FORMATION OF IMMOBILIZED BIOLOGICAL LAYERS FOR SENSING

• US 20090159442A1
• Filed: 12/20/2007
• Published: 06/25/2009
• Est. Priority Date: 12/20/2007
• Status: Active Grant

• Current Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: i STAT Corporation (Abbott Laboratories Incorporated)
• Inventors: Collier, Gordon Bruce, Nemeth, Attila Csaba, Wong, Anjulia, Macleod, Jason Andrew
• Granted Patent: US 8,241,697 B2
• Time in Patent Office: Days
• US Class Current: 204/403.100
• CPC Class Codes: B05C 11/1034 specially designed for conducting intermittent application of small quantities, e.g. drops, of coating material B05C5/0225, B05C11/1026 take precedence
• CPC Class Codes: B05C 5/0291 the material being discharged on the work through discrete orifices as discrete droplets, beads or strips that coalesce on the work or are spread on the work so as to form a continuous coating
• CPC Class Codes: B05C 9/12 the auxiliary operation being performed after the application B05C9/14 takes precedence
• 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: C12Q 1/003 Functionalisation
• CPC Class Codes: C12Q 1/005 involving specific analytes or enzymes including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence
• CPC Class Codes: C12Q 1/006 for glucose
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/28 involving peroxidase
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/58 involving urea or urease

Abstract

The invention is directed to enzyme immobilization compositions comprising: one or more enzymes, a humectant, an acrylic-based monomer, a water-soluble organic photo-initiator and a water-soluble acrylic-based cross-linker in a substantially homogeneous aqueous mixture. The invention is also directed to methods for forming sensors comprising such compositions and to apparati for forming arrays of immobilized layers on an array of sensors by dispensing such compositions onto a substrate.

Claims

1. A method of forming a sensor comprising the steps of:

(a) forming a substantially homogeneous aqueous mixture comprising one or more enzymes, an acrylic-based monomer, a water soluble organic photo-initiator and a water soluble acrylic-based cross-linker in an aqueous mixture,

(b) applying a controlled volume of said mixture onto a base sensor sufficient to cover said base sensor, and

(c) exposing said applied volume to sufficient UV radiation to form an immobilized enzyme layer adhered to said base sensor.

2. The method of claim 1, wherein said enzyme is selected from urease, glucose oxidase, lactate oxidase, creatinase, creatininase, sarcosine oxidase, catalase, carbonic anhydrase, NAD(P)H oxidase, cholesterol oxidase, alcohol oxidase, choline oxidase, glycerol-3-phosphate oxidase, thiamine oxidase, pyruvate oxidase, pyridoxal oxidase, D-amino acid oxidase, L-amino acid oxidase, alkaline phosphatase, horseradish peroxidase and combinations thereof.

3. The method of claim 1, wherein the monomer is selected from acrylamide, methacrylamide, N-[3-(dimethylamino)propyl]methacrylamide, hydroxyethylmethacrylate and combinations thereof.

4. The method of claim 1, wherein the organic photo-initiator is selected from 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone, (insert means for claim 8) and combinations thereof.

5. The method of claim 1, wherein the cross-linker is selected from 1,4-bisacryloyl piperazine, N,N′

-(1,2-dihydroxyethylene)bis-acrylamide,N,N′

-bis(acryloyl)cystamine, N,N′

-methylenebisacrylamide, ethylene glycol diacrylate, (+)-N,N′

-diallyltartramide and combinations thereof.

6. The method of claim 1, wherein the aqueous mixture further comprises one or more stabilizing components selected from the group consisting of pH buffer, disulfide bond reducing agent, divalent ion chelating agent, protease inhibitor, bovine serum albumin, salts, biocide and humectant.

7. The method of claim 1, wherein the base sensor is selected from the group consisting of electrode, ion-selective electrode, potentiometer electrode, amperometric electrode, conductimetric electrode, enzyme electrode, biosensor, optical sensor, fiber optic sensor, surface acoustic wave sensor, evanescent sensor, surface plasmon resonance sensor and optical wave guide sensor.

8. The method of claim 1, wherein the enzyme is urease and the base sensor is an ammonium ion-selective membrane.

9. The method of claim 1, wherein said method of applying the mixture is by microdispensing a controlled volume in the range of about 5 nL to about 1 μ

L.

10. The method of claim 1, wherein said method of applying the mixture is by means selected from the group consisting of spin-coating, dip-coating, spray coating, screen printing, ink-jet printing, laser printing, painting and contact printing.

11. The method of claim 1, wherein said aqueous mixture comprises urease, 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone, bisacryloyl piperazine, glycerol and acrylamide monomer.

12. The method of claim 1, wherein said UV radiation is in the wavelength range of about 185 to 400 nm.

13. The method of claim 1, wherein said UV radiation intensity is in the range of about 100 to 400 mW/cm².

14. The method of claim 1, wherein said UV radiation step is a spot cure performed immediately after the dispensing step in a dispensing cycle.

15. The method of claim 1, wherein said UV radiation step is a spot cure performed with a pre-selected time delay after the dispensing step.

16. The method of claim 1, wherein said UV radiation step is a spot cure performed for a duration of about 0.1 to 10 seconds.

17. A method of forming a urea sensor comprising the steps of:

(a) forming a substantially homogeneous aqueous mixture comprising urease, glycerol, 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone, bisacryloyl piperazine and acrylamide monomer,

(b) dispensing a controlled volume of said mixture onto an ammonium ion-selective membrane sufficient to cover said membrane, and

(c) exposing said dispensed volume to sufficient UV radiation to form an immobilized urease layer adhered to said membrane.

18. An apparatus for forming an immobilized enzyme layer on a substantially planar surface, the apparatus comprising a dispensing head for dispensing a controlled volume of a photoformable enzyme-containing matrix at a pre-selected location on said surface and a UV radiation source with a registration and alignment means capable of focusing a beam of radiation onto an area substantially covering said pre-selected location at a predetermined time and for a predetermined duration, at a predetermined intensity, after said matrix has been dispensed.

19. The apparatus of claim 18, wherein said substantially planar surface is selected from the group consisting of silicon wafer, alumina wafer, liquid crystal substrate, glass substrate and plastic substrate and flexible plastic substrate.

20. The apparatus of claim 18, wherein said dispensing head comprises a syringe needle with a reservoir for said matrix, and a displacement means for controlling the dispensed volume from said syringe onto said surface.

21. The apparatus of claim 18, wherein the photoformable matrix is the composition comprising one or more enzymes, a humectant, an acrylic-based monomer, a water soluble organic photo-initiator and a water soluble acrylic-based cross-linker in a substantially homogeneous aqueous mixture.

22. The apparatus of claim 18, wherein the controlled volume is in the range of about 5 nL to about 1 μ

L.

23. The apparatus of claim 18, wherein said pre-selected location has an area in the range of about 10 square microns to about 75 square millimeters.

24. The apparatus of claim 18, wherein said pre-selected location is substantially circular with radial dimensions in the range of about 5 μ

m to about 5 mm.

25. The apparatus of claim 18, wherein said UV radiation source is a mercury lamp.

26. The apparatus of claim 18, wherein registration and alignment means permit the beam to be focused on a selected area of said surface and illuminate an area in the range of about 10 square microns to about 75 square millimeters.

27. The apparatus of claim 18, comprising a computer program for controlling the timing and location of dispensing.

28. The apparatus of claim 18, comprising a computer program for controlling the timing and location of the application of UV radiation with respect to the timing of dispensing.

29. The apparatus of claim 18, further comprising a step and repeat means for changing the relative position of said surface with respect to said dispensing head and said UV radiation source for forming an array of immobilized enzyme layers at a set of pre-selected locations.

30. The apparatus of claim 18, for forming an array of immobilized layers on said substantially planar surface where the dispensing head is capable of dispensing a sequence of controlled volumes of a photoformable matrix at a pre-selected set of locations on said surface, and said UV radiation source is capable of focusing a beam of radiation onto an area substantially covering each said pre-selected locations, in sequence at a predetermined time after each controlled volume is dispensed, for a predetermined duration.

31. An apparatus for forming an array of immobilized enzyme layers on an array of sensors on a substantially planar surface comprising:

a dispensing head for dispensing a sequence of controlled volumes of a photoformable enzyme-containing matrix at a pre-selected set of locations on said surface, and a UV radiation source with a registration and alignment means capable of focusing a beam of radiation onto an area substantially covering each said pre-selected location, in sequence at a predetermined time after each controlled volume is dispensed, for a predetermined duration.

32. A method of forming an immobilized layer on a sensor on a substantially planar surface comprising the steps of:

(a) dispensing a controlled volume of a photoformable matrix at a pre-selected location on said surface, wherein the photoformable matrix comprises a biologically active material; and

(b) applying a UV radiation beam onto an area substantially covering said pre-selected location, starting at a predetermined time after said volume has been dispensed and for a predetermined duration at a predetermined intensity, to form said immobilized layer.

33. The method claim 32, wherein the photoformable matrix comprises one or more enzymes, an acrylic-based monomer, a water soluble organic photo-initiator and a water soluble acrylic-based cross-linker in an aqueous mixture.

34. The method claim 32, wherein the biologically active material is selected from the group consisting of protein, enzyme, antibody, antibody fragment, RNA, single stranded DNA and double stranded DNA.

35. The method claim 32, wherein the immobilized layer is an enzyme layer.

36. A method of forming an array of immobilized layers on an array of sensors on a substantially planar surface comprising the steps of:

(a) dispensing a sequence of controlled volumes of a photoformable matrix at a pre-selected set of locations on said surface, and

(b) applying a UV radiation beam onto an area substantially covering each said pre-selected location, in sequence, starting at a predetermined time after each controlled volume has been dispensed, and applying said radiation at a predetermined intensity for a predetermined duration, to form said immobilized array of layer.

37. The method of claim 36, wherein the predetermined time is in the range of about 0.1 to about 10 seconds.

38. The method of claim 36, wherein the predetermined duration is in the range of about 0.1 to about 10 seconds.

39. The method of claim 36, wherein said UV radiation is in the wavelength range of about 185 to 400 nm.

40. The method of claim 36, wherein said UV radiation intensity is in the range of about 100 to 400 mW/cm².

41. The method of claim 36, wherein said planar surface is a silicon wafer and the pre-selected set of locations is an array of sensors on said wafer.

42. The method of claim 36, wherein the UV radiation beam is applied to the Nth minus X pre-selected location while dispensing occurs at the Nth pre-selected location, where X is equal to an integer from 1 to 10.

43. A sensor, comprising an electrode with a first layer covering said electrode comprising an ion-selective membrane, and a second layer covering said ion-selective membrane comprising a UV cured matrix formed from a substantially homogeneous aqueous mixture of one or more enzymes, a humectant, an acrylic-based monomer, a water-soluble organic photo-initiator and a water-soluble acrylic-based cross-linker.