TWO-DIMENSIONAL FLUORESCENCE DIFFERENCE SPECTROSCOPY CHARACTERIZATION OF NANOPARTICLES AND THEIR INTERACTIONS

• US 20170131272A1
• Filed: 11/04/2016
• Published: 05/11/2017
• Est. Priority Date: 11/05/2015
• Status: Active Grant

• Current Assignee: Kansas State University Research Foundation
• Original Assignee: Kansas State University Research Foundation
• Inventors: DeLong, Robert, Hurst, Miranda
• Granted Patent: US 9,977,016 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Y 113/12007 Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase
• CPC Class Codes: C12Y 302/01023 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• 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 33/54346 Nanoparticles

Abstract

Methods of characterizing nanoparticles, nanoparticle complexes, and their biomolecular interactions are provided. Concurrent excitation and emission wavelength scans are performed and the optimal fluorescence intensity for the intersect of these wavelengths is determined. The intersect is dependent upon the physical characteristics of the particle and can be used to verify, for example, attachment of biomolecules, amount of biomolecules present, and structure of the attached biomolecule.

Claims

1. A method of detecting the attachment of an organic species onto the surface of one or more nanoparticles within a sample comprising the nanoparticles and the organic species, wherein the method comprises performing concurrent scans of the fluorescence excitation and emission wavelengths for the nanoparticles within the sample and determining an intersect between the excitation and emission wavelengths yielding the highest fluorescence intensity, the intersect indicating the presence or absence within the sample of a nanoparticle complex comprising the one or more nanoparticles having the organic species attached to the surface of the nanoparticle.

2. The method of claim 1, wherein the method further comprises comparing the intersect to one or more known standards for the nanoparticle and/or nanoparticle complex.

3. The method of claim 2, wherein the one or more known standards comprise the intersect between the excitation and emission wavelengths yielding the highest intensity for the nanoparticle without the organic species attached to the surface of the nanoparticle.

4. The method of claim 1, wherein the organic species comprises a member selected from the group consisting of proteins, protein enzymes, RNA, DNA, oligonucleotides, aptamers, pharmaceutical compounds, and combinations thereof.

5. The method of claim 4, wherein the organic species comprises a member selected from the group consisting of B-Galactosidase, Luciferase, reverse transcriptase enzymes, deoxyribonuclease, polyinosinic-polycytidylic acid RNA, RNAse, protamine sulfate, Ras binding domain (RBD) peptide, torula yeast RNA, splice switching oligonucleotide (SSO), polyethylene glycol (PEG), glycol chitosan, and polyacrylic acid (PAA).

6. The method of claim 1, wherein the organic species comprises a polymer.

7. The method of claim 6, wherein the polymer at least partially coats the surface of the nanoparticle.

8. The method of claim 1, wherein the concurrent scans of the fluorescence excitation and emission wavelengths for the nanoparticles are performed using two-dimensional fluorescence difference spectroscopy.

9. The method of claim 1, wherein the nanoparticle comprises a member selected from the group consisting of metal oxides, elemental metals, and combinations thereof.

10. The method of claim 9, wherein the nanoparticle is selected from the group consisting of the lanthanides, ZnO, ZnS doped Mn, ZnS doped Fe, MgO, MnO, CoO, Si₃N₄, CSi, B₄C, NiO, Ni/NiO, Ni/ZnO, Co/ZnO, Au, FeO, and Fe₂O₃.

11. A method of quantifying the attachment of an organic species onto the surface of one or more nanoparticles a nanoparticle complex, wherein the method comprises:

performing concurrent scans of the fluorescence excitation and emission wavelengths for the nanoparticle complex;

determining an intersect between the excitation and emission wavelengths yielding the highest fluorescence intensity; and

comparing the intersect to one or more known standards for the nanoparticle complex to determine an amount of the organic species that is attached to the nanoparticle.

12. The method of claim 11, wherein the concurrent scans of the fluorescence excitation and emission wavelengths for the nanoparticle complex are performed using two-dimensional fluorescence difference spectroscopy.

13. The method of claim 11, wherein the organic species comprises a member selected from the group consisting of proteins, protein enzymes, RNA, DNA, oligonucleotides, aptamers, pharmaceutical compounds, and combinations thereof.

14. The method of claim 13, wherein the organic species comprises a member selected from the group consisting of B-Galactosidase, Luciferase, reverse transcriptase enzymes, deoxyribonuclease, polyinosinic-polycytidylic acid RNA, RNAse, protamine sulfate, Ras binding domain (RBD) peptide, splice switching oligonucleotide (SSO), torula yeast RNA, polyethylene glycol (PEG), glycol chitosan, and polyacrylic acid (PAA).

15. The method of claim 11, wherein the organic species comprises a polymer.

16. The method of claim 15, wherein the polymer at least partially coats the surface of the nanoparticle.

17. The method of claim 11, wherein the nanoparticle comprises a member selected from the group consisting of metal oxides, elemental metals, and combinations thereof.

18. The method of claim 17, wherein the nanoparticle is selected from the group consisting of the lanthanides, ZnO, ZnS doped Mn, ZnS doped Fe, MgO, MnO, CoO, Si₃N₄, CSi, B₄C, NiO, Ni/NiO, Ni/ZnO, Co/ZnO, Au, FeO, and Fe₂O₃.

19. A method of detecting changes in the structure of a biomolecule associated with attachment of the biomolecule to a nanoparticle thereby forming a nanoparticle complex, wherein the method comprises:

performing concurrent scans of the fluorescence excitation and emission wavelengths for the nanoparticle complex;

determining the intersect between the excitation and emission wavelengths yielding the highest fluorescence intensity; and

comparing the intersect to one or more known standards for the nanoparticle complex to detect changes in the biomolecule structure occurring during attachment to the nanoparticle.

20. The method of claim 19, wherein the concurrent scans of the fluorescence excitation and emission wavelengths for the nanoparticle complex are performed using two-dimensional fluorescence difference spectroscopy.

21. The method of claim 19, wherein the biomolecule comprises a member selected from the group consisting of proteins, protein enzymes, RNA, DNA, oligonucleotides, aptamers, pharmaceutical compounds, and combinations thereof.

22. The method of claim 21, wherein the biomolecule comprises a member selected from the group consisting of B-Galactosidase, Luciferase, reverse transcriptase enzymes, deoxyribonuclease, polyinosinic-polycytidylic acid RNA, RNAse, protamine sulfate, Ras binding domain (RBD) peptide, splice switching oligonucleotide (SSO), torula yeast RNA, polyethylene glycol (PEG), glycol chitosan, and polyacrylic acid (PAA).

23. The method of claim 19, wherein the nanoparticle comprises a member selected from the group consisting of metal oxides, elemental metals, and combinations thereof.

24. The method of claim 23, wherein the nanoparticle is selected from the group consisting of the lanthanides, ZnO, ZnS doped Mn, ZnS doped Fe, MgO, MnO, CoO, Si₃N₄, CSi, B₄C, NiO, Ni/NiO, Ni/ZnO, Co/ZnO, Au, FeO, and Fe₂O₃.