Intra-droplet surface engineering to capture a molecular target

Abstract

The present invention relates to a method for capturing a molecular target present in the aqueous phase of a water-in-oil emulsion, said method being based on the use of a binding system comprising (a) a surfactant bearing a functional moiety on its hydrophilic head group and (b) a chemoprobe that acts as a molecular staple between the functionalized surfactant and the molecular target and comprises at least two distinct domains namely (i) at least one capture moiety which is able to specifically bind the molecular target and (ii) at least one binding domain which is able to interact with the functional group of the surfactant through covalent or non-covalent interactions, directly or through a binding intermediary.

We claim: 
     
         1 . A functionalized surfactant comprising or consisting of a moiety of formula (Ib):    
       wherein:
 a and b are integers independently selected from 1 to 5, 
 each LIPO is a lipophilic tail independently selected from the group consisting of a saturated or unsaturated hydrocarbon chain, optionally interrupted by one or several heteroatoms and optionally substituted by one or several groups selected from C 1 -C 3  alkyl groups, halogens, —OH, —OMe, and —CF 3 , a perfluoropolyether chain, a perfluorocarbon chain and combinations thereof, 
 HYDRO is a hydrophilic head comprising a moiety independently selected from a dimorpholinophosphate group, a polyether, a polyetheramine, a polyglycerol, and combinations thereof, 
 each FUNCT is a functional moiety comprising a group selected from a biotin, a streptavidin, an avidin and a chemical reactive group able to create a covalent bond by click-chemistry or by bioconjugation reaction, and 
 LIPO and HYDRO on one hand and HYDRO and FUNCT on the other hand are directly linked, or linked via a connector group or a linker. 
 
     
     
         2 . The functionalized surfactant of  claim 1 , wherein each FUNCT comprising a group selected from an azido group and an alkynyl group. 
     
     
         3 . The functionalized surfactant of  claim 1 , wherein:
 each LIPO comprises, or consists in, the following moiety (L 1 ):   
       
         
           
           
               
               
           
         
         wherein n is an integer from 25 to 45, and 
         HYDRO comprises one or several polyethylene glycol chains comprising from 2 to 30. 
       
     
     
         4 . The functionalized surfactant of  claim 1 , which comprises
 one or two LIPO comprising, or consisting of the moiety of formula (L 1 ):   
       
         
           
           
               
               
           
         
       
       wherein n is an integer from 25 to 45 and
 a HYDRO bearing at least one moiety of formula (H 2 ) 
 
       
         
           
           
               
               
           
         
         wherein d is an integer from 1 to 12. 
       
     
     
         5 . The functionalized surfactant of  claim 4 , which has one of the following formula: 
       
         
           
           
               
               
           
         
       
       wherein e is an integer from 1 to 12 and X 1  and Y 1  are independently selected from NH, CH 2  and O; 
       
         
           
           
               
               
           
         
       
       wherein Y 2  and Y 3  are independently selected from NH, CH 2  or O and X 2 , X 3  and X 4  are independently selected from CH and N, and 
       
         
           
           
               
               
           
         
       
       wherein
 X 2 , X 3  and X 4  are independently selected from CH and N, 
 Y 8  is CH or N, 
 Y 4 , Y 5 , Y 6  and Y 7  are independently selected from NH, O and CH 2  and, 
 e1 and e2 are integers independently selected from 1 to 12, 
 wherein L 1  and H 2  being 
 
       
         
           
           
               
               
           
         
       
       wherein n is an integer from 25 to 45 and
 and H 2  being 
 
       
         
           
           
               
               
           
         
         wherein d is an integer from 1 to 12. 
       
     
     
         6 . The functionalized surfactant of  claim 4 , which is selected from the group consisting of: 
       
         
           
           
               
               
           
         
         wherein n is an integer from 25 to 45 and d is an integer from 1 to 12. 
       
     
     
         7 . The functionalized surfactant of  claim 1  which is of formula (Ic):  
 wherein and a is 1, 2 or 3. 
 
     
     
         8 . The functionalized surfactant of  claim 7 , which is a diblock surfactant of formula (II): 
       
         
           
           
               
               
           
         
       
       wherein
 n is an integer from 25 to 45, 
 p and q are integers independently selected from 1 to 10, 
 Y 1  and Y 2  are selected from the group consisting of —O—, —NH 2 , —C(═O)—, OC(═O), (C═O)O, NHC(═O), C(═O)NH, NHC(═O)NH, NHC(═O)O, and OC(═O)NH, and 
 FUNCT comprises a moiety selected from a biotin, an azido group, and an alkynyl group, or a strained alkynyl group. 
 
     
     
         9 . The functionalized surfactant of  claim 8 , which is selected from the group consisting of: 
       
         
           
           
               
               
           
         
         (i) with n=35-40, p=5 and q=4, 
       
       
         
           
           
               
               
           
         
         (ii) with n=35-40, p=q=5 
       
       
         
           
           
               
               
           
         
         (iii) with n=35-40, p=q=5, and 
       
       
         
           
           
               
               
           
         
         (iv) with n=35-40, p=5, q=4 
       
     
     
         10 . The functionalized surfactant of  claim 7  which is of formula (IIIa): 
       
         
           
           
               
               
           
         
       
       (IIIa) 
       wherein
 p and q are integers independently selected from 1 to 10, 
 Krytox is 
 
       
         
           
           
               
               
           
         
       
       with n is from 25 to 45,
 FUNCT comprises a moiety selected from a biotin, an azido group, an alkynyl group, or a strained alkynyl group. 
 
     
     
         11 . The functionalized surfactant of  claim 10 , which is selected from the group consisting of: 
       
         
           
           
               
               
           
         
         (i) with n=35-40 and p=6, 5, or 4, and 
       
       
         
           
           
               
               
           
         
         (ii) with n=35-40 and p=6, 5, or 4. 
       
     
     
         12 . A kit to be used in a method for capturing a molecular target which comprises
 (i) a functionalized surfactant comprising or consisting of a moiety of formula (Ib):      
       wherein:
 a and b are integers independently selected from 1 to 5, 
 each LIPO is a lipophilic tail independently selected from the group consisting of a saturated or unsaturated hydrocarbon chain, optionally interrupted by one or several heteroatoms and optionally substituted by one or several groups selected from C 1 -C 3  alkyl groups, halogens, —OH, —OMe, and —CF 3 , a perfluoropolyether chain, a perfluorocarbon chain and combinations thereof, 
 HYDRO is a hydrophilic head comprising a moiety independently selected from a dimorpholinophosphate group, a polyether, a polyetheramine, a polyglycerol, and combinations thereof, 
 each FUNCT is a functional moiety comprising a group selected from a biotin, a streptavidin, an avidin and a chemical reactive group able to create a covalent bond by click-chemistry or by bioconjugation reaction, and 
 LIPO and HYDRO on one hand and HYDRO and FUNCT on the other hand are directly linked, or linked via a connector group or a linker; and 
 (ii) a chemoprobe comprising at least (i) one capture moiety capable of specific binding to a molecular target and at least (ii) one binding domain capable of direct or indirect binding to the functionalized surfactant. 
 
     
     
         13 . The kit of  claim 12 , wherein the capture moiety of the chemoprobe is selected from the group consisting of an antibody, a spiegelmer, a peptide aptamer, an aptamer, a ligand or a substrate of the molecular target, a nucleic acid capable of hybridizing the molecular target, and a receptor fragment able to bind the molecular target. 
     
     
         14 . The kit of  claim 12 , wherein:
 the functionalized hydrophilic head of the surfactant comprises an azido group and the binding domain of the chemoprobe comprises a strained cycloalkynyl group, or vice-versa, or   the functionalized hydrophilic head of the surfactant comprises an avidin or a streptavidin group and the binding domain of the chemoprobe comprises a biotin, or vice-versa.   
     
     
         15 . The kit of  claim 12 , further comprising:
 a binding intermediate which is able to bind both the chemoprobe and the functionalized surfactant thereby acting as a bridge between said two entities and/or   non-functionalized surfactant(s); and/or   an aqueous phase; and/or   an oil phase; and/or   a leaflet providing guidelines to use said kit, and/or   a microfluidic chip comprising:
 a module for generating water-in-oil emulsion droplets or re-injecting water-in-oil emulsion, 
 a module for creating double emulsion in fluid communication and downstream of the module for generating water-in-oil emulsion droplets or re-injecting water-in-oil emulsion, and 
 a phase inversion module in fluid communication and downstream of the module for creating double emulsion. 
   
     
     
         16 . A method for capturing a molecular target, said method comprising:
 a) providing water-in-oil emulsion droplets comprising a functionalized surfactant at the interface of droplets, said functionalized surfactant comprising or consisting of a moiety of formula (Ib):      
       wherein:
 a and b are integers independently selected from 1 to 5, 
 each LIPO is a lipophilic tail independently selected from the group consisting of a saturated or unsaturated hydrocarbon chain, optionally interrupted by one or several heteroatoms and optionally substituted by one or several groups selected from C 1 -C 3  alkyl groups, halogens, —OH, —OMe, and —CF 3 , a perfluoropolyether chain, a perfluorocarbon chain and combinations thereof, 
 HYDRO is a hydrophilic head comprising a moiety independently selected from a dimorpholinophosphate group, a polyether, a polyetheramine, a polyglycerol, and combinations thereof, 
 each FUNCT is a functional moiety comprising a group selected from a biotin, a streptavidin, an avidin and a chemical reactive group able to create a covalent bond by click-chemistry or by bioconjugation reaction, and
 LIPO and HYDRO on one hand and HYDRO and FUNCT on the other hand are directly linked, or linked via a connector group or a linker; 
 
 b) contacting said functionalized surfactant with a chemoprobe present or added in the aqueous phase of the droplets, said chemoprobe comprising at least (i) one capture moiety capable of specific binding to a molecular target and at least (ii) one binding domain capable of direct or indirect binding to the functionalized hydrophilic head of the functionalized surfactant; and 
 c) contacting said functionalized surfactant with the molecular target present or added in the aqueous phase of the droplets, 
 thereby capturing the molecular target at the inner interface of the emulsion droplets through (i) the direct or indirect binding of the chemoprobe to the functionalized surfactant and (ii) the specific binding of the chemoprobe to the molecular target, and 
 wherein steps b) and c) are performed simultaneously or sequentially, in any order. 
 
     
     
         17 . The method of  claim 16 , which is implemented using a microfluidic system. 
     
     
         18 . The method of  claim 16 , which further comprises inverting the phase of water-in-oil emulsion droplets thereby producing oil-in-water emulsion droplets and exposing the captured molecular target at the outer surface of the emulsion droplets. 
     
     
         19 . The method of  claim 16 , which further comprises a step of recovering, detecting, and/or quantifying the captured molecular target. 
     
     
         20 . The method of  claim 16 , wherein the molecular target is a protein or a nucleic acid.