Fluorescent polymeric sensor for the detection of urea

• US 5,945,343 A
• Filed: 08/05/1997
• Issued: 08/31/1999
• Est. Priority Date: 08/05/1997
• Status: Expired due to Fees

• Current Assignee: Chiron Corporation (Bayer AG)
• Original Assignee: Bayer Corporation (Bayer AG)
• Inventors: Munkholm, Christiane
• Primary Examiner(s): WALLENHORST, MAUREEN
• Application Number:
  US08/906,518
• Time in Patent Office: 756 Days
• Field of Search: 436/106, 436/108, 436/111, 436/113, 436/164, 436/169, 436/170, 436/172, 436/800, 422/55, 422/56, 422/82.05, 422/82.07, 422/82.08, 422/82.11
• US Class Current: 436/108
• CPC Class Codes: C12Q 1/58 involving urea or urease
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/17 Nitrogen containing
• CPC Class Codes: Y10T 436/171538 Urea or blood urea nitrogen
• CPC Class Codes: Y10T 436/175383 Ammonia

Abstract

The present invention provides a urea sensor material having a first layer of a protonated pH sensitive fluorophore immobilized in a hydrophobic polymer, wherein the fluorophore can react quantitatively with ammonia and the transducing moiety of the fluorophore is neutrally charged when deprotonated; a second layer of urease and a polymer; and a third layer of a polymer. The present invention also provides a method for detecting urea using the urea sensor material and optical sensing devices that incorporate the urea sensor material.

Claims

1. A urea sensor material comprising:

a first layer comprising a pH-sensitive fluorophore immobilized non-aqueously within a first, hydrophobic polymer, wherein the fluorophore is protonated within the first hydrophobic polymer, can react quantitatively with ammonia, and has a transducing moiety that is neutrally charged when deprotonated;

a second layer comprising urease and a second polymer, a first side of the second layer contacting one side of the first layer; and

a third layer comprising a third polymer, the third layer contacting a second side of the second layer.

2. The sensor material of claim 1 wherein the first layer further comprises an onium compound.

3. The sensor material of claim 2 wherein the onium compound is a quaternary ammonium compound.

4. The sensor material of claim 2 wherein the onium compound is tetrabutylammonium hydroxide.

5. The sensor material of claim 1 wherein the fluorophore is acridine orange.

6. The sensor material of claim 1 wherein the fluorophore is a rhodamine dye.

7. The sensor material of claim 1 wherein the first hydrophobic polymer is ethyl cellulose.

8. The sensor material of claim 1 wherein the polymer of the second layer is polyvinyl alcohol.

9. The sensor material of claim 1 wherein the third polymer is polyhydroxyethylmethacrylate.

10. A method for measuring the concentration of urea, comprising:

measuring a fluorescence of the sensor material of any of claims 1-9,

exposing the sensor material to a solution comprising urea,

measuring the fluorescence of the sensor material after the exposing step,

determining a fluorescence change, and

determining the concentration of the urea from the fluorescence change.

11. A process for preparing a urea sensor material comprising:

non-aqueously combining a pH-sensitive fluorophore and a first, hydrophobic polymer to form a first mixture,

wherein the fluorophore is protonated within the first, hydrophobic polymer, can react quantitatively with ammonia, and has a transducing moiety that is neutrally charged when deprotonated;

forming a first layer of the first mixture on a substrate;

combining urease and a second polymer to form a second mixture;

forming a second layer of the second mixture on the first layer; and

forming a third layer of a third polymer on the second layer.

12. The process of claim 11 wherein the combining of the fluorophore and the first hydrophobic polymer is performed in the presence of a solvent.

13. The process of claim 11 wherein the fluorophore is acridine orange.

14. The process of claim 11 wherein the fluorophore is a rhodamine dye.

15. The process of claim 11 wherein the first, hydrophobic polymer is ethyl cellulose.

16. The process of claim 11 wherein the polymer of the second layer is polyvinyl alcohol.

17. The process of claim 11 wherein the polymer of the third layer is polyhydroxyethylmethacrylate.

18. The process of claim 11 wherein the substrate is glass, plastic or ceramic.

19. The process of claim 11 wherein the first mixture further comprises an onium compound.

20. The process of claim 19 wherein the onium compound is a quaternary ammonium compound.

21. The process of claim 19 wherein the onium compound is tetrabutylammonium hydroxide.

22. An optical sensing device for measuring ammonia concentration in a solution comprising urea comprising:

a first layer comprising a pH-sensitive fluorophore immobilized non-aqueously in a first, hydrophobic polymer, wherein the fluorophore is protonated within the first, hydrophobic polymer, can react quantitatively with ammonia, and has a transducing moiety that is neutrally charged when deprotonated, a first side of the first layer contacting a surface of an optical component, which is transparent to incident and emissive electromagnetic waves;

a second layer comprising a urease and a second polymer, a first side of the second layer contacting a second side of the first layer;

a third layer comprising a third polymer, one side of the third layer contacting a second side of the second layer;

wherein the optical component is optically connected to means for collecting radiant emission to measure fluorescence indicative of ammonia concentration.

23. The device of claim 22 wherein the fluorophore is acridine orange.

24. The device of claim 22 wherein the fluorophore is a rhodamine dye.

25. The device of claim 22 wherein the first, hydrophobic polymer is ethyl cellulose.

26. The device of claim 22 wherein the polymer of the second layer is polyvinyl alcohol.

27. The device of claim 22 wherein the third polymer is polyhydroxyethylmethacrylate.

28. The device of claim 22 wherein the optical component is an optical fiber.

29. The device of claim 22 wherein the optical component is a planar waveguide.

30. The device of claim 22 wherein the optical component is an evanescent wave sensor.

31. The device of claim 22 further comprising one or more sensors capable of detecting analytes other than ammonia.

32. A urea sensor material comprising:

a first layer consisting essentially of a pH-sensitive fluorophore immobilized non-aqueously within a first, hydrophobic polymer, wherein the fluorophore is protonated within the first, hydrophobic polymer, can react quantitatively with ammonia, and has a transducing moiety that is neutrally charged when deprotonated;

a second layer comprising urease and a second polymer, a first side of the second layer contacting one side of the first layer; and

a third layer comprising a third polymer, the third layer contacting a second side of the second layer.

34. An optical sensing device for measuring ammonia concentration in a solution comprising urea, comprising the urea sensor material of claim 32 or 33, deposited on a surface of an optical component that is transparent to incident and emissive electromagnetic waves.

33. A urea sensor material comprising:

a first layer consisting essentially of a pH-sensitive fluorophore and an onium compound immobilized non-aqueously within a first, hydrophobic polymer, wherein the fluorophore is protonated within the first, hydrophobic polymer, can react quantitatively with ammonia, and has a transducing moiety that is neutrally charged when deprotonated;

a second layer comprising urease and a second polymer, a first side of the second layer contacting one side of the first layer; and

a third layer comprising a third polymer, the third layer contacting a second side of the second layer.