US 11,754,562 B2
Methods for multiplex imaging using labeled nucleic acid imaging agents
Stephanie Rae Hennek, Medford, MA (US); Mael Manesse, Medford, MA (US); Abdul Mohammed, Cambridge, MA (US); Xi Chen, West Newton, MA (US); and Michael Jules Natan, Weston, MA (US)
Assigned to Ultivue, Inc., Cambridge, MA (US)
Filed by Ultivue, Inc., Cambridge, MA (US)
Filed on Aug. 13, 2020, as Appl. No. 16/992,654.
Application 16/992,654 is a continuation of application No. 16/794,686, filed on Feb. 19, 2020.
Application 16/794,686 is a continuation of application No. 15/836,322, filed on Dec. 8, 2017, abandoned.
Claims priority of provisional application 62/445,896, filed on Jan. 13, 2017.
Claims priority of provisional application 62/432,511, filed on Dec. 9, 2016.
Prior Publication US 2020/0371097 A1, Nov. 26, 2020
Int. Cl. C12Q 1/6804 (2018.01); C12Q 1/6832 (2018.01); C12Q 1/6874 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/6823 (2018.01); G01N 33/543 (2006.01); G01N 33/58 (2006.01); C12Q 1/68 (2018.01); C12Q 1/6818 (2018.01); C12Q 1/6816 (2018.01); C12Q 1/682 (2018.01)
CPC G01N 33/54386 (2013.01) [C12Q 1/68 (2013.01); C12Q 1/682 (2013.01); C12Q 1/6804 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6816 (2013.01); C12Q 1/6818 (2013.01); C12Q 1/6823 (2013.01); C12Q 1/6832 (2013.01); C12Q 1/6874 (2013.01); G01N 33/543 (2013.01); G01N 33/54306 (2013.01); G01N 33/54333 (2013.01); G01N 33/58 (2013.01); G01N 33/587 (2013.01)] 15 Claims
OG exemplary drawing
 
1. A method for detecting two or more targets, comprising:
(1) contacting a sample being tested for the presence of two or more targets with two or more target-specific binding partners, wherein each target-specific binding partner is linked to a docking strand and wherein target-specific binding partners of different specificity are linked to different docking strands;
(2) optionally removing unbound target-specific binding partners;
(3) contacting the sample with enzyme-labeled strands capable of binding a docking strand, wherein the enzyme-labeled strands comprise horseradish peroxidase (HRP)-labeled strands;
(4) optionally removing unbound enzyme-labeled strands;
(5) contacting the sample with a substrate for the enzyme comprising a tyramide moiety linked to a fluorophore;
(6) allowing an enzymatic reaction to produce an amplification product;
(7) quenching the enzymatic reaction by removing the bound enzyme-labeled strands;
(8) repeating steps (3)-(7),
(9) after step (8), imaging the sample to detect the presence or absence of two or more targets; and
(10) optionally repeating at least some of steps (1)-(9).