Microfluidic determination of low abundance events

Abstract

Provided are microfluidic systems and methods for detecting, sorting, and dispensing of low abundance events such as single cells and particles, including a variety of eukaryotic and bacterial cells, for a variety of bioassay applications. The systems and methods described herein, when implemented in whole or in part, will make relevant microfluidic based tools available for a variety of applications in biotechnology including antibody discovery, immuno-therapeutic discovery, high-throughput single cell analysis, target-specific compound screening, and synthetic biology screening.

1 .- 20 . (canceled) 
     
     
         21 . A system for processing droplets, the system comprising:
 (a) a microfluidic device comprising a first channel connected to a second channel by a first sorting junction;   (b) one or more droplets, wherein a droplet of the one or more droplets comprises at least one cell, at least one particle, or both;   (c) a first sensor corresponding to a first point of detection disposed along the first channel configured to detect one or more signals generated from the droplet of the one or more droplets;   (d) a second sensor corresponding to a second point of detection disposed along the second channel, wherein the second sensor is configured to detect one or more signals generated from the droplet of the one or more droplets; and   (e) a processor configured to index the droplet of the one or more droplets with the one or more signals of the first and the second sensor at the first and second points of detection.   
     
     
         22 . The system of  claim 21 , further comprising one or more lasers or laser-like light sources to generate illumination at the first point of detection. 
     
     
         23 . The system of  claim 21 , further comprising an optical element configured to provide dual focusing along the first channel at the first point of detection. 
     
     
         24 . The system of  claim 23 , wherein the optical element comprises an optical fiber splitter or a birefringent polarizer configured to split an energy beam generated by the one or more lasers or laser-like sources into a first beam and a second beam and direct the first and second beams to the first point of detection 
     
     
         25 . The system of  claim 21 , wherein the first sensor comprises a fast-response optical detector. 
     
     
         26 . The system of  claim 25 , wherein the fast-response optical detector comprises a photo multiplier tube (PMT) or an avalanche photodiode detector (APD). 
     
     
         27 . The system of  claim 21 , wherein the second sensor comprises a camera or a camera-like detector. 
     
     
         28 . The system of  claim 21 , wherein the second sensor is configured to generate two or more images of the droplet of the one or more droplets, wherein the two or images comprise a signal generated by stroboscopic illumination. 
     
     
         29 . The system of  claim 21 , wherein the processor is configured to synchronize a dispensing nozzle with one or more of the first signal and the second signal. 
     
     
         30 . The system of  claim 21 , further comprising an optical assembly configured to provide stroboscopic illumination at the second point of detection. 
     
     
         31 . The system of  claim 30 , further comprising an upstream sensor corresponding to a third point of detection disposed along the second channel between the sorting junction and the second point of detection, wherein the upstream sensor is configured to provide a precise timing trigger to the optical assembly to trigger the stroboscopic illumination. 
     
     
         32 . The system of  claim 30 , wherein the first sensor is configured to provide a precise timing trigger to the optical assembly to trigger the stroboscopic illumination. 
     
     
         33 . The system of  claim 21 , wherein the microfluidic device further comprises a waste channel in fluid communication with the first channel. 
     
     
         34 . The system of  claim 21 , wherein the one or more droplets comprise water-in-oil droplets. 
     
     
         35 . The system of  claim 21 , wherein (e) is performed in a sorting module disposed downstream from the second point of detection. 
     
     
         36 . The system of  claim 30 , wherein the stroboscopic illumination provides a short illumination within a range of about 0.5 to about 50 milliseconds. 
     
     
         37 . The system of  claim 21 , wherein the one or more droplets comprise a droplet volume from about 0.01 nanoliters (nL) to about 10 nL. 
     
     
         38 . The system of  claim 21 , wherein the first channel comprises a size of at least 35 micrometers. 
     
     
         39 . The system of  claim 27 , wherein the camera or camera-like detector comprise a charge coupled device (CCD), photodiodes, complementary metal-oxide semiconductor (CMOS) cameras, or any combination thereof. 
     
     
         40 . The system of  claim 32 , wherein the stroboscopic illumination is generated by modulating a continuous-wave (CW) laser.