Systems and methods related to continuous flow droplet reaction

Abstract

Described herein are systems relating to a continuous-flow instrument that includes all necessary components for digital droplet quantification without the need to introduce key reagents or collect and transfer droplets between stages of instrument operation. Digital quantification can proceed without any additional fluid or consumable handling and without exposing fluids to risk of external contamination.

What is claimed is: 
     
         1 . A system comprising:
 (a) a reservoir comprising a continuous phase in fluid communication with an injector;   (b) the injector configured to receive a sample comprising nucleic acids and direct the sample to an outlet of the injector;   (c) wherein the outlet of the injector is in fluid communication with a flow path comprising:
 (i) a separator configured to introduce the continuous phase into a droplet stream to separate droplets from one another prior to flowing the droplets into an inlet of a detector; 
 (ii) the detector comprising the inlet, a detection stage, and an outlet, wherein the detector is configured to measure one or more signals of each droplet as the droplets flow from the inlet through the detection stage to the outlet; and 
 (iii) a waste reservoir in fluid communication with the outlet of the detector; 
   wherein the system is configured to measure a quantity of nucleic acids in the sample.   
     
     
         2 . The system of  claim 1 , wherein the injector comprises a zero dead volume injector. 
     
     
         3 . The system of  claim 1 , wherein the surfaces of the injector have greater affinity for the continuous phase than the sample. 
     
     
         4 . The system of claim  0 , wherein the surfaces of the injector are at least one of hydrophobic or fluorophilic. 
     
     
         5 . The system of  claim 1 , wherein the signal comprises at least one of an optical, electrical, mechanical, or magnetic signal. 
     
     
         6 . The system of  claim 1 , further comprising a droplet generator. 
     
     
         7 . The system of  claim 1 , further comprising a reactor configured to induce a chemical reaction in the droplets. 
     
     
         8 . The system of  claim 7 , wherein the reactor comprises a thermocycler. 
     
     
         9 . The system of  claim 1 , wherein the continuous phase comprises an oil comprising a fluorinated oil, silicone oil, hydrocarbon oil, or mineral oil. 
     
     
         10 . The system of  claim 1 , wherein the continuous phase further comprises one or more surfactants. 
     
     
         11 . A method comprising:
 (a) providing a sample comprising nucleic acids to a system comprising:
 (i) a reservoir comprising a continuous phase in fluid communication with an injector; 
 (ii) the injector configured to receive the sample comprising nucleic acids and direct the sample to an outlet of the injector; 
 (iii) wherein the outlet of the injector is in fluid communication with a flow path comprising:
 (1) a separator configured to introduce the continuous phase into a droplet stream to separate droplets from one another prior to flowing the droplets into an inlet of a detector; 
 (2) the detector comprising the inlet, a detection stage, and an outlet, wherein the detector measures one or more signals of each droplet as the droplets flow from the inlet through the detection stage to the outlet; and 
 (3) a waste reservoir in fluid communication with the outlet of the detector; 
 
 wherein the system is configured to measure a quantity of nucleic acids in the sample; 
   (b) detecting a signal from each of the plurality of droplets; and   (c) measuring the quantity of nucleic acids in the sample.   
     
     
         12 . The method of  claim 11 , further comprising calculating the quantity of nucleic acids in the sample by counting a total number of droplets that comprise the signal (N[1+]) and a total number of droplets that do not comprise the signal (N[0]) and calculating the quantity using a Poisson distribution. 
     
     
         13 . The method of claim  0 , wherein the quantity of nucleic acids (μ) is calculated using the formula μ=−ln ([N[0]/(N[0]+N[1+])), wherein ln is the natural logarithm. 
     
     
         14 . The method of claim  0 , further comprising calculating a concentration of nucleic acids in the sample. 
     
     
         15 . The method of claim  0 , wherein the concentration of nucleic acids (C) in the sample is calculated using the formula C=μΣV d , wherein V d  is the droplet volume and ΣV d  is the total volume of the sample analyzed. 
     
     
         16 . The method of  claim 11 , wherein the injector comprises a zero dead volume injector. 
     
     
         17 . The method of  claim 11 , wherein the signal comprises at least one of an optical, electrical, mechanical, or magnetic signal. 
     
     
         18 . The method of  claim 11 , wherein the system further comprises a droplet generator. 
     
     
         19 . The method of  claim 11 , wherein the system further comprises a reactor configured to induce a chemical reaction in the droplets. 
     
     
         20 . The method of  claim 11 , wherein the continuous phase comprises an oil comprising a fluorinated oil, silicone oil, hydrocarbon oil, or mineral oil.