Single-molecule detection system and methods

• US 9,482,615 B2
• Filed: 03/11/2011
• Issued: 11/01/2016
• Est. Priority Date: 03/15/2010
• Status: Active Grant

• Current Assignee: Industrial Technology Research Institute
• Original Assignee: Industrial Technology Research Institute
• Inventors: Kuo, Chein-Shiu, Chiou, Chung-Fan, Pu, Ying-Chih, Pan, Chao-Chi, Shih, Chih-Tsung, Li, Ming-Chia, Chien, Hung-Chi, Chu, Chang-Sheng
• Primary Examiner(s): Warden, Jill
• Assistant Examiner(s): Fisher, Brittany
• Application Number:
  US13/046,457
• Publication Number:
  US 20110223590A1
• Time in Patent Office: 2,062 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• 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 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6454 using an integrated detector array
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G01N 21/7703 using reagent-clad optical fibres or optical waveguides using measurement of total internal reflection or attenuated total reflection G01N21/552; optical fibres or waveguides per se G02B
• CPC Class Codes: G01N 2201/08 Optical fibres; light guides
• CPC Class Codes: G01N 2201/12 Circuits of general importance; Signal processing

Abstract

Embodiments encompass a single-molecule detection system and methods of using the detection system to detect an object. Further, embodiments encompass a detection system comprising a movable light coupler, a waveguide, and a light detector. Embodiments further encompass methods of single-molecule detection, including methods of single-molecule nucleic acid sequencing.

Claims

1. A single-molecule detection system, comprising:

a movable light coupler;

a waveguide comprising;

a core layer,

a first cladding layer formed on the core layer, and

a second cladding layer formed below the core layer,

wherein at least one adapter site for the movable light coupler is formed in the first cladding layer without extending into the core layer and the second cladding layer; and

a light detector;

wherein the movable light coupler is able to localize a single-molecule object at the at least one adapter site, positionable adjacent the core layer, within an evanescent field generated by a light wave propagating along the core layer,

wherein the movable light coupler is configured to couple the evanescent field when the movable light coupler is positioned within the evanescent field, thereby allowing formation of an induced light field around the surface of the light coupler,

wherein the refractive index of the movable light coupler is higher than the refractive index of the first cladding layer, and the refractive index of the core layer is higher than the refractive indices of the first cladding layer and the second cladding layer, and

wherein the single-molecule object is not linked to the surface of the waveguide.

2. The detection system of claim 1, further comprising a light source.

3. The detection system of claim 1, wherein the at least one adapter site for the movable light coupler is a nanowell.

4. The detection system of claim 1, wherein the movable light coupler is a nano-scale particle.

5. The detection system of claim 4, wherein the nano-scale particle has a length of from about 1/10 to twice the wavelength of a light wave propagating along the core layer.

6. The detection system of claim 1, wherein the at least one adapter site extends through the full thickness of the first cladding layer.

7. The detection apparatus of claim 1, wherein the core layer is made from a material selected from at least one of Si_xTi_1-xO₂, TiO₂, Ta₂O₅, Nb₂O₅, HfO₂, Al₂O₃, ZrO₂, ZnS, SiN_x, AlN_x, TiN_x, PC, PMMA, Or Su8.

8. The detection apparatus of claim 1, wherein the movable light coupler is made from a material selected from an oxide, a polymer, a metal, a sulfide, a selenide, or a nitride.

9. The detection apparatus of claim 1, wherein the movable light coupler is made from an oxide.

10. The detection apparatus of claim 9, wherein the oxide is selected from SiO₂, TiO₂, Ta₂O₅, or Fe₃O₄.

11. The detection apparatus of claim 8, wherein the movable light coupler is made from a polymer.

12. The detection apparatus of claim 11, wherein the movable light coupler is made from polystyrene.

13. The detection apparatus of claim 1, wherein the movable light coupler has a core-shell structure, comprising a core material and a shell material.

14. The detection apparatus of claim 13, wherein the core material and shell material are different and are each independently selected from an oxide, a polymer, a metal, a sulfide, a selenide, or a nitride.

15. The detection apparatus of claim 1, wherein the movable light coupler is able to carry a target molecule on at least a portion of its surface which is adjacent to the core layer in the adapter site.

16. The detection apparatus of claim 15, wherein the movable light coupler has a property by which the light coupler can be located at the adapter site in a specific orientation.

17. The detection system of claim 16, wherein the movable light coupler is a magnetic particle.

18. The detection apparatus of claim 15, wherein one or more regions of the surface of the movable light coupler are modified.

19. The detection apparatus of claim 18, wherein one region of the surface of the movable light coupler is modified.

20. The detection apparatus of claim 19, wherein the modified region of the surface of the movable light coupler is from about 10 to 90% of the surface of the movable light coupler.

21. The detection apparatus of claim 18, wherein two regions of the surface of the movable light coupler are modified.

22. The detection apparatus of claim 18, wherein the modification comprises chemical modification.

23. The detection apparatus of claim 22, wherein the chemical modification is modification with a functional group selected from a hydrophobic group or a hydrophilic group.

24. The detection apparatus of claim 22, wherein the Chemical modification is modification with a hydrophilic group, wherein the hydrophilic group is a charged group.

25. The detection apparatus of claim 22, wherein the chemical modification is modification with a ligand-coordinating group.

26. The detection apparatus of claim 22, wherein the chemical modification is a coating with a polymer.

27. The detection apparatus of claim 22, wherein the chemical modification is modification with a magnetic material.

28. The detection apparatus of claim 18, wherein the movable light coupler comprises a region of surface modification that has an affinity for the adapter site.

29. The detection apparatus of claim 18, wherein the movable light coupler comprises a region of surface modification that is repelled by the adapter site.

30. The detection system of claim 18, wherein the movable light coupler is modified with an oligonucleotide.

33. The detection system of claim 1, wherein the refractive index of the movable light coupler is lower than the refractive index of the core layer.

34. The detection system of claim 1, wherein the refractive index of the movable light coupler approximately equals the refractive index of the core layer.

35. The detection system of claim 3, wherein the nanowell has a cone shape.

36. The detection system of claim 3, wherein an angle of a sidewall of the nanowell relative to a direction perpendicular to a bottom of the nanowell is less than about 60 degree.

37. The detection system of claim 1, wherein the first cladding layer is made form a material including at least one of silicon oxide, magnesium fluoride, calcium fluoride, aluminum oxide, Su8, PMMA, or polycarbonate.

31. A method of detecting a single-molecule object, comprising:

introducing an incident light from a light source into a waveguide, thereby forming an excitation light in the waveguide;

localizing a single-molecule object on a movable light coupler;

localizing the movable light coupler at an adapter site for movable light coupler formed in the waveguide, without linking the single-molecule object to the surface of the waveguide, the waveguide including a first cladding layer, a core layer formed below the first cladding layer, and a second cladding layer formed below the core layer, and the adapter site being formed in the first cladding layer without extending into the core layer and the second cladding layer;

exciting, by the excitation light, the single-molecule object localized on the movable light coupler, causing the single-molecule object to emit a light to be detected by a light detector;

generating an evanescent field introduced by a light wave propagating along the core layer; and

coupling the evanescent field to the movable light coupler, and thereby allowing formation of induced light field around the surface of the light coupler,

wherein the refractive index of the movable light coupler is higher than the refractive index of the first cladding layer, and the refractive index of the core layer is higher than the refractive indices of the first cladding layer and the second cladding layer.

32. A method of detecting a single-molecule object, comprising:

providing a detection apparatus comprising

i) a movable light coupler;

ii) a waveguide comprising;

a core layer,

a first cladding layer formed on the core layer, and

a second cladding layer formed below the core layer,

wherein at least one adapter site for the movable light coupler is formed in the first cladding layer without extending into the core layer and the second cladding layer; and

a light detector;

providing at least one binding moiety capable of binding a single-molecule object;

localizing the at least one binding moiety individually on the movable light coupler;

providing a single-molecule object molecule to the at least one binding moiety localized on the movable light coupler;

localizing at the adapter site the movable light coupler on which the at least one binding moiety and single-molecule object are localized, without linking the single-molecule object to the surface of the waveguide;

introducing an incident light from a light source into the waveguide, thereby forming an excitation light in the waveguide;

exciting, by the excitation light, the single-molecule object molecule bound to the at least one binding moiety localized on the movable light coupler, causing the single-molecule object to emit a light to be detected by a light detector;

generating an evanescent field induced by a light wave propagating along the core layer at the adapter site; and

coupling the evanescent field to the movable light coupler, and thereby allowing formation of an induced light field around the surface of the light coupler,

wherein the refractive index of the movable light coupler is higher than a refractive index of the first cladding layer, and the refractive index of the core layer is higher than the refractive indices of the first cladding layer and the second cladding layer.