Evanescent wave immunoassay system

• US 5,492,674 A
• Filed: 03/17/1995
• Issued: 02/20/1996
• Est. Priority Date: 03/17/1995
• Status: Expired due to Term

• Current Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Original Assignee: Boehringer Mannheim Corporation (Roche Holding AG)
• Inventors: Meserol, Peter
• Primary Examiner(s): Snay, Jeffrey R.
• Application Number:
  US08/406,065
• Time in Patent Office: 340 Days
• Field of Search: 436/518, 436/527, 436/165, 422/82.05, 422/82.08, 422/55, 422/56, 422/58, 422/82.11, 422/102, 356/244, 385/12, 385/137
• US Class Current: 422/82.08
• 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

Abstract

Apparatus for assaying a fluid sample with radiation capable of exciting fluorescence in fluorescent material comprises a totally internally reflecting, unitary elongated substrate transmissive to both the excitation radiation and to the fluorescence. The fluorescent material includes at least a moiety of an antibody-antigen complex that includes a tag that will provide the fluorescence when excited by an evanescent wave generated by the excitation radiation. The substrate includes an elongated fiber and an integral lens formed to guide the optical radiation into the fiber within the bounds of a critical angle to assure total internal reflectance. A hollow elongated enclosure is disposed concentrically about and spaced from the fiber so as to provide an interspace of capillary dimensions. A test implement supports the elongated substrate, has a reservoir for receiving a fluid sample to be assayed, and is selectively movable to an operative position whereat the fluid sample is caused to flow onto the fiber and, by capillary action, into the interspace between the fiber and the enclosure. The apparatus further includes a source of the excitation radiation, refractive element for focusing the radiation toward the lens, detector for detecting fluorescence radiation emitted from an end of the fiber responsively to the excitation radiation, and optics frame for mounting all components in fixed relation to the lens for introducing the excitation radiation into the lens within the critical angle.

Claims

1. Single use disposable apparatus for assaying a fluid sample with excitation radiation from a radiation source, which radiation is capable of exciting fluorescence in fluorescent material, said apparatus comprising:

a totally internally reflecting, unitary elongated substrate transmissive to both the excitation radiation and to the fluorescence, said substrate including an elongated fiber and integral refractive means into which the radiation is to be introduced and at least a portion of the surface of which is adapted to contact the sample, said refractive means being formed to guide the optical radiation into said fiber within the bounds of a critical angle to assure total internal reflectance;

a coating of the fluorescent material on at least a portion of the surface of said fiber;

a hollow elongated enclosure disposed about and spaced from said fiber so as to provide an interspace of capillary dimension between said fiber and said enclosure, said elongated enclosure extending from a location adjacent said lens to an extreme end distant therefrom;

test implement means for supporting said elongated substrate and having reservoir means for receiving a fluid sample to be assayed and being movable between an inoperative position whereat the fluid sample is spaced from the elongated substrate and an operative position whereat the fluid sample is caused to flow onto said fiber and, by capillary action, into the interspace between said fiber and said enclosure.

2. Apparatus as set forth in claim 1 wherein said integral refractive means includes a prism.

3. Apparatus as set forth in claim 1 wherein said integral refractive means is of the same material as said elongated fiber.

4. Apparatus as set forth in claim 1 wherein said test implement means includes:

an internal region defining a first reservoir for initially containing the fluid sample;

support means for supporting said elongated substrate distant from the fluid sample in the first reservoir;

boss means defining a second reservoir for temporarily containing the fluid sample; and

channel means extending between the first and second reservoirs for directing flow of the fluid sample from the first reservoir to the second reservoir when said test implement means is moved from the inoperative position to the operative position.

5. Apparatus as set forth in claim 1 wherein the fluorescent material includes at least a moiety of an antibody-antigen complex that includes a tag that will provide the fluorescence when excited by an evanescent wave generated by the excitation radiation.

6. Apparatus as set forth in claim 1 wherein said fiber is disposed coaxially with said enclosure.

7. Apparatus as set forth in claim 1 wherein said elongated enclosure is opaque and includes at least three longitudinally extending ridges equally spaced, circumferentially, for supportive engagement with said fiber.

8. Apparatus as set forth in claim 1 wherein said elongated enclosure is opaque and includes at least three longitudinally extending similarly dimensioned sidewalls for supportive tangential engagement with said fiber.

9. Apparatus as set forth in claim 1 wherein said integral refractive means includes a lens.

10. Apparatus as set forth in claim 9 wherein said lens includes a spherical lens having an index of refraction matched to that of said fiber.

11. Apparatus as set forth in claim 1 including:

a source of the excitation radiation;

said refractive means being operable for focusing said radiation toward said lens;

detector means for detecting fluorescence radiation emitted from an end of said fiber responsively to the excitation radiation; and

optics frame means for mounting said source, said refractive means, and said detector means in fixed relation to said lens for introducing the excitation radiation into said lens within the critical angle.

12. Apparatus as set forth in claim 11 including:

test implement means for operatively supporting said substrate on said optics frame means.

13. Apparatus as set forth in claim 1 wherein said test implement means includes:

a housing having opposed first and second endwalls, said first endwall having an aperture therethrough for slidably receiving said elongated fiber and for supporting said elongated fiber adjacent said refractive means;

an internal region intermediate said first and second endwalls defining a first reservoir for initially containing the fluid sample;

boss means on said second endwall facing said first endwall having a cylindrical internal surface for slidable reception of said extremity of said elongated enclosure, said cylindrical internal surface defining a second reservoir therein for temporarily containing the liquid sample; and

channel means extending between the first and second reservoirs for directing flow of the fluid sample from the first reservoir to the second reservoir when said test implement means moves from the inoperative position to the operative position.

14. Apparatus as set forth in claim 13 wherein said elongated enclosure has an annular rim at said extreme end;

and including;

bibulous mass means received in the second reservoir and fittingly engaged with said cylindrical internal surface of said boss means, said bibulous mass means being initially distant from said annular rim, said bibulous mass means being expandable by infusion therein of the fluid sample when said test implement is moved from the inoperative position to the operative position and thereby caused to expand to engage said annular end, such that the fluid is caused to flow onto said fiber and, by capillary action, into the interspace between said fiber and said enclosure.

15. Apparatus as set forth in claim 13 including:

bibulous mass means received in the second reservoir and fittingly engaged with said cylindrical internal surface of said boss means, said elongated enclosure having an annular rim at said extreme end in engagement with said bibulous mass means.

16. Apparatus as set forth in claim 15 wherein said bibulous mass means includes a polymeric open cell foam cylinder.

17. Apparatus as set forth in claim 1 wherein said test implement means includes:

a housing having a bottom wall defining a first reservoir for initially containing the fluid sample, opposed first and second endwalls upstanding from and integral with said bottom wall, said first endwall having an aperture therethrough for slidably receiving said elongated fiber and for supporting said elongated fiber adjacent said refractive means, and opposed first and second side walls upstanding from and integral with said bottom wall, said sidewalls and said endwalls being integral with one another;

c-shaped boss means on said second endwall projecting toward said first endwall having a cylindrical internal surface for slidable reception of said extremity of said elongated enclosure, said cylindrical internal surface defining a second reservoir therein; and

channel means including a transverse slot in said boss means extending between the first and second reservoirs for directing flow of the fluid sample from the first reservoir to the second reservoir when said test implement means moves from the inoperative position to the operative position.

18. Apparatus as set forth in claim 17 including:

bibulous mass means received in the second reservoir and fittingly engaged with said cylindrical internal surface of said boss means, said elongated enclosure having an annular rim at said extreme end in engagement with said bibulous mass means.

19. Apparatus as set forth in claim 18 wherein said bibulous mass means includes a polymeric open cell foam cylinder.

20. Apparatus as set forth in claim 1 wherein said test implement means includes:

a housing including;

a bottom wall defining a first reservoir for initially containing the fluid sample;

a first endwall upstanding from and integral with said bottom wall, said first endwall having an aperture therethrough for slidably receiving said elongated fiber and for supporting said elongated fiber adjacent said refractive means;

opposed first and second side walls upstanding from and integral with said bottom wall, said sidewalls and said first endwall being integral with one another;

a second endwall;

hinge means pivotally mounting said second endwall to said bottom wall for movement between a lowered position generally coplanar with said bottom wall and an upright position upstanding from said bottom wall;

mutually engageable locking means on said sidewalls and on said second endwall for maintaining said second endwall in the upright position;

boss means on said second endwall facing said first endwall having a cylindrical internal surface for slidable reception of said extremity of said elongated enclosure, said cylindrical internal surface defining a second reservoir therein; and

channel means extending between the first and second reservoirs for directing flow of the fluid sample from the first reservoir to the second reservoir when said second endwall moves from the lowered position to the upright position.

21. Apparatus as set forth in claim 20 including:

bibulous mass means received in the second reservoir and fittingly engaged with said cylindrical internal surface of said boss means.

22. Apparatus as set forth in claim 21 wherein said bibulous mass means includes a polymeric open cell foam cylinder.

23. A single use disposable system for assaying a fluid sample with excitation radiation from a radiation source, which radiation is capable of exciting fluorescence in fluorescent material and recovering a coupled evanescent wave, said apparatus comprising:

a totally internally reflecting, unitary elongated substrate transmissive to both the excitation radiation and to the fluorescence, said substrate including an elongated fiber and integral refractive means into which the radiation is to be introduced and at least a portion of the surface of which is adapted to contact the sample, said refractive means being formed to guide the optical radiation into said fiber within the bounds of a critical angle to assure total internal reflectance;

a coating of the fluorescent material on at least a portion of the surface of said fiber;

a hollow elongated enclosure disposed about and spaced from said fiber so as to provide an interspace of capillary dimension between said fiber and said enclosure, said elongated enclosure extending from a location adjacent said lens to an extreme end distant therefrom;

test implement means for supporting said elongated substrate and having reservoir means for receiving a fluid sample to be assayed and being movable between an inoperative position whereat the fluid sample is spaced from the elongated substrate and an operative position whereat the fluid sample is caused to flow onto said fiber and, by capillary action, into the interspace between said fiber and said enclosure;

optics frame means including a platform for supporting said test implement means and being selectively movable to move said test implement means between the inoperative position and the operative position;

collimated light source means for directing an interrogating beam into said refractive means when said test implement means is in the operative position; and

detector means for receiving reflected light radiation from said substrate through said refractive means coupled to an evanescent wave.

24. A system as set forth in claim 23 wherein said collimated light source means includes a laser.

25. A system as set forth in claim 23 wherein said detector means includes a photodetector.