Nucleic acid biosensor diagnostics

• US 6,503,711 B1
• Filed: 02/16/2000
• Issued: 01/07/2003
• Est. Priority Date: 06/18/1997
• Status: Expired due to Term

• Current Assignee: Andre H. Uddin, Masad Damha, Paul A. Piunno, Robert H.E. Hudson, Ulrich J. Krull
• Original Assignee: Andre H. Uddin, Masad Damha, Paul A. Piunno, Robert H.E. Hudson, Ulrich J. Krull
• Inventors: Krull, Ulrich J., Damha, Masad, Piunno, Paul A., Hudson, Robert H. E., Uddin, Andre H.
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US09/446,222
• Time in Patent Office: 1,056 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 422/68.1, 422/82.05, 422/82.07, 422/82.08, 422/82.09, 250/458.1, 654/09, 536/23.1
• US Class Current: 506/17
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6839 Triple helix formation or other higher order conformations in hybridisation assays
• 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 21/648 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: Y10S 977/924 using nanostructure as support of dna analysis

Abstract

A biosensor for direct analysis of nucleic acid hybridazation by use of an optical fiber functionalized with nucleic acid molecules and fluorescence transduction is disclosed. Nucleic acid probes are immobilized onto the surface of optical fibers and undergo hybridization with complementary nucleic acids introduced into the local environment of the sensor. Hybridization events are detected by the use of fluorescent compounds which bind into nucleic acid hybrids. The invention finds uses in detection and screening of genetic disorders, viruses, and pathogenic micoorganisms. Biotechnology applications include monitoring of gene cultures and gene expression and the effectiveness (e.g. dose-response) of gene therapy pharmaceuticals. The invention includes biosensor systems in which fluorescent molecules are connected to the immobilized nucleic acid molecules. The preferred method for immobilization of nucleic acids is by in situ solid phase nucleic acid synthesis. Control of the refractive index of the immobilized nucleic acid is achieved by the support derivatization chemistry and the nucleic acid synthesis. The preferred optical fiber derivation yields a DNA coating of higher refractive index than the fiber core onto the fiber surface.

Claims

1. A biosensor for detecting a target nucleic acid in the presence of a fluorophore by detection of fluorescence from the fluorophore which comprises:

(a) an optical element having an index of refraction and which comprises an interaction surface;

(b) an immobilized layer having an index of refraction which comprises a nucleic acid or nucleic acid analogue covalently attached to the interaction surface of the optical element, the nucleic acid or nucleic acid analogue capable of hybridizing to the target nucleic acid to form a hybridized nucleic acid complex;

(c) a light source for introducing light capable of stimulating fluorescence of the fluorophore into the optical element in contact with the interaction surface; and

(d) a detector for detecting fluorescence emitted by the fluorophore on binding to a hybridized nucleic acid complex;

wherein the index of refraction of the immobilized layer is equal to or greater than the refractive index of the interaction surface of the optical element such that direct excitation of the fluorophore in the immobilization layer to emit fluorescence results in the detection of the target nucleic acid.

2. The biosensor of claim 1 wherein the index of refraction of the immobilized layer is controlled by changing the density of molecules immobilized on the interaction surface of the optical element.

3. The biosensor of claim 2 wherein the index of refraction of the immobilized layer is controlled by changing the density of nucleic acids or nucleic acid analogs immobilized on the interaction surface.

4. The biosensor of claim 1 wherein the nucleic acid or nucleic acid analogue is attached to the interaction surface through a linker molecule.

5. The biosensor of claim 4 wherein the index of refraction of the immobilized layer is controlled by changing the density of linker molecules immobilized on the interaction surface of the optical element.

6. The biosensor of claim 4 wherein the index of refraction of the immobilized layer is increased by increasing the density of linker molecules immobilized on the interaction surface of the optical element.

7. The biosensor of claim 4 wherein the linker is at least about 25 Å

in length.

8. The biosensor of claim 4 wherein the linker contains ethylene glycol subunits.

9. The biosensor of claim 4 wherein the linker is hexaethylene glycol.

10. The biosensor of claim 4 wherein the nucleic acid or nucleic acid analogue is attached to the linker by in situ synthesis.

11. The biosensor of claim 10 wherein the nucleic acid is attached to the linker by in-situ solid-phase oligonucleotide synthesis.

12. The biosensor of claim 11 wherein the nucleic acid is attached to the linker by in situ oligonucleotide synthesis using the β

-cyanoethylphosphoramidite method.

13. The biosensor of claim 10 wherein the nucleic acid or nucleic acid analogue has 50 or fewer bases.

14. The biosensor of claim 4 wherein a free strand of nucleic acid or nucleic acid analogue is covalently attached to the linker.

15. The biosensor of claim 1 wherein the immobilization layer comprises a fluorophore.

16. The biosensor of claim 1 wherein the fluorophore is covalently tethered to the nucleic acid or nucleic acid analogue in the immobilization layer.

17. The biosensor of claim 1 wherein the fluorophore is provided in a solution into which the biosensor is immersed.

18. The biosensor of claim 1 wherein the index of refraction of the immobilized layer is in the range 1.4 to 1.6.

19. The biosensor of claim 1 wherein the index of refraction of the immobilized layer is 1.62.

20. The biosensor of claim 1 wherein the optical element is an optical waveguide.

21. The biosensor of claim 1 wherein the optical element is an optical fiber.

22. The biosensor of claim 21 wherein the optical fiber is a fused silica fiber.

23. The biosensor of claim 1 wherein the detection limit is below 10⁶ molecules of target nucleic acid.

24. The biosensor of claim 1 for detecting more than one different target nucleic acid wherein the immobilized layer includes, for each target nucleic acid, a nucleic acid or nucleic acid analogue capable of hybridizing to that target nucleic acid.

25. The biosensor of claim 24 wherein a fluorophore is covalently tethered to nucleic acids or nucleic acid analogues.

26. The biosensor of claim 25 wherein the fluorophores covalently tethered to different nucleic acids or nucleic acid analogues emit fluorescence at different wavelengths.

27. The biosensor of claim 1 wherein the index of refraction of the immobilized layer is in the range 1.4 to 1.6 and the nucleic acid or nucleic acid analogue is attached to the interaction surface through a linker molecule which contains an ethylene glycol subunit.

28. The biosensor of claim 27 wherein the linker molecule is hexaethylene glycol.

29. A method for detecting a target nucleic acid in a sample which comprises the steps:

(a) contacting the immobilized layer of the biosensor of claim 1 with the sample such that target nucleic acids in the sample can hybridize to the nucleic acids or nucleic acid analogues of the immobilization layer;

(b) contacting the immobilization layer of the biosensor with a fluorophore and allowing the fluorophore to bind to hybridization complexes of the nucleic acids or nucleic acid analogues with the target nucleic acid in the immobilization layer;

(c) introducing light into the optical element of the biosensor in contact with the interaction surface of the optical element to stimulate emission from bound fluorophore; and

(d) detecting the fluorescence emitted by bound fluorophore

whereby the target nucleic acid is detected.

30. The method of claim 29 wherein the immobilization layer is contacted with the fluorophore by covalent bonding within the immobilization layer.

31. The method of claim 29 wherein the immobilization layer is contacted with the fluorophore by covalently tethering the fluorophore to nucleic acids or nucleic acid analogues of the immobilization layer.

32. The method of claim 29 wherein the target nucleic acid is a nucleic acid of bacteria, viruses, fungi, unicellular or multicellular organisms.

33. The method of claim 29 wherein the target nucleic acid is a nucleic acid of a cell, a cellular homogenate, a tissue or an organ.

34. The method of claim 29 wherein the biosensor is internally calibrated by comparing fluorescence emission from the fluorophore in the immobilization layer before and after the biosensor is contacted with the sample.

35. The method of claim 34 wherein the biosensor is internally calibrated using time-resolved fluorescence measurements.

36. A method for detecting more than one target nucleic acid in a sample which comprises the steps:

(a) contacting the immobilized layer of the biosensor of claim 24 with the sample such that target nucleic acids in the sample can hybridize to the nucleic acids or nucleic acid analogues of the immobilization layer;

(b) allowing the covalently tethered fluorophores of the immobilization layer to bind to hybridization complexes of the nucleic acids or nucleic acid analogues with the target nucleic acid in the immobilization layer;

(c) introducing light into the optical element of the biosensor in contact with the interaction surface of the optical element to stimulate emission from bound fluorophore; and

(d) detecting the fluorescence emitted by bound fluorophore

whereby the target nucleic acid is detected.

37. The method of claim 36 wherein the target nucleic acids are nucleic acids of a bacterium, a virus, a fungus, a unicellular or a multicellular organism.

42. A method for making a biosensor of claim 1 which comprises the steps of:

(a) activating the interaction surface of the optical element of the biosensor;

(b) attachment of nucleic acids or nucleic acid analogues, which can hybridize to the target nucleic acid, to the activated interaction surface;

(c) measurement of the refractive index of the immobilized layer by angularly dependent light scattering; and

(d) adjusting the conditions of step b, if necessary, to obtain an immobilized layer having an index of refraction equal to or greater than the index of refraction of the optical element.

43. The method of claim 42 wherein the optical element is an optical fiber.

44. The method of claim 42 further comprising the step of covalently bonding a fluorophore in the immobilization layer.

45. The method of claim 42 wherein the fluorophore is covalently tethered to a nucleic acid or nucleic acid analogue in the immobilization layer.

46. The method of claim 42 wherein the optical element is a fused silica optical fiber.

47. The method of claim 42 wherein the interaction surface of the optical fiber is activated by treatment with methanesulfonyl chloride or an organosilane prior to attachment of the linker.

48. The method of claim 47 wherein the organosilanes are glycidoxypropyltrimethoxysilane or aminopropyltriethoxysilane.

49. The method of claim 42 wherein one terminus of the linker is protected prior to reaction with the activated interaction surface of the optical element.

50. The method of claim 42 wherein the nucleic acid or nucleic acid analogue is attached to the linker by in situ synthesis.

51. The method of claim 42 wherein free strands of nucleic acid or nucleic acid analogue are covalently attached to the linker.

52. The biosensor of claim 1 which operates in the intrinsic mode.

53. The biosensor of claim 1 wherein emitted fluorescence is captured by the optical element and conveyed to the detector.

54. The biosensor of claim 53 wherein the optical element is an optical fiber.

55. The biosensor of claim 1 wherein the immobilized layer is formed on an interaction surface along the length of the optical element and not at an end of the optical element.

56. The biosensor of claim 55 wherein the optical element is an optical fiber.

57. A biosensor of claim 1 wherein the optical element is a single optical fiber.

58. The method of claim 42 wherein nucleic acids or nucleic acid analogues are attached to the activated interaction surface by in situ synthesis.

59. The method of claim 42 wherein nucleic acids or nucleic acid analogues are attached to the activated interaction surface by initial covalent attachment of linker molecules to the activated interaction surface followed by covalent attachment of the nucleic acids or nucleic acid analogues to the linker molecules.

60. The method of claim 59 wherein the linker molecules contain ethylene glycol subunits.

61. The method of claim 59 wherein the linker is hexaethylene glycol.

38. A method for detecting triplex formation or multistranded nucleic acid formation between one or more target nucleic acids or nucleic acid analogues in a sample and an immobilized nucleic acid or nucleic acid analogue which comprises the steps:

(a) providing a biosensor which comprises

an optical element having an index of refraction and comprising an interaction surface, and an immobilized layer having an index of refraction and including a nucleic acid or nucleic acid analogue covalently attached to the interaction surface of the optical element, wherein the index of refraction of the immobilized layer is equal to or greater than the refractive index of the optical element;

(b) contacting the immobilization layer with a fluorophore;

(b) introducing light into the optical element in contact with the interaction surface such that fluorescence is stimulated and emitted from the fluorophore in the immobilization layer;

(c) detecting fluorescence emitted by the fluorophore in the immobilization layer;

(d) contacting the sample with the immobilization layer of the biosensor such that triplexes or multistranded nucleic acid complexes can be formed and such that fluorophores of the immobilization layer can bind to triplexes or multistranded nucleic acid complexes;

(e) introducing light into the optical element in contact with the interaction surface such that fluorescence is stimulated and emitted from bound fluorophore;

(f) detecting fluorescence emitted by the bound fluorophore;

(g) detecting the difference in fluorescence emitted by the fluorophore before and after triplex or multistranded nucleic acid complex formation

to thereby detect triplex formation or multistranded nucleic acid formation.

39. The method of claim 38 wherein the immobilized layer is contacted with fluorophore by covalently tethering fluorophores to the nucleic acid or nucleic acid analogue in the immobilization layer.

40. The method of claim 38 wherein the optical element is an optical waveguide.

41. The method of claim 38 wherein the target nucleic acid or nucleic acid analogues in the sample is a branched nucleic acid.