Calibrations and controls for droplet-based assays

• US 9,417,190 B2
• Filed: 07/18/2013
• Issued: 08/16/2016
• Est. Priority Date: 09/23/2008
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Hindson, Benjamin J., Colston, Billy W. Jr., Ness, Kevin D., Masquelier, Donald A.
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US13/945,661
• Publication Number:
  US 20130302792A1
• Time in Patent Office: 1,125 Days
• Field of Search: 435/6.1
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 2201/1242 Validating, e.g. range invalidation, suspending operation

Abstract

System, including methods and apparatus, for performing droplet-based assays that are controlled and/or calibrated using signals detected from droplets.

Claims

1. A method of performing a droplet-based assay, comprising:

forming droplets from a same mixture, wherein only a subset of the droplets contain a target;

irradiating a region of a channel;

detecting a forward scattering signal and a fluorescence signal from a plurality of the droplets as each droplet passes through the region of the channel, wherein the fluorescence signal is detected from a label present in each droplet and varies according to whether the target is present in the droplet;

identifying accepted droplets of the plurality for which the forward scattering signal meets a condition and rejected droplets of the plurality for which the forward scattering signal does not meet the condition, wherein the condition corresponds to a permitted size of a droplet; and

determining a concentration of the target based on the fluorescence signal from the accepted droplets, and without any contribution of the fluorescence signal from the rejected droplets.

2. The method of claim 1, wherein the forward scattering signal has an intensity that is at least substantially independent of whether or not the target is present in the droplet.

3. The method of claim 1, wherein the forward scattering signal detected from each droplet forms a peak, and wherein the condition corresponds to a permitted width of the peak.

4. The method of claim 1, wherein the step of determining includes a step of determining a fraction of the accepted droplets that are positive or a fraction that are negative for the target.

5. The method of claim 1, wherein the forward scattering signal is produced by refraction of incident radiation.