Molecular nanotags

Patent metadata

Field	Value
Publication number	US-11536719-B2
Application number	US-201716342345-A
Country	US
Kind code	B2
Filing date	Oct 23, 2017
Priority date	Oct 21, 2016
Publication date	Dec 27, 2022
Grant date	Dec 27, 2022

Abstract

A molecular nanotag is disclosed that includes a core nanoparticle with a diameter of less than about 100 nm, with an optional shell surrounding the core, and an armor bound to the surface of the core nanoparticle, or if present, to the surface of the shell. The molecular nanotag also includes a functionalized end with a fixed number of binding sites that can selectively bind to a molecular targeting ligand. Any one of, or any combination of, the core, the shell and the armor contribute to fluorescence, light scattering and/or ligand binding properties of the molecular tag that are detectable by microscopy or in a devices that measures intensity or power of fluorescence and light scattering. The light scattering intensity or power of the assembled structure is detectable above the specific level of the reference noise of a device detecting the light scattering intensity or power, its fluorescence intensity or power has sufficient brightness for detection above the limit of detection for the instrument, and ligand specificity is conferred by the ligand binding component. Methods of biomarker and biosignature detection using the molecular tags are also disclosed.

First claim

The invention claimed is:

1. A method for detecting in a flow cytometer a single target molecule in a sample, comprising:
contacting the sample with a nanoscale molecular tag, wherein the nanoscale molecular tag comprises:
(i) a core nanoparticle with a diameter of 30-80 nm and wherein the core nanoparticle comprises a noble metal;
(ii) an optional shell surrounding the core wherein said shell is selected from the group consisting of a layer of gold, silver, or both, noble metals; or includes nucleic acids or PEG; and
(iii) an armor comprising a first portion and a second portion, wherein the first portion reduces the valency of the nanoparticle to only one functional binding site and is bound to the surface of the core nanoparticle, or if present, to the surface of the shell, and the second portion is not bound to the core nanoparticle or shell, and comprises a functionalized end with one binding site, wherein the functionalized end comprises a first binding partner that is capable of specifically binding to a second binding partner or a target ligand and wherein the first and second binding partners are, respectively, selected from:
benzylguanine and a SNAP-Tag;
benzylguanine and a CLIP-Tag;
biotin and streptavidin;
a single-stranded oligonucleotide and a complementary single-stranded oligonucleotide;
a single-stranded oligonucleotide and an aptamer;
DCFPyL and prostate specific membrane antigen (PMSA);
a receptor and a ligand;
a ligand and a receptor;
an antibody and an antigen; or
an antigen and an antibody;
wherein any one of, or any combination of, the core, the shell and the armor contribute to fluorescence, light scattering and/or ligand binding properties of the molecular tag that are detectable by microscopy or an instrument that measures fluorescence and/or light scattering intensity or power; and
wherein components (i) and (iii) or (i), (ii) and (iii) together provide the following functionalities:
(a) light scattering intensity or power of the assembled structure of components (i) and (iii) or (i), (ii), and (iii) is detectable above the specific level of the reference noise of the instrument detecting the light scattering intensity or power;
(b) fluorescence intensity has sufficient brightness for detection above the limit of detection for the instrument; and/or
(c) ligand specificity is conferred by a ligand binding component,

wherein the functionalized end of the nanoscale molecular tag specifically binds the target molecule if present in the sample; and
analyzing the sample using the instrument that measures light scattering intensity or power, wherein the instrument is configured for resolution of small particles to detect individual nanoscale molecular tags bound to the target molecule by detection of side scatter or forward light scatter or detection of fluorescence, or any combination thereof.

2. The method of claim 1 , wherein the core nanoparticle is comprised of a nanomaterial having a high refractive index, surface geometry, or other attributes that contribute to light scattering properties that are detectable by the device that measures light scattering intensity or power.

3. The method of claim 1 , wherein, a single assembled molecular nanotag is detectable with microscopy or the device that measures light scattering intensity or power.

4. The method of claim 1 , wherein the core nanoparticle comprises gold or silver.

5. The method of claim 1 , wherein cumulative optical properties of the components of the nanoscale molecular tag, result in a collected power greater than a detection device's limit of sensitivity (Y limit ) for one or more light scattering wavelengths, wherein the cumulative optical properties comprise one or more of refractive index, extinction coefficient, diameter, resonance, transmittance, and reflectivity.

6. The method of claim 1 , wherein the constituent components have a parameter N RAQD =N Refractive index,Angular and Quantum properties, and Diameter , where N RAQD must be greater than the limit of detection (Y limit ) for one or more wavelengths using a device that measures light scattering intensity or power, wherein intensity or power is defined as a unit of power per unit area.

7. The method of claim 1 , wherein the armor comprises a polymer.

8. The method of claim 7 , wherein the polymer comprises phosphorothioate DNA molecule.

9. The method of claim 7 , wherein the polymer has attributes for fluorescent or light scattering properties or both.

10. The method of claim 9 , wherein the polymer contributes to the fluorescence, light scattering and/or ligand binding properties of the molecular tag.

11. The method of claim 1 , wherein the antigen comprises a tumor-associated antigen.

12. The method of claim 1 , wherein the antigen comprises a protein tag.

13. The method of claim 1 , wherein the nanoscale molecular tag comprises a fluorophore.

14. The method of claim 1 , wherein the sample is analyzed in the device that measures light scattering intensity or power using at least two angles of detection from respective side scatter channels.

15. The method of claim 14 , wherein a first side scatter channel is used as a trigger and a second side scatter channel is used as a detector.

16. The method of claim 1 , comprising detecting parallel subthreshold events.

17. The method of claim 1 , wherein the sample is a biological sample.

18. The method of claim 17 , wherein the biological sample comprises a biological membrane.

19. The method of claim 17 , wherein the biological sample comprises extracellular vesicles.

20. The method of claim 1 , wherein the target molecule comprises a tumor antigen.

21. The method of claim 20 , wherein the tumor antigen comprises prostate specific membrane antigen (PSMA), epidermal growth factor receptor (EGFR), HER-2/neu, epithelial cell adhesion molecule (EpCAM), CD24, CD133, CD47, CD147, PD-L1, GPC-1, Muc-1, CD44, CD26, CD147, EpCAM, PSMA, or PD-L1.

22. The method of claim 1 , wherein the core nanoparticle has a diameter of less than about 75 nm or less than about 40 nm.

Assignees

• The Us Secretary Dept Of Health & Human Services
• Us Health

Inventors

• Jones Jennifer C
• Morales-Kastresana Aizea
• Berzofsky Jay A
• Welsh Joshua
• Rosner Ari

Classifications

• G01N33/533

  with fluorescent label · CPC title

• C12Q1/6834

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title

• B82Y15/00

  Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors · CPC title

• G01N21/6428

  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 title

• G01N2021/4726

  Detecting scatter at 90° · CPC title