (a) to (c) are iteratively repeated, such that after the first cycle, step (a) comprises compartmentalising subsets of primary compounds identified in step (c) into microcapsules and, optionally, compartmentalising additional sets of compounds.  
     
     
         17 . A method for identifying individual compounds which bind to or modulate the activity of the target, comprising the steps of: 
 (a) compartmentalising into microcapsules a primary compound identified in step (c) of the second or third aspect of the invention and additional sets of primary compounds;    (b) forming secondary compounds in the microcapsules by chemical reactions between primary compounds from different sets; and    (c) identifying subsets of primary compounds which react to form secondary compounds which bind to or modulate the activity of the target;wherein one or more of steps (a) and (b) and (c) are performed under microfluidic control.    
     
     
         18 . A method according to  claim 14 , wherein the activity of the target is a binding activity.  
     
     
         19 . A method according to  claim 14 , wherein the activity of the target is a catalytic activity.  
     
     
         20 . A method according to  claim 14 , wherein the secondary compound reacts with the target to generate a reaction product.  
     
     
         21 . A method according to  claim 14 , wherein the target is compartmentalised into microcapsules together with the compounds.  
     
     
         22 . A method according to  claim 14 , wherein a scaffold molecule is compartmentalised into microcapsules together with the compounds.  
     
     
         23 . A method according to  claim 14 , wherein the compounds are attached to beads, and in step (a) the primary molecules are released from the beads and react with other beads in the compartment to facilitate the identification of the primary compounds which react to form a secondary compounds which bind to and modulate the activity of the target in step (c).  
     
     
         24 . A method according to  claim 23 , wherein the compound having a desired activity causes a change in the microcapsule or microbead which allows the microcapsule or microbead to be identified, sorted or selected.  
     
     
         25 . A method according to  claim 23 , wherein the activity of the compound within the microcapsule results, directly or indirectly, in the modification of the microcapsule or microbead to enable the isolation of the microcapsule or microbead.  
     
     
         26 . A method according to  claim 23 , wherein the primary compounds and the target are bound to microbeads, the target is a ligand and the secondary compound within the microcapsule binds, directly or indirectly, to said ligand to enable the isolation of the microbead.  
     
     
         27 . A method according to  claim 23 , wherein microbeads are isolated by affinity purification.  
     
     
         28 . A method according to  claim 14 , wherein a substrate is present in the microcapsules and the desired activity of the compound within the microcapsule results, directly or indirectly, in the regulation of conversion of said substrate into product.  
     
     
         29 . A method according to  claim 28 , wherein the substrate is converted into the product by the catalytic action of the target.  
     
     
         30 . A method according to  claim 28 , wherein the substrate and the product have different optical properties.  
     
     
         31 . A method according to  claim 30 , wherein the substrate and product have different fluorescent properties.  
     
     
         32 . A method according to  claim 28 , wherein the primary compound and the substrate are bound to a microbead, and the desired activity of the secondary compound within the microcapsule results, directly or indirectly, in the regulation of conversion of said substrate into a product which remains part of the microbead and enables its identification and, optionally, isolation.  
     
     
         33 . A method according to  claim 32 , wherein the product and, optionally, the unreacted substrate are subsequently complexed with the microbead in the microcapsule.  
     
     
         34 . A method according to  claim 28 , wherein the desired activity enhances the conversion of substrate into product.  
     
     
         35 . A method according to  claim 28 , wherein the desired activity inhibits the conversion of substrate into product.  
     
     
         36 . A method according to  claim 28 , wherein the compound having a desired activity causes a change in the microcapsule which allows the microcapsule to be identified, sorted or selected.  
     
     
         37 . A method according to  claim 28 , wherein the modification of the target within the microcapsule induces a change in the optical properties of the microcapsule.  
     
     
         38 . A method according to  claim 24 , wherein the modification of the microbead enables it to be further modified outside the microcapsule so as to induce a change in its optical properties.  
     
     
         39 . A method according to  claim 24 , wherein the change in optical properties of the microbead is due to binding of a compound with distinctive optical properties to the microbead.  
     
     
         40 . A method according to  claim 24 , wherein the change in optical properties of the microbead is due to binding of a target with distinctive optical properties by the compound.  
     
     
         41 . A method according to  claim 24 , wherein the change in optical properties of the microbead is due to a change in the optical properties of the compound when bound to target.  
     
     
         42 . A method according to  claim 24 , wherein the change in optical properties of the microbead is due to a change in the optical properties of the target when bound by the compound.  
     
     
         43 . A method according to  claim 24 , wherein the change in optical properties of the microbead is due to a change in the optical properties of both target and compound on binding.  
     
     
         44 . A method according to  claim 24 , wherein the compound acts to cause a change in the target, and the change in optical properties of the microbead is due to the different optical properties of the target and the product of the action of the compound on the target.  
     
     
         45 . A method according to  claim 24 , wherein the compound acts to cause a change in the target without altering its optical properties, but only the product of the action, and not the target, binds to, or reacts with, the microbead, thereby changing the optical properties of the microbead.  
     
     
         46 . A method according to  claim 24 , wherein further reagents specifically bind to, or specifically react with, the product (and not the substrate) of the regulated reaction, which is attached to the microbead, thereby altering the optical properties of the microbead.  
     
     
         47 . A method according to  claim 24 , wherein further reagents specifically bind to, or specifically react with, the substrate (and not the product) of the regulated reaction, which is attached to the microbead, thereby altering the optical properties of the microbead.  
     
     
         48 . A method according to  claim 24 , wherein the compound in a microcapsule is identified by a difference in the optical properties of the microcapsule.  
     
     
         49 . A method according to  claim 48  wherein the difference in the optical properties of the microcapsule is a difference in fluorescence.  
     
     
         50 . A method according to  claim 49  wherein the difference in the fluorescence of the microcapsule is due to the presence of quantum dots.  
     
     
         51 . A method according to  claim 24 , wherein the compounds are present in different microcapsules at different concentrations.  
     
     
         52 . A method according to  claim 51 , wherein the concentration of the compound in a microcapsule is identified by a difference in the optical properties of the microcapsule.  
     
     
         53 . A method according to  claim 52 , wherein the difference in the optical properties of the microcapsule is a difference in fluorescence.  
     
     
         54 . A method according to  claim 53 , wherein the difference in the fluorescence of the microcapsule is due to the presence of quantum dots.  
     
     
         55 . A method according to  claim 24  wherein the compound on a microbead is identified by a difference in the optical properties of the microbead.  
     
     
         56 . A method according to  claim 55  wherein the difference in the optical properties of the microbead is a difference in fluorescence.  
     
     
         57 . A method according to  claim 56  wherein the difference in the fluorescence of the microbead is due to the presence of quantum dots.  
     
     
         58 . A method according to  claim 24 , wherein a second activity of a compound results in a change in the microbead or the microcapsule which is distinct from that resulting from a first activity.  
     
     
         59 . A method according to  claim 58 , wherein the change resulting from the second activity is used to positively select the microbeads or microcapsules.  
     
     
         60 . A method according to  claim 58 , wherein the change resulting from the second activity is used to negatively select the microbeads or microcapsules.  
     
     
         61 . A method according to  claim 60 , wherein negative selection is combined with positive selection to improve reaction specificity.  
     
     
         62 . A method according to  claim 61 , wherein the improved reaction specificity is an improvement in binding specificity.  
     
     
         63 . A method according to  claim 61 , wherein the improved reaction specificity is an improvement in regio- and/or stereo-selectivity for the target.  
     
     
         64 . A method according to  claim 24 , wherein microbeads modified directly or indirectly by the activity of the compound are further modified by Tyramide Signal Amplification (TSA™; NEN), resulting directly or indirectly in a change in the optical properties of said microbeads thereby enabling their identification and, optionally, isolation.  
     
     
         65 . Method according to  claim 24 , wherein the compound or compounds are low molecular weight compounds.  
     
     
         66 . Method according to  claim 24 , wherein each microbead has attached thereto multiple molecules of a single compound.  
     
     
         67 . A method according to  claim 24 , wherein microencapsulation is achieved by forming a water-in-oil emulsion.  
     
     
         68 . A method according to  claim 67 , wherein the water-in-oil emulsion is made using a microfluidic system.  
     
     
         69 . A method according to  claim 68 , wherein the emulsion is formed by aqueous droplet break off in a co-flowing steam of an immiscible liquid.  
     
     
         70 . A method according to  claim 68  or  claim 69  wherein microcapsules are transported for analysis by laminar-flow of aqueous microdroplets dispersed in a stream of oil in microfluidic channels.  
     
     
         71 . A method according to  claim 70 , wherein the microcapsules are of aqueous microdroplets dispersed in a stream of oil in microfluidic channels.  
     
     
         72 . A method according to  claim 71 , wherein the compound in a microcapsule is identified by the relative position of the microcapsule compared to other microdroplets in a microfluidic channel.  
     
     
         73 . A method according to  claim 71 , wherein the concentration of a compound in a microcapsule is identified by the relative position of the microcapsule compared to other microdroplets in a microfluidic channel.  
     
     
         74 . A method according to  claim 71 , wherein the target in a microcapsule is identified by the relative position of the microcapsule compared to other microdroplets in a microfluidic channel.  
     
     
         75 . A method according to  claim 64 , wherein the identified microcapsules are sorted using a microfluidic device.  
     
     
         76 . A method according to  claim 67 , wherein microencapsulation is achieved by forming an emulsion with a fluorocarbon or perfluorocarbon continuous phase.  
     
     
         77 . A method according to  claim 76 , wherein microencapsulation is achieved by forming an emulsion with a fluorocarbon or perfluorocarbon continuous phase and a non-aqueous discontinuous phase.  
     
     
         78 . A method according to  claim 76 , wherein microencapsulation is achieved by forming an emulsion with a fluorocarbon or perfluorocarbon continuous phase and an aqueous discontinuous phase.  
     
     
         79 . A method according to  claim 76 , wherein the fluorocarbon is perfluorooctyl bromide or perfluorooctylethane.  
     
     
         80 . A method according to  claim 78 , wherein the emulsion is formed using F-alkyl dimorpholinophosphate(s).  
     
     
         81 . A method according to  claim 80 , wherein the F-alkyl dimorpholinophosphate (s) have the general formula C n F 2n+1 C m H 2m OP(O)[N(CH 2 CH 2 ) 2 O] 2 .  
     
     
         82 . A method according to  claim 81 , wherein the F-alkyl dimorpholinophosphate is C 8 F 17 C 11 H 22 OP(O)[N(CH 2 CH 2 ) 2 O] 2 .  
     
     
         83 . A method according to  claim 24 , wherein the microcapsules are identified and, optionally, sorted by detection of a change in their fluorescence.  
     
     
         84 . A method according to  claim 83 , wherein the change in fluorescence is due to fluorescence resonance energy transfer (FRET).  
     
     
         85 . A method according to  claim 7 , wherein the microbead is nonmagnetic, magnetic or paramagnetic.  
     
     
         86 . A method according to  claim 24 , wherein the microbeads are identified and, optionally, sorted by detection of a change in their fluorescence.  
     
     
         87 . A method according to  claim 86 , wherein the identification of microbeads is by flow cytometry.  
     
     
         88 . A method according to  claim 87 , wherein the sorting of microbeads is performed using a fluorescence activated cell sorter (FACS).  
     
     
         89 . A method according to  claim 88 , wherein the change in fluorescence is due to fluorescence resonance energy transfer (FRET).  
     
     
         90 . A method according to  claim 1 , wherein the internal environment of the microcapsules is modified by the addition of one or more reagents to the oil phase.  
     
     
         91 . A method according to  claim 7 , wherein the primary compounds are coupled to beads with different optical properties.  
     
     
         92 . A method according to  claim 91 , wherein the beads have different fluorescent properties.  
     
     
         93 . A method according to  claim 92 , wherein the different optical properties of the beads are due to incorporation of different levels of two or more fluorochromes into the beads.  
     
     
         94 . A method according to  claim 93 , wherein the different optical properties of the beads are due to incorporation of different numbers of quantum dots with different emission spectra.  
     
     
         95 . A method according to  claim 94 , wherein the different optical properties of the beads are used to identify the compound bound to the beads.  
     
     
         96 . A method according to  claim 7 , wherein the compounds on the isolated beads are identified by releasing the compounds from the beads and direct analysis.  
     
     
         97 . A method according to  claim 96 , wherein the compounds on the isolated beads are identified by mass-spectroscopy.  
     
     
         98 . A method according to  claim 1 , further comprising the step of isolating the secondary compound produced by reaction of the primary compounds.  
     
     
         99 . A method according to  claim 1 , further comprising the step of manufacturing one or more secondary compounds.  
     
     
         100 . A method according to  claim 14 , wherein a compound and a target are initially located in different microcapsules which are subsequently fused.  
     
     
         101 . A method according to  claim 1  wherein microcapsules containing different subsets of compounds are fused.  
     
     
         102 . A method according to  claim 1 , wherein a microcapsule containing a compound is split to generate two or more microcapsules.  
     
     
         103 . A method according to  claim 14 , wherein a microcapsule containing a target is split to generate two or more microcapsules.  
     
     
         104 . A method according to  claim 1 , wherein a cell or cells are located in a microcapsule.  
     
     
         105 . A method according to  claim 1 , wherein the compound modulates the activity of a target in the cell(s).  
     
     
         106 . A method according to  claim 1 , wherein the compound modulates an activity of the cell(s).  
     
     
         107 . A method according to  claim 1 , wherein one or both of steps (a) and (b) is performed under electronic microfluidic control.  
     
     
         108 . A method according to  claim 106 , wherein the technique of electronic microfluidic control involves generating an electric charge in at least a proportion of the microcapsules.  
     
     
         109 . A method according to  claim 107 , wherein the step of generating an electric charge in at least a proportion of the microcapsules comprises applying an electric field to at least a proportion of those microcapsules.  
     
     
         110 . A method according to  claim 107 , erein the electric field is generated within a microfluidic channel.  
     
     
         111 . A method according to  claim 109 , wherein the electric field is at least 0.1V/microlitre.  
     
     
         112 . A method according to  claim 111 , wherein the electric field is generated using at least one electrode within the microfluidic channel.