Compartmentalised screening by microfluidic control

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) compartmentalising the compounds into microcapsules together with the target, such that only a subset of the repertoire is represented in multiple copies in any one microcapsule; and b) identifying the compound which binds to or modulates the activity of the target; wherein at least one step is performed under microfluidic control. The invention enables the screening of large repertoires of molecules which can serve as leads for drug development.

1 . A method for identifying a compound or compounds in a repertoire of compounds, which compound or compound(s) possess(es) a desired activity, comprising the steps of:
 a) compartmentalising the compounds into microcapsules, such that only a subset of the repertoire is represented in multiple copies in any one microcapsule; and   b) identifying the compounds which possess the desired activity,   wherein one or both of steps a) and b) is performed under microfluidic control.   
     
     
         2 . A method according to  claim 1 , wherein step (a) comprises forming groups of microcapsules comprising individual compounds and mixing the groups of microcapsules to form an emulsified compound repertoire wherein a subset of the repertoire is represented in multiple copies in any one microcapsule. 
     
     
         3 . A method according to  claim 1 , wherein step (a) comprises
 i. attaching the repertoire of compounds onto microbeads, such that only a subset of the repertoire is represented on any one microbead; and   ii. compartmentalising the microbeads into microcapsules;   
       such that a subset of the repertoire is represented in multiple copies in any one microcapsule; and wherein wherein one or both of steps i) and ii) is performed under microfluidic control. 
     
     
         4 . A method according to  claim 1 , wherein the desired activity is selected from the group consisting of a binding activity and the modulation of the activity of a target. 
     
     
         5 . A method according to  claim 4 , wherein the binding activity is binding to a target. 
     
     
         6 . A method according to  claim 4 , wherein the modulated activity is a binding activity. 
     
     
         7 . A method according to  claim 4 , wherein the modulated activity is a catalytic activity. 
     
     
         8 . A method according to  claim 1 , wherein the compound reacts with a target to generate a reaction product. 
     
     
         9 . A method according to  claim 3 , wherein the compounds are coupled to the microbeads via a non-cleavable linker. 
     
     
         10 . A method according to  claim 3 , wherein the compounds are coupled to the microbeads via a cleavable linker. 
     
     
         11 . A method according to  claim 10 , wherein the compounds are coupled to the microbeads via a photochemically cleavable linker. 
     
     
         12 . A method according to  claim 3 , wherein the compounds are coupled to the microbeads by a combinations of a cleavable linker and a non-cleavable linker. 
     
     
         13 . A method according to  claim 4 , wherein the target is compartmentalised into microcapsules together with the compound(s). 
     
     
         14 . A method according to  claim 1 , wherein the compound having a desired activity causes a change in the microbead or microcapsule with which it is associated which allows the microbead or microcapsule to be identified, sorted or selected. 
     
     
         15 . A method according to  claim 14 , wherein the compound is bound to a microbead and the desired activity within the microcapsule results, directly or indirectly, in the modification of the microbead to which the compound is attached to enable the isolation of the microbead. 
     
     
         16 . A method according to  claim 15 , wherein the target is bound to the microbead and the compound having the desired activity within the microcapsule binds, directly or indirectly, to the target to enable the isolation of the microbead. 
     
     
         17 . A method according to  claim 15  or  claim 16 , wherein the microbeads are isolated by affinity purification. 
     
     
         18 . A method according to  claim 7 , 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. 
     
     
         19 . A method according to  claim 18 , wherein the substrate is converted into the product by the catalytic action of the target. 
     
     
         20 . A method according to  claim 18  or  19 , wherein the substrate and the product have different optical properties. 
     
     
         21 . A method according to  claim 20 , wherein the the substrate and the product have different fluorescence properties. 
     
     
         22 . A method according to  claim 18 , wherein the substrate is bound to the microbead, and the desired activity of the 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. 
     
     
         23 . A method according to  claim 18 , wherein the product and, optionally, the unreacted substrate are subsequently complexed with the microbead in the microcapsule. 
     
     
         24 . A method according to  claim 18 , wherein the desired activity enhances the conversion of substrate into product. 
     
     
         25 . A method according to  claim 18 , wherein the desired activity inhibits the conversion of substrate into product. 
     
     
         26 . A method according to  claim 7 , wherein the desired activity of the compound within the microcapsule results, directly or indirectly, in the generation of a product which is subsequently complexed with the microbead and enables its isolation. 
     
     
         27 . A method according to  claim 1 , wherein the compound having a desired activity causes a change in the microcapsule which allows the microcapsule to be identified, sorted or selected. 
     
     
         28 . A method according to  claim 1 , wherein the desired activity of the compound within the microcapsule induces a change in the optical properties of the microcapsule. 
     
     
         29 . A method according to  claim 28 , wherein the microcapsules are identified by detection of a change in their fluorescence. 
     
     
         30 . A method according to  claim 29 , wherein the change in fluorescence is due to fluorescence resonance energy transfer (FRET). 
     
     
         31 . A method according to  claim 28 , wherein the compound in a microcapsule is identified by a difference in the optical properties of the microcapsule. 
     
     
         32 . A method according to  claim 31 , wherein the difference in the optical properties of the microcapsule is a difference in fluorescence. 
     
     
         33 . A method according to  claim 32 , wherein the difference in the fluorescence of the microcapsule is due to the presence of quantum dots. 
     
     
         34 . A method according to  claim 1 , wherein the compounds are present in different microcapsules at different concentrations. 
     
     
         35 . A method according to  claim 34 , wherein the concentration of the compound in a microcapsule is identified by a difference in the optical properties of the microcapsule. 
     
     
         36 . A method according to  claim 35 , wherein the difference in the optical properties of the microcapsule is a difference in fluorescence. 
     
     
         37 . A method according to  claim 36 , wherein the difference in the fluorescence of the microcapsule is due to the presence of quantum dots. 
     
     
         38 . A method according to  claim 28 , wherein the target in a microcapsule is identified by a difference in the optical properties of the microcapsule. 
     
     
         39 . A method according to  claim 38 , wherein the difference in the optical properties of the microcapsule is a difference in fluorescence. 
     
     
         40 . A method according to  claim 39 , wherein the difference in the fluorescence of the microcapsule is due to the presence of quantum dots. 
     
     
         41 . A method according to  claim 15 ; wherein the modification of the microbead comprises a change in its optical properties. 
     
     
         42 . A method according to  claim 41 , wherein the difference in the optical properties of the microbead is a difference in fluorescence. 
     
     
         43 . A method according to  claim 41 , wherein the compound on a microbead is identified by a difference in the optical properties of the microbead. 
     
     
         44 . A method according to  claim 43 , wherein the difference in the optical properties of the microbead is a difference in fluorescence. 
     
     
         45 . A method according to  claim 44 , wherein the difference in the fluorescence of the microbead is due to the presence of quantum dots. 
     
     
         46 . A method according to  claim 3 , wherein the microbead is nonmagnetic, magnetic or paramagnetic. 
     
     
         47 . A method according to  claim 42 , wherein the microbeads are sorted by detection of a change in their fluorescence. 
     
     
         48 . A method according to  claim 47 , wherein the identification of microbeads is by flow cytometry. 
     
     
         49 . A method according to  claim 47 , wherein the sorting of microbeads is performed using a fluorescence activated cell sorter (FACS). 
     
     
         50 . A method according to  claim 47 , wherein the change in fluorescence is due to fluorescence resonance energy transfer (FRET). 
     
     
         51 . A method according to  claim 42 , 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. 
     
     
         52 . A method according to  claim 42 , wherein the change in optical properties of the microbead is due to binding of a compound with distinctive optical properties to the microbead. 
     
     
         53 . A method according to  claim 42  or  claim 43 , wherein the change in optical properties of the microbead is due to binding of a target with distinctive optical properties by the compound. 
     
     
         54 . A method according to  claim 42  or  claim 43 , 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. 
     
     
         55 . A method according to  claim 42  or  claim 43 , 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. 
     
     
         56 . A method according to  claim 42  or  claim 43 , 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. 
     
     
         57 . A method according to  claim 42  or  claim 43 , wherein the compound with the desired activity 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. 
     
     
         58 . A method according to  claim 42  or  claim 43 , wherein the compound with the desired activity 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. 
     
     
         59 . A method according to  claim 42  or  claim 43 , 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. 
     
     
         60 . A method according to  claim 42  or  claim 43 , 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. 
     
     
         61 . A method according to  claim 1 , wherein a non-desired activity of a compound results in a change in the microbead or the microcapsule which is distinct from that resulting from the desired activity. 
     
     
         62 . A method according to  claim 61 , wherein the change resulting from the non-desired activity is used to negatively select the microcapsules or the microbeads. 
     
     
         63 . A method according to  claim 62 , wherein negative selection is combined with positive selection to improve reaction specificity. 
     
     
         64 . A method according to  claim 63 , wherein the improved reaction specificity is an improvement in binding specificity. 
     
     
         65 . A method according to  claim 64 , wherein the improved reaction specificity is an improvement in regio- and/or stereo-selectivity for the target. 
     
     
         66 . A method according to  claim 42 , wherein microbeads modified directly or indirectly by the activity of the desired 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. 
     
     
         67 . A method according to  claim 1 , wherein each microbead or microcapsule comprises two or more compounds and each compound must have a desired activity in order for the microbead or the microcapsule to be selected. 
     
     
         68 . A method according to  claim 1 , wherein the compound or compounds are low molecular weight compounds. 
     
     
         69 . A method according to  claim 1 , wherein the subset of the repertoire is a single compound. 
     
     
         70 . A method according to  claim 69 , wherein each microcapsule or microbead has associated therewith multiple molecules of a single compound. 
     
     
         71 . A method according to  claim 1  further comprising iteratively repeating one or more of steps (a) to (b). 
     
     
         72 . A method according to  claim 1 , wherein microencapsulation is achieved by forming a water-in-oil emulsion. 
     
     
         73 . A method according to  claim 72 , wherein the water-in-oil emulsion is made using a microfluidic system. 
     
     
         74 . A method according to  claim 73 , wherein the emulsion is formed by aqueous droplet break off in a co-flowing steam of an immiscible liquid. 
     
     
         75 . A method according to  claim 73 , wherein the emulsion is formed by subjecting a first fluid surrounded by a second fluid to an electric charge. 
     
     
         76 . A method according to  claim 1 , wherein microcapsules are transported by laminar-flow in a stream of fluid in microfluidic channels. 
     
     
         77 . A method according to  claim 76  wherein the microcapsules are aqueous microdroplets dispersed in a stream of oil in microfluidic channels. 
     
     
         78 . A method according to  claim 76  wherein the compound in a microcapsule is identified by the relative position of of the microcapsule compared to other microdroplets in a microfluidic channel. 
     
     
         79 . A method according to  claim 76  wherein the concentration of a compound in a microcapsule is identified by the relative position of of the microcapsule compared to other microdroplets in a microfluidic channel. 
     
     
         80 . A method according to  claim 76  wherein the target in a microcapsule is identified by the relative position of of the microcapsule compared to other microdroplets in a microfluidic channel. 
     
     
         81 . A method according to  claim 1  wherein the identified microcapsules are sorted using a microfluidic device. 
     
     
         82 . A method according to  claim 81 , wherein the sorting of identified microcapsules is achieved using a microfluidic flow sorting device by steering charged microcapsules using an electric field. 
     
     
         83 . A method according to  claim 82 , wherein the microfluidic device is equipped with a sensor which detects a signal emitted by the microcapsules in the microfluidic channels. 
     
     
         84 . A method according to  claim 1 , wherein microcapsules are fused or split using a microfluidic device. 
     
     
         85 . A method according to  claim 84 , wherein a microcapsule is split by application of an electric field. 
     
     
         86 . A method according to  claim 84 , wherein first and second microcapsules are fused by application of opposite electric charges thereto. 
     
     
         87 . A method according to  claim 84 , wherein microcapsules are fused by the induction of dipoles therein which cause the microcapsules to coalesce. 
     
     
         88 . A method according to  claim 72 , wherein microencapsulation is achieved by forming a water-in-fluorocarbon or water-in-perfluorocarbon emulsion. 
     
     
         89 . A method according to  claim 88 , wherein the fluorocarbon is perfluorooctyl bromide or perfluorooctylethane. 
     
     
         90 . A method according to  claim 88 , wherein the emulsion is formed using F-alkyl dimorpholinophosphate(s). 
     
     
         91 . A method according to  claim 90 , wherein the F-alkyl dimorpholinophosphate(s) have the general formula C—F 2n+1 C m H 2m OP(O)[N(CH 2 CH 2 ) 2 O] 2 . 
     
     
         92 . A method according to  claim 91  wherein the F-alkyl dimorpholinophosphate is C 8 F 17 C 11 H 22 OP(O)[N(CH 2  CH 2 ) 2 O] 2 . 
     
     
         93 . 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. 
     
     
         94 . A method according to  claim 3 , wherein the compounds are coupled to beads with different optical properties. 
     
     
         95 . A method according to  claim 94 , wherein the beads have different fluorescent properties. 
     
     
         96 . A method according to  claim 94 , wherein the different optical properties of the beads are due to incorporation of different levels of two or more fluorochromes into the beads. 
     
     
         97 . A method according to  claim 94 , wherein the different optical properties of the beads are due to incorporation of different numbers of quantum dots with different emission spectra. 
     
     
         98 . A method according to  claim 94 , wherein the different optical properties of the beads are used to identify the compound bound to the beads. 
     
     
         99 . A method according to  claim 15  or  16 , wherein the compounds on the isolated beads are identified by releasing the compounds from the beads and direct analysis. 
     
     
         100 . A method according to  claim 99 , wherein the compounds on the isolated beads are identified by mass-spectroscopy. 
     
     
         101 . A method for preparing a compound, comprising the steps of:
 (a) compartmentalising the compounds into microcapsules, such that only a subset of the repertoire is represented in multiple copies in any one microcapsule;   (b) identifying the compounds which possess the desired activity.   (c) identifying and producing the compound having the desired activity,   wherein one or more of steps a, b and c is performed under microfluidic control.   
     
     
         102 . A method for preparing a compound, comprising the steps of:
 (a) compartmentalising the compounds into microcapsules, such that only a subset of the repertoire is represented in multiple copies in any one microcapsule;   (b) identifying and, optionally, sorting the microcapsules which have contain the compound(s) having the desired activity using a change in their optical properties; and   (c) identifying and producing the compound having the desired activity.   
     
     
         103 . A method for screening a compound or compounds capable of modulating the activity of a target, comprising the steps of:
 (a) compartmentalising the compounds into microcapsules, such that only a subset of the repertoire is represented in multiple copies in any one microcapsule;   (b) identifying and, optionally, sorting the microcapsules which contain the compound(s) having the desired activity using a change in their optical properties; and   (c) contacting a target having a desired activity with the compound or compounds and monitoring the modulation of an activity of the target by the compound or compounds,   
       wherein one or more of steps a, b and c is performed under microfluidic control. 
     
     
         104 . A method for preparing a target comprising the steps of:
 (a) providing a synthesis protocol for a target wherein at least one step is facilitated by a compound;   (b) preparing variants of the compound which facilitates this step;   (c) compartmentalising the compounds into microcapsules, such that only a subset of the repertoire is represented in multiple copies in any one microcapsule;   (d) identifying and, optionally, sorting the microcapsules which contain the compound(s) having the desired activity using a change in their optical properties; and   (e) preparing the target using the compound identified in (d) to facilitate the relevant step of the synthesis,   
       wherein one or more of steps a to e is performed under microfluidic control. 
     
     
         105 . A method according to any one of  claims 102  to  105 , wherein the compounds are attached to microbeads, and the compounds are optionally released from the microbeads inside the microcapsules. 
     
     
         106 . A method according to  claim 13 , wherein a compound and a target are initially located in different microcapsules which are subsequently fused. 
     
     
         107 . A method according to  claim 13 , wherein a microcapsule containing a compound and a target is fused with a microcapsule containing a molecule or molecules which are required to detect the desired activity of the compound on the target. 
     
     
         108 . A method according to  claim 13  or  106 , wherein the microcapsule resulting from the fusion of a microcapsule containing a compound is fused with a microcapsule containing a target, is later fused with a microcapsule containing a molecule or molecules which are required to detect the desired activity of the compound(s) on the target(s). 
     
     
         109 . A method according to  claim 107 , wherein the molecule or molecules which are required to detect the desired activity of the compound on the target is an enzyme substrate. 
     
     
         110 . A method according to  claim 109 , wherein the substrate is a substrate for the target. 
     
     
         111 . A method according to  claim 1  wherein a microcapsule containing a compound is split to generate two or more microcapsules. 
     
     
         112 . A method according to  claim 13  wherein a microcapsule containing a target is split to generate two or more microcapsules. 
     
     
         113 . A method according to  claim 1  wherein a cell or cells are located in a microcapsule. 
     
     
         114 . A method according to  claim 113  wherein the compound modulates the activity of a target in the cell(s). 
     
     
         115 . A method according to  claim 114  wherein the compound modulates an activity of the cell(s).