Detection chip and detection method

1. A detection chip for use in detecting an analyte utilizing surface plasmon resonance, the chip comprising:

a housing section for housing a liquid;

a metal film disposed in a bottom portion of the housing section so that one surface of the metal film faces inside the housing section;

a first reaction site where a ligand for capturing an analyte is immobilized, the first reaction site being disposed on the one surface of the metal film; and

a second reaction site where the ligand is immobilized, the second reaction site being disposed in a different region from the first reaction site on the one surface of the metal film, wherein

when a liquid is housed inside the housing section, a depth of the liquid above the first reaction site is different from a depth of the liquid above the second reaction site, and irradiating the metal film with excitation light while liquid is present generates surface plasmon resonance and different amounts of detectable fluorescence above each of the first and second reaction sites.

2. The detection chip according to claim 1 , wherein

the housing section includes a first bottom surface and a second bottom surface disposed at a different height from a height of the first bottom surface,

the first reaction site is disposed on the metal film above the first bottom surface, and

the second reaction site is disposed on the metal film above the second bottom surface.

3. The detection chip according to claim 1 , wherein

the housing section includes a first upper surface and a second upper surface disposed at a different height from a height of the first upper surface,

the first reaction site is disposed on the metal film in a position facing the first upper surface, and

the second reaction site is disposed on the metal film in a position facing the second upper surface.

4. The detection chip according to claim 1 , wherein

a bottom surface of the housing section contains a sloping surface, and

the first reaction site and the second reaction site are disposed on the metal film above the sloping surface.

5. The detection chip according to claim 1 , wherein

the housing section includes an upper surface containing a sloping surface relative to a bottom surface, and

the first reaction site and the second reaction site are disposed on the metal film in a position facing the sloping surface.

6. The detection chip according to claim 1 , wherein the metal film includes a diffraction grating in a region corresponding to the first reaction site and the second reaction site.

7. A detection method for detecting an analyte utilizing surface plasmon resonance, the method comprising:

a first step of binding an analyte labeled with a fluorescent substance to the ligand in the first reaction site and in the second reaction site inside the housing section of the detection chip according to claim 6 ;

a second step of irradiating the metal film positioned under the first reaction site with excitation light so as to generate surface plasmon resonance while the liquid is present inside the housing section in a first depth of the liquid above the first reaction site, and detecting fluorescence emitted from the fluorescent substance present above the first reaction site;

a third step of irradiating the metal film positioned under the second reaction site with excitation light so as to generate surface plasmon resonance while the liquid is present inside the housing section in a second depth of the liquid different from the first depth above the second reaction site, and detecting fluorescence emitted from the fluorescent substance present above the second reaction site; and

a fourth step of calculating a signal value which indicates the presence or an amount of an analyte based on a detected value obtained in each of the second step and the third step.

8. The detection method according to claim 7 , wherein

in the second step and in the third step, the diffraction grating is irradiated with excitation light; and

in the fourth step, a signal value I i which is represented by the following equation 1 and indicates the presence or an amount of an analyte is calculated based on a detected value I a obtained in the second step and a detected value I b obtained in the third step:

(

Equation

⁢

⁢

1

)

I

1

=

m

×

I

a

-

I

b

m

-

1

[

1

]

wherein m is a ratio of the second depth to the first depth, and is a positive real number excluding 1.

9. The detection method according to claim 8 , wherein

in the fourth step, a noise value I 2 originated from light which is contained in the fluorescence and generated without being influenced by an enhanced electric field due to the surface plasmon resonance is further calculated according to the following equation 3:

(

Equation

⁢

⁢

3

)

I

2

=

I

b

-

I

a

m

-

1

.

[

3

]

10. The detection chip according to claim 1 , further comprising a prism formed from a dielectric, wherein the metal film is disposed on one surface of the prism which constitutes a bottom surface of the housing section.

11. A detection method for detecting an analyte utilizing surface plasmon resonance, the method comprising:

a first step of binding an analyte labeled with a fluorescent substance to the ligand in the first reaction site and in the second reaction site inside the housing section of the detection chip according to claim 10 ;

a second step of irradiating the metal film positioned under the first reaction site with excitation light so as to generate surface plasmon resonance while the liquid is present inside the housing section in a first depth of the liquid above the first reaction site, and detecting fluorescence emitted from the fluorescent substance present above the first reaction site;

a third step of irradiating the metal film positioned under the second reaction site with excitation light so as to generate surface plasmon resonance while the liquid is present inside the housing section in a second depth of the liquid different from the first depth above the second reaction site, and detecting fluorescence emitted from the fluorescent substance present above the second reaction site; and

a fourth step of calculating a signal value which indicates the presence or an amount of an analyte based on a detected value obtained in each of the second step and the third step.

12. The detection method according to claim 11 , wherein

in the second step, a rear surface of the metal film corresponding to the first reaction site is irradiated with excitation light through the prism;

in the third step, a rear surface of the metal film corresponding to the second reaction site is irradiated with excitation light through the prism; and

in the fourth step, a signal value Ii which is represented by the following equation 2 and indicates the presence or an amount of an analyte is calculated based on a detected value I a obtained in the second step and a detected value I b obtained in the third step:

(

Equation

⁢

⁢

2

)

I

1

=

m

×

I

a

-

I

b

m

-

1

[

2

]

wherein m is a ratio of the second depth to the first depth, and is a positive real number excluding 1.

13. The detection method according to claim 12 , wherein in the fourth step, a noise value I 2 originated from light which is contained in the fluorescence and generated without being influenced by an enhanced electric field due to the surface plasmon resonance is further calculated according to the following equation 3:

(

Equation

⁢

⁢

3

)

I

2

=

I

b

-

I

a

m

-

1

.

[

3

]

14. A detection method for detecting an analyte utilizing surface plasmon resonance, the method comprising:

a first step of binding an analyte labeled with a fluorescent substance to the ligand in the first reaction site and in the second reaction site inside the housing section of the detection chip according to claim 1 ;

a second step of irradiating the metal film positioned under the first reaction site with excitation light so as to generate surface plasmon resonance while the liquid is present inside the housing section in a first depth of the liquid above the first reaction site, and detecting fluorescence emitted from the fluorescent substance present above the first reaction site;

a third step of irradiating the metal film positioned under the second reaction site with excitation light so as to generate surface plasmon resonance while the liquid is present inside the housing section in a second depth of the liquid different from the first depth above the second reaction site, and detecting fluorescence emitted from the fluorescent substance present above the second reaction site; and

a fourth step of calculating a signal value which indicates the presence or an amount of an analyte based on a detected value obtained in each of the second step and the third step.

15. The detection method according to claim 14 , wherein

in the second step and in the third step, the fluorescence is detected continually; and

in the fourth step, the signal value is calculated continually based on a detected value obtained in each of the second step and the third step.