Nanodiamond counting

Patent metadata

Field	Value
Publication number	US-9759719-B1
Application number	US-201414336589-A
Country	US
Kind code	B1
Filing date	Jul 21, 2014
Priority date	Jul 21, 2014
Publication date	Sep 12, 2017
Grant date	Sep 12, 2017

Abstract

Methods and systems for detecting the locations of individual instances of an analyte (e.g., individual cells, individual molecules) in an environment are provided. The environment includes functionalized fluorophores that are configured to selective interact with (e.g., bind with) the analyte and that have a fluorescent property that can be modulated (e.g., a fluorescence intensity that can be affected by the presence of a magnetic field). Detecting the location of individual instances of the analyte includes illuminating the environment and detecting signals emitted from the fluorophores in response to the illumination during first and second periods of time. Detecting the location of individual instances of the analyte further includes modulating the modulatable fluorescent property of the fluorophores during the second period of time and determining which individual fluorophores in the environment are bound to the analyte based on the signals detected during the first and second periods of time.

First claim

What is claimed is:

1. A method comprising:
exposing a detection environment to first illumination during a first period of time, wherein the detection environment includes a plurality of functionalized fluorophores, wherein each of the functionalized fluorophores has a fluorescent property that is modulatable, wherein each of the functionalized fluorophores is functionalized to selectively bind to an analyte, and wherein the first illumination causes individual functionalized fluorophores to emit respective first-period signals;
detecting the first-period signals emitted by the functionalized fluorophores during the first period of time in response to the first illumination;
modulating the modulatable fluorescent property of the functionalized fluorophores during a second period of time;
exposing the detection environment to second illumination during the second period of time, wherein the second illumination causes individual functionalized fluorophores to emit respective second-period signals;
detecting the second-period signals emitted by the functionalized fluorophores during the second period of time in response to the second illumination; and
determining which individual functionalized fluorophores in the plurality of functionalized fluorophores are bound to the analyte based at least on the detected first-period signals and the detected second-period signals, wherein the determining is further based on at least one characteristic that distinguishes between bound and unbound functionalized fluorophores.

2. The method of claim 1 , wherein modulating the modulatable fluorescent property of the functionalized fluorophores comprises exposing the detection environment to microwave radiation.

3. The method of claim 1 , wherein each of the functionalized fluorophores comprises at least one nanodiamond comprising at least one nitrogen-vacancy defect.

4. The method of claim 3 , wherein the first illumination and the second illumination comprise light having wavelengths in the range of about 500 nanometers to about 650 nanometers, and wherein detecting the first-period signals and the second-period signals comprises detecting light having wavelengths in the range of about 650 nanometers to about 800 nanometers.

5. The method of claim 3 , wherein modulating the modulatable fluorescent property of the functionalized fluorophores comprises exposing a region of the detection environment to a high-strength magnetic field.

6. The method of claim 1 , wherein each of the functionalized fluorophores comprises at least one quantum dot.

7. The method of claim 1 , further comprising:
extracting the analyte from a living body; and
introducing the extracted analyte into the detection environment, wherein the detection environment is external to the living body.

8. The method of claim 1 , further comprising:
adding a fixative to the detection environment, wherein the fixative is configured to substantially reduce motion of the analyte and the functionalized fluorophores in the detection environment.

9. The method of claim 1 , further comprising:
determining locations of individual instances of the analyte in the detection environment based on determining which individual functionalized fluorophores in the plurality of functionalized fluorophores are bound to the analyte.

10. The method of claim 9 , further comprising:
determining a concentration of the analyte based on determining the locations of individual instances of the analyte in the detection environment.

11. The method of claim 1 , wherein determining which individual functionalized fluorophores in the plurality of functionalized fluorophores are bound to the analyte comprises determining a degree of blurring of images of the individual functionalized fluorophores, wherein the images of the individual functionalized fluorophores are based on the detected first-period signals and the detected second-period signals.

12. The method of claim 1 , wherein determining which individual functionalized fluorophores in the plurality of functionalized fluorophores are bound to the analyte comprises determining that the individual functionalized fluorophores are proximate to other individual functionalized fluorophores based on the detected first-period signals and the detected second-period signals.

13. The method of claim 1 , wherein the detection environment is a substantially planar region containing the analyte, the functionalized fluorophores, and a medium containing the analyte and the functionalized fluorophores.

14. The method of claim 13 , wherein the substantially planar region has a thickness between approximately 10 microns and approximately 20 microns.

15. The method of claim 13 , wherein the medium is an aqueous medium.

16. The method of claim 1 , wherein the analyte is a cell.

Assignees

• Verily Life Sciences Llc

Inventors

• Acosta Victor Marcel
• Schwartz Jerrod
• Demas Vasiliki
• Bajaj Vikram Singh
• Thompson Jason Donald
• Askew Mark West

Classifications

• G01N33/569 Primary

  for microorganisms, e.g. protozoa, bacteria, viruses · CPC title

• G01N33/543

  with an insoluble carrier for immobilising immunochemicals · CPC title

• G01N33/582 Primary

  with fluorescent label · CPC title

• G01N33/588

  with semiconductor nanocrystal label, e.g. quantum dots · CPC title