Process for the manufacture of wholly microfabricated biosensors

• US 5,554,339 A
• Filed: 08/19/1993
• Issued: 09/10/1996
• Est. Priority Date: 11/14/1988
• Status: Expired due to Term

• Current Assignee: i STAT Corporation (Abbott Laboratories Incorporated)
• Original Assignee: i STAT Corporation (Abbott Laboratories Incorporated)
• Inventors: Davis, Graham, Lauks, Imants R., Wieck, Henry J., Smit, Nicolaas, Cozzette, Stephen N., Mier, Randall M., Piznik, Sylvia, Van Der Werf, Paul
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/109,507
• Time in Patent Office: 1,118 Days
• Field of Search: 435/6, 435/810, 435/287, 435/288, 435/291, 436/501, 536/22.1, 935/78, 935/88, 422/50, 422/63, 422/68.1, 422/69, 422/78, 422/79, 422/82.05
• US Class Current: 422/50
• CPC Class Codes: B01L 2200/12 Specific details about manufacturing devices
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 3/0268 using pulse dispensing or spraying, eg. inkjet type, piezo actuated ejection of droplets from capillaries
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/5088 confining liquids at a location by surface tension, e.g. virtual wells on plates, wires B01L3/50857 takes precedence
• CPC Class Codes: C12Q 1/001 Enzyme electrodes
• CPC Class Codes: G01N 2035/00059 vacuum chucks
• CPC Class Codes: G01N 2035/00237 Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 2035/00455 Controlling humidity in analyser
• CPC Class Codes: G01N 2035/00534 Mixing by a special element, e.g. stirrer
• CPC Class Codes: G01N 2035/1034 Transferring microquantities of liquid
• CPC Class Codes: G01N 2035/1076 plurality or independently movable heads
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 35/0099 comprising robots or similar manipulators robots per se B25J
• CPC Class Codes: G01N 35/109 with two horizontal degrees of freedom
• CPC Class Codes: G01V 9/00 Prospecting or detecting by methods not provided for in groups G01V1/00 - G01V8/00
• CPC Class Codes: G10K 11/006 Transducer mounting in underwater equipment, e.g. sonobuoys
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode
• CPC Class Codes: Y10S 436/806 : Electrical property or magnetic property

Abstract

An efficient method for the microfabrication of electronic devices which have been adapted for the analyses of biologically significant analyte species is described. The techniques of the present invention allow for close control over the dimensional features of the various components and layers established on a suitable substrate. Such control extends to those parts of the devices which incorporate the biological components which enable these devices to function as biological sensors. The materials and methods disclosed herein thus provide an effective means for the mass production of uniform wholly microfabricated biosensors. Various embodiments of the devices themselves are described herein which are especially suited for real time analyses of biological samples in a clinical setting. In particular, the present invention describes assays which can be performed using certain ligand/ligand receptor-based biosensor embodiments. The present invention also discloses a novel method for the electrochemical detection of particular analyte species of biological and physiological significance using an substrate/label signal generating pair which produces a change in the concentration of electroactive species selected from the group consisting of dioxygen and hydrogen peroxide.

Claims

1. A method of establishing a dispensed layer onto a substantially planar surface comprising:

(a) preparing a fluid composition suitable for loading into a microsyringe assembly;

(b) loading said fluid composition into said microsyringe assembly, which assembly comprises (i) a reservoir for holding said fluid composition, (ii) a microsyringe needle, including an elongated member and a needle tip, (iii) means for delivering said fluid composition from said reservoir to said microsyringe needle, (iv) means for forcing controlled amounts of said fluid composition through said elongated member to form an emerging droplet of a predetermined volume on said needle tip, and (v) means for controlling the multidirectional movement of said assembly such that said droplet may be brought into contact with a preselected area of a substantially planar surface;

(c) treating said surface under conditions sufficient to bring its surface free energy, in terms of its hydrophilicity or hydrophobicity, within a desired range such that the contact angle and thickness of the dispensed fluid composition are controlled;

(d) contacting said droplet on said needle tip with a preselected area of said surface; and

(e) retracting said assembly away from said surface such that said droplet disengages from said needle tip in a manner that provides a dispensed layer of said fluid composition having predictable and reproducible dimensions on said surface.

2. The method of claim 1 in which said substantially planar surface comprises a wafer having an array of unit cells of uniform dimensions.

3. The method of claim 2 in which said unit cells include a base sensor selected from the group consisting of amperometric and potentiometric sensors.

4. The method of claim 2 in which said unit cells include a base sensor selected from the group consisting of acoustic wave sensing devices, thermistors, gas-sensing electrodes, field-effect transistors, optical waver guides, evanescent field sensors, and conductimetric sensors.

5. The method of claim 1 in which said fluid composition includes a film-forming latex.

6. The method of claim 5 in which said film-forming latex comprises an aqueous emulsion of a polymer or copolymer derived from synthetic or natural sources.

7. The method of claim 5 in which said film-forming latex further comprises a porosity-altering substance selected from the group consisting of polyhydroxylated compounds, salts, and mixtures thereof.

8. The method of claim 5 in which said film-forming latex further comprises a crosslinking agent.

9. The method of claim 1 in which said fluid composition includes a photoformable proteinaceous mixture.

10. The method of claim 9 in which said proteinaceous mixture includes a substance selected from the group consisting of albumin, casein, gamma-globulin, collagen, derivatives, and mixtures thereof.

11. The method of claim 9 in which said proteinaceous mixture includes fish gelatin.

12. The method of claim 9 in which said proteinaceous mixture includes a photosensitizer selected from the group consisting of ferric chloride, ferric ammonium citrate, ferric potassium citrate, ferrric ammonium oxalate, ferric sodium oxalate, ferric potassium oxalate, ferric oxalate, potassium dichromate, and ammonium dichromate.

13. The method of claim 9 in which said proteinaceous mixture further includes a porosity-altering substance selected from the group consisting of polyhydroxylated compounds, salts, and mixtures thereof.

14. The method of claim 5 or 9 in which said fluid composition further includes one or more bioactive molecules.

15. The method of claim 14 in which said bioactive molecule is an enzyme selected from the group consisting of glucose oxidase, glucose dehydrogenase, NADH oxidase, uricase, urease, creatininase, sarcosine oxidase, creatinase, creatine kinase, creatine amidohydrolase, cholesterol esterase, cholesterol oxidase, glycerol kinase, hexokinase, glycerol-3-phosphate oxidase, lactate dehydrogenase, alkaline phosphatase, alanine transaminase, aspartate transaminase, amylase, lipase, esterase, gamma-glutamyl transpeptidase, L-glutamate oxidase, pyruvate oxidase, diaphorase, bilirubin oxidase, and their mixtures.

16. The method of claim 14 in which said bioactive molecule is selected from the group consisting of ionophores, cofactors, polypeptides, proteins, glycoproteins, enzymes, immunoglobulins, antibodies, antigens, lectins, neurochemical receptors, oligonucleotides, polynucleotides, molecules of DNA, molecules of RNA, active fragments or subunits or single strands of the preceding molecules, and mixtures thereof.

17. The method of claim 14 in which said bioactive molecule is glucose oxidase.

18. The method of claim 14 in which said bioactive molecule is urease.

19. The method of claim 6 in which said fluid composition includes a polymer matrix, a plasticizer, and an ionophore.

20. The method of claim 19 in which said polymer matrix includes a polymeric substance selected from the group consisting of polyurethane, poly(vinyl chloride), poly(tetrafluoroethylene), cellulose acetate, cellulose nitrate, silicone rubber, derivatives, and mixtures thereof.

21. The method of claim 19 in which said ionophore is selected from the group consisting of crown ethers, trialkylamines, phosphate esters, valinomycin, nonactin, monensin, methylmonensin, and mixtures of monensin and methylmonensin.

22. The method of claim 1 in which a plurality of independently controlled microsyringe assemblies are used to dispense different layers on the same planar surface.

23. The method of claim 2 in which the microsyringe needle is aligned to a preselected area of the wafer prior to dispensing, and after dispensing is advanced automatically to another preselected area of the wafer for dispensing until all areas of the wafer that require a dispensed layer have received said dispensed layer.

24. The method of claim 23 in which the initial alignment of said microsyringe needle is performed manually with a reticle and camera.

25. The method of claim 23 in which the initial alignment of said microsyringe needle is performed automatically with an artificial intelligence system capable of recognizing one or more preselected areas of the wafer.

26. The method of claim 1 in which said surface is pretreated at step (c) with a plasma.

27. The method of claim 26 in which said plasma is selected from the group consisting of oxygen, hydrogen, water, carbon tetrafluoride, trifluoromethane, argon and nitrogen.

28. The method of claim 26 in which said plasma is selected to provide a hydrophilic surface.

29. The method of claim 26 in which said plasma is selected to provide a hydrophobic surface.

30. The method of claim 26 in which said pretreatment etches said surface.

31. The method of claim 26 in which said pretreatment results in the deposition of a plasma species on said surface.

32. The method of claim 1 in which the surface is selected from the group consisting of silicon dioxide, silver, silver chloride, iridium, gold, polyimide, a photoformable proteinaceous mixture, a film forming latex and a siloxane-nonsiloxane copolymer.

33. The method of claim 32 in which the thickness of said dispensed layer falls in the range of about 1-200 microns.

34. The method of claim 32 in which the thickness of said dispensed layer falls in the range of about 1-50 microns.

35. The method of claim 1 in which the predetermined volume of said emerging droplet falls in the range of about 5-500 nanoliters.

36. The method of claim 1 in which said means for forcing controlled amounts of said fluid comprises a compressed gas regulated by a solenoid valve,

37. The method of claim 1 in which said microsyringe needle has an internal diameter of 150 microns and an external diameter of 300 microns.

38. The method of claim 1 in which said microsyringe needle has an external coating.

39. The method of claim 38 in which said external coating is hydrophilic.

40. The method of claim 38 in which said external coating is hydrophobic.

41. The method of claim 38 in which said external coating is poly (tetrafluoroethylene).

42. The method of claim 38 in which said external coating is poly(vinyl alcohol).

43. The method of claim 1 in which said fluid is an organic liquid.

44. The method of claim 1 in which said fluid is an aqueous liquid.

45. The method of claim 44 in which said aqueous liquid contains dissolved salts and detergents to adjust the surface tension of said aqueous liquid.

46. The method of claim 19 in which said dispensed layer is sensitive to ionic species selected from the group K⁺, Na⁺, Cl^-, NH₄⁺, H⁺, and Ca²⁺.

47. The method of claim 46 in which said dispensed layer is sensitive to K⁺, Na⁺, NH₄⁺ or H⁺ and includes a plasticizer, polymer matrix and ionophore having a solids content of 60-80 percent, 15-40 percent and 0.5-3 percent, respectively.

48. The method of claim 46 in which said dispensed layer is sensitive to Cl and includes a plasticizer, PVC matrix and ionophore having a solids content of 25-40 percent, 25-45 percent and 25-35 percent, respectively.

49. The method of claim 46 in which said surface is pretreated at step (c) first with an argon plasma and, subsequently, with a carbon tetrafluoride plasma.

50. The method of claim 9 in which said photoformable proteinaceous mixture is established on said surface to provide a dispensed layer which further comprises exposing said dispensed layer to ultra-violet light through a mask, and which further comprises developing said exposed dispensed layer to leave on said surface only those portions of said dispensed layer that had been exposed to ultra-violet light.

51. The method of claim 1 which is carried out under a controlled low humidity environment.

52. The method of claim 19 in which said plasticizer is selected from the group consisting of N-methylpyrrolidone, propriophenone, cyclohexanone, Bis(2-ethylhexyl) sebacate, phenyl-1-pentanol, Tris(2-ethylhexyl)phosphate and mixtures thereof.

53. The method of claim 1 in which the substantially planar surface is heat-treated with a silane compound having the formula R'"'"'_n Si(OR)_4-n in which n is an integer selected from the group consisting of 0, 1 and 2;

R'"'"' is a hydrocarbon radical comprising 3 to 12 carbon atoms; and

R is a hydrogen radical or a lower alkyl radical comprising 1 to 4 carbon atoms.

54. The method of claim 53 in which said hydrocarbon radical further comprises at least one heteroatom selected from the group consisting of oxygen, nitrogen, phosphorous, sulfur, halogen and stable combinations thereof.

55. The method of claim 53 in which said hydrocarbon radical is selected from the group consisting of 3-aminopropyl, N-(2-aminoethyl)-3-aminopropyl, 3-methacryloxypropyl, 3-mercaptopropyl, 3-hydroxypropyl, 3-isocyanatopropyl, 10-aminodecyl, 11-aminoundecyl, 2-[p-(N-(2-aminoethyl)aminomethyl)phenyl]ethyl, n-propyl, phenyl, diethylphosphatoethyl and N,N-bis(2-hydroxyethyl)aminopropyl groups.

56. The method of claim 53 in which said silane compound is selected from the group consisting of 3-aminopropyltriethoxysilane, N-(2-aminoethyl)-3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, 3-isocyanatopropyltriethoxysilane, 10-aminodecyltrimethoxysilane, 11-aminoundecyltrimethoxysilane, 2-[p-(N-(2-aminoethyl)aminomethyl)phenyl]ethyltrimethoxysilane, n-propyltrimethoxysilane, phenyltrimethoxysilane, diethylphosphatoethyltriethoxysilane, N,N-bis(2-hydroxyethyl)aminopropyltriethoxysilane, 3-chloropropyltriethoxysilane and mixtures thereof.

57. The method of claim 53 in which said silane compound is N-(2-aminoethyl)-3-aminopropyltrimethoxysilane.

58. The method of claim 53 in which said silane compound is a tetraalkyl or tetrahydroxy orthosilicate.

59. A method of establishing a dispensed layer which is sensitive to potassium ion onto a silver-silver chloride planar base sensor comprising:

(a) preparing a fluid composition suitable for loading into a microsyringe assembly, said fluid composition, comprising N-methylpyrrolidone, propiophenone, cyclohexanone, bis(2-ethylhexyl)sebacate, poly(vinyl chloride) and valinomycin, in an amount sufficient to function as a potassium ion sensor;

(b) loading said fluid composition into said microsyringe assembly, which assembly comprises (i) a reservoir for holding said fluid composition, (ii) a microsyringe needle, including an elongated member and a needle tip, (iii) means for delivering said fluid composition from said reservoir to said microsyringe needle, (iv) means for forcing controlled amounts of said fluid composition through said elongated member to form an emerging droplet of about ten nanoliters on said needle tip, and (v) means for controlling the multidirectional movement of said assembly such that said droplet may be brought into contact with a preselected area of said base sensor;

(c) treating said base sensor first with an argon plasma and, subsequently, with a carbon tetrafluoride plasma under conditions sufficient to bring its surface free energy within a desired hydrophilic range;

(d) contacting said droplet on said needle tip with a preselected area of said base sensor; and

(e) retracting said assembly away from said base sensor such that said droplet disengages from said needle tip in a manner that provides a dispensed layer of said fluid composition which is sensitive to potassium ion and having a thickness falling in the range of about 20-60 microns.

60. A method of establishing a dispensed layer which is sensitive to sodium ion onto a silver-silver chloride planar base sensor comprising:

(a) preparing a fluid composition suitable for loading into a microsyringe assembly, said fluid composition, comprising N-methylpyrrolidone, propiophenone, Tris-(2-ethylhexyl) phosphate, cyclohexanone, poly(vinyl chloride) and methyl monensin, in an amount sufficient to function as a sodium sensor;

(b) loading said fluid composition into said microsyringe assembly, which assembly comprises (i) a reservoir for holding said fluid composition, (ii) a microsyringe needle, including an elongated member and a needle tip, (iii) means for delivering said fluid composition from said reservoir to said microsyringe needle, (iv) means for forcing controlled amounts of said fluid composition through said elongated member to form an emerging droplet of about ten nanoliters on said needle tip, and (v) means for controlling the multidirectional movement of said assembly such that said droplet may be brought into contact with a preselected area of said base sensor;

(c) treating said base sensor first with an argon plasma and, subsequently, with a carbon tetrafluoride plasma under conditions sufficient to bring its surface free energy within a desired hydrophilic range;

(d) contacting said droplet on said needle tip with a preselected area of said base sensor; and

(e) retracting said assembly away from said base sensor such that said droplet disengages from said needle tip in a manner that provides a dispensed layer of said fluid composition which is sensitive to sodium ion and having a thickness falling in the range of about 20-40 microns.

61. A method of establishing a dispensed layer which is sensitive to chloride ion onto a silver-silver chloride planar base sensor comprising:

(a) preparing a fluid composition suitable for loading into a microsyringe assembly, said fluid composition, comprising propiophenone, cyclohexanone, 5-phenyl-1-pentanone, poly(vinyl chloride), tridodecylmethylammonium chloride and kemamine BQ-9702C fatty amine, in an amount sufficient to function as a chloride sensor;

(b) loading said fluid composition into said microsyringe assembly, which assembly comprises (i) a reservoir for holding said fluid composition, (ii) a microsyringe needle, including an elongated member and a needle tip, (iii) means for delivering said fluid composition from said reservoir to said microsyringe needle, (iv) means for forcing controlled amounts of said fluid composition through said elongated member to form an emerging droplet of about ten nanoliters on said needle tip, and (v) means for controlling the multidirectional movement of said assembly such that said droplet may be brought into contact with a preselected area of said base sensor;

(c) treating said base sensor first with an argon plasma and, subsequently, with a carbon tetrafluoride plasma under conditions sufficient to bring its surface free energy within a desired hydrophilic range;

(d) contacting said droplet on said needle tip with a preselected area of said base sensor; and

(e) retracting said assembly away from said base sensor such that said droplet disengages from said needle tip in a manner that provides a dispensed layer of said fluid composition which is sensitive to chloride ion and having a thickness falling in the range of about 20-60 microns.

62. A method of establishing a dispensed layer which is sensitive to ammonium ion onto a planar silver-silver chloride base sensor comprising:

(a) preparing a fluid composition suitable for loading into a microsyringe assembly, said fluid composition, comprising N-methylpyrrolidone, propiophenone, cyclohexanone, Tris-(2 ethylhexyl)phosphate, poly(vinyl chloride) and nonactin, in an amount sufficient to function as an ammonium ion sensor;

(b) loading said fluid composition into said microsyringe assembly, which assembly comprises (i) a reservoir for holding said fluid composition, (ii) a microsyringe needle, including an elongated member and a needle tip, (iii) means for delivering said fluid composition from said reservoir to said microsyringe needle, (iv) means for forcing controlled amounts of said fluid composition through said elongated member to form an emerging droplet of about ten nanoliters on said needle tip, and (v) means for controlling the multidirectional movement of said assembly such that said droplet may be brought into contact with a preselected area of said base sensor;

(c) treating said base sensor first with an argon plasma and, subsequently, with a carbon tetrafluoride plasma under conditions sufficient to bring its surface free energy within a desired hydrophilic range;

(d) contacting said droplet on said needle tip with a pressletted area of said base sensor; and

(e) retracting said assembly away from said base sensor such that said droplet disengages from said needle tip in a manner that provides a dispensed layer of said fluid composition which is sensitive to ammonium ion and having a thickness falling in the range of about 10-20 microns.

63. The method of claim 62 which further comprises establishing a biolayer over said dispensed layer, said biolayer comprising urease and ELVACE™

.