	US 12,392,779 B2
	Method of determining the presence and/or amount of target molecules
Christian Hennig, Hannover (DE)
Assigned to Canopy Biosciences, LLC, St. Louis, MO (US)
Filed by Zellkraftwerk GmbH, Leipzig (DE)
Filed on Feb. 12, 2021, as Appl. No. 17/174,917.
Application 17/174,917 is a continuation of application No. 15/708,221, filed on Sep. 19, 2017, granted, now 10,955,418.
Claims priority of application No. 16189483 (EP), filed on Sep. 19, 2016.
Prior Publication US 2021/0231671 A1, Jul. 29, 2021
Int. Cl. G01N 33/542 (2006.01); G01N 15/1433 (2024.01); G01N 33/53 (2006.01); G01N 33/537 (2006.01); G01N 33/543 (2006.01); G01N 33/566 (2006.01); G01N 33/569 (2006.01); G01N 33/58 (2006.01); G01N 15/10 (2006.01); G01N 15/14 (2006.01)

CPC G01N 33/582 (2013.01) [G01N 15/1433 (2024.01); G01N 33/5308 (2013.01); G01N 33/537 (2013.01); G01N 33/542 (2013.01); G01N 33/543 (2013.01); G01N 33/54353 (2013.01); G01N 33/5436 (2013.01); G01N 33/54366 (2013.01); G01N 33/566 (2013.01); G01N 33/56966 (2013.01); G01N 33/56972 (2013.01); G01N 2015/1006 (2013.01); G01N 2015/1486 (2013.01); G01N 2015/1488 (2013.01)]

17 Claims

1. A system for single cell analysis by determining the presence and/or amount of one or more target molecules in a plurality of cells, comprising:

a solid substrate;

means for immobilizing the plurality of cells on the solid substrate, wherein the plurality of cells are immobilized in a monolayer form, wherein each individual immobilized cell is sufficiently spaced apart so as to allow the position and focal plane of each individual cell to be determined;

means for determining a position of each immobilized cell of the plurality of cells on the solid substrate;

means for measuring fluorescence of each immobilized cell of the plurality of cells on the solid substrate;

means for contacting each immobilized cell immobilized on the solid substrate with at least a first detection reagent comprising:

a moiety that specifically recognizes and binds a first target molecule; and

a fluorescent label;

wherein the contacting occurs under conditions that allow binding of the at least a first detection reagent to the first target molecule;

means for measuring fluorescence of the fluorescent label of each of the at least a first detection reagent bound to the first target molecule of each immobilized cell of the plurality of cells; and

means for determining the presence and/or amount of the first target molecule of each individual immobilized cell of the plurality of cells, wherein the means for determining:

determines an auto-fluorescence background signal of each immobilized cell of the plurality of cells on the solid substrate before the cell is contacted with the at least a first detection reagent;

measures the fluorescence of the fluorescent label of the at least a first detection reagent on a cell-by-cell basis; and

compares the auto-fluorescence background signal with the fluorescence of the fluorescent label of each individual immobilized cell on the solid substrate.