Multi-color fluorescence enhancement from a photonic crystal surface

• US 20120007000A1
• Filed: 06/28/2011
• Published: 01/12/2012
• Est. Priority Date: 07/08/2010
• Status: Active Grant

• Current Assignee: Board of Trustees of The University of Illinois
• Original Assignee: Board of Trustees of The University of Illinois
• Inventors: Schulz, Stephen C., Cunningham, Brian T., Lu, Meng, Pokhriyal, Anusha
• Granted Patent: US 8,344,333 B2
• Time in Patent Office: Days
• US Class Current: 250/458.1
• 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 21/7743 the reagent-coated grating coupling light in or out of the waveguide

Abstract

A photonic crystal substrate exhibiting resonant enhancement of multiple fluorophores has been demonstrated. The device, which can be fabricated uniformly from plastic materials over a ˜3×5 in² surface area by nanoreplica molding, features a 1-D periodic grating structure which utilizes two distinct resonant modes to enhance electric field stimulation of a first dye excited by a first laser (e.g., λ=632.8 nm laser exciting cyanine-5) and a second dye excited by a second laser (e.g., λ=532 nm laser exciting cyanine-3). The first and second lasers could be replaced by a single variable wavelength (tunable) laser. Resonant coupling of the laser excitation to the photonic crystal surface is obtained for each wavelength at a distinct incident angle θ. The photonic crystal is capable of amplifying the output of any fluorescent dye with an excitation wavelength in a given wavelength range (e.g., the range 532 nm <λ<660 nm) by selection of an appropriate incident angle. The device can be used for biological assays that utilize multiple fluorescent dyes within a single imaged area, such as gene expression microarrays.

Claims

1. Detection apparatus comprising, in combination:

a photonic crystal sensor having a 1-D periodic grating structure designed with a single resonant wavelength that is between the wavelengths of at least two different excitation wavelengths of at least two different fluorescent dyes applied to the sensor surface, and

one or more lasers emitting light at the excitation wavelengths, light from the lasers impinging on the photonic crystal sensor at an incident angle θ

,

wherein a tuning of the incident angle θ

of light from the one or more lasers produces electric field enhancement of the fluorescence from the at least two different fluorescent dyes.

2. The apparatus of claim 1, wherein the lasers emit light at a wavelength of between 530 and 660 nanometers.

3. The apparatus of claim 1, wherein the sensor grating structure has a period Λ

of between 350-400 nm, and a grating depth d of between 20 and 100 nm.

20. The apparatus of claim 1, wherein at least one of the fluorescent dyes are selected from the group of fluorophores consisting of Rhodamine, Texas Red, Cy-5, Cy-3 dyes, and Alexa fluor 532 to 647.

4. A photonic crystal and instrument combination, comprising:

a photonic crystal sensor having a 1-D periodic grating structure and having fluorophores 1 and 2 deposited thereon,

an instrument having first and second lasers emitting light at wavelengths λ

1 and λ

2, respectively, wavelengths λ

1 and λ

2 selected to excite the fluorophores 1 and 2, respectively;

wherein the sensor produces a resonance for incident radiation at wavelengths between λ

1 and λ

2, inclusive within a given range of angles of incidence, and

wherein the angle of incidence θ

of the first and second lasers is selected so as to produce enhanced electric field stimulation of the fluorophores 1 and 2.

5. The sensor and instrument combination of claim 4, wherein the instrument includes a focusing lens and wherein the angle of incidence θ

is selected by changing the orientation of the first and second lasers relative to the focusing lens and thereby changing the location where laser light intersects the focusing lens.

6. The sensor and instrument combination of claim 4, further comprising an imaging device generating images of the sensor under conditions of the enhanced electric field stimulation of the fluorophores 1 or 2 or both.

7. A photonic crystal and instrument combination, comprising:

a photonic crystal sensor having a 1-D periodic grating structure and having fluorophores 1 and 2 deposited thereon,

a detection instrument having a tunable laser emitting light at wavelengths λ

1 and λ

2, wavelengths λ

1 and λ

2 selected to excite the fluorophores 1 and 2, respectively;

wherein the sensor produces a resonance for incident radiation at a wavelength between λ

1 and λ

2 within a given range of incident angles, and wherein the angle of incidence of light from tunable laser onto the sensor is changed to angles of incidence θ

1 and θ

2 so as to produce enhanced electric field stimulation of the fluorophores 1 and 2 at wavelengths λ

1 and λ

2, respectively.

8. The sensor and instrument combination of claim 7, wherein the instrument includes a focusing lens and wherein the angle of incidence θ

is selected by changing the orientation of the tunable laser relative to the focusing lens and thereby changing the location where laser light intersects the focusing lens.

9. The sensor and instrument combination of claim 7, further comprising an imaging device generating images of the sensor under conditions of the enhanced electric field stimulation of the fluorophores 1 or 2.

10. A method of conducting an assay with a photonic crystal sensor, comprising the steps of:

providing a photonic crystal sensor having a 1-D periodic grating structure and having fluorophores 1 and 2 deposited thereon, illuminating the sensor with a first laser emitting light at wavelength λ

1 at a first angle of incidence θ

1 so as to produce enhanced electric field excitation of the first fluorophore;

illuminating the sensor with a second laser emitting light at wavelength λ

2 at a second angle of incidence θ

2 so as to produce enhanced electric field excitation of the second fluorophore.

11. The method of claim 10, further comprising the steps of:

generating an image of the sensor during the enhanced electric field excitation of the first and second fluorophores.

12. The method of claim 10, wherein the assay comprises a gene expression assay.

13. The method of claim 10 wherein fluorophore 1 and fluorophore 2 are excited by light at a wavelength between 530 and 660 nm.

14. The method of claim 10, wherein fluorophore 1 or fluorophore 2 is selected from the group of fluorophores consisting of Rhodamine, Texas Red, Cy-5, Cy-3 dyes, and Alexa fluor 532 to 647.

15. A method of conducting an assay with a photonic crystal sensor, comprising the steps of:

providing a photonic crystal sensor having a 1-D periodic grating structure and having fluorophores 1 and 2 deposited thereon, illuminating the sensor with a tunable laser emitting light at wavelength λ

1 at a first angle of incidence so as to produce enhanced electric field excitation of the first fluorophore;

illuminating the sensor with the tunable laser emitting light at wavelength λ

2 at a second angle of incidence so as to produce enhanced electric field excitation of the second fluorophore.

16. The method of claim 15, further comprising the steps of generating images of the sensor during the enhanced electric field excitation of the first and second fluorophores.

17. The method of claim 15, wherein the assay comprises a gene expression assay.

18. The method of claim 15, wherein fluorophore 1 and fluorophore 2 are excited by light at a wavelength between 530 and 660 nm.

19. The method of claim 15, wherein fluorophore 1 or fluorophore 2 is selected from the group of fluorophores consisting of Rhodamine, Texas Red, Cy-5, Cy-3 dyes, and Alexa fluor 532 to 647.

21. A method of conducting an assay with a photonic crystal sensor, comprising the steps of:

(a) providing a photonic crystal sensor having a 1-D periodic grating structure and having fluorophores 1, 2, . . . N deposited thereon where N is an integer greater than or equal to 3;

(b) illuminating the sensor with a first laser emitting light at wavelength λ

1 at a angle of incidence so as to produce enhanced electric field excitation of the first fluorophore and the second fluorophore; and

(c) illuminating the sensor with light from a second laser emitting light at wavelength λ

2 at a angle of incidence so as to produce enhanced electric field excitation of the third fluorophore.

22. The method of claim 21, wherein N=4 and the method further comprises the step (d) illuminating the sensor with light from the second laser so as to produce enhanced electric field excitation of the fourth fluorophore.

23. The method of claim 22, wherein the fluorophores are bound to nucleotides of DNA or RNA.

24. The method of claim 23, further comprising the step of imaging the photonic crystal sensor during the performing of steps (b), (c), and (d).

25. A photonic crystal sensor and detection arrangement comprising, in combination:

a photonic crystal sensor having a 1-D periodic grating structure and having fluorophores 1, 2, 3 and 4 deposited thereon,

an instrument having first and second lasers emitting light at wavelengths λ

1 and λ

2, respectively, wavelength a λ

1 selected to excite the fluorophores 1 and 2, respectively and wavelength λ

2 selected to excite the fluorophores 3 and 4, respectively;

wherein the sensor produces a resonance for incident radiation at wavelengths between λ

1 and λ

2, inclusive, within a given range of angles of incidence;

wherein the angle of incidence θ

1 of the first laser is selected so as to produce enhanced electric field stimulation of the fluorophores 1 and 2; and

wherein the angle of incidence θ

2 of the second laser is selected so as to produce enhanced electric field stimulation of the fluorophores 3 and 4.