SYSTEMS AND METHODS FOR ENHANCING FLUORESCENT DETECTION OF TARGET MOLECULES IN A TEST SAMPLE

• US 20110053278A1
• Filed: 07/09/2008
• Published: 03/03/2011
• Est. Priority Date: 07/09/2007
• Status: Active Grant

• Current Assignee: Fio Corporation
• Original Assignee: Fio Corporation
• Inventors: Chan, Warren Che Wor, Klostranec, Jesse M., Jennings, Travis Leon
• Granted Patent: US 8,551,786 B2
• Time in Patent Office: Days
• US Class Current: 436/64
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C40B 30/10 by measuring physical properties, e.g. mass
• CPC Class Codes: C40B 70/00 Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
• CPC Class Codes: G01N 2021/6432 Quenching
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/588 with semiconductor nanocrystal label, e.g. quantum dots

Abstract

Systems and methods for enhancing fluorescent detection of target molecules in a test sample are for use with an irradiating device. First fluorophores are provided for absorption of EMF radiation, and emission of a first signal. Second fluorophores are provided for partial absorption of the first signal, and emission of a second signal distinguishable from the first signal. The fluorophores are combined with the test sample, and secured to the target molecules and relative to one another. After the first fluorophores receive the EMF radiation from the irradiating device, the first signal is detected, together with the second spectral signal if the target molecules are present in the test sample.

Claims

1. A method of enhancing fluorescent detection of target molecules in a test sample, for use with an irradiating device, the method comprising the steps of:

(a) providing one or more first fluorophores operatively adapted for absorption of electromagnetic frequency (EMF) radiation, and for emission of a first fluorescent signal following absorption of the EMF radiation; and

(b) providing one or more second fluorophores operatively adapted for absorption of a first incident portion of the first fluorescent signal, and for emission of a second fluorescent signal following absorption of the first incident portion, with the second fluorescent signal being distinguishable from the first fluorescent signal;

wherein the first fluorophores and the second fluorophores are adapted for operative combination with the test sample, and for securement relative to the target molecules, if present in the test sample, so as to secure the first fluorophores relative to the second fluorophores; and

wherein, following operative irradiation of at least the first fluorophores with the EMF radiation via the irradiating device, the first fluorophores emit the first fluorescent signal and, if the target molecules are present in the test sample, the second fluorophores absorb the first incident portion of the first fluorescent signal and emit the second fluorescent signal;

whereby the first fluorescent signal is operatively detectable, together with the second fluorescent signal if the target molecules are present in the test sample.

2. A method according to claim 1, wherein in step (a), the first fluorophores are characterized by a first fluorophore emission profile corresponding to the first fluorescent signal;

and wherein in step (b), the second fluorophores are characterized by a second fluorophore absorption profile which substantially overlaps with the first fluorophore emission profile.

4. A method according to claim 1, wherein in step (a), the first fluorophores are characterized by a first fluorophore absorption profile substantially corresponding to the EMF radiation;

and wherein in step (b), the second fluorophores are characterized by a second fluorophore emission profile, corresponding to the second fluorescent signal, which is substantially removed from the first fluorophore absorption profile.

5. A method according to claim 1, wherein in step (a), the first fluorophores are bound by microbeads;

and further comprising step (c), after step (a), of providing biorecognition molecules (BRMs) adapted to operatively bind with the microbeads and the target molecules, so as to secure the first fluorophores relative to the target molecules if present in the test sample.

8. A method according to claim 5, wherein in step (a), the first fluorophores comprise quantum dots of one or more quantum dot types.

11. A method according to claim 1, wherein in step (b), the second fluorophores are adapted for substantially direct operative binding with the target molecules.

12. A method according to claim 1, further comprising step (d), after step (b), of providing marker molecules adapted to operatively bind with the second fluorophores and the target molecules, so as to secure the second fluorophores relative to the target molecules if present in the test sample.

18. A method according to claim 1, wherein the first fluorophores have a higher emission wavelength than the second fluorophores.

19. A method according to claim 1 for use with a laser as the irradiating device, and wherein in step (a), the EMF radiation has a wavelength of about 488 nanometers (nm).

20. A method according to claim 1, wherein following operative combination of the first fluorophores and the second fluorophores with the test sample, the target molecules, if present in the test sample, secure the second fluorophores within a predetermined maximum range of the first fluorophores, with a radiative flux of the first spectral signal being substantially unabated over the predetermined maximum range.

23. A method according to claim 1, for detection of infectious diseases.

24. A method according to claim 1, for detection of cancer.

25. A method according to claim 1, for detection of cystic fibrosis.

26. A method according to claim 1, for use in a biomolecular assay.

28. A method according to claim 1, wherein in step (b), the second fluorophores are also operatively adapted for absorption of the EMF radiation, and for emission of the second fluorescent signal following absorption of the EMF radiation.

29. A method according to claim 1, further comprising step (e), after step (b), of operatively combining the first fluorophores with the test sample and the second fluorophores.

30. A method according to claim 5, further comprising step (e), after step (c), of operatively combining the microbeads with the BRMs, the test sample, and the second fluorophores.

31. A method according to claim 12, further comprising step (e), after step (d), of operatively combining the first fluorophores with the test sample, the marker molecules, and the second fluorophores.

32. A method according to claim 29, further comprising step (f), after step (e), of operatively irradiating at least the first fluorophores with the EMF radiation via the irradiating device.

33. A method according claim 29, wherein in step (b), the second fluorophores are also operatively adapted for absorption of the EMF radiation, and for emission of the second fluorescent signal following absorption of the EMF radiation;

and further comprising step (0, after step (e), of operatively irradiating the first fluorophores and the second fluorophores with the EMF radiation via the irradiating device.

34. A method according to claim 32, further comprising step (g), after step (f), of operatively detecting the first spectral signal, together with the second spectral signal if the target molecules are present in the test sample.

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35. A system for enhancing fluorescent detection of target molecules in a test sample, for use with an irradiating device, the system comprising:

(a) one or more first fluorophores operatively adapted for absorption of electromagnetic frequency (EMF) radiation, and for emission of a first fluorescent signal following absorption of the EMF radiation; and

(b) one or more second fluorophores operatively adapted for absorption of a first incident portion of the first fluorescent signal, and for emission of a second fluorescent signal following absorption of the first incident portion, with the second fluorescent signal being distinguishable from the first fluorescent signal;

wherein the first fluorophores and the second fluorophores are adapted for operative combination with the test sample, and for securement relative to the target molecules, if present in the test sample, so as to secure the first fluorophores relative to the second fluorophores; and

wherein, following operative irradiation of at least the first fluorophores with the EMF radiation via the irradiating device, the first fluorophores emit the first fluorescent signal and, if the target molecules are present in the test sample, the second fluorophores absorb the first incident portion of the first fluorescent signal and emit the second fluorescent signal;

whereby the first fluorescent signal is operatively detectable, together with the second fluorescent signal if the target molecules are present in the test sample.

36. A system according to claim 35, wherein the first fluorophores are characterized by a first fluorophore emission profile corresponding to the first fluorescent signal;

and the second fluorophores are characterized by a second fluorophore absorption profile which substantially overlaps with the first fluorophore emission profile.

38. A system according to claim 35, wherein the first fluorophores are characterized by a first fluorophore absorption profile substantially corresponding to the EMF radiation;

and the second fluorophores are characterized by a second fluorophore emission profile, corresponding to the second fluorescent signal, which is substantially removed from the first fluorophore absorption profile.

39. A system according to claim 35, wherein the first fluorophores are bound by microbeads;

and further comprising biorecognition molecules (BRMs) adapted to operatively bind with the microbeads and the target molecules, so as to secure the first fluorophores relative to the target molecules if present in the test sample.

42. A system according to claim 39, wherein the first fluorophores comprise quantum dots of one or more quantum dot types.

45. A system according to claim 35, wherein the second fluorophores are adapted for substantially direct operative binding with the target molecules.

46. A system according to claim 35, further comprising marker molecules adapted to operatively bind with the second fluorophores and the target molecules, so as to secure the second fluorophores relative to the target molecules if present in the test sample.

52. A system according to claim 35, wherein the first fluorophores have a higher emission wavelength than the second fluorophores.

53. A system according to claim 35, for use with a laser as the irradiating device, and wherein the EMF radiation has a wavelength of about 488 nanometers (nm).

54. A system according to claim 35, wherein following operative combination of the first fluorophores and the second fluorophores with the test sample, the target molecules, if present in the test sample, secure the second fluorophores within a predetermined maximum range of the first fluorophores, with a radiative flux of the first spectral signal being substantially unabated over the predetermined maximum range.

57. A system according to claim 35, for detection of infectious diseases.

58. A system according to claim 35, for detection of cancer.

59. A system according to claim 35, for detection of cystic fibrosis.

60. A system according to claim 35, for use in a biomolecular assay.

62. A system according to claim 35, wherein the second fluorophores are also operatively adapted for absorption of the EMF radiation, and for emission of the second fluorescent signal following absorption of the EMF radiation.

63. A system according to claim 39, wherein the microbeads are operatively combined with the BRMs, the test sample, and the second fluorophores.

64. A system according to claim 46, wherein the first fluorophores are operatively combined with the test sample, the marker molecules, and the second fluorophores.

65. A system according to claim 63, wherein the second fluorophores are also operatively adapted for absorption of the EMF radiation, and for emission of the second fluorescent signal following absorption of the EMF radiation;

and wherein the first fluorophores and the second fluorophores are operatively irradiated with the EMF radiation via the irradiating device.

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