Systems and methods for particle multiplexing in droplets

Abstract

Described herein are systems and methods for multiplexed analysis of two or more targets in a test sample including a first set of particles including a first set of target-specific reagents and a first optically detectable identifier capable of emitting a first wavelength indicative of a first target, and at least one second set of particles including a second set of target-specific reagents and a second optically detectable identifier capable of emitting a second wavelength indicative of a second target; and at least one optically detectable reporter probe capable of constitutively emitting a third wavelength in response to reaction of the first set of target-specific reagents with the first target in the test sample and/or reaction of the second set of target-specific reagents with the second target in the test sample, wherein the first wavelength, the second wavelength, and the third wavelength are optically discernable from one another.

1 - 167 . (canceled) 
     
     
         168 . A method for multiplexed analysis of two or more targets in a test sample comprising:
 combining in an aqueous medium the test sample, one or more reaction reagents, a first set of particles, at least one second set of particles, and at least one optically detectable reporter probe, wherein each particle of the first set of particles is degradable in response to a first environmental condition and has associated therewith a first set of one or more target-specific reagents specific for a first target and a first optically detectable identifier capable of emitting a first wavelength indicative of the first set of one or more target-specific reagents, wherein each particle of the at least one second set of particles is degradable in response to a second environmental condition and has associated therewith a second set of one or more target-specific reagents specific for a second target and a second optically detectable identifier emitting a second wavelength indicative of the second set of one or more target-specific reagents, and wherein the at least one optically detectable reporter probe constitutively emits a third wavelength in response to reaction of the first set of one or more target-specific reagents with the first target in the test sample and/or to reaction of the second set of one or more target-specific reagents with the second target in the test sample;   forming a plurality of reaction droplets by adding an immiscible carrier fluid to the aqueous medium;   performing a reaction with the plurality of formed reaction droplets;   interrogating at least a portion of the plurality of formed reaction droplets for emission of the first wavelength indicative of the first set of one or more target-specific reagents, the second wavelength indicative of the second set of one or more target-specific reagents, and the third wavelength indicative of constitutive emission from the at least one optically detectable reporter probe;   reporting a number of the formed reaction droplets emitting both the first wavelength and the third wavelength; and   reporting a number of the formed reaction droplets emitting both the second wavelength and the third wavelength.   
     
     
         169 . A method for multiplexed analysis of two or more nucleic acid sequence targets in a test sample comprising:
 combining in an aqueous medium the test sample, one or more reaction reagents, a first set of particles, at least one second set of particles, and at least one optically detectable reporter probe, wherein each particle of the first set of particles is degradable in response to a first environmental condition and has associated therewith a first amplification primer set selected to interact with a first nucleic acid sequence and a first optically detectable identifier capable of emitting a first wavelength indicative of the first amplification primer set, wherein each particle of the at least one second set of particles is degradable in response to a second environmental condition and has associated therewith a second amplification primer set selected to interact with a second nucleic acid sequence and a second optically detectable identifier emitting a second wavelength indicative of the second amplification primer set, and wherein the at least one optically detectable reporter probe is capable of constitutively emitting a third wavelength in response to amplification of the first nucleic acid sequence and/or the second nucleic acid sequence;   forming a plurality of reaction droplets by adding an immiscible carrier fluid to the aqueous medium;   performing an amplification reaction with the plurality of formed reaction droplets;   interrogating at least a portion of the plurality of formed reaction droplets for emission of the first wavelength indicative of the first amplification primer set, the second wavelength indicative of the second amplification primer set, and the third wavelength indicative of constitutive emission from the at least one optically detectable reporter probe in response to amplification of the first and/or the second nucleic acid sequence in the formed reaction droplets;   reporting a number of the formed reaction droplets emitting both the first wavelength and the third wavelength; and   reporting a number of the formed reaction droplets emitting both the second wavelength and the third wavelength.   
     
     
         170 . A method for multiplexed analysis of antibiotic resistance in a bacterial sample, comprising:
 combining in an aqueous medium the bacterial sample, one or more reaction reagents, a first set of particles, at least one second set of particles, and at least one optically detectable reporter probe, wherein each particle of the first set of particles is degradable in response to a first environmental condition and has associated therewith a first antibiotic having at least one of bactericidal or bacteriostatic activity against a first subset of bacteria and a first optically detectable identifier capable of emitting a first wavelength indicative of the first antibiotic, wherein each particle of the at least one second set of particles is degradable in response to a second environmental condition and has associated therewith a second antibiotic having at least one of bactericidal or bacteriostatic activity against a second subset of bacteria and a second optically detectable identifier capable of emitting a second wavelength indicative of the second antibiotic, and wherein the at least one optically detectable reporter probe is capable of constitutively emitting a third wavelength in response to viability of bacteria in the bacterial sample;   forming a plurality of reaction droplets by adding an immiscible carrier fluid to the aqueous medium;   performing a reaction with the plurality of formed reaction droplets;   interrogating at least a portion of the plurality of formed reaction droplets for emission of the first wavelength indicative of the first antibiotic, the second wavelength indicative of the second antibiotic, and the third wavelength indicative of constitutive emission from the at least one optically detectable reporter probe in response to viability of bacteria in the bacterial sample;   reporting a number of the formed reaction droplets emitting both the first wavelength and the third wavelength; and   reporting a number of the formed reaction droplets emitting both the second wavelength and the third wavelength.   
     
     
         171 . A method for multiplexed analysis of two or more cell types in a test sample, comprising:
 combining in an aqueous medium the test sample, one or more reaction reagents, a first set of particles, at least one second set of particles, and at least one optically detectable reporter probe, wherein each particle of the first set of particles is degradable in response to a first environmental condition and has associated therewith a first antibody set and a first optically detectable identifier capable of emitting a first wavelength, the first antibody set including two or more antibodies specific for proximal targets on a first antigen, the two or more antibodies of the first antibody set including modifications capable of interacting in an antibody-based proximity assay, and the first optically detectable identifier indicative of the first antibody set, wherein each particle of the second set of particles is degradable in response to a second environmental condition and has associated therewith a second antibody set and a second optically detectable identifier capable of emitting a second wavelength, the second antibody set including two or more antibodies specific for proximal targets on a second antigen, the two or more antibodies of the second antibody set including modifications capable of interacting in an antibody-based proximity assay, and the second optically detectable identifier indicative of the second antibody set, and wherein the at least one optically detectable reporter probe is capable of constitutively emitting a third wavelength in response to the two or more antibodies of the first antibody set binding to their proximal targets on the first antigen and/or the two or more antibodies of the second antibody set binding to their proximal targets on the second antigen.   forming a plurality of reaction droplets by adding an immiscible carrier fluid to the aqueous medium;   performing a reaction with the plurality of formed reaction droplets;   interrogating at least a portion of the plurality of formed reaction droplets for emission of the first wavelength indicative of the first antibody set, the second wavelength indicative of the second antibody set, and the third wavelength indicative of constitutive emission from the at least one optically detectable reporter probe in response to the two or more antibodies of the first antibody set binding to the first antigen and/or the two or more antibodies of the second antibody set binding to the second antigen;   reporting a number of the formed reaction droplets emitting both the first wavelength and the third wavelength; and   reporting a number of the formed reaction droplets emitting both the second wavelength and the third wavelength.   
     
     
         172 . The method of  claim 168 , wherein the particles of the first set of particles and of the at least one second set of particles include a structure at least partially formed from a hydrophilic material and degradable in response to at least a first temperature condition or a second temperature condition, wherein at least a portion of the particles of the first set of particles and of the at least one second set of particles are distributable into an aqueous portion of an aqueous-in-oil droplet. 
     
     
         173 . The method of  claim 168 , wherein the first wavelength emitted by the first optically detectable identifier is a first detectable color, the second wavelength emitted by the second optically detectable identifier is a second detectable color, and the third wavelength constitutively emitted by the at least one optically detectable reporter probe is a third detectable color; and wherein the first wavelength, the second wavelength, and the third wavelength are optically discernable from one another. 
     
     
         174 . The method of  claim 168 , wherein the first optically detectable identifier includes a first fluorophore capable of emitting fluorescence at the first wavelength and the second optically detectable identifier includes a second fluorophore capable of emitting fluorescence at the second wavelength; and wherein the at least one optically detectable reporter probe comprises a fluorescent DNA intercalating agent capable of constitutively emitting the third wavelength. 
     
     
         175 . The method of  claim 169 , wherein the first amplification primer set and the second amplification primer set are selected to specifically interact with at least one of single stranded DNA sequences, double stranded DNA sequences, or RNA sequences. 
     
     
         176 . The method of  claim 169 , wherein the particles of the first set of particles and of the at least one second set of particles include a structure at least partially formed from a hydrophilic material and degradable in response to at least a first temperature condition or a second temperature condition, wherein at least a portion of the particles of the first set of particles and of the at least one second set of particles are distributable into an aqueous portion of an aqueous-in-oil droplet. 
     
     
         177 . The method of  claim 169 , wherein the first wavelength emitted by the first optically detectable identifier is a first detectable color, the second wavelength emitted by the second optically detectable identifier is a second detectable color, and the third wavelength constitutively emitted by the at least one optically detectable reporter probe is a third detectable color; and wherein the first wavelength, the second wavelength, and the third wavelength are optically discernable from one another. 
     
     
         178 . The method of  claim 169 , wherein the first optically detectable identifier includes a first fluorophore capable of emitting fluorescence at the first wavelength and the second optically detectable identifier includes a second fluorophore capable of emitting fluorescence at the second wavelength; and wherein the at least one optically detectable reporter probe comprises a fluorescent DNA intercalating agent capable of constitutively emitting the third wavelength. 
     
     
         179 . The method of  claim 170 , wherein the first subset of bacteria or the second subset of bacteria includes one or more strains of  Escherichia coli.    
     
     
         180 . The method of  claim 170 , wherein the first subset of bacteria or the second subset of bacteria includes  Mycobacterium tuberculosis.    
     
     
         181 . The method of  claim 170 , wherein the first subset of bacteria and the second subset of bacteria are identical subsets of bacteria. 
     
     
         182 . The method of  claim 170 , wherein the first subset of bacteria or the second subset of bacteria includes one or more of methicillin resistant  Staphylococcus aureus,  methicillin susceptible  Staphylococcus aureus, Escherichia coli, Streptococcus pneumonia, Pseudomonas aeruginosa, Staphylococcus epidermidis, Salmonella enterica, Klebsiella pneumonia, Streptococcus pyogenes, Acinetobacter baumannii,  or  Enterococcus faecalis.    
     
     
         183 . The method of  claim 170 , wherein the first subset of bacteria or the second subset of bacteria includes one or more of  Enterococcus faecium, Proteus mirabilis, Streptococcus agalactiae, Staphylococcus saprophyticus, Corynebacterium urealyticum, Serratia marcescens, Klebsiella oxytoca,  or  Actinobaculum schaalii.    
     
     
         184 . The method of  claim 171 , wherein at least the first antibody set or the second antibody set comprises at least one polyclonal antibody. 
     
     
         185 . The method of  claim 171 , wherein at least the first antibody set or the second antibody set comprises two or more monoclonal antibodies. 
     
     
         186 . The method of  claim 171 , wherein the two or more antibodies of the first antibody set and the two or more antibodies of the second antibody set include oligonucleotide modifications. 
     
     
         187 . The method of  claim 171 , wherein the first wavelength emitted by the first optically detectable identifier is a first detectable color, the second wavelength emitted by the second optically detectable identifier is a second detectable color, and the third wavelength constitutively emitted by the at least one optically detectable reporter probe is a third detectable color; and wherein the first wavelength, the second wavelength, and the third wavelength are optically discernable from one another. 
     
     
         188 . The method of  claim 171 , wherein the first optically detectable identifier includes a first fluorophore capable of emitting fluorescence at the first wavelength and the second optically detectable identifier includes a second fluorophore capable of emitting fluorescence at the second wavelength; and wherein the at least one optically detectable reporter probe comprises a fluorescent DNA intercalating agent capable of constitutively emitting the third wavelength.