Process for the manufacture of wholly microfabricated biosensors

• US 5,837,454 A
• Filed: 06/07/1995
• Issued: 11/17/1998
• Est. Priority Date: 11/14/1988
• Status: Expired due to Fees

• Current Assignee: i STAT Corporation (Abbott Laboratories Incorporated)
• Original Assignee: i STAT Corporation (Abbott Laboratories Incorporated)
• Inventors: Mier, deceased, Randall M., Davis, Graham, Lauks, Imants R., Wieck, Henry J., Smit, Nicolaas, Cozzette, Stephen N., Piznik, Sylvia, Van Der Werf, Paul, Steiner, Susan, Itak, Jeanne
• Primary Examiner(s): Chin, Christopher L.
• Application Number:
  US08/484,095
• Time in Patent Office: 1,259 Days
• Field of Search: 204/403, 204/411, 204/412, 204/414-419, 204/430-432, 422/57, 422/82.08, 427/2.11, 427/2.13, 427/58, 427/96, 623/24, 435/6, 435/14, 435/25, 435/177, 435/817, 435/287.1, 435/287.2, 435/287.9, 436/518, 436/524, 436/528, 436/531, 436/806
• US Class Current: 435/6.11
• 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 manufacturing a plurality of uniform microfabricated sensing devices which comprises:

(a) establishing a plurality of base sensors on a substrate wafer;

(b) establishing a permselective layer, superimposed over at least a portion of each base sensor, having a thickness sufficient to exclude substantially molecules with a molecular weight of about 120 or more while allowing free permeation of molecules with a molecular weight of about 50 or less; and

(c) establishing a topmost layer comprising an immobilized ligand receptor in an amount sufficient to be superimposed over said permselective layer.

2. The method of claim 1 in which said permselective layer comprises a polymer film with available reactive functional groups on its outer surface.

3. The method of claim 1 in which said permselective layer comprises a heat-treated film of 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 3-12 carbon atoms; and

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

4. The method of claim 1 in which said permselective layer comprises a copolymer of a siloxane compound and a nonsiloxane compound.

5. The method of claim 4 in which said copolymer is selected from the group consisting of dimethylsiloxane-alkene oxide, tetramethyldisiloxane-divinylbenzene, tetramethyldisiloxane-ethylene, dimethylsiloxane-silphenylene, dimethylsiloxane-silphenylene oxide, dimethylsiloxane, methylstyrene, and dimethylsiloxane-bisphenol A carbonate, and mixtures thereof.

6. The method of claim 4 in which said copolymer is dimethylsiloxane-bisphenol A carbonate.

8. The method of claim 1 or 7 which further comprises establishing an electrolyte layer over at least a portion of each base sensor, prior to establishing said permselective layer.

9. The method of claim 1 or 7 in which said ligand receptor is an immunoreactive species.

10. The method of claim 1 or 7 in which said ligand receptor is selected from the group consisting of ionophores, cofactors, enzymes, antibodies, antigens, lectins, neurochemical receptors, active fragments or subunits of the preceding molecules and mixtures thereof.

11. The method of claim 1 or 7 in which said ligand receptor is an antibody.

12. The method of claim 1 or 7 in which said ligand receptor is an antigen.

13. The method of claim 1 or 7 in which said ligand receptor is a polypeptide or protein.

14. The method of claim 1 or 7 in which said ligand receptor is an oligonucleotide or polynucleotide or a single strand thereof.

15. The method of claim 1 or 7 in which said ligand receptor is immobilized over a preselected area of each base sensor.

16. The method of claim 1 or 7 in which each base sensor comprises an indicator electrode and said ligand receptor is immobilized over the indicator electrode of each base sensor.

17. The method of claim 1 or 7 in which said ligand receptor is immobilized by covalent attachment.

18. The method of claim 1 or 7 in which said ligand receptor is immobilized by absorption.

19. The method of claim 1 or 3 in which said silane compound is N-(2-aminoethyl)-3-aminopropyltrimethoxysilane.

20. The method of claim 17 in which said covalent attachment is accomplished with the benefit of a crosslinking agent.

21. The method of claim 20 in which said crosslinking agent is a polyfunctional compound having at least two functional groups selected from the group consisting of formyl, vinyl, carboxyl, anhydride, amine, amide, epoxy, hydroxyl, cyano, isocyanato, thio, halo and stable combinations thereof.

22. The method of claim 1 or 7 in which said base sensor comprises an electrochemical transducer.

23. The method of claim 8 in which said base sensor comprises an electrochemical transducer.

24. The method of claim 22 in which said electrochemical transducer is amperometric.

25. The method of claim 22 in which said electromechanical transducer is potentiometric.

26. The method of claim 1 or 7 in which said base sensor comprises an amperometric electrochemical transducer comprising an indicator electrode which includes an electrocatalyst selected from the group consisting of carbon, platinum, gold, silver, rhodium, iridium, ruthenium, mercury, palladium, and osmium.

27. The method of claim 8 in which said base sensor comprises an amperometric electrochemical transducer comprising an indicator electrode which includes an electrocatalyst selected from the group consisting of carbon, platinum, gold, silver, rhodium, iridium, ruthenium, mercury, palladium, and osmium.

28. The method of claim 1 or 7 in which said base sensor comprises a potentiometric electrochemical transducer comprising an indicator electrode which includes a mixed metal oxide alloy electrocatalyst.

29. The method of claim 8 in which said base sensor comprises a potentiometric electrochemical transducer comprising an indicator electrode which includes a mixed metal oxide alloy electrocatalyst.

30. The method of claim 1 or 7 in which said base sensor comprises a potentiometric electrochemical transducer comprising an indicator electrode which includes a silver/silver halide electrocatalyst.

31. The method of claim 8 in which said base sensor comprises a potentiometric electrochemical transducer comprising an indicator electrode which includes a silver/silver halide electrocatalyst.

32. The method of claim 22 in which said electrochemical transducer further comprises a reference electrode which includes an electrocatalyst metal selected from the group consisting of silver, gold, and platinum.

33. The method of claim 22 in which said electrochemical transducer further comprises a silver/silver halide reference electrode.

34. The method of claim 1 or 7 in which said ligand receptor is a glycoprotein.

7. A method of manufacturing a plurality of uniform microfabricated sensing devices which comprises:

(a) establishing a plurality of base sensors on a substrate wafer;

(b) establishing an adhesion promoting layer, localized over preselected areas of said base sensor, which layer comprises a film of a silane compound having the formula R'"'"'_n Si(OR)_4-n, in which n is an integer with a value of 1 or 2;

R'"'"' is a hydrocarbon radical, comprising 3-12 carbon atoms, having a terminal reactive functional group; and

R is a hydrogen radical or a lower alkyl radical comprising 1-4 carbon atoms; and

(c) establishing a topmost layer comprising an immobilized ligand receptor in an amount sufficient to be superimposed over said permselective layer.