Methods for electronically-controlled enzymatic reactions

• US 7,172,864 B1
• Filed: 01/24/2000
• Issued: 02/06/2007
• Est. Priority Date: 11/01/1993
• Status: Expired due to Fees

• Current Assignee: Gamida for Life B.V.
• Original Assignee: Nanogen
• Inventors: Tu, Eugene, Evans, Glen A., Heller, Michael J., Sosnowski, Ronald G.
• Primary Examiner(s): Brusca; John S
• Assistant Examiner(s): DeJong; Eric S.
• Application Number:
  US09/490,965
• Time in Patent Office: 2,570 Days
• Field of Search: 422/50, 422/68.1, 435/6, 435/7.1, 435/4, 702/19, 702/20, 702/27, 703/11
• US Class Current: 435/6.11
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 19/0093 Microreactors, e.g. miniaturised or microfabricated reactors laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00686 Automatic
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00713 Electrochemical synthesis
• CPC Class Codes: B01J 2219/0072 : Organic compounds
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• CPC Class Codes: B01J 2219/00731 : Saccharides
• CPC Class Codes: B01L 2200/0647 : Handling flowable solids, e.g. microscopic beads, cells, particles
• 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 3/502707 : characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: B01L 3/5085 : for multiple samples, e.g. microtitration plates
• CPC Class Codes: B82Y 10/00 : Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 5/00 : Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07H 21/00 : Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 1/04 : on carriers C07K1/003, C07K1/006 take precedence
• CPC Class Codes: C07K 1/045 : using devices to improve synthesis, e.g. reactors, special vessels
• CPC Class Codes: C07K 1/047 : Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C12Q 1/6813 : Hybridisation assays
• CPC Class Codes: C12Q 1/6816 : characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 : Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2565/515 : characterised by the interaction between or sequential use of two or more arrays
• CPC Class Codes: C12Q 2565/607 : being a sensor, e.g. electrode
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 40/12 : Libraries containing saccharides or polysaccharides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G11C 13/0014 : comprising cells based on organic memory material
• CPC Class Codes: G11C 13/0019 : comprising bio-molecules
• CPC Class Codes: G11C 19/00 : Digital stores in which the information is moved stepwise, e.g. shift registers
• CPC Class Codes: H01L 2224/45144 : Gold (Au) as principal constituent
• CPC Class Codes: H01L 2224/95145 : Electrostatic alignment, i.e. polarity alignment with Coulomb charges
• CPC Class Codes: H01L 25/50 : Multistep manufacturing processes of assemblies consisting of devices, each device being of a type provided for in group H01L27/00 or H01L29/00 H01L21/50 takes precedence
• CPC Class Codes: H01L 2924/00 : Indexing scheme for arrangements or methods for connecting or disconnecting semiconductor or solid-state bodies as covered by H01L24/00
• CPC Class Codes: H01L 2924/10253 : Silicon [Si]

Abstract

A self-addressable, self-assembling microelectronic device is designed and fabricated to actively carry out and control multi-step and multiplex molecular biological reactions in microscopic formats. These reactions include nucleic acid hybridizations, antibody/antigen reactions, diagnostics, and biopolymer synthesis. The device can be fabricated using both microlithographic and micro-machining techniques. The device can electronically control the transport and attachment of specific binding entities to specific micro-locations. The specific binding entities include molecular biological molecules such as nucleic acids and polypeptides. The device can subsequently control the transport and reaction of analytes or reactants at the addressed specific micro-locations. The device is able to concentrate analytes and reactants, remove non-specifically bound molecules, provide stringency control for DNA hybridization reactions, and improve the detection of analytes. The device can be electronically replicated.

Claims

1. A method for an electronically controlled enzymatic reaction at an addressable location, comprising the steps of:

providing an array of microlocations comprising a permeation layer coupled to a plurality of electrodes, wherein each microlocation comprises a distinct electrode within the plurality of electrodes;

contacting a biomolecule in solution with the permeation layer at a microlocation;

concentrating the biomolecule at the microlocation by placing the electrode of the microlocation at an opposite charge to the biomolecule;

attaching the biomolecule to the permeation layer at the microlocation; and

reacting an enzyme with the biomolecule at the microlocation, wherein the enzyme is selected from the group consisting of a restriction enzyme, a ligase, a proteinase, a glycosidase, a DNA polymerase, a RNA polymerase, and a phosphorylase.

2. The method of claim 1, wherein said biomolecule comprises a nucleic acid.

3. The method of claim 2, wherein the addressable location includes a first sequence that is complementary to a first portion of the biomolecule, further comprising the steps of:

contacting a second sequence, the second sequence being complementary to a second portion of the biomolecule, with the biomolecule at said location, the second sequence being capable of being ligated with the first sequence,

enzymatically ligating the first sequence with the second sequence, and

placing the electrode of the microlocation at similar charge to said biomolecule to remove said biomolecule from the ligated first sequence and second sequence.

4. The method of claim 3, wherein the steps are repeated for amplification.

5. The method of claim 4 wherein the amplification is of the biomolecule.

6. The method of claim 3, further including the step of placing the electrode of the microlocation at an opposite charge to said enzyme, thereby concentrating said enzyme on said location.

7. The method of claim 6, wherein the steps are repeated for amplification of the biomolecule.

8. The method of claim 3, wherein the second sequence is labeled.

9. The method of claim 1, wherein said enzyme comprises a DNA polymerase.

10. The method of claim 1, wherein said enzyme comprises an RNA polymerase.

11. The method of claim 1, wherein said enzymatic reaction comprises an enzymatic digestion of a nucleic acid.

12. The method of claim 1, wherein said enzymatic reaction comprises synthesis of a nucleic acid.

13. The method of claim 1, further including the step of placing the electrode of the microlocation at an opposite charge to said enzyme, thereby concentrating said enzyme on said location.

14. A method for electronically controlled amplification of nucleic acid, comprising the steps of:

(1) providing a location comprising a permeation layer coupled to an electrode;

(2) providing an oligonucleotide primer Y attached to said permeation layer;

(3) contacting a single stranded nucleic acid X with said primer Y, wherein said primer Y specifically hybridizes to said nucleic acid X;

(4) placing the electrode of the location at an opposite charge to said nucleic acid X, thereby concentrating said nucleic acid X on said location and hybridizing said nucleic acid X to said primer Y;

(5) contacting a nucleic acid polymerase with said nucleic acid X and said primer Y;

(6) placing the electrode of the location at an opposite charge to said polymerase, thereby concentrating said polymerase on said location and allowing said polymerase to synthesize a nucleic acid Y from said primer Y on said location;

(7) placing the electrode of the location at a negative potential for a sufficient time to remove said nucleic acid X from said location;

(8) contacting an oligonucleotide primer X with said nucleic acid Y, wherein said primer X specifically hybridizes to said nucleic acid Y;

(9) placing the electrode of the location at an opposite charge to said primer X, thereby concentrating said primer X on said location and hybridizing said primer X to said nucleic acid Y; and

(10) placing the electrode of the location at an opposite charge to said polymerase, thereby concentrating said polymerase on said location and allowing said polymerase to synthesize a nucleic acid from said primer X on said location.