Optochemical fluorescence sensor and method for measuring the concentration of at least one analyte in a sample

• US 5,866,433 A
• Filed: 03/14/1996
• Issued: 02/02/1999
• Est. Priority Date: 03/17/1995
• Status: Expired due to Fees

• Current Assignee: Alfred Leitner, Franz Aussenegg, Fritz Pittner, Georg Bauer, Harald Brunner, Thomas Schalkhammer
• Original Assignee: Avl Medical Instruments AG (Roche Holding AG)
• Inventors: Pittner, Fritz, Schalkhammer, Thomas, Brunner, Harald, Leitner, Alfred, Aussenegg, Franz
• Primary Examiner(s): Chin, Christopher L.
• Application Number:
  US08/616,153
• Time in Patent Office: 1,055 Days
• Field of Search: 422/57, 422/82.05, 422/82.08, 422/82.11, 435/6, 435/287.1, 435/287.2, 435/287.9, 435/288.7, 435/808, 436/518, 436/524, 436/525, 436/527, 436/164, 436/165, 436/172, 436/800, 436/805, 436/807, 385/12, 385/129, 385/131
• US Class Current: 436/525
• CPC Class Codes: G01N 2021/6432 Quenching
• 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 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: Y10S 435/808 Optical sensing apparatus
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures

Abstract

An optochemical fluorescence sensor with a biorecognitive layer for measuring the concentration of one or more analytes in a sample is provided with at least one island layer which is applied on a sensor substrate. The islands of the island layer are in the form of electrically-conductive material and have a diameter of less than 300 nm, the biorecognitive layer being directly applied on the island layer or bound via a spacer film. In addition, an analyte-specific fluorescent compound is provided which may be added to the sample or is provided in the sensor itself. The biorecognitive layer can bind the analyte to be measured directly or by means of analyte-binding molecules, the originally low quantum yield of the fluorescent compound increasing strongly in the vicinity of the island layer.

Claims

1. A method for measuring the concentration of at least one analyte in a sample, comprising the steps of

(a) contacting the sample with a biorecognitive sensor layer, which is applied on or in close vicinity of at least one island layer consisting of islands of electrically-conductive material,

(b) contacting the sample with an analyte-specific fluorescent compound of low quantum yield,

(c) binding the analyte-specific fluorescent compound to the analyte, which in turn is bound by the biorecognitive layer, the quantum yield of the analyte-specific fluorescent compound increasing strongly in the vicinity of the island layer,

(d) radiating excitation radiation into the at least one island layer, which is suitable for excitation of the analyte-specific fluorescent compound,

(e) determining the fluorescence radiation emitted by the bound analyte-specific fluorescent compound as a measure for the analyte concentration.

2. Method according to claim 1, wherein as an alternative to step (c) both the analyte-specific fluorescent compound and the analyte to be measured are bound by the biorecognitive layer, the quantum yield of the analyte-specific fluorescent system increasing strongly in the vicinity of the island layer.

3. A combination of an optochemical fluorescence measuring apparatus and an analyte-specific fluorescent compound for measuring the concentration of an analyte in a sample,

said optochemical fluorescence measuring sensor comprising a substrate, an island layer consisting of islands of electrically-conductive material on the substrate, said islands having a diameter of less than 300 nm;

a biorecognitive layer applied to said island layer;

said biorecognitive layer being capable of binding with said analyte; and

said analyte-specific fluorescent compound having a quantum yield which increases strongly in the vicinity of said island layer.

4. A sensor according to claim 3, wherein said analyte-specific fluorescent compound is a fluorescence-labeled biorecognitive molecule which is able to bind said analyte.

5. A sensor according to claim 4, wherein said analyte-specific fluorescent compound includes a fluorophor having a quantum yield of less than 30%.

6. A sensor according to claim 4, wherein said analyte-specific fluorescent compound comprises two coupled molecules, at least one of which is a fluorophor whose quantum yield is reduced by spatial vicinity to the other molecule quenching the fluorescence of said fluorophor.

7. A sensor according to claim 6, wherein said fluorophor is a fluorescein or a rhodamine.

8. A sensor according to claim 4, wherein the molecules of said fluorophor of said analyte-specific fluorescent compound have a spatial density which leads to strong self-quenching.

9. A sensor according to claim 8, wherein said fluorophor is a fluorescein.

10. A sensor according to claim 3, wherein said analyte-specific fluorescent compound is a fluorescence-labeled analyte analogue which can be bound by said biorecognitive layer.

11. A sensor according to claim 10, wherein said fluorophor of said analyte-specific fluorescent compound has a quantum yield of less than 30%.

12. A sensor according to claim 10, wherein said analyte-specific fluorescent compound comprises two coupled molecules, at least one of which is a fluorophor whose quantum yield is reduced by spatial vicinity to the other molecule quenching the fluorescence of said fluorophor.

13. A sensor according to claim 10, wherein the molecules of said fluorophor of said analyte-specific fluorescent compound have a spatial density which leads to strong self-quenching.

14. A sensor according to claim 3, wherein said islands of said island layer are made of gold or silver.

15. A sensor according to claim 14, wherein the diameter of said islands is smaller than 100 nm.

16. A sensor according to claim 3, wherein the biorecognitive layer on or above said island layer consists of proteins, lipids, nucleic acids, or artificial ligands.

17. A sensor according to claim 3, wherein said island layer has a thickness of less than 25 nm.

18. A sensor according to claim 3, wherein said analyte-specific fluorescent compound contains at least one fluorophor from a group consisting of fuchsines, erythrosines and rhodamines.