Sol-gel encapsulated enzyme

• US 5,200,334 A
• Filed: 08/13/1991
• Issued: 04/06/1993
• Est. Priority Date: 08/13/1991
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Nishida, Fumito, Zink, Jeffrey I., Ellerby, Lisa, Dunn, Bruce S., Yamanaka, Stacey A., Valentine, Joan S., Nishida, Clinton
• Primary Examiner(s): Griffin, Ronald W.
• Assistant Examiner(s): GITOMER, RALPH J
• Application Number:
  US07/744,524
• Time in Patent Office: 602 Days
• Field of Search: 435/176, 435/177, 435/180, 435/182, 435/25, 435/12, 501/12
• US Class Current: 435/182
• CPC Class Codes: C03C 4/0007 for biologically-compatible glass
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: G01N 33/5436 with ligand physically entrapped within the solid phase liposomes G01N33/5432; immunological test elements G01N33/54386
• CPC Class Codes: G01N 33/552 Glass or silica
• CPC Class Codes: G01N 33/585 with a particulate label, e.g. coloured latex

Abstract

An active biological material encapsulated in a glass is formed using a sol-gel process. A metal alkoxide is mixed with water and exposed to ultrasonic energy at a pH≦2 to form a single phase solution which is then buffered to a pH between about 5 and 7. The buffered solution is then mixed with the active biological material and the resultant gel is aged and dried. The dried product is a transparent porous glass with substantially all of the added active biological material encapsulated therein, the biological material retaining a high level of activity.

Claims

1. A process for the production of a porous, transparent sol-gel glass containing an alcohol sensitive active biological material entrapped therein comprising:

(a) forming a single phase sol by mixing a metal alkoxide in a non-alcoholic medium comprising water and an acid catalyst in a container exposed to ultrasonic energy, the mixture having a pH not greater than about 2;

(b) removing the ultrasonic energy and raising the pH of the sol to about 5 to 7 by the addition of a buffering agent;

(c) adding said active biological material to the buffered sol;

(d) forming a gel and allowing the gel to age; and

(e) allowing at least a portion of the water in the gel to evaporate so that the volume of the product produced in step (d) is decreased and the active biological material is trapped in a monolith of the gel having a reduced volume.

2. The process of claim 1 wherein a pre-buffer aging step is added after exposing the mixture to ultrasound but prior to buffering the mixture.

3. The process of claim 1 wherein the gel is aged for at least about ten days.

4. The process of claim 1 wherein step(e) continues for at least about ten days.

5. The process of claim 1 wherein the active biological material is a protein.

6. The process of claim 1 wherein the active biological material is selected from the group consisting of nuclease, protease, oxidase, esterase, isomerase, metal and metal ion binders, bicarbonate binders, free radical inhibitors, reversible oxygen binders and combinations thereof.

7. The process of claim 1 wherein the active biological material is selected from the group consisting of RNase A, RNase T1, protease k, chymotrypsin, alcohol oxidase, glucose oxidase, acetylcholine esterase, phosphodiesterase II, aldolase, glucose isomerase, hemoglobin, myoglobin, cytochrome c, aequorin, transferase, urease, superoxide dismutase and combinations thereof.

8. The process of claim 1 wherein the resultant product is a three dimensional object with a smallest dimension being from about 1000 Angstroms to about 0.5 cm.

9. The process of claim 1 wherein the volume of the product of step (e) is reduced to about twelve percent (12%) of the volume of the product of step (d).

10. The process of claim 1 wherein the gel is aged for about 2 days to about 21 days.

11. The process of claim 1 wherein the aged gel is dried for about 8 days to about 60 days.

12. A porous, transparent sol-gel glass having entrapped therein an active biological material wherein the glass is prepared by:

(a), forming a single phase sol by mixing a metal alkoxide in a non-alcoholic medium comprising water and an acid catalyst and exposing the mixture to ultrasonic energy, the mixture having a pH not greater than about 2;

(b) removing the ultrasonic energy and raising the pH of the sol to about 5 to 7 by the addition of a buffering agent;

(c) adding an active biological material to the buffered sol;

(d) forming a gel and allowing the gel to age; and

(e) allowing at least a portion of the water in the gel to evaporate so that external dimensions of the gel produced in step (d) are reduced and the active biological material is held within the structure after evaporation.

13. The porous transparent sol-gel glass of claim 12 wherein a pre-buffer aging step is added after exposing the mixture to ultrasound but prior to buffering the mixture.

14. The porous transparent glass of claim 12 wherein the gel is aged for at least about ten days.

15. The porous transparent glass of claim 14 wherein the aged gel is dried for at least about ten days.

16. The porous transparent glass of claim 12 wherein the active biological material is a protein.

17. The porous transparent glass of claim 12 wherein the active biological material is selected from the group consisting of nuclease, protease, oxidase, esterase, isomerase, metal and metal ion binders, bicarbonate binders, free radical inhibitors, reversible oxygen binders and combinations thereof.

18. The porous transparent glass of claim 12 wherein the active biological material is selected from the group consisting of RNase A, RNase T1, protease k, chymotrypsin, alcohol oxidase, glucose oxidase, acetylcholine esterase, phosphodiesterase II, aldolase, glucose isomerase, hemoglobin, myoglobin, cytochrome c, aequorin, transferase, urease, superoxide dismutase and combinations thereof.

19. The porous transparent sol-gel glass of claim 12 wherein the volume of the structure produced by evaporation is reduced to about twelve percent (12%) of the volume of the gel produced in step (d).

20. The porous transparent glass of claim 12 wherein the gel is aged for about 2 days to about 21 days.

21. The porous glass of claim 12 wherein the aged gel is dried for about 8 days to about 60 days.

22. A porous, transparent sol-gel glass having an apparent density of 1.2 g/cc, a specific surface area of about 400 to 800 m² /g, a median pore radius of about 15 Angstroms and a maximum pore radius of about 100 Angstroms, the porous, transparent glass having an active biological material entrapped therein.

23. The porous, transparent sol-gel glass of claim 22 wherein the active biological material is selected from the group consisting of nuclease, protease, oxidase, esterase, isomerase, metal and metal ion binders, bicarbonate binders, free radical inhibitors, reversible oxygen binders and combinations thereof.

24. The porous transparent sol-gel glass of claim 22 wherein the active biological material is selected from the group consisting of RNase A, RNase T1, protease k, chymotrypsin, alcohol oxidase, glucose oxidase, acetylcholine esterase, phosphodiesterase II, aldolase, glucose isomerase, hemoglobin, myoglobin, cytochrome c, aequorin, transferase, urease, superoxide dismutase and combinations thereof.

25. A method for the qualitative or quantitative detection of a substance which reacts with or whose reaction is catalyzed by an active biological material which comprises:

(1) The production of a porous, transparent sol-gel glass containing an active biological material entrapped therein comprising;

(a) forming a single phase sol by mixing a metal alkoxide in a non-alcoholic medium comprising water and an acid catalyst in a container exposed to ultrasonic energy, the mixture having a pH not greater than about 2;

(b) removing the ultrasonic energy and raising the pH of the sol to about 5 to 7 by the addition of a buffering agent;

(c) adding an active biological material to the buffered sol;

(d) forming a gel and allowing the gel to age; and

(e) allowing at least a portion of the water in the gel to evaporate causing the gel to shrink and the active biological material to be trapped in the porous glass.

(2) Brining the porous, transparent glass containing the active biological material into contact with an aqueous solution of the substance; and

(3) Observing the change in optical characteristics of the porous, transparent glass containing an active biological material.

26. The method of claim 25 wherein the change in optical characteristics is observed using spectroscopic techniques.

27. The method of claim 26 wherein the spectroscopic techniques are selected from the group consisting of UV, IR, visible light, fluorescence, luminescence, absorption, emission and reflection techniques.

28. The method of claim 25 wherein a pre-buffer aging step is added after exposing the mixture to ultrasound but prior to buffering the mixture.

29. The method of claim 25 wherein the gel is aged for at least about ten days

30. The method of claim 29 wherein the aged gel is dried for at least about ten days.

31. The method of claim 25 wherein the active biological material is a protein.

32. The method of claim 25 wherein the active biological material is selected from the group consisting of nuclease, protease, oxidase, esterase, isomerase, metal and metal ion binders, bicarbonate binders, free radical inhibitors, reversible oxygen binders and combinations thereof.

33. The method of claim 25 wherein the active biological material is selected from the group consisting of RNase A, RNase T1, chymotrypsin, alcohol oxidase, glucose oxidase, acetylcholine esterase, phosphodiesterase II, aldolase, glucose isomerase, hemoglobin, myoglobin, cytochrome c, aequorin, transferase, urease, superoxide dismutase and combinations thereof.

34. The method of claim 25 wherein the porous transparent sol-gel glass is prepared in the form of a film with a thickness of greater than about 1000 Angstroms.

35. The method of claim 25 wherein the porous transparent sol-gel glass is prepared in the form of a monolith with a smallest dimension less than about 0.5 centimeters.

36. The method of claim 26 wherein the porous transparent sol-gel glass is prepared in the form of a three dimensional structure having a shortest dimension between about 1000 Angstroms and about 0.5 centimeters.

37. The method of claim 25 wherein the porous glass formed in step (1) (e) has a volume of about twelve percent (12%) of the volume of the gel formed in step (1) (d).

38. The method of claim 25 wherein the gel is aged for about 2 days to about 21 days.

39. The method of claim 25 wherein the aged gel is dried for about 8 days to about 60 days.

40. A process for the production of a porous, transparent aged gel containing an alcohol sensitive active biological material entrapped therein comprising:

(a) forming a single phase sol by mixing a metal alkoxide in a non-alcoholic medium comprising water and an acid catalyst in a container exposed to ultrasonic energy, the mixture having a pH not greater than about 2;

(b) removing the ultrasonic energy and raising the pH of the sol to about 5 to 7 by the addition of a buffering agent;

(c) adding an active biological material to the buffered sol; and

(d) forming a gel and allowing the gel to age.