Method for ultra-high performance screening of biological objects

Abstract

The invention relates to the field of biotechnology and pharmaceutics. Proposed is a method for ultra-high performance screening of biological objects which is based on microfluidic generation of droplets of a biocompatible water-in-oil-in-water double emulsion, and also a method for producing a monodisperse biocompatible water-in-oil-in-water double emulsion. The invention can be used in diagnosing conditions and diseases in mammals, as well as for investigating biological objects.

1 - 2 . (canceled) 
     
     
         3 . A method for ultrahigh-throughput screening of a mixture of microbiological objects to isolate microbiological objects having a specific enzymatic or biocatalytic activity, the method comprising:
 generating a double microfluidic water-in-oil-in-water (W/O/W) emulsion comprising monodisperse droplets encapsulating individual microbiological objects or mixtures of said objects in individual droplet microcompartments, which monodisperse droplets have controlled diameters ranging from 20 to 90 µm,   wherein said double water-in-oil-in-water (W/O/W) emulsion is generated by sequential microfluidic emulsification of a water phase comprising mixed streams of the microbiological objects in the mixture and an indicator that changes in fluorescence when activated by an enzyme or biocatalyst in the mixture of said objects that have the specific enzymatic or biocatalytic activity, and an oil phase to produce a water-in-oil emulsion, followed by microfluidic emulsification of water-in-oil emulsion in outer aqueous phase to produce monodisperse double water-in-oil-in-water (W/O/W) emulsion;   optionally, incubating or culturing microbiological objects in the monodisperse droplets;   isolating a population of the monodisperse droplets containing microbiological objects having the enzymatic or biocatalytic activity by an ultrahigh-throughput method comprising fluorescence-activated cell sorting (FACS) as applied to the microbiological objects;   optionally, regenerating the microbiological objects from isolated droplets while maintaining the viability of microbiological objects;   analyzing the genome or metabolome of the microbiological objects having the specific enzymatic or biocatalytic activity.   
     
     
         4 . The method of  claim 3 , wherein the mixture of biological objects comprises a library of phenotypes having specific enzymatic or biocatalytic activities. 
     
     
         5 . The method of  claim 3 , wherein the mixture of biological objects comprises a library of phenotypes having new artificial enzymatic or biocatalytic activities. 
     
     
         6 . The method of  claim 3 , wherein the microbiological objects carry anchored enzymes or biocatalysts on their surface including display libraries. 
     
     
         7 . The method of  claim 3 , wherein the microbiological objects comprise microspheres, ribosomes, phages, bacteria, yeasts, or mammalian cells providing specific enzymes or biocatalysts. 
     
     
         8 . The method of  claim 3 , wherein the microbiological objects are cells or living cells that retain their viability while generating a double microfluidic water-in-oil-in-water (W/O/W) emulsion, incubating in droplets, isolating by an ultrahigh-throughput method, and regenerating from isolated droplets. 
     
     
         9 . The method of  claim 3 , wherein the microbiological objects are cells and wherein the method further comprises isolating and high-throughput sequencing of cells having the specific enzymatic or biocatalytic activity. 
     
     
         10 . The method of  claim 3 , wherein the microbiological objects are cells and wherein the method further comprises isolating and culturing cells having the specific enzymatic or biocatalytic activity. 
     
     
         11 . The method of  claim 3 , wherein the mixture of microbiological objects comprises an enzyme or biocatalyst that is a phosphodiesterase, protease, esterase, or organophosphate hydrolase. 
     
     
         12 . The method of  claim 3 , wherein the microbiological objects comprise objects exhibiting different types or different levels of enzymatic or biocatalytic activities. 
     
     
         13 . The method of  claim 3 , wherein the indicator comprises a fluorogenic/fluorescent compound changing its fluorescence (level or spectrum) by interacting with the microbiological objects having a specific enzymatic or biocatalytic activity. 
     
     
         14 . The method of  claim 3 , wherein the indicator comprises a fluorogenic/fluorescent compound changing its fluorescence (level or spectrum) by interacting with the product of the reaction catalyzed by microbiological objects having a specific enzymatic or biocatalytic activity. 
     
     
         15 . The method of  claim 3 , wherein the indicator comprises a reporter target cell, wherein fluorescence of the reporter target cell changes when it interacts with the microbiological objects having the enzymatic or biocatalytic activity. 
     
     
         16 . The method of  claim 3 , wherein a combination of different fluorescent indicators is used for isolation of microbiological objects with the enzymatic or biocatalytic activity. 
     
     
         17 . The method of  claim 3 , wherein the water-in-oil emulsion is generated in a first hydrophobic chip and the resulting single oil-in-water emulsion is reemulsified in external aqueous phase in a second hydrophilic chip to generate the double water-in-oil-in-water emulsion. 
     
     
         18 . The method of  claim 3 , wherein the chips have channels that range in diameter from 20 to 60 µm that generate a monodisperse double microfluidic emulsion having droplets with a controlled diameter ranging from 20 to 90 µm. 
     
     
         19 . The method of  claim 3 , wherein the ultrahigh-throughput method has a productivity of 1000-20000 events per second.