Systems and methods for enhancing fluorescent detection of target molecules in a test sample

• US 8,551,786 B2
• Filed: 07/09/2008
• Issued: 10/08/2013
• Est. Priority Date: 07/09/2007
• Status: Expired due to Fees

• Current Assignee: Fio Corporation
• Original Assignee: Fio Corporation
• Inventors: Chan, Warren Che Wor, Klostranec, Jesse M., Jennings, Travis Leon
• Primary Examiner(s): Wallenhorst, Maureen
• Application Number:
  US12/668,264
• Publication Number:
  US 20110053278A1
• Time in Patent Office: 1,917 Days
• Field of Search: 436/501, 436/518, 436/531, 436/532, 436/533, 436/534, 436/535, 436/64, 436/164, 436/172, 436/805, 436/811, 436/813, 422/82.05, 422/82.08, 422/82.09, 435/6.1, 435/6.11, 435/6.14, 435/6.19, 435/7.1, 435/287.1, 435/287.2, 435/288.7, 435/808, 435/968, 356/432, 356/317, 250/458.1, 250/459.1
• US Class Current: 436/172
• 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:

combining one or more first fluorophores and one or more second fluorophores with the test sample, wherein the first fluorophores comprise quantum dots of one or more quantum dot types, so as to secure the first fluorophores and the second fluorophores relative to the target molecules if present in the test sample, and so as to secure the second fluorophores within a predetermined maximum range of the first fluorophores, wherein the predetermined maximum range is less than about 10 micrometers (μ

m); and

irradiating at least the first fluorophores with electromagnetic frequency (EMF) radiation via the irradiating device, such that the first fluorophores absorb the EMF radiation and thereafter emit a first fluorescent signal, such that a radiative flux of the first fluorescent signal is substantially unabated over the predetermined maximum range, and such that if the target molecules are present in the test sample, the second fluorophores absorb a first incident portion of the first fluorescent signal and thereafter emit a second fluorescent signal, with the second fluorescent signal being distinguishable from the first fluorescent signal;

such that the first fluorescent signal is operatively detectable, together with the second fluorescent signal if the target molecules are present in the test sample;

wherein the first fluorophores are characterized by a first fluorophore emission profile corresponding to the first fluorescent signal; and

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

wherein the first fluorophores are bound by microbeads; and

further comprising a step of binding biorecognition molecules (BRMs) with the microbeads and the target molecules, so as to as aforesaid secure the first fluorophores relative to the target molecules if present the test sample; and

further comprising a step of binding marker molecules with the second fluorophores and the target molecules, so as to as aforesaid secure the second fluoro hones relative to the target molecules if present in the test sample.

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

and wherein 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.

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

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

5. A method according to claim 1, wherein the predetermined maximum range is in the order of about 300 nanometers (nm).

6. A method according to claim 1, wherein the target molecules are known to be associated with, and wherein the method is for detection of infectious diseases.

7. A method according to claim 1, wherein the target molecules are known to be associated with, and wherein the method is for detection of cancer.

8. A method according to claim 1, wherein the target molecules are known to be associated with, and wherein the method is for detection of cystic fibrosis.

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

10. A method according to claim 1, wherein the second fluorophores also absorb the EMF radiation, and emit the second fluorescent signal following absorption of the EMF radiation.

11. A method according to claim 1, further comprising a step of combining the microbeads with the BRMs, the test sample, and the second fluorophores.

12. A method according to claim 1, further comprising a step of combining the first fluorophores with the test sample, the marker molecules, and the second fluorophores.

13. A method according claim 1, wherein the second fluorophores also absorb the EMF radiation, and emit the second fluorescent signal following absorption of the EMF radiation;

and further comprising a step of irradiating the first fluorophores and the second fluorophores with the EMF radiation via the irradiating device.

14. A method according to claim 1, further comprising a step of operatively detecting the first fluorescent signal, together with the second fluorescent signal if the target molecules are present in the test sample.

15. 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 characterized by an ability to absorb electromagnetic frequency (EMF) radiation, and by an ability to emit a first fluorescent signal following absorption of the EMF radiation, wherein the first fluorophores comprise quantum dots of one or more quantum dot types; and

(b) one or more second fluorophores characterized by an ability to absorb a first incident portion of the first fluorescent signal, and by an ability to emit 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 further characterized in that;

(i) when operatively combined with the test sample, the first fluorophores and the second fluorophores are secured relative to the target molecules if present in the test sample, such that the second fluorophores are secured within a predetermined maximum range of the first fluorophores, wherein the predetermined maximum range is less than about 10 micrometers (μ

m);

(ii) when at least the first fluorophores are operatively irradiated with the EMF radiation via the irradiating device, the first fluorophores emit the first fluorescent signal, such that a radiative flux of the first fluorescent signal is substantially unabated over the predetermined maximum range, 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;

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;

wherein the first fluorophores are bound by microbeads; and

further comprising biorecognition molecules (BRMs) characterized by an ability 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; and

further comprising marker molecules characterized by an ability 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.

16. A system according to claim 15, 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.

17. A system according to claim 15, wherein the first fluorophores have a higher emission wavelength than the second fluorophores.

18. A system according to claim 15, for use with a laser as the irradiating device, and wherein the EMF radiation has a wavelength of about 488 nanometers (nm).

19. A system according to claim 15, wherein the predetermined maximum range is in the order of about 300 nanometers (nm).

20. A system according to claim 15, wherein the target molecules are known to be associated with, and wherein the system is for detection of infectious diseases.

21. A system according to claim 15, wherein the target molecules are known to be associated with, and wherein the system is for detection of cancer.

22. A system according to claim 15, wherein the target molecules are known to be associated with, and wherein the system is for detection of cystic fibrosis.

23. A system according to claim 15, for use in a biomolecular assay.

24. A system according to claim 15, wherein the second fluorophores are further characterized by an ability to operatively absorb the EMF radiation, and by an ability to operatively emit the second fluorescent signal following absorption of the EMF radiation.

25. A system according to claim 15, wherein the microbeads are operatively combined with the BRMs, the test sample, and the second fluorophores.

26. A system according to claim 25, 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.

27. A system according to claim 15, wherein the first fluorophores are operatively combined with the test sample, the marker molecules, and the second fluorophores.