Electrostatic enzyme biosensor

• US 6,096,497 A
• Filed: 07/07/1998
• Issued: 08/01/2000
• Est. Priority Date: 06/15/1998
• Status: Expired due to Fees

• Current Assignee: Biosensor Systems Design, Inc.
• Original Assignee: Biosensor Systems Design, Inc.
• Inventors: Bauer, Alan Joseph
• Primary Examiner(s): Leary, Louise N.
• Application Number:
  US09/110,686
• Time in Patent Office: 756 Days
• Field of Search: 435/4, 435/283.1, 435/289.1, 435/817, 204/403, 204/164, 204/193, 205/81, 205/372, 422/82.01, 422/50, 257/40, 257/43
• US Class Current: 435/4
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/001 Enzyme electrodes
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode

Abstract

The device described herein is an enzyme-based biosensor for detecting and/or quantifying molecules of interest. The biosensor relies on the following properties shared by all enzymes: (1) that enzymes are highly specific molecules designed to bind with only one analyte type or one class of analyte molecules; (2) that enzymes contain charges; (3) that enzymes undergo significant spacial fluctuation during periods of interaction with substrates; and (4) that these spacial fluctuations cause the charged moieties on the enzyme to move and thus generate a measurable electrostatic potential (voltage) in both the enzyme and support layers. The instant device determines analyte presence/concentration through measurement of changes in voltage or current in a conducting or semiconducting support material as a result of changes in the position of immobilized charged enzyme molecules during their interaction with analyte. More particularly, the instant device immobilizes enzyme molecules sufficiently close to a conducting or semiconducting layer to insure that any alteration in the position of charged groups of the enzyme during interaction with analyte will cause the generation of a voltage in the base layer. When the enzymes encounter the targeted analyte the enzymes move, thus causing the enzyme charges to move. The movement of charges, in turn, changes the electrostatic field around the enzymes and results in a corresponding generation of a dipole (as measured in a generated voltage) in the conducting or semiconducting base layer. The presence and quantity of generated voltage is used to signal the presence and quantity of analyte.

Claims

1. A biosensor for detecting the presence and/or concentration of an analyte based on the physical change in position of immobilized enzyme molecules in the presence of said analyte, comprising:

(i) a multilayer sensor comprising a conducting or semiconducting layer and an optional self-assembled monolayer or other chemical entity directly or indirectly bound to this layer;

(ii) enzyme molecules bound to or coordinated with the conducting or semiconducting layer of the multilayer substrate either through their interaction with the self-assembled monolayer/chemical entity or their interaction with the conducting or semiconducting support itself; and

(iii) a means for measuring directly or indirectly electrostatic effects that result from motions of the immobilized enzyme molecules during their contact with analyte.

2. The biosensor of claim 1 wherein the self-assembled monolayer is directly and chemically bound to the conducting or semiconducting layer and wherein the enzyme molecules are directly and chemically bound to the self-assembled monolayer.

3. The biosensor of claim 1, wherein the enzyme molecules rest close enough to the conducting or semiconducting layer so as to enable changes in the location of charged portions of the enzyme molecules to generate a voltage and current in or across the conducting or semiconducting layer.

4. The biosensor of claim 1, wherein the enzyme molecules rest approximately 0-3 nanometers from the conducting or semiconducting layer.

5. The biosensor of claim 1 in which the detection of the voltage or current produced in the conducting or semiconducting material in response to enzyme motions is effected by the connection of the sensor to an appropriate voltage meter device and/or current meter device.

6. The biosensor of claim 1 in which a voltage or current meter device is connected to either the same or opposite sides of the sensor.

7. The biosensor of claim 1 wherein the multilayer substrate comprises the following layers:

(a) a non-conducting base layer;

(b) an optional intermediate self-assembled monolayer positioned above the base layer;

(c) an intermediate conductive layer positioned above the base layer and, if present, above the intermediate self-assembled monolayer; and

(d) an outer self-assembled monolayer positioned above the intermediate conductive layer comprising one or more compounds than can bind or be made to bind with enzymes and/or provide physical stabilization to chemically bound enzymes.

8. The biosensor of claim 3 wherein the multilayer substrate comprises the following layers:

(a) a base layer containing silicon dioxide functionality chosen from the group consisting of glass, quartz, silicon, and the reaction product of an organic polymer, silicon tetrachloride and water;

(b) an optional intermediate self-assembled monolayer formed from compounds containing either an organic sulfide, disulfide or thiol group and a group that can react with silicon dioxide positioned above the base layer;

(c) an intermediate conductive layer positioned above the base layer and, if present, above the intermediate self-assembled monolayer, formed by deposition by any means of a metallic layer to the base layer;

(d) an optional outer self-assembled monolayer positioned above the intermediate conductive layer formed from one or more compounds having an organic sulfide, disulfide or thiol group and terminal functional groups that are either reactive with amino acid functionalities (or can be made reactive with said functionalities) and/or act to stabilize the enzyme molecules associated with the device.

9. The biosensor of claim 8, wherein the base layer is a reaction product of organic polymer and the vapors of silicon tetrachloride and water.

10. The biosensor of claim 8, wherein the intermediate self-assembled monolayer is formed from a compound having the formula (CH₃ O)_y --Si--(CH₂)_z --SH wherein y and z, independently, may be zero or any whole integer.

11. The biosensor of claim 8, wherein the intermediate conductive layer is a silver or gold layer.

12. The biosensor of claim 8, where the outer self-assembled monolayer is formed from a mercapto acid (HS--R--COOH) and a mercapto alcohol (HS--R'"'"'--OH) wherein R and R'"'"' may be any organic groups and are chosen to balance insulation with electrical connectivity between the enzymes and the conductive layer.

18. The biosensor of claim 1 in which the enzyme used is lysozyme and the conducting support material is aluminum foil.

13. A method for detecting and/or quantifying the presence of an analyte through the motion of enzymes in contact with said analyte comprising the following steps:

i) forming a multilayer sensor comprising a conductive or semiconductive layer and an optional self-assembled monolayer directly or indirectly bound to this layer;

ii) immobilizing enzyme molecules on the conductive or semiconductive layer of the multilayer substrate through direct or indirect binding with either the conductive/semiconductive layer or with the self-assembled monolayer; and

(iii) providing a means for measuring a voltage or current generated by the movement of the enzymes in response to their contact with analyte.

14. The method of claim 13 wherein the self-assembled monolayer is directly and chemically bound to the conductive layer and wherein the enzyme molecules are directly and chemically bound to the self-assembled monolayer.

15. The method of claim 13, wherein the enzyme molecules rest close enough to the conducting or semiconducting layer to enable changes in the position of charged portions of enzyme molecules to be detected through the generation of a voltage or current in the conducting or semiconducting support material.

16. The method of claim 13, wherein the enzyme molecules rest approximately 0-3 nanometers from the conductive layer.

17. The method of claim 13 in which the leads of a voltage and/or current meter are brought into contact with the sensor on either the same or opposite sides of the sensor.

19. The method of claim 13 wherein the multilayer substrate comprises the following layers:

(a) a base layer;

(b) an optional intermediate self-assembled monolayer positioned above the base layer;

(c) an intermediate conductive layer positioned above the base layer and, if present, above the intermediate self-assembled monolayer; and

(d) an optional outer self-assembled monolayer positioned above the intermediate conductive layer comprising one or more compounds that chemically bind with enzymes and/or provide environmental stabilization to immobilized enzymes.

20. The method of claim 19 wherein the multilayer substrate comprises the following layers:

(a) a base layer containing an outer silicon dioxide layer chosen from the group consisting of glass, quartz, silicon, and the reaction product of an organic polymer, silicon tetrachloride and water;

(b) an optional intermediate self-assembled monolayer formed from compounds containing either a sulfide or thiol group and a group that is reactive with silicon dioxide positioned above the base layer;

(c) an intermediate conductive layer positioned above the base layer and, if present, above the intermediate self-assembled monolayer, formed by depositing a metallic solution to the base layer/SAM;

(d) an outer self-assembled (optionally mixed) monolayer positioned above the intermediate conductive layer formed from a compound having an organic sulfide, disulfide or thiol group and a terminal functional group that is reactive with amino acid functionalities (or can be made so) and a compound that has an organic sulfide, disulfide or thiol group and a terminal functional group that stabilizes enzymes through hydrophilic and/or hydrophobic interactions.

21. The method of claim 20, wherein the base layer is a reaction product of an organic polymer and the vapors of silicon tetrachloride and water.

22. The method of claim 20, wherein the intermediate self-assembled monolayer is formed from a compound having the formula (CH₃ O)_y --Si--(CH₂)_z --SH wherein y and z, independently, may be zero or any whole integer.

23. The method of claim 20, wherein the intermediate conducting layer is a silver and/or gold layer.

24. The method of claim 20, where the outer self-assembled monolayer is formed from a mercapto-acid (HS--R--COOH) and a compound having the formula HS--R'"'"'--OH wherein R'"'"' may be any organic group and is chosen to balance insulation with electrical connectivity between the enzymes and the conductive layer.

32. The method of claim 20 in which the enzyme is bound or coordinated directly to the conducting or semiconducting support layer.

25. A method for detecting and/or quantifying the presence of an analyte in a sample comprising the following steps:

(i) immobilization of enzyme molecules by any means to a solid support; and

(ii) direct or indirect measurement of the changes in enzyme electrostatic fields generated by the motions of enzyme molecules during their interaction with said analyte.

28. The method of claim 25 in which the enzymes are part of an Enzyme-Linked Immunosorbent Assay (ELISA).

36. The method of claim 25 in which the energy generated by the moving enzymes is used to provide electrical energy.

26. A biosensor for detecting and/or determining the concentration of an analyte, comprising:

(i) one or more immobilized enzyme molecules; and

(ii) means for measuring the voltage or current generated by the change in location of said immobilized enzyme molecules as a result of an interaction of said immobilized enzyme molecules with said analyte.

27. The biosensor of claim 26 in which the enzymes are derived from natural sources, mutagenic experiments, chemical synthesis and/or chemical modification.

29. The biosensor of claim 26 in which the support material used is semiconducting in nature.

30. The biosensor of claim 26 in which the enzyme is physically absorbed directly or indirectly to a conducting or semiconducting base material.

31. The biosensor of claim 26 in which the enzyme molecule is coordinated to a self-assembled monolayer attached to a conducting/semiconducting support material.

33. The biosensor of claim 26 in which the sensor is part of a solid-state electronic device.

34. The biosensor of claim 26 in which more than one enzyme is attached to a single sensor device and used in the monitoring of more than one analyte type.

35. The biosensor of claim 34 in which the device is referred to as an "enzyme chip" or an "enzyme biochip".