Detection of Promiscuous Small Submicrometer Aggregates

• US 20100015721A1
• Filed: 06/04/2009
• Published: 01/21/2010
• Est. Priority Date: 06/04/2008
• Status: Abandoned Application

• Current Assignee: Sru Biosystems Incorporated
• Original Assignee: Sru Biosystems Incorporated
• Inventors: Laing, Lance
• Application Number:
  US12/478,534
• Publication Number:
  US 20100015721A1
• Time in Patent Office: Days
• US Class Current: 436/172
• CPC Class Codes: G01N 2021/7773 Reflection
• CPC Class Codes: G01N 2021/7776 Index
• CPC Class Codes: G01N 21/253 for batch operation, i.e. multisample apparatus analytical automats G01N35/00
• CPC Class Codes: G01N 21/7743 the reagent-coated grating coupling light in or out of the waveguide
• CPC Class Codes: G01N 21/78 producing a change of colour
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Abstract

The invention provides methods for the detection of aggregating molecules that are capable of promiscuous or non-specific binding to proteins in a time efficient manner without the use of labels.

Claims

1. A method of detecting aggregate-forming particles or promiscuous inhibitor molecules comprising:

(a) applying test species to a colorimetric resonant reflectance biosensor or a grating-based waveguide biosensor; and

(b) illuminating the biosensor with light and determining peak wavelength value shifts or refractive index changes over time;

wherein, discontinuous, non-linear, or slope of greater than 2 pm/minute peak wavelength value shifts or refractive index changes over time indicates that the test species are aggregate-forming particles or promiscuous binding molecules.

2. The method of claim 1, wherein the biosensor has one or more specific binding substances, binding partners or linkers immobilized on a surface of the biosensor.

4. The method of claim 1, wherein the biosensor has a hydrophilic coating on the biosensor surface.

5. The method of claim 1, wherein the refractive index change or peak wavelength value shift is continuously measured over an about 15 second to about a 10 minute time period.

6. The method of claim 1, wherein the biosensor is a bottom surface of one or more microtiter wells.

7. The method of claim 6, wherein the test species is added at several different concentrations to several different microtiter wells.

8. The method of claim 1, wherein the peak wavelength values or refractive index values are determined at a distance of about 100 up to about 300 nm off of the surface of the biosensor.

3. The method of claim 3, wherein the stoichiometry of a binding reaction between the test species and the one or more specific binding substances, binding partners or linkers greater than about 1:

1, thereby indicating that the test species are aggregate-forming particles or promiscuous binding molecules.

9. A method of detecting non-specific binding of a test species comprising:

(a) applying test species to a colorimetric resonant reflectance biosensor or a grating-based waveguide biosensor, wherein the biosensor has one or more specific binding substances, binding partners or linkers immobilized on a surface of the biosensor; and

(b) illuminating the biosensor with light and determining peak wavelength value shifts or refractive index changes over time;

wherein, discontinuous, non-linear, or slope of greater than 2 pm/minute peak wavelength value shifts or refractive index changes over time indicates that the test species is non-specifically binding.

10. The method of claim 9, wherein the stoichiometry of a binding reaction between the test species and the one or more specific binding substances, binding partners or linkers greater than about 1:

1, thereby indicating that the test species is non-specifically binding.

11. The method of claim 9, wherein the refractive index change or peak wavelength value shift is continuously measured over an about 15 second to about a 10 minute time period.

12. The method of claim 9, wherein the peak wavelength values or refractive index values are determined at a distance of about 100 up to about 300 nm off of the surface of the biosensor.

13. The method of claim 9, wherein the biosensor is a bottom surface of one or more microtiter wells.

14. The method of claim 13, wherein the test species is added at several different concentrations to several different microtiter wells.

15. A method of detecting non-specific binding of a test species comprising:

(a) applying a test species at varying concentrations to two or more locations on a calorimetric resonant biosensor or a grating-based waveguide biosensor, wherein the biosensor has one or more specific binding substances, binding partners, or linkers immobilized to the biosensor surface; and

(b) illuminating the biosensor with light and detecting peak wavelength values or refractive index values for each of the two or more locations;

wherein, a discontinuous, non-linear, or slope of greater than 2 pm/minute refractive index change with regard to increasing concentration of the test species indicates that the test species is non-specifically binding.

16. The method of claim 15, wherein the stoichiometry of a binding reaction between the test species and the one or more specific binding substances, binding partners or linkers greater than about 1:

1, thereby indicating that the test species is non-specifically binding.

17. The method of claim 15, wherein the refractive index change or peak wavelength value shift is continuously measured over an about 15 second to about a 10 minute time period.

18. The method of claim 15, wherein the peak wavelength values or refractive index values are determined at a distance of about 100 up to about 300 nm off of the surface of the biosensor.

19. The method of claim 15, wherein the biosensor is a bottom surface of one or more microtiter wells.

20. The method of claim 19, wherein the test species is added at several different concentrations to several different microtiter wells.

21. A method of detecting aggregate-forming particles or promiscuous inhibitor molecules comprising:

(a) applying a test species at varying concentrations to two or more locations on a calorimetric resonant biosensor or a grating-based waveguide biosensor; and

(b) illuminating the biosensor with light and detecting peak wavelength values or refractive index values for each of the two or more locations;

wherein, discontinuous, non-linear or slope of greater than 2 pm/minute peak wavelength shift or refractive index change with regard to increasing concentration of the test species indicates that the test species is an aggregate-forming particle or a promiscuous inhibitor.

22. The method of claim 21, wherein the biosensor has one or more specific binding substances, binding partners, or linkers immobilized to the biosensor surface.

23. The method of claim 22, wherein the stoichiometry of a binding reaction between the test species and the one or more specific binding substances, binding partners or linkers greater than about 1:

1, thereby indicating that the test species are aggregate-forming particles or promiscuous binding molecules.

24. The method of claim 21, wherein the biosensor has a hydrophilic coating on the biosensor surface.

25. The method of claim 21, wherein the peak wavelength values or refractive index values are determined at a distance of about 100 up to about 300 nm off of the surface of the biosensor.

26. The method of claim 21, wherein the refractive index change or peak wavelength value shift is continuously measured over an about 15 second to about a 30 minute time period.

27. The method of claim 21, wherein the biosensor is a bottom surface of one or more microtiter wells.

28. The method of claim 27, wherein the test species is added at several different concentrations to several different microtiter wells.

29. A method of detecting promiscuous inhibitor molecules or aggregate-forming particles comprising:

(a) applying a ligand to a first location of a biosensor, wherein the biosensor has a target molecule comprising a specific binding site for the ligand immobilized on the first location and a second location of the biosensor;

(b) applying a test species to the first and second locations of the biosensor;

(c) applying a molecule known to bind to the target molecule at the specific binding site of the target molecule to the first and the second locations of the biosensor;

(d) illuminating the biosensor with light and determining peak wavelength value shifts or refractive index changes at steps (a)-(c) and determining stoichiometric ratios of binding reactions at the first and second locations of the biosensor;

thereby detecting promiscuous inhibitor molecules or aggregate-forming particles.

30. The method of claim 29, wherein the peak wavelength values or refractive index values are determined at a distance of about 100 up to about 300 nm off of the surface of the biosensor.