U.S. patent application number 10/371066 was filed with the patent office on 2003-08-28 for self-addressable self-assembling microelectronic systems and devices for molecular biological analysis and diagnostics.
This patent application is currently assigned to Nanogen, Inc.. Invention is credited to Evans, Glen A., Heller, Michael J., Sosnowski, Ronald G., Tu, Eugene.
Application Number | 20030162214 10/371066 |
Document ID | / |
Family ID | 27386408 |
Filed Date | 2003-08-28 |
United States Patent
Application |
20030162214 |
Kind Code |
A1 |
Heller, Michael J. ; et
al. |
August 28, 2003 |
Self-addressable self-assembling microelectronic systems and
devices for molecular biological analysis and diagnostics
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.
Inventors: |
Heller, Michael J.;
(Encinitas, CA) ; Tu, Eugene; (San Diego, CA)
; Evans, Glen A.; (Plano, TX) ; Sosnowski, Ronald
G.; (Coronado, CA) |
Correspondence
Address: |
O'MELVENY & MEYERS
114 PACIFICA, SUITE 100
IRVINE
CA
92618
US
|
Assignee: |
Nanogen, Inc.
10398 Pacific Center Court
San Diego
CA
92121
|
Family ID: |
27386408 |
Appl. No.: |
10/371066 |
Filed: |
February 21, 2003 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10371066 |
Feb 21, 2003 |
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09490965 |
Jan 24, 2000 |
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09490965 |
Jan 24, 2000 |
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08271882 |
Jul 7, 1994 |
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6017696 |
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08271882 |
Jul 7, 1994 |
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08146504 |
Nov 1, 1993 |
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5605662 |
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Current U.S.
Class: |
435/6.11 ;
205/777.5; 257/E21.705; 435/287.2 |
Current CPC
Class: |
C12Q 2565/515 20130101;
H01L 2224/45144 20130101; B01J 2219/00653 20130101; H01L 2924/00
20130101; H01L 2924/00 20130101; B01J 2219/0072 20130101; C12Q
1/6813 20130101; C40B 60/14 20130101; B01J 19/0046 20130101; B01J
2219/00637 20130101; H01L 2924/10253 20130101; C07B 2200/11
20130101; B01L 2200/0647 20130101; B01J 2219/00605 20130101; B01J
2219/00612 20130101; B01J 2219/00713 20130101; B01L 2300/0645
20130101; C07H 21/00 20130101; C07K 1/047 20130101; B01J 2219/00585
20130101; B01J 2219/00641 20130101; B01J 2219/00659 20130101; B01J
2219/00722 20130101; C12Q 1/6816 20130101; G11C 13/0014 20130101;
C12Q 2565/607 20130101; G11C 13/0019 20130101; B01J 2219/00317
20130101; B01L 3/5085 20130101; C07K 1/045 20130101; B01J
2219/00315 20130101; C07K 1/04 20130101; B01J 2219/00626 20130101;
B01L 2300/0636 20130101; H01L 2224/45144 20130101; B01J 2219/00621
20130101; B01J 2219/0061 20130101; B01J 2219/00725 20130101; B01J
2219/00596 20130101; C40B 40/10 20130101; C12Q 1/6837 20130101;
B82Y 10/00 20130101; G11C 19/00 20130101; B01L 3/502707 20130101;
B82Y 5/00 20130101; B01J 2219/00689 20130101; H01L 25/50 20130101;
C40B 40/12 20130101; B01J 2219/00686 20130101; B01J 2219/00527
20130101; B01L 2300/0877 20130101; C12Q 1/6825 20130101; C12Q
1/6837 20130101; H01L 2224/95145 20130101; C40B 40/06 20130101;
B01J 19/0093 20130101; B01J 2219/0059 20130101; B01J 2219/00731
20130101; H01L 2924/10253 20130101 |
Class at
Publication: |
435/6 ;
435/287.2; 205/777.5 |
International
Class: |
C12Q 001/68; C12M
001/34 |
Claims
What is claimed is:
1. An electronically self-addressable device, comprising: a
substrate; an electrode, said electrode being supported by said
substrate; a current source operatively connected to said
electrode; and an attachment layer adjacent to said electrode,
wherein said layer is permeable to a counterion but not permeable
to a molecule capable of insulating or binding to said electrode
and said layer is capable of attaching a macromolecule.
2. The electronic device of claim 1, further comprising a
permeation layer, said permeation layer being disposed between said
attachment layer and said electrode.
3. The electronic device of claim 1, wherein said current source
comprises a direct current source.
4. The electronic device of claim 1, wherein said substrate
comprises a member selected from a group consisting of silicon,
glass, silicon dioxide, plastic, and ceramic.
5. The electronic device of claim 1, wherein said substrate
comprises a base and an overlying insulator.
6. The electronic device of claim 5, wherein said base comprises a
member selected from a group consisting of silicon, glass, silicon
dioxide, plastic, and ceramic.
7. The electronic device of claim 5, wherein said base consists of
silicon.
8. The electronic device of claim 5, wherein said insulator
comprises silicon dioxide.
9. The electronic device of claim 1, wherein said substrate
comprises a circuit pattern or circuit board.
10. The electronic device of claim 1, wherein said electrode is
capable of moving a charged macromolecule to said attachment
layer.
11. The electronic device of claim 1, wherein said electrode is
capable of simultaneously moving a charged first macromolecule to
said attachment layer and removing a second macromolecule having
the opposite charge to said first macromolecule from said
attachment layer.
12. The electronic device of claim 2, wherein said permeation layer
comprises aminopropyltriethoxy silane.
13. The electronic device of claim 2, further comprising a buffer
reservoir disposed between said permeation layer and said
electrode.
14. The electronic device of claim 1, wherein the attachment of
said macromolecule to said attachment layer does not insulate said
electrode.
15. The electronic device of claim 1, wherein said electrode
comprises a material selected from a group consisting of aluminum,
gold, silver, tin, copper, platinum, palladium, carbon, and
semiconductor materials.
16. The electronic device of claim 1, wherein the electrode
comprises a material selected from a group consisting of aluminum,
gold, silver, tin, copper, platinum, palladium, carbon, and
semiconductor materials.
17. The electronic device of claim 1, wherein the attachment of a
binding entity to said attachment layer does not insulate said
electrode.
18. An electronically self-addressable device, comprising: a
substrate; a plurality of electrodes, each said electrode being
disposed upon said substrate; a current source operatively
connected to said plurality of electrodes; and an attachment layer
adjacent each said electrode, wherein said layer is permeable to a
counterion but not permeable to a molecule capable of insulating or
binding to each said electrode and said layer is capable of
attaching a macromolecule.
19. The electronic device of claim 18, further comprising a switch
controller which connects said current source to said plurality of
electrodes.
20. The electronic device of claim 18, further comprising a
permeation layer disposed between said attachment layer and each
said electrode.
21. The electronic device of claim 18, wherein each said electrode
comprises a material selected from a group consisting of aluminum,
gold, silver, tin, copper, platinum, palladium, carbon, and
semiconductor material.
22. The electronic device of claim 18, further comprising an
electronic insulative material disposed between said plurality of
electrodes.
23. The electronic device of claim 18, wherein said plurality of
electrodes arranged in an array.
24. The electronic device of claims 18, further comprising a cavity
for holding a solution comprising an entity selected from a group
consisting of binding entities, reagents, and analytes.
25. The electronic device of claim 18, wherein specific binding
entities have been selectively transported and bound to said
plurality of addressable binding locations, forming an addressed
active location device.
26. The electronic device of claim 18, wherein the width of the
binding locations on the device is between 0.5 microns and 200
microns.
27. The electronic device of claim 18, wherein the width of
the.binding locations on the device is between 5 microns and 100
microns.
28. The electronic device of claim 18, wherein said plurality of
binding locations is arranged in a two dimensional array.
29. The electronic device of claim 18, wherein said plurality of
binding locations is arranged in a three dimensional array.
30. The electronic device of claim 18, further comprising a
computation system, wherein said system is electronically connected
to said plurality of binding locations.
31. The electronic device of claim 30, wherein said computation
system is connected to said electrodes.
32. Method for electronically controlling nucleic acid
hybridization, comprising the steps of: providing a location
connected to an electrical source; contacting a first nucleic acid
with a second nucleic acid, wherein said second nucleic acid is
attached to said location; and placing said location at a negative
potential for a sufficient time,,wherein said first nucleic acid is
removed from said second nucleic acid if said first nucleic acid is
a non-specific nucleic acid sequence to said second nucleic acid,
but not removed if said first nucleic acid is a specific nucleic
acid sequence to said second nucleic acid.
33. The method of claim 32, wherein both said first nucleic acid
and said second nucleic acid are in a solution.
34. The method of claim 32, further comprising the step of placing
said location at a positive potential before placing said location
at a negative potential, thereby concentrating said first nucleic
acid on said location.
35. The method of claim 32, wherein said negative potential is
increased or decreased incrementally.
36. The method of claim 32, wherein said non-specific nucleic acid
sequence has one mismatch with the sequence of said second nucleic
acid.
37. The method of claim 32, wherein said first nucleic acid
consists of no more than seven nucleotides.
38. The method of claim 32, wherein said first nucleic acid
consists of no less than 22 nucleotides.
39. The method of claim 32, wherein said first nucleic acid
consists of between 7 and 22 nucleotides.
40. The method of claim 32, wherein said first nucleic acid
comprises a detectable element.
41. The method of claim 32, wherein said first nucleic acid
comprises a fluorophore.
42. The method of claim 41, wherein said fluorophore is selected
from a group consisting of Texas Red and fluorescein.
43. The method of claim 32, wherein said first nucleic acid
comprises a deoxyribonucleotide.
44. The method of claim 32, wherein said first nucleic acid
comprises a ribonucleotide.
45. The method of claim 32, wherein said first nucleic acid
comprises a modified nucleotide.
46. The method of claim 33, further comprising the steps of: adding
a detectable dye in said solution, wherein said dye binds to
double-stranded nucleic acid with higher affinity than
single-stranded nucleic acid; and determining the level of
hybridization between said first nucleic acid and said second
nucleic acid at said location by measuring the level of said dye at
said location.
47. The method of claim 46, wherein said dye comprises ethidium
bromide.
48. The method of claim 33, further comprising the steps of: adding
a detectable dye in said solution, wherein said dye gives a
stronger detectable signal when in contact with a double-stranded
nucleic acid than with a single-stranded nucleic acid; and
determining the level of hybridization between said first nucleic
acid and said second nucleic acid at said location by measuring the
level of said detectable signal of said dye at said location.
49. The method of claim 48, wherein said dye comprises ethidium
bromide.
50. The method of claim 33, wherein said solution comprising a
third nucleic acid consisting of a non-pecific nucleic acid
sequence to said second nucleic acid.
51. The method of claim 50, wherein the concentration of said third
nucleic acid is more than 1,000 fold of the concentration of said
first nucleic acid.
52. The method of claim 32, wherein said first nucleic acid
consists of seven nucleotides.
53. The method of claim 32, wherein said first nucleic acid
consists of between 5 and 7 nucleotides.
54. The method of claim 32, wherein said first nucleic acid
consists of 22 nucleotides.
55. Method for electronically controlling nucleic acid
hybridization, comprising the steps of: providing a location
connected to an electrical source; contacting a plurality of
nucleic acids with a target nucleic acid, wherein said target
nucleic acid is attached to said location; and placing said
location at a negative potential for a sufficient time, wherein a
non-specific nucleic acid sequence to said target nucleic acid but
not a specific nucleic acid sequence from said plurality of nucleic
acids is removed from said target nucleic acid.
56. Method for electronically concentrating an electrically charged
entity in a solution at a location, comprising the steps of;
contacting said solution with a first location including a first
underlying electrode, and a second location including a second
underlying electrode; and placing said first location at an
opposite charge to said entity, relative to said second location,
thereby concentrating said entity on said first location but not
said second location.
57. The method of claim 56, further comprising the step of placing
said second location at the same charge to said entity.
58. The method of claim 56, further comprising the step of forming
a covalent bond between said entity and an attachment layer at said
first location.
59. The method of claim 56, wherein said entity is a nucleic acid
and said first location is charged with positive potential.
60. The method of claim 59, wherein said second location is charged
with negative potential.
61. The method of claim 56, wherein the concentration of said
entity at said first location is more than 10 times of that of said
entity at said second location.
62. The method of claim 56, wherein the concentration of said
entity at said first location is more than 1,000 times of that of
said entity at said second location.
63. The method of claim 56, wherein the concentration of said
entity at said first location is more than 10.sup.6 times of that
of said entity at said second location.
64. The method of claim 56, further comprising the step of
attaching said entity to said first location.
65. Method for electronically transporting a charged entity in a
solution from a first location to a second location, comprising the
steps of: contacting said solution with said first and second
locations; placing said first location at an opposite charge to
said entity, relative to said second location, thereby transporting
said entity to said first location; and thereafter, placing said
second location at an opposite charge to said entity, relatively to
said first location, thereby transporting said nucleic acid from
said first location to said second location.
66. The method of claim 65, wherein said entity is a nucleic
acid.
67. The method of claim 65, further comprising the step of
attaching said entity to said second location.
68. Method for electronically controlled synthesis of biopolymers
on a plurality of locations, comprising the steps of: providing a
plurality of reaction locations on a substrate, wherein each
reaction location is individually electronically addressable;
forming an attachment layer upon each reaction location; contacting
said plurality of reaction locations with a solution comprising a
charged monomer-A; selectively biasing a designated A location at
which reaction A is to occur at an opposite charge to monomer-A,
and biasing another location at which no reaction A is to occur the
same charge as monomer-A, thereby concentrating and reacting
monomer A on said A location; thereafter, removing the unreacted
monomer-A from said plurality of reaction locations; contacting
said plurality of reaction locations with a solution comprising a
charged monomer-B; selectively biasing said A location at the
opposite charge of monomer-B, and biasing another location at which
no reaction B is to occur the same charge as monomer-B, thereby
concentrating and reacting monomer B on said A location to form
dimer A-B.
69. The method of claim 68, wherein said monomer-A consists of a
nucleotide and said monomer-B consists of a nucleotide.
70. The method of claim 68, wherein said monomer-A consists of an
amino acid and said monomer-B consists of an amino acid.
71. Method for replicating a master self-addressable electronic
device addressed with specific nucleic acid sequences, comprising
the steps of: providing complimentary sequences to said specific
nucleic acid sequences and hybridizing said complimentary sequences
to said specific nucleic acid sequences addressed on said master
device; aligning unaddressed locations on a recipient
self-addressable electronic device with the addressed locations on
said master device; and biasing the locations on said master device
negative and the locations on said recipient device positive,
thereby transporting said complimentary sequences to said
unaddressed locations on said recipient device.
72. The method for replicating patterned sequences of claim 71,
further comprising the step of providing a positively charged
chaotropic agent or denaturant of denature the complimentary
sequences from the master template.
73. A self-addressable electronic device for genetic typing,
comprising: a plurality of electronically addressable locations
each comprising an electrode; and a binding entity attached to each
of said plurality of locations, wherein each said entity is capable
of detecting the presence of a genetic sequence.
74. The self-addressable electronic device of claim 73, wherein
said genetic sequence is a nucleotide sequence.
75. The self-addressable electronic device of claim 74, wherein
said nucleotide sequence is a cDNA sequence.
76. The self-addressable electronic device of claim 74, wherein
said nucleotide sequence is a genomic DNA sequence.
77. The self-addressable electronic device of claim 74, wherein
said nucleotide sequence is an mRNA sequence.
78. The self-addressable electronic device of claim 74, wherein
said nucleotide sequence is a cRNA sequence.
79. The self-addressable electronic device of claim 73, wherein
said genetic sequence is an amino acid sequence.
80. The self-addressable electronic device of claim 73, wherein
each said binding entity attached to each said plurality of
locations is the same.
81. The self-addressable electronic device of claim 73, wherein a
said binding entity is different from another said binding
entity.
82. Method for electronically controlled genetic typing, comprising
the steps of: providing a plurality of electronically addressable
locations each comprising an electrode; attaching a binding entity
to each of said plurality of locations, wherein each said entity is
capable of detecting the presence of a genetic sequence; contacting
a sample with said plurality of locations; determining the genetic
profile of said sample by detecting the presence or absence of said
genetic sequence at each of said plurality of locations.
83. Method for electronically controlled enzymatic reaction at an
addressable location, comprising the steps of: providing an
electronically addressable location comprising an electrode;
contacting a substrate with said location; placing said location at
an opposite charge to said substrate, thereby concentrating said
substrate on said location; attaching said substrate to said
location; contacting an enzyme with said location; placing said
location at an opposite charge to said enzyme, thereby
concentrating said enzyme on said location; and allowing said
enzyme to react with said substrate on said location.
84. The method of claim 83, wherein said substrate comprises a
nucleic acid.
85. The method of claim 83, wherein said enzyme comprises a
restriction enzyme, a ligase, a proteinase, a glycosidase, or a
phosphorylase.
86. The method of claim 83, wherein said enzyme comprises a DNA
polymerase.
87. The method of claim 83, wherein said enzyme comprises an RNA
polymerase.
88. The method of claim 83, wherein said enzymatic reaction
comprises an enzymatic digestion of a nucleic acid.
89. The method of claim 83, wherein said enzymatic reaction
comprises synthesis of a nucleic acid.
90. The method of claim 83, wherein said enzymatic reaction
comprises synthesis of a polypeptide.
91. Method for electronically controlled amplification of nucleic
acid, comprising the steps of: (1) providing an electronically
addressable location comprising an electrode; (2) providing an
oligonucleotide primer Y attached to said location; (3) contacting
a single stranded nucleic acid X with said location, wherein said
primer Y specifically hybridizes to said nucleic acid X; (4)
placing said 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 location; (6) placing said
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 said 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
location, wherein said primer X specifically hybridizes to said
nucleic acid Y; (9) placing said 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; (10) placing
said 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.
92. Method for electronically controlling binding between
macromolecules, comprising the steps of: contacting a charged first
macromolecule with a second macromolecule in a direct current
electric field, wherein said second macromolecule is attached to a
location; placing said location at a potential opposite to the
charge of said first macromolecule for a sufficient time, wherein
said first macromolecule is removed from said second molecule if
said first macromolecule does not specifically bind to said second
macromolecule, but not removed if said first macromolecule
specifically binds to said second macromolecule.
93. The method of claim 92, wherein said first macromolecule is a
polypeptide.
94. The method of claim 92, wherein said first macromolecule is a
nucleic acid.
Description
FIELD OF THE INVENTION
[0001] This invention pertains to the design, fabrication, and uses
of a self-addressable, self-assembling microelectronic system which
can actively carry out and control multi-step and multiplex
reactions in microscopic formats. In particular, these reactions
include molecular biological reactions, such as nucleic acid
hybridizations, nucleic acid amplification, sample preparation,
antibody/antigen reactions, clinical diagnostics, and biopolymer
synthesis.
BACKGROUND OF THE INVENTION
[0002] Molecular biology comprises a wide variety of techniques for
the analysis of nucleic acids and proteins, many of which form the
basis of clinical diagnostic assays. These techniques include
nucleic acid hybridization analysis, restriction enzyme analysis,
genetic sequence analysis, and separation and purification of
nucleic acids and proteins (See, e.g., J. Sambrook, E. F. Fritsch,
and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2 Ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York,
1989).
[0003] Many molecular biology techniques involve carrying out
numerous operations on a large number of samples. They are often
complex and time consuming, and generally require a high degree of
accuracy. Many a technique is limited in its application by a lack
of sensitivity, specificity, or reproducibility. For example,
problems with sensitivity and specificity have so far limited the
practical applications of nucleic acid hybridization.
[0004] Nucleic acid hybridization analysis generally involves the
detection of a very small number of specific target nucleic acids
(DNA or RNA) with probes among a large amount of non-target nucleic
acids. In order to keep high specificity, hybridization is normally
carried out under the most stringent conditions, achieved through
various combinations of temperature, salts, detergents, solvents,
chaotropic agents, and denaturants.
[0005] Multiple sample nucleic acid hybridization analysis has been
conducted on a variety of filter and solid support formats (see G.
A. Beltz et al., in Methods in Enzymology, Vol. 100, Part B, R. Wu,
L. Grossmam, K. Moldave, Eds., Academic Press, New York, Chapter
19, pp. 266-308, 1985). One format., the so-called "dot blot"
hybridization, involves the non-covalent attachment of target DNAs
to a .filter, which are subsequently hybridized with a radioisotope
labeled probe(s). "Dot blot" hybridization gained wide-spread use,
and many versions were developed (see M. L., M. Anderson and B. D.
Young, in Nucleic Acid Hybridization--A Practical Approach, B. D.
Hames and S. J. Higgins, Eds., IRL Press, Washington DC, Chapter 4,
pp. 73-111, 1985). The "dot blot" hybridization has been further
developed for multiple analysis of genomic mutations (D.
Nanibhushan and D. Rabin, in EPA 0228075, Jul. 8, 1987) and for the
detection of overlapping clones and the construction of genomic
maps (G. A. Evans, in U.S. Pat. No. 5,219,726, Jun. 15, 1993).
[0006] Another format, the so-called "sandwich" hybridization,
involves attaching oligonucleotide probes covalently to a solid
support and using them to capture and detect multiple nucleic acid
targets. (M. Ranki et al., Gene, 21, pp. 77-85, 1983; A. M. Palva,
T. M. Ranki, and H. E. Soderlund, in UK Patent Application GB
2156074A, Oct. 2, 1985; T. M. Ranki and H. E. Soderlund in U.S.
Pat. No. 4,563,419, Jan. 7, 1986; A. D. B. Malcolm and J. A.
Langdale, in PCT WO 86/03782, Jul. 3, 1986; Y. Stabinsky, in U.S.
Pat. No. 4,751,177, Jan. 14, 1988; T. H. Adams et al., in PCT WO
90/01564, Feb. 22, 1990; R. B. Wallace et al. 6 Nucleic Acid Res.
11, p. 3543, 1979; and B. J. Connor et al., 80 Proc. Natl. Acad.
Sci. U.S.A pp. 278-282, 1983). Multiplex versions of these formats
are called "reverse dot blots".
[0007] Using the current nucleic acid hybridization formats and
stringency control methods, it remains difficult to detect low copy
number (i.e., 1-100,000) nucleic acid targets even with the most
sensitive reporter groups (enzyme, fluorophores, radioisotopes,
etc.) and associated detection systems (fluorometers, luminometers,
photon counters, scintillation counters, etc.).
[0008] This difficulty is caused by several underlying problems
associated with direct probe hybridization. One problem relates to
the stringency control of hybridization reactions. Hybridization
reactions are usually carried out under the stringent conditions in
order to achieve hybridization specificity. Methods of stringency
control involve primarily the optimization of temperature, ionic
strength, and denaturants in hybridization and subsequent washing
procedures. Unfortunately, the application of these stringency
conditions causes a significant decrease in the number of
hybridized probe/target complexes for detection.
[0009] Another problem relates to the high complexity of DNA in
most samples, particularly in human genomic DNA samples. When a
sample is composed of an enormous number of sequences which are
closely related to the specific target sequence, even the most
unique probe sequence has a large number of partial hybridizations
with non-target sequences.
[0010] A third problem relates to the unfavorable hybridization
dynamics between a probe and its specific target. Even under the
best conditions, most hybridization reactions are conducted with
relatively low concentrations of probes and target molecules. In
addition, a probe often has to compete with the complementary
strand for the target nucleic acid.
[0011] A fourth problem for most present hybridization formats is
the high level of non-specific background signal. This is caused by
the affinity of DNA probes to almost any material.
[0012] These problems, either individually or in combination, lead
to a loss of sensitivity and/or specificity for nucleic acid
hybridization in the above described formats. This is unfortunate
because the detection of low copy number nucleic acid targets is
necessary for most nucleic acid-based clinical diagnostic
assays.
[0013] Because of the difficulty in detecting low copy number
nucleic acid targets, the research community relies heavily on the
polymerase chain reaction (PCR) for the amplification of target
nucleic acid sequences (see M. A. Innis et al., PCR Protocols: A
Guide to Methods and Applications, Academic Press, 1990). The
enormous number of target nucleic acid sequences produced by the
PCR reaction improves the subsequent direct nucleic acid probe
techniques, albeit at the cost of a lengthy and cumbersome
procedure.
[0014] A distinctive exception to the general difficulty in
detecting low copy number target nucleic acid with a direct probe
is the in-situ hybridization technique. This technique allows low
copy number unique nucleic acid sequences to be detected in
individual cells. In the in-situ format, target nucleic acid is
naturally confined to the area of a cell (.about.20-50 .mu.m.sup.2)
or a nucleus (.about.10 .mu.m.sup.2) at a relatively high local
concentration. Furthermore, the probe/target hybridization signal
is confined to a microscopic and morphologically distinct area;
this makes it easier to distinguish a positive signal from
artificial or non-specific signals than hybridization on a solid
support.
[0015] Mimicking the in-situ hybridization in some aspects, new
techniques are being developed for carrying out multiple sample
nucleic acid hybridization analysis on micro-formatted multiplex or
matrix devices (e.g., DNA chips) (see M. Barinaga, 253 Science, pp.
1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992). These
methods usually attach specific DNA sequences to very small
specific areas of a solid support, such as micro-wells of a DNA
chip. These hybridization formats are micro-scale versions of the
conventional "reverse dot blot" and "sandwich" hybridization
systems.
[0016] The micro-formatted hybridization can be used to carry out
"sequencing by hybridization" (SBH) (see M. Barinaga, 253 Science,
pp. 1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992).
SBH makes use of all possible n-nucleotide oligomers (n-mers) to
identify n-mers in an unknown DNA sample, which are subsequently
aligned by algorithm analysis to produce the DNA sequence (R.
Drmanac and R. Crkvenjakov, Yugoslav Patent Application #570/87,
1987; R. Drmanac et al., 4 Genomics, 114, 1989; Strezoska et al.,
88 Proc. Natl. Acad. Sci. U.S.A 10089, 1991; and R. Drmanac and R.
B. Crkvenjakov, U.S. Pat. No. 5,202,231, Apr. 13, 1993).
[0017] There are two formats for carrying out SBH. One format
involves creating an array of all possible n-mers on a support,
which is then hybridized with the target sequence. This is a
version of the reverse dot blot. Another format involves attaching
the target sequence to a support, which is sequentially probed with
all possible n-mers. Both formats have the fundamental problems of
direct probe hybridizations and additional difficulties related to
multiplex hybridizations.
[0018] Southern, United Kingdom Patent-Application GB 8810400,
1988; E. M. Southern et al., 13 Genomics 1008, 1992, proposed using
the "reverse dot blot" format to analyze or sequence DNA. Southern
identified a known single point mutation using PCR amplified
genomic DNA. Southern also described a method for synthesizing an
array of oligonucleotides on a solid support for SBH. However,
Southern did not address how to achieve optimal stringency
condition for each oligonucleotide on an array.
[0019] Fodor et al., 364 Nature, pp. 555-556, 1993, used an array
of 1,024 8-mer oligonucleotides on a solid support to sequence DNA.
In this case, the target DNA was a fluorescently labeled
single-stranded 12-mer oligonucleotide containing only the A and C
bases. A concentration of 1 pmol (.about.6.times.10.sup.11
molecules) of the 12-mer target sequence was necessary for the
hybridization with the 8-mer oligomers on the array. The results
showed many mismatches. Like Southern, Fodor et al., did not
address the underlying problems of direct probe hybridization, such
as stringency control for multiplex hybridizations. These problems,
together with the requirement of a large quantity of the simple
12-mer target, indicate severe limitations to this SBH format.
[0020] Concurrently, Drmanac et al., 260 Science 1649-1652, 1993,
used the above discussed second format to sequence several short
(116 bp) DNA sequences. Target DNAs were attached to membrane
supports ("dot blot" format). Each filter was sequentially
hybridized with 272 labeled 10-mer and 11-mer oligonucleotides. A
wide range of stringency conditions were used to achieve specific
hybridization for each n-mer probe; washing times varied from 5
minutes to overnight, and temperatures from 0.degree. C. to
16.degree. C. Most probes required 3 hours of washing at 16.degree.
C. The filters had to be exposed for 2 to 18 hours in order to
detect hybridization signals. The overall false positive
hybridization rate was 5% in spite of the simple target sequences,
the reduced set of oligomer probes, and the use of the most
stringent conditions available.
[0021] Fodor et al., 251 Science 767-773, 1991, used
photolithographic techniques to synthesize oligonucleotides on a
matrix. Pirrung et al., in U.S. Pat. No. 5,143,854, Sep. 1, 1992,
teach large scale photolithographic solid phase synthesis of
polypeptides in an array fashion on silicon substrates.
[0022] In another approach of matrix hybridization, Beattie et al.,
in The 1992 San Diego Conference: Genetic Recognition, November,
1992, used a microrobotic system to deposit micro-droplets
containing specific DNA sequences into individual microfabricated
sample wells on a glass substrate. The hybridization in each sample
well is detected by interrogating miniature electrode test
fixtures, which surround each individual microwell with an
alternating current (AC) electric field.
[0023] Regardless of the format, all current micro-scale DNA
hybridizations and SBH approaches do not overcome the underlying
problems associated with nucleic acid hybridization reactions. They
require very high levels of relatively short single-stranded target
sequences or PCR amplified DNA, and produce a high level of false
positive hybridization signals even under the most stringent
conditions. In the case of multiplex formats using arrays of short
oligonucleotide sequences, it is not possible to optimize the
stringency condition for each individual sequence with any
conventional approach because the arrays or devices used for these
formats can not change or adjust the temperature, ionic strength,
or denaturants at an individual location, relative to other
locations. Therefore, a common stringency condition must be used
for all the sequences on the device. This results in a large number
of non-specific and partial hybridizations and severely limits the
application of the device. The problem becomes more compounded as
the number of different sequences on the array increases, and as
the length of the sequences decreases below 10-mers or increases
above 20-mers. This is particularly troublesome for SBH, which
requires a large number of short oligonucleotide probes.
[0024] Nucleic acids of different size, charge, or conformation are
routinely separated by electrophoresis techniques which can
distinguish hybridization species by their differential mobility in
an electric field. Pulse field electrophoresis uses an arrangement
of multiple electrodes around a medium (e.g., a gel) to separate
very large DNA fragments which cannot be resolved by conventional
gel-electrophoresis systems (see R. Anand and E. M. Southern in Gel
Electrophoresis of Nucleic Acids--A Practical Approach, 2 ed., D.
Rickwood and B. D. Hames Eds., IRL Press, New York, pp. 101-122,
1990).
[0025] Pace, U.S. Pat. No. 4,908,112, Mar. 13, 1990, describes
using micro-fabrication techniques to produce a capillary gel
electrophoresis system on a silicon substrate. Multiple electrodes
are incorporated into the system to move molecules through the
separation medium within the device.
[0026] Soane and Soane, U.S. Pat. No. 5,126,022, Jun. 30, 1992,
describe that a number of electrodes can be used to control the
linear movement of charged molecules in a mixture through a gel
separation medium contained in a tube. Electrodes have to be
installed within the tube to control the movement and position of
molecules in the separation medium.
[0027] Washizu, M. and Kurosawa, O., 26 IEEE Transactions on
Industry Applications 6, pp. 1165-1172, 1990, used high-frequency
alternating current (AC) fields to orient DNA molecules in electric
field lines produced between microfabricated electrodes. However,
the use of direct current (DC) fields is prohibitive for their
work. Washizu Journal of Electrostatics 109-123, 1990, describes
the manipulation of cells and biological molecules using
dielectrophoresis. Cells can be fused and biological molecules can
be oriented along the electric fields lines produced by AC voltages
between the micro-electrode structures. However, the
dielectrophoresis process requires a very high frequency AC (1 MHz)
voltage and a low conductivity medium. While these techniques can
orient DNA molecules of different sizes along the AC field lines,
they cannot distinguish between hybridization complexes of the same
size.
[0028] As is apparent from the preceding discussion, numerous
attempts have been made to provide effective techniques to conduct
multi-step, multiplex molecular biological reactions. However, for
the reasons stated above, these techniques have been proved
deficient. Despite the long-recognized need for effective
technique, no satisfactory solution has been proposed
previously.
SUMMARY OF THE INVENTION
[0029] The present invention relates to the design, fabrication,
and uses of programmable, self-addressable and self-assembling
microelectronic systems and devices which can actively carry out
controlled multi-step and multiplex reactions in microscopic
formats. These reactions include, but are not limited to, most
molecular biological procedures, such as nucleic acid
hybridizations, anti-body/antigen reactions, and related clinical
diagnostics. In addition, the claimed devices are able to carry out
multi-step combinational biopolymer synthesis, including, but not
limited to, the synthesis of different oligo-nucleotides or
peptides at specific micro-locations.
[0030] The claimed devices are fabricated using both
microlithographic and micro-machining techniques. The devices have
a matrix of addressable microscopic locations on their surface;
each individual micro-location is able to electronically control
and direct the transport and attachment of specific binding
entities (e.g., nucleic acids, antibodies) to itself. All
micro-locations can be addressed with their specific binding
entities. Using these devices, the system can be self-assembled
with minimal outside intervention.
[0031] The addressed devices are able to control and actively carry
out a variety of assays and reactions. Analytes or reactants can be
transported by free field electrophoresis to any specific
micro-location where the analytes or reactants are effectively
concentrated and reacted with the specific binding entity at said
micro-location. The sensitivity for detecting a specific analyte or
reactant is improved because of the concentrating effect. Any
un-bound analytes or reactants can be removed by reversing the
polarity of a micro-location. Thus, the devices also improve the
specificity of assays and reactions.
[0032] The active nature of the devices provides independent
electronic control over all aspects of the hybridization reaction
(or any other affinity reaction) occurring at each specific
micro-location. These devices provide a new mechanism for affecting
hybridization reactions which is called electronic stringency
control (ESC). For DNA hybridization reactions which require
different stringency conditions, ESC overcomes the inherent
limitation of conventional array technologies. The active devices
of this invention can electronically produce "different stringency
conditions" at each micro-location. Thus, all hybridizations can be
carried out optimally in the same bulk solution. These active
devices are fundamentally different from conventional multiplex
hybridization arrays and DNA chips. While conventional arrays have
different probes or target DNA's located at each site; all the
sites on the array have the same common reaction or stringency
conditions of temperature, buffer, salt concentration, and pH. Any
change in the reaction or stringency condition, affects all sites
on the array. While sophisticated photolithographic techniques may
be used to make an array, or microelectronic sensing elements are
incorporated for detection, conventional devices are passive and do
not control or influence the actual hybridization process. The
active devices of this invention allow each micro-location to
function as a completely independent test or analysis site (i.e.
they form the equivalent of a "test tube" at each location).
Multiple hybridization reactions can be carried out with minimal
outside physical manipulations. Additionally, it is unnecessary to
change temperatures, to exchange buffers, and the need for multiple
washing procedures is eliminated.
[0033] Thus, the claimed devices can carry out multi-step and
multiplex reactions with complete and precise electronic control,
preferably with overall micro-processor control (i.e. run by a
computer). The rate, specificity, and sensitivity of multi-step and
multiplex reactions are greatly improved at each specific
micro-location on the claimed device.
[0034] The device also facilitates the detection of hybridized
complexes at each micro-location by using an associated optical
(fluorescent, chemiluminescent, or spectrophotometric) imaging
detector system. Integrated optoelectronic or electronic sensing
components which directly detect DNA, can also be incorporated
within the device itself.
[0035] If desired, a master device addressed with specific binding
entities can be electronically replicated or copied to another base
device.
[0036] This invention may utilize micro-locations of any size or
shape consistent with the objective of the invention. In the
preferred embodiment of the invention, micro-locations in the
sub-millimeter range are used.
[0037] By "specific binding entity" is generally meant a biological
or synthetic molecule that has specific affinity to another
molecule, macromolecule or cells, through covalent bonding or
non-covalent bonding. Preferably, a specific binding entity
contains (either by nature or by modification) a functional
chemical group (primary amine, sulfhydryl, aldehyde, etc.), a
common sequence (nucleic acids), an epitope (antibodies), a hapten,
or a ligand, that allows it to covalently react or non-covalently
bind to a common functional group on the surface of a
micro-location. Specific binding entities include, but are not
limited to: deoxyribonucleic acids (DNA), ribonucleic acids (RNA),
synthetic oligonucleotides, antibodies, proteins, peptides,
lectins, modified polysaccharides, cells, synthetic composite
macromolecules, functionalized nanostructures, synthetic polymers,
modified/blocked nucleotides/nucleosides, modified/blocked amino
acids, fluorophores, chromophores, ligands, chelates and
haptens.
[0038] By "stringency control" is meant the ability to discriminate
specific and non-specific binding interactions by changing some
physical parameter. In the case of nucleic acid hybridizations,
temperature control is often used for stringency. Reactions are
carried out at or near the melting temperature (Tm) of the
particular double-stranded hybrid pair.
[0039] Thus, the first and most important aspect of the present
invention is a device with an array of electronically programmable
and self-addressable microscopic locations. Each microscopic
location contains an underlying working direct current (DC)
micro-electrode supported by a substrate. The surface of each
micro-location has a permeation layer for the free transport of
small counter-ions, and an attachment layer for the covalent
coupling of specific binding entities. These unique design features
provide the following critical properties for the device: (1) allow
a controllable functioning DC electrode to be maintained beneath
the microlocation; (2) allow electrophoretic transport to be
maintained; and (3) separate the affinity or binding reactions from
the electrochemical and the adverse electrolysis reactions
occurring at the electrode (metal) interfaces. It should be
emphasized that the primary function of the micro-electrodes used
in these devices is to provide electrophoretic propulsion of
binding and reactant entities to specific locations.
[0040] By "array" or "matrix" is meant an arrangement of
addressable locations on the device. The locations can be arranged
in two dimensional arrays, three dimensional arrays, or other
matrix formats. The number of locations can range from several to
at least hundreds of thousands. Each location represents a totally
independent reaction site.
[0041] In a second aspect, this invention features a method for
transporting the binding entity to any specific micro-location on
the device. When activated, a micro-location can affect the free
field electrophoretic transport of any charged functionalized
specific binding entity directly to itself. Upon contacting the
specific micro-location, the functionalized specific binding entity
immediately becomes covalently attached to the attachment layer
surface of that specific micro-location. Other micro-locations can
be simultaneously protected by maintaining them at the opposite
potential to the charged molecules. The process can be rapidly
repeated until all the micro-locations are addressed with their
specific binding entities.
[0042] By "charged functionalized specific binding entity" is meant
a specific binding entity that is chemically reactive (i.e.,
capable of covalent attachment to a location) and carries a net
charge (either positive or negative).
[0043] In a third aspect, this invention features a method for
concentrating and reacting analytes or reactants at any specific
micro-location on the device. After the attachment of the specific
binding entities, the underlying microelectrode at each
micro-location continues to function in a direct current (DC) mode.
This unique feature allows relatively dilute charged analytes or
reactant molecules free in solution to be rapidly transported,
concentrated, and reacted in a serial or parallel manner at any
specific micro-locations which are maintained at the opposite
charge to the analyte or reactant molecules. Specific
micro-locations can be protected or shielded by maintaining them at
the same charge as the analytes or reactant molecules. This ability
to concentrate dilute analyte or reactant molecules at selected
micro-locations greatly accelerates the reaction rates at these
micro-locations.
[0044] When the desired reaction is complete, the micro-electrode
potential can be reversed to remove non-specific analytes or
unreacted molecules from the micro-locations.
[0045] Specific analytes or reaction products may be released from
any micro-location and transported to other locations for further
analysis; or stored at other addressable locations; or removed
completely from the system.
[0046] The subsequent analysis of the analytes at the specific
micro-locations is also greatly improved by the ability to repulse
non-specific entities from these locations.
[0047] In a fourth aspect, this invention features a method for
improving stringency control of nucleic acid hybridization
reactions, comprising the steps of:
[0048] rapidly concentrating dilute target DNA and/or probe DNA
sequences at specific micro-location(s) where hybridization is to
occur;
[0049] rapidly removing non-specifically bound target DNA sequences
from specific micro-location(s) where hybridization has
occurred;
[0050] rapidly removing competing complementary target DNA
sequences from specific micro-location(s) where hybridization has
occurred;
[0051] adjusting electronic stringency control (ESC) to remove
partially hybridized DNA sequences (more than one base
mis-match);
[0052] adjusting ESC to improve the resolution of single mis-match
hybridizations using probes in the 8-mer to 21-mer range(e.g., to
identify point mutations);
[0053] using ESC to efficiently hybridize oligonucleotide point
mutation probes outside of the ranges used in conventional
procedures (e.g., probes longer than 21-mers and shorter than
8-mers);
[0054] applying independent ESC to individual hybridization events
occurring in the same bulk solution and at the same temperature;
and
[0055] using ESC to improve hybridization of un-amplified target
DNA sequences to arrays of capture oligonucleotide probes.
[0056] In a fifth aspect, this invention features a method for the
combinaorial synthesis of biopolymers at micro-locations.
[0057] In a sixth aspect, this invention features a method for
replicating a master device.
[0058] In a seventh aspect, this invention-features a device which
electronically carries out sample preparation and transports target
DNA to the analytical component of the device.
[0059] In an eighth aspect, this invention features a device which
electronically delivers reagents and reactants with minimal use of
fluidics.
[0060] In a ninth aspect, this invention features a device which
carries out molecular biology and DNA amplification reactions (e.g.
restriction cleavage reactions; and DNA/RNA polymerase and DNA
ligase target amplification reactions.
[0061] In a tenth aspect, this invention features a device which
can electronically size and identify restriction fragments (e.g.
carry out electronic restriction fragment length polymorphism and
DNA finger printing analysis).
[0062] In a eleventh aspect, this invention features a device which
carries out antibody-antigen and immunodiagnostic reactions.
[0063] In a twelveth aspect, this invention features a device which
is able to carry out combinatorial synthesis of oligonucleotides
and peptides.
[0064] In a thirteenth aspect, this invention features a device
which selectively binds cells, processes cells for hybridization,
removes DNA from cells, or carries out electronic in-situ
hybridization within the cells.
[0065] In a fourteenth aspect, this invention features methods for
detecting and analyzing reactions that have occurred at the
addressed micro-locations using self-addressed microelectronic
devices with associated optical, optoelectronic or electronic
detection systems or self-addressed microelectronic devices with
integrated optical, optoelectronic or electronic detection
systems.
[0066] Because the devices of this invention are active
programmable electronic matrices, the acronym "APEX" is used to
describe or designate the unique nature of these devices. The APEX
acronym is used for both the microlithographically produced "chips"
and micro-machined devices.
[0067] The active nature of APEX microelectronic devices and chips
allows us to create new mechanisms for carrying out a wide variety
of molecular biological reactions. These include novel methods for
achieving both the linear and exponential multiplication or
amplification of target DNA and RNA molecules.
[0068] The device provides electronic mechanisms to: (1)
selectively denature DNA hybrids in common buffer solutions at room
temperature (e.g. well below their Tm points); (2) to rapidly
transport or move DNA back and forth between two or more
micro-locations; and (3) to selectively concentrate specific
reactants, reagents, and enzymes at the desired micro-locations.
These all involve new physical parameters for carrying out
molecular biological and target amplification type reactions.
[0069] A number of examples of electronically controlled molecular
biology reactions have been developed, these include: (1)
Electronically Directed Restriction Enzyme cleavage of Specific
ds-DNA Sequences; (2) Electronic Restriction Fragment Analysis; (3)
Electronic Multiplication of Target DNA By DNA Polymerases; (4)
Electronic Ligation and Multiplication of Target DNA Sequences By
DNA and RNA Ligases; and (5) Electronic Multiplication of Target
DNA By RNA Polymerases. These examples are representative of the
types of molecular biological reactions and procedures which can be
carried out on the APEX devices.
[0070] Other features and advantages of the invention will be
apparent from the following detailed description of the invention,
and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0071] FIG. 1 is the cross-section of three self-addressable
micro-locations fabricated using microlitho-graphic techniques.
[0072] FIG. 2 is the cross-section of a microlithographically
fabricated micro-location.
[0073] FIG. 3 is a schematic representation of a self-addressable
64 micro-location chip which was actually fabricated, addressed
with oligonucleotides, and tested.
[0074] FIG. 4 shows particular attachment chemistry procedure which
allows rapid covalent coupling of specific oligonucleotides to the
attachment surface of a micro-location.
[0075] FIG. 5 is a blown-up schematic diagram of a micro-machined
96 micro-locations device.
[0076] FIG. 6 is the cross-section of a micro-machined device.
[0077] FIG. 7 shows the mechanism the device uses to electronically
concentrate analyte or reactant molecules at a specific
micro-location.
[0078] FIG. 8 shows the self-directed assembly of a device with
three specific oligonucleotide binding entities (SSO-A, SSO-B, and
SSO-C).
[0079] FIG. 9 shows an electronically controlled hybridization
process with sample/target DNA being concentrated at
micro-locations containing specific DNA capture sequences.
[0080] FIG. 10 shows an electronically directed serial
hybridization process.
[0081] FIG. 11 shows the electronic stringency control (ESC) of a
hybridization process for determining single point mutations.
[0082] FIG. 12 shows a scheme for the detection of hybridized DNA
without using labeled DNA probe, i.e., electronically controlled
fluorescent-dye detection process.
[0083] FIG. 13 shows a scheme of electronically controlled
replication of devices.
[0084] FIG. 14 shows a scheme of electronically directed
combinatorial synthesis of oligonucleotides.
[0085] FIG. 15 shows a graph comparing the results for 15-mer Ras
12 point mutation hybridizations carried out using electronic
stringency control and conventional techniques.
[0086] FIG. 16 shows a scheme for electronically controlled
restriction fragment cleavage of DNA.
[0087] FIG. 17 shows a scheme for the electronically controlled
amplification of DNA using DNA polymerase.
[0088] FIG. 18 shows a diagram of an APEX device which is designed
to carry out sample preparation and DNA analysis.
DETAILED DESCRIPTION OF THE INVENTION
[0089] The devices and the related methodologies of this invention
allow molecular biology and diagnostic reactions to be carried out
under "complete electronic control". The meaning of "electronic
control" as referred to in this invention goes beyond the
conventional connotation of the term. Most conventional
microelectronic devices, instruments, and detector systems are
always at some level under electronic control. The microelectronic
devices of this invention are not only under conventional
electronic control, but more importantly they also provide further
direct electronic control over the physical aspects of carrying out
molecular biological and diagnostic reactions. The basic concept of
this invention is a microelectronic device with programmable and
addressable microscopic locations. Each micro-location has a
derivatized upper surface for the covalent attachment of specific
binding entities (i.e., an attachment layer), an intermediate
permeation layer, and an underlying direct current (DC)
micro-electrode. After the initial fabrication of the basic
microelectronic structure, the device is able to self-direct the
addressing of each specific micro-location with specific binding
entities. In this sense, the device self-assembles itself. The
self-addressed device is subsequently able to actively carry out
individual multi-step and combinatorial reactions at any of its
micro-locations. The device is able to carry out multiplex
reactions, but with the important advantage that each reaction
occurs at the equivalent of a truly independent test site. The
device is able to electronically direct and control the rapid
movement and concentration of analytes and reactants to or from any
of its micro-locations. The ability of the device to electronically
control the dynamic aspects of various reactions provides a number
of new mechanisms and important advantages and improvements.
[0090] The concepts and embodiments of this invention are described
in three sections. The first section, "Design and Fabrication of
the Basic Devices," describes the design of the basic underlying
microelectronic device and the fabrication of devices using both
microlithographic and micromachining techniques. The second
section, "Self-Directed Addressing of the Devices," describes the
self-addressing and self-assembly of the device, specifically the
rapid transport and attachment of specific binding entities to each
micro-location. The third section, "Applications of the Devices,"
describes how the device provides electronic control of various
multi-step, combinatorial, and multiplex reactions. This section
also describes the various uses and applications of the device.
I. DESIGN AND FABRICATION OF THE BASIC DEVICES
[0091] In order for a device to carry out multi-step and multiplex
reactions, its electronic components must be able to maintain
active operation in aqueous solutions. To satisfy this requirement,
each micro-location must have an underlying controllable and
functioning DC mode micro-electrode. However, it is important for
device performance, particularly sensitivity (signal to noise
ratio), that binding and affinity reactions are not affected by the
electrolysis reactions occurring on the active DC electrode
surfaces. Other considerations for the design and fabrication of a
device include, but are not limited to, materials compatibilities,
nature of the specific binding entities and the subsequent
reactants and analytes, and the number of micro-locations.
[0092] By "a controllable and functioning DC mode micro-electrode"
is meant a micro-electrode biased either positively or negatively,
operating in a direct current mode (either continuous or pulse),
which can in a controllable manner affect or cause the free field
electrophoretic transport of charged specific binding entities,
reactants, or analytes to or from any location on the device, or
from the sample solution.
[0093] Within the scope of this invention, the free field
electrophoretic transport of molecules, is not actually dependent
on the electric field produced being bounded or confined by an
insulating material. Conventional electrophoretic separation
technologies require confinement or enclosure of electric field
lines by insulating (non-conducting) materials. In the case of free
field electrophoretic transport, charged molecules are moved from
one micro-location to any other micro-location, or from the bulk
solution to specific micro-locations. Therefore, special
arrangements or confinement by insulating materials is not required
for this aspect of the invention.
[0094] A device can be designed to have as few as two addressable
micro-locations or as many as hundreds of thousands of
micro-locations. In general, a complex device with a large number
of micro-locations is fabricated using microlithography techniques.
Fabrication is carried out on silicon or other suitable substrate
materials, such as glass, silicon dioxide, piastic, or ceramic
materials. These microelectronic "chip" designs would be considered
large scale array or multiplex analysis devices. A device with a
small number of micro-locations or macro-locations would be
fabricated using micro-machining techniques.
[0095] Addressable micro-locations can be of any shape, preferably
round, square, or rectangular. The size of an addressable
micro-location can be of any size, preferably range from sub-micron
(.about.0.5 .mu.m) to several centimeters (cm), with 5 .mu.m to 100
.mu.m being the most preferred size range for devices fabricated
using microlithographic techniques, and 100 .mu.m to 10 millimeters
being the most preferred size range for devices fabricated using
the micro-machining techniques. To make micro-locations smaller
than the resolution of microlithographic methods would require
techniques such as electron beam lithography, ion beam lithography,
or molecular beam epitaxy. While microscopic locations are
desirable for analytical and diagnostic type applications, larger
addressable locations or macro-locations (e.g., larger than 5 mm)
are desirable for applications such as, but not limited to,
preparative scale biopolymer synthesis, sample preparation,
electronically dispensing of reagents.
[0096] After micro-locations have been created by using
microlithographic and/or micro-machining techniques, chemical
modification, polymerization, or even further microlithographic
fabrication techniques are used to create the specialized
attachment and permeation layers. These important layers separate
the binding entities from the metal surface of the electrode. These
important structures allow the DC mode micro-electrodes under the
surface of each micro-location to: (1) affect or cause the free
field electrophoretic transport of specific (charged) binding
entities from the surface of one micro-location to the surface of
another micro-location, or from the bulk solution to specific
micro-locations; (2) concentrate and covalently attach the specific
binding entities to the specially modified surface of the specific
micro-location; (3) continue to actively function in the DC mode
after the attachment of specific binding entities so that other
reactants and analytes can be transported in a controlled manner to
or from the micro-locations; and (4) not adversely affect the
binding or affinity reactions with electrochemical reactions and
products.
I(a). DESIGN PARAMETERS (MICROLITHOGRAPHY)
[0097] FIG. 1 shows a basic design of self-addressable
micro-locations fabricated using microlithographic techniques. The
three micro-locations (10) (ML-1, ML-2, ML-3) are formed on the
surface of metal sites (12) which have been deposited on an
insulator layer/base material. The metal sites (12) serve as the
underlying micro-electrode structures (10). An insulator material
separates the metal sites (12) from each other. Insulator materials
include, but are not limited to, silicon dioxide, silicon nitride,
glass, resist, polyimide, rubber, plastic, or ceramic
materials.
[0098] FIG. 2 shows the basic features of an individual
micro-location (10) formed on a microlithographically produced
metal site (12). The addressable micro-location is formed on the
metal site (12), and incorporates an oxidation layer (20), a
permeation layer (22), an attachment layer (24), and a binding
entity layer (26). The metal oxide layer provides a base for the
covalent coupling of the permeation layer. Metal oxide and hydroxyl
groups (either alone or in combination), and other materials known
to those skilled in the art of surface coating chemistries may
provide covalent sites from which to construct or hold the
permeations layer. It is not absolutely essential that the
permeation layer actually be covalently attached to the metal
electrode surface. The physical overlaying of permeable materials
represents an alternative method which is within the scope of this
invention.
[0099] The permeation layer provides spacing between the metal
surface and the attachment/binding entity layers and allows solvent
molecules, small counter-ions, and electrolysis reaction gases to
freely pass to and from the metal surface. It is possible to
include within the permeation layer substances which can reduce the
adverse physical and chemical effects of electrolysis reactions,
including, but not limited to, redox reaction trapping substances,
such as palladium for H.sub.2, and iron complexes for O.sub.2 and
peroxides. The thickness of the permeation layer for
microlithographically produced devices can range from approximately
1 nanometers (nm) to 100 microns (.mu.m), with 2 nm to 10 .mu.m
being the most preferred.
[0100] The attachment layer provides a base for the covalent
binding of the binding entities. The thickness of tht attachment
layer for microlithographically produced devices can range from 0.5
nm to 5 .mu.m, with 1 nm to 500 nm being the most preferred. In
some cases, the permeation and attachment layers can be formed from
the same material. Certain permeation layer materials which can be
further activated for the coupling of binding entities are included
within the scope of this invention.
[0101] The specific binding entities are covalently coupled to the
attachment layer, and form the specific binding entity layer.
Ideally, the specific binding entity layer is usually a mono-layer
of the specific binding molecules. However, in some cases the
binding entity layer can have several or even many layers of
binding molecules.
[0102] Certain design and functional aspects of the permeation and
attachment layer are dictated by the physical (e.g., size and
shape) and chemical properties of the specific binding entity
molecules. They are also dictated to some extent by the physical
and chemical properties of the reactant and analyte molecules,
which will be subsequently transported and bound to the
micro-locations. For example, oligonucleotide binding entities can
be attached to one type of a micro-location surface without causing
a loss of the DC mode function, i.e., the underlying
micro-electrode can still cause the rapid free field
electrophoretic transport of other analyte molecules to or from the
surface to which the oligonucleotide binding entities are attached.
However, if large globular protein binding entities (e.g.,
antibodies) are attached to the same type of surface, they might
insulate the surface and cause a decrease or a complete loss of the
DC mode function. Appropriate modification of the attachment layer
would have to be carried out so as to either reduce the number of
large binding entities (e.g., large globular proteins) or provide
spacing between the binding entities on the surface.
[0103] The spacing between micro-locations is determined by the
ease of fabrication, the requirement for detector resolution
between micro-locations, and the number of micro-locations desired
on a device. However, particular spacings between micro-locations,
or spacial arrangement or geometry of the micro-locations is not
necessary for device function, in that any combination of
micro-locations (i.e., underlying micro-electrodes) can operate
over the complete device area. Nor is it actually necessary to
enclose the device or completely confine the micro-locations with
dielectric or insulating barriers. This is because complex
electronic field patterns or dielectric boundaries are not required
to selectively move, separate, hold, or orient specific molecules
in the space or medium between any of the electrodes. The device
accomplishes this by attaching the specific binding molecules and
subsequent analytes and reactants to the surface of an addressable
micro-location. Free field electrophoretic propulsion provides for
the rapid and direct transport of any charged molecule between any
and all locations on the device; or from the bulk solution to
microlocations. However, it should be pointed out that the devices
would be enclosed for fluid containment and for bio-hazard
purposes.
[0104] As the number of micro-locations increases beyond several
hundred, the complexity of the underlying circuitry of the
micro-locations increases. In this case the micro-location grouping
patterns have to be changed and spacing distances increased
proportionally, or multi-layer circuitry can be fabricated into the
basic device.
[0105] In addition to micro-locations which have been addressed
with specific binding entities, a device will contain
non-analytical micro-locations and macro-locations which serve
other functions. These micro-locations or macro-locations can be
used to store reagents, to temporarily hold reactants, analytes, or
cells; and as disposal units for excess reactants, analytes, or
other interfering components in samples (i.e., reagent dispensing
and sample preparation systems). Other un-addressed micro-locations
can be used in combination with the addressed micro-locations to
affect or influence the reactions that are occurring at these
specific micro-locations. These micro-locations add to both
inter-device and intra-device activity and control. Thus, it is
also possible for the micro-locations to interact and transport
molecules between two separate devices. This provides a mechanism
for loading a working device with binding entities or reactants
from a storage device, for sample preparations and for copying or
replicating a device.
[0106] FIG. 3 shows a matrix type device containing 64 addressable
micro-locations (30). A 64 micro-location device is a convenient
design, which fits with standard microelectronic chip packaging
components. Such a device is fabricated on a silicon chip substrate
approximately 1.5 cm.times.1.5 cm, with a central area
approximately 750 .mu.m.times.750 .mu.m containing the 64
micro-locations. Each micro-location (32) is approximately 50 .mu.m
square with 50 .mu.m spacing between neighboring micrd-locations.
Connective circuitry for each individual underlying micro-electrode
runs to an outside perimeter (10 mm.times.10 mm) of metal contact
pads (300 .mu.m square) (34). A raised inner perimeter can be
formed between the area with the micro-locations and the contact
pads, producing a cavity which can hold approximately 2 to 10
microliters (.mu.l) of a sample solution. The "chip" can be mounted
in a standard quad package, and the chip-contact-pads (34) wired to
the quad package pins. Systems containing more than one chip and
additional packaging and peripheral components may be designed to
address problems related to clinical diagnostics, i.e., addition of
sample materials, fluid transfer, and containment of bio-hazardous
materials. The packaged chip can then be plugged into a
microprocessor controlled DC power supply and multimeter apparatus
which can control and operate the device. It is contemplated by
this invention that device manufacture (prior to addressing) will
ultimately involve the incorporation of three basic components
which would be essentially sandwiched together. The basic chip
device to which the binding entities are attached, would be in the
middle position; a sample or fluid containment component, would be
annealed over the top of the basic chip device; and a
microelectronic detector and on board controller component would be
annealed to the bottom of the basic chip device. This strategy
solves a number of problems related to fabrication techniques and
materials compatibilities.
I(b). MICROLITHOGRAPHY FABRICATION PROCEDURES
[0107] I(b)(1) Fabrication Steps
[0108] General microlithographic or photolithographic techniques
can be used for the fabrication of the complex "chip" type device
which has a large number of small micro-locations. While the
fabrication of devices does not require complex photolithography,
the selection of materials and the requirement that an electronic
device function actively in aqueous solutions does require special
considerations.
[0109] The 64 micro-location device (30) shown in FIG. 3 can be
fabricated using relatively simple mask design and standard
microlithographic techniques. Generally, the base substrate
material would be a 1 to 2 centimeter square silicon wafer or a
chip approximately 0.5 millimeter in thickness. The silicon chip is
first overcoated with a 1 to 2 .mu.m thick silicon dioxide
(SiO.sub.2) insulation coat, which is applied by plasma enhanced
chemical vapor deposition (PECVD).
[0110] In the next step, a 0.2 to 0.5 .mu.m metal layer (e.g.,
aluminum) is deposited by vacuum evaporation. It is also possible
to deposit metals by sputtering techniques. In addition to
aluminum, suitable metals and materials for circuitry include gold,
silver, tin, titanium, copper, platinum, palladium, polysilicon,
carbon, and various metal combinations. Special techniques for
ensuring proper adhesion to the insulating substrate materials
(SiO.sub.2) are used with different metals. Different metals and
other materials may be used for different conductive components of
the device, for example, using aluminum for the perimeter contact
pads, polysilicon for the interconnect circuitry, and a noble metal
(gold or platinum) for the micro-electrodes.
[0111] The chip is next overcoated with a positive photoresist
(Shipley, Microposit AZ 1350 J), masked (light field) with the
circuitry pattern, exposed and developed. The photosolubilized
resist is removed, and the exposed aluminum is etched away. The
resist island is now removed, leaving the aluminum circuitry
pattern on the-chip. This includes an outside perimeter of metal
contact pads,. the connective circuitry (wires), and the center
array of micro-electrodes which serve as the underlying base for
the addressable micro-locations.
[0112] Using PECVD, the chip is overcoated first with a 0.2 to 0.4
micron layer of SiO.sub.2, and then with a 0.1 to 0.2 micron layer
of silicon nitride (Si.sub.3N.sub.4). The chip is then covered with
positive photoresist, masked for the contact pads and
micro-electrode locations, exposed, and developed. Photosolubilized
resist is removed, and the SiO.sub.2 and Si.sub.3N.sub.4 layers are
etched away to expose the aluminum contact pads and
micro-electrodes. The surrounding island resist is then removed,
the connective wiring between the contact pads and the
micro-electrodes remains insulated by the SiO.sub.2 and
Si.sub.3N.sub.4 layers.
[0113] The SiO.sub.2 and Si.sub.3N.sub.4 layers provide important
properties for the functioning of the device. The second SiO.sub.2
layer provides better contact and improved sealing with the
aluminum circuitry. It is also possible to use resist materials to
insulate and seal. This prevents undermining of the circuitry due
to electrolysis effects when the micro-electrodes are operating.
The final surface layer coating of Si.sub.3N.sub.4 is used because
it has much less reactivity with the subsequent reagents used to
modify the micro-electrode surfaces for the attachment of specific
binding entities.
[0114] I(b)(2) Permeation and Attachment Layer Formation Steps
[0115] At this point the micro-electrode locations on the device
are ready to be modified with a specialized permeation and
attachment layer. This is an important aspect of the invention. The
objective is to create on the micro-electrode an intermediate
permeation layer with selective diffusion properties and an
attachment surface layer with optimal binding properties.
[0116] Optimally, the attachment layer has from 10.sup.5 to
10.sup.7 functionalized locations per square micron (.mu.m.sup.2)
for the attachment of specific binding entities. The attachment of
specific binding entities should not overcoat or insulate the
surface so as to prevent the underlying micro-electrode from
functioning. A functional device requires some fraction (.about.5%
to 25%) of the actual metal micro-electrode surface to remain
accessible to solvent (H.sub.2O) molecules, and to allow the
diffusion of counter-ions (e.g., Na.sup.+ and Cl.sup.-) and
electrolysis gases (e.g., O.sub.2 and H.sub.2) to occur.
[0117] The intermediate permeation layer is also designed to allow
diffusion to occur. Additionally, the permeation layer should have
a pore limit property which inhibits or impedes the larger binding
entities, reactants, and analytes from physical contact with the
micro-electrode surface. The permeation layer keeps the active
micro-electrode surface physically distinct from the binding entity
layer of the micro-location.
[0118] This design allows the electrolysis reactions required for
electrophoretic transport to occur on micro-electrode surface, but
avoids adverse electrochemical effects to the binding entities,
reactants, and analytes.
[0119] The permeation layer can also be designed to include
substances which scavenge adverse materials produced in the
electrolysis reactions (H.sub.2, O.sub.2, free radicals, etc.). A
sub-layer of the permeation layer may be designed for this
purpose.
[0120] A variety of designs and techniques can be used to produce
the permeation layer. The general designs include: (1) "Lawns", (2)
"Meshes", and (3) "Porous" structures.
[0121] Lawn type permeation layers involve the arrangement of
linear molecules or polymers in a vertical direction from the metal
surface, in a way resembling a thick lawn of grass. These
structures can be formed by attaching linear or polymeric
hydrophilic molecules directly to the metal surface, with minimum
cross linkages between the vertical structures. Ideally these
hydrophilic linear molecules are bifunctional, with one terminal
end suited for covalent attachment to the metal pad, and the other
terminal end suited for covalent attachment of binding
entities.
[0122] Mesh type permeation layers involve random arrangements of
polymeric molecules which form mesh like structures having an
average pore size determined by the extent of cross-linking. These
structures can be formed by hydrogel type materials such as, but
not limited to polyacrylamide, agarose, and a variety of other
biological and non-biological materials which can be polymerized
and cross-linked.
[0123] Pore type permeation layers involve the use of materials
which can form a channel or hole directly from the top surface of
the layer to the metal pad, including, but not limited to,
polycarbonates, polysulfone, or glass materials. In all cases the
permeation layer must be secured either physically or chemically to
the metal surface, and must contain functional groups or be capable
of being functionalized for the attachment of binding entities to
its surface.
[0124] One preferred procedure which produces a lawn type structure
involves the derivatization of the metal micro-electrode surface
uses aminopropyltriethoxy silane (APS). APS reacts readily with the
oxide and/or hydroxyl groups on metal and silicon surfaces. APS
provides a combined permeation layer and attachment layer, with
primary-amine groups for the subsequent covalent coupling of
binding entities. In terms of surface binding sites, APS produces a
relatively high level of functionalization (i.e., a large number of
primary amine groups) on slightly oxidized aluminum surfaces, an
intermediate level of functionalization on SiO.sub.2 surfaces, and
very limited functionalization of Si.sub.3N.sub.4 surfaces.
[0125] The APS reaction is carried out by treating the whole device
(e.g., a chip) surface for 30 minutes with a 10% solution of APS in
toluene at 50.degree. C. The chip is then washed in toluene,
ethanol, and then dried for one hour at 50.degree. C. The
micro-electrode metal surface is functionalized with a large number
of primary amine groups (10.sup.5 to 10.sup.6 per square micron).
Binding entities can now be covalently bound to the. derivatized
micro-electrode surface. The depth of this "Lawn Type" permeation
layer may be increased by using polyoxyethylene bis(amine),
bis(polyoxyethylene bis(amine)), and other polyethylene glycols or
similar compounds.
[0126] The APS procedure works well for the attachment of
oligonucleotide binding entities. FIG. 4 shows the mechanism for
the attachment of 3'-terminal aldehyde derivatized oligonucleotides
(40) to an APS functionalized surface (42). While this represents
one of the approaches, a variety of other approaches for forming
permeation and attachment layers are possible. These include the
use of self-directed addressing by the base electrode itself to:
(1) form secondary metal layers by electroplating to the base
micro-electrode; (2) to form permeation layers by
electropolymerization to the micro-electrode location, or (3) to
transport by the free field electrophoresis process activated
polymers and reagents to the micro-electrode surface to form
subsequent permeation and attachment layers.
I(c). MICRO-MACHINED DEVICE DESIGN AND FABRICATION
[0127] This section describes how to use micro-machining techniques
(e.g., drilling, milling, etc.) or non-lithographic techniques to
fabricate devices. In general, these devices have relatively larger
micro-locations (>100 microns) than those produced by
microlithography. These devices can be used for analytical
applications, as well as for preparative type applications, such as
biopolymer synthesis, sample preparation, reagent dispenser,
storage locations, and waste disposal. Large addressable locations
can be fabricated in three dimensional formats (e.g., tubes or
cylinders) in order to carry a large amount of binding entities.
Such devices can be fabricated using a variety of materials,
including, but not limited to, plastic, rubber, silicon, glass
(e.g., microchannelled, microcapillary, etc.), or ceramics. Low
fluorescent materials are more ideal for analytical applications.
In the case of micro-machined devices, connective circuitry and
larger electrode structures can be printed onto materials using
standard circuit board printing techniques known to those skilled
in the art.
[0128] Addressable micro-location devices can be fabricated
relatively easily using micro-machining techniques. FIG. 5 is a
schematic of a representative 96 micro-location device. This
micro-location device is fabricated from a suitable material stock
(2 cm.times.4 cm.times.1 cm), by drilling 96 proportionately spaced
holes (1 mm in diameter) through the material. An electrode circuit
board (52) is formed on a thin sheet of plastic material stock,
which fits precisely over the top of the micro-location component
(54). The underside of the circuit board contains the individual
wires (printed circuit) to each micro-location (55). Short platinum
electrode structures (.about.3-4 mm) (62) are designed to extend
down into the individual micro-location chambers (57). The printed
circuit wiring is coated with a suitable water-proof insulating
material. The printed circuit wiring converges to a socket, which
allows connection to a multiplex switch controller (56) and DC
power supply (58). The device is partially immersed and operates in
a common buffer reservoir (59).
[0129] While the primary function of the micro-locations in devices
fabricated by micro-machining and microlithography techniques is
the same, their designs are different. In devices fabricated by
microlithography, the permeation and attachment layers are formed
directly on the underlying metal micro-electrode. In devices
fabricated by micro-machining techniques, the permeation and
attachment layers are physically separated from their individual
metal electrode structure (62) by a buffer solution in the
individual chamber or reservoir (57) (see FIG. 6). In
micro-machined devices the permeation and attachment layers can be
formed using functionalized hydrophilic gels, membranes, or other
suitable porous materials.
[0130] In general, the thickness of the combined permeation and
attachment layers ranges from 10 .mu.m to 30 mm. For example, a
modified hydrophilic gel of 20% to 35 % polyacrylamide (with 0.1%
polylysine), can be used to partially fill (.about.0.5 mm) each of
the individual micro-location chambers in the device. These
concentrations of gel form an ideal permeation layer with a pore
limit of from 2 nm to 10 nm. The polylysine incorporated into the
gel provides primary amine functional groups for the subsequent
attachment of specific binding entities. This type of gel
permeation layer allows the electrodes to function actively in the
DC mode. When the electrode is activated, the gel permeation layer
allows small counter-ions to pass through it, but the larger
specific binding entity molecules are concentrated on the outer
surface. Here they become covalently bonded to the outer layer of
primary amines, which effectively becomes the attachment layer.
[0131] An alternative technique for the formation of the permeation
and attachment layers is to incorporate into the base of each
micro-location chamber a porous membrane material. The outer
surface of the membrane is then derivatized with chemical
functional groups to form the attachment layer. Appropriate
techniques and materials for carrying out this approach are known
to those skilled in the art.
[0132] The above descriptions for the design and fabrication of
both the microlithographic and micromachined devices should not be
considered as a limit to other variations or forms of the basic
device. Many variations of the device with larger or smaller
numbers of addressable micro-locations or combinations of devices
can be for different analytical and preparative applications.
Variations of the device with larger addressable locations can be
designed for preparative biopolymer synthesis applications, sample
preparation, cell sorting systems, in-situ hybridization, reagent
dispensers, storage systems, and waste disposal systems.
II. SELF-DIRECTED ADDRESSING OF THE DEVICES
[0133] The devices of this invention are able to electronically
self-address each micro-location with a specific binding entity.
The device itself directly affects or causes the transport of a
charged specific binding entity to a specific micro-location. The
binding entities are generally functionalized so that they readily
react and covalently bond to the attachment layer. The device
self-assembles in the sense that no outside process, mechanism, or
equipment is needed to physically direct, position, or place a
specific binding-entity at a specific micro-location. This
self-addressing process is both rapid and specific, and can be
carried out in either a serial or parallel manner.
[0134] A device can be serially addressed with specific binding
entities by maintaining the selected micro-location in a DC mode
and at the opposite charge (potential) to that of a specific
binding entity. If a binding entity has a net negative charge, then
the micro-location to which the binding entity is to be transported
would be biased positive. Conversely, a negatively charged
micro-location would be used to transport a positively charged
binding entity. Options for biasing the remaining micro-locations
in the serial addressing process include: biasing all other
micro-locations at the opposite charge (counter to the
micro-location being addressed); biasing a limited group of
micro-locations at the opposite charge; or biasing just one
micro-location (or other electrode) at the opposite charge. In some
cases, it will be desirable to strongly bias one or more
micro-locations at the opposite charge, while other groups of
micro-locations are biased only weakly. This process allows
previously addressed micro-locations to be protected during the
addressing of the remaining micro-locations. In cases where the
binding entity is not in excess of the attachment sites on the
micro-location, it may be necessary to activate only one other
micro-electrode to affect the free field electrophoretic transport
to the specific micro-location. Specific binding entities can be
rapidly transported through the bulk solution, and concentrated
directly at the specific micro-location(s) where they immediately
become covalently bonded to the special surface of the attachment
layer. Transportation rates are dependent on the size and charge of
the binding entities, and the voltage and current levels used
between the micro-locations. In general, transportation rates can
range from several seconds to several minutes. The ability to
electronically concentrate binding entities, reactants, or analytes
(70) on a specific micro-location (72) is shown in FIG. 7. All
other micro-locations can be protected and remain unaffected during
the specific binding entity addressing process. Any unreacted
binding entity is removed by reversing the polarity of that
specific micro-location, and electrophoresing it to a disposal
location. The cycle is repeated until all desired micro-locations
are addressed with their specific binding entities. FIG. 8 shows
the serial process.for addressing specific micro-locations (81, 83,
85) with specific oligonucleotide binding entities (82, 84,
86).
[0135] The parallel process for addressing micro-locations involves
simultaneously activating more than one micro-location (a
particular group) so that the same specific binding entity is
transported, concentrated, and reacted with more than one specific
micro-location. The subsequent parallel processing is similar to
the serial process.
III. APPLICATIONS OF THE DEVICES
[0136] Once a device has been self-addressed with specific binding
entities, a variety of molecular biology type multi-step and
multiplex reactions and analyses can be carried out on the device.
The devices of this invention are able to electronically provide
active and dynamic control over a number of important reaction
parameters. This electronic control leads to new physical
mechanisms for controlling reactions, and significant improvements
in reaction rates, specificities, and sensitivities. The
improvements in these parameters come from the ability of the
device to electronically control and directly affect: (1) the rapid
transport of reactants or analytes to a specific micro-location
containing attached specific binding entities; (2) an increase in
reaction rate due to the concentration of reactants or analytes
with the specific binding entities on the surface of the specific
micro-location; (3) the rapid and selective removal of un-reacted
and non-specifically bound components from the micro-location; and
(4) the stringency for optimal binding conditions.
[0137] The self-addressed devices of this invention are able to
rapidly carry out a variety of micro-formatted multi-step and/or
multiplex reactions and procedures; which include, but are not
limited to:
[0138] DNA and RNA hybridizations procedures and analysis in
conventional formats; e.g., attached target DNA/probe DNA, attached
probe DNA/target DNA, attached capture DNA/target DNA/probe
DNA;
[0139] multiple or multiplexed hybridization reactions in both
serial and parallel fashion;
[0140] restriction fragment and general DNA/RNA fragment size
analysis;
[0141] molecular biology reactions, e.g., restriction enzyme
reactions and analysis, ligase reactions, kinasing reactions, and
DNA/RNA amplification;
[0142] antibody/antigen reactions involving large or small antigens
and haptens;
[0143] diagnostic assays, e.g., hybridization analysis (including
in-situ hybridization), gene analysis, fingerprinting, and
immunodiagnostics;
[0144] sample preparation, cell sorting,- selection, and
analysis;
[0145] biomolecular conjugation procedures (i.e. the covalent and
non-covalent labeling of nucleic acids, enzymes, proteins, or
antibodies with reporter groups, including fluorescent,
chemiluminescent, calorimetric, and radioisotopic labels);
[0146] biopolymer synthesis, e.g., combinatorial synthesis of
oligonucleotides or peptides;
[0147] water soluble synthetic polymer synthesis, e.g.,
carbohydrates or linear polyacrylates; and
[0148] macromolecular and nanostructure (nanometer size particles
and structures) synthesis and fabrication.
III(a) NUCLEIC ACID HYBRIDIZATION
[0149] Nucleic acid hybridizations are used as main examples of
this invention because of their importance in diagnostics, and
because they characterize one of the more difficult types of
binding (affinity) reactions. This is particularly true when they
are carried out in multiplex formats, where each individual
hybridization reaction requires a different stringency
condition.
[0150] The claimed device and methods allow nucleic acid
hybridization to be carried out in a variety of conventional and
new formats. The ability of the device to electronically control
reaction parameters greatly improves nucleic acid hybridization
analysis, particularly the ability of the device to provide
electronic stringency control (ESC) to each individual
micro-location on an array. In essence, this allows each individual
hybridization reaction on a common array to be carried out as a
single test tube assay.
[0151] The term "nucleic acid hybridization" is meant to include
all hybridization reactions between all natural and synthetic forms
and derivatives of nucleic acids, including: deoxyribonucleic acid
(DNA), ribonucleic acid (RNA), polynucleotides and
oligonucleotides.
[0152] Conventional hybridization formats, such as "dot blot"
hybridization and "sandwich" hybridization, can be carried out with
the claimed device as well as large scale array or matrix
formats.
[0153] As an example, an APEX device for DNA hybridization analysis
is designed, fabricated, and used in the following manner. Arrays
of micro-locations are first fabricated using microlithographic (or
micromechining) techniques. The number of addressable
micro-locations on an array depends on the final use. The device is
rapidly self-addressed in a serial manner with a group of specific
oligonucleotides. In this case, the specific oligonucleotides are
3'-terminal aldehyde functionalized oligonucleotides in the range
of 6-mers to 100-mers, larger polynucleotides can be attached if
desired. The aldehyde functional group allows for covalent
attachment to the specific micro-location attachment surface (see
FIG. 4). This group of specific oligonucleotides can be readily
synthesized on a conventional DNA synthesizer using conventional
techniques. The synthesis of each specific oligonucleotide is
initiated from a ribonucleotide controlled pore glass (CPG)
support. Thus, the 3'-terminal position contains a ribonucleotide,
which is then easily converted after synthesis and purification to
a terminal dialdehyde derivative by periodate oxidation. The
aldehyde containing oligonucleotides (40) will react readily with
the primary amine functional groups on the surface of
micro-locations by a Schiff's base reaction process.
[0154] The electronic addressing of the device with specific
oligonucleotides is shown in FIG. 8. The addressing of the first
specific micro-location (ML-1) (81) with its specific sequence
oligonucleotide (SSO-1) (82) is accomplished by maintaining the
specific microelectrode (ML-1) at a positive DC potential, while
all other micro-electrodes are maintained at a negative potential
(FIG. 8(A)). The aldehyde functionalized specific sequence (SSo-1)
in aqueous buffered solution is free field electrophoresed to the
ML-1 address, where it concentrates (>10.sup.6 fold) and
immediately becomes covalently bound to the surface of ML-1 (81).
All other microelectrodes are maintained negative, and remain
protected or shielded from reacting with SSO-1 sequence (82). The
ML-1 potential is then reversed to negative (-) to electrophorese
any unreacted SSO-1 to a disposal system. The cycle is repeated,
SSO-2 (84) ---> ML-2 (83), SSO-3 (86) ---> ML-3 (85), SSO-n
---> ML-n until all the desired micro-locations are addressed
with their specific DNA sequences (FIG. 8(D)).
[0155] Another method for addressing the device is to transport
specific binding entities such as specific oligonucleotides from an
electronic reagent supply device. This supply device would hold a
large quantity of binding entities or reagents and would be used to
load analytical devices. Binding entities would be electronically
transported between the two devices. This system eliminates the
need for physical manipulations, such as micro-pipetting, and for
complicated fluidic delivery systems within or between devices.
[0156] Yet another method for addressing the device is to carry out
the combinatorial synthesis of the specific oligonucleotides at the
specific micro-locations. Combinatorial synthesis is described in a
later section.
[0157] After the device is addressed with specific DNA sequences,
it is important that the micro-electrodes beneath the
micro-locations on the array device remain as independent working
direct current (DC) electrodes. This is made possible because the
attachment to the electrode surface is carried out in such a manner
that the underlying micro-electrode does not become chemically or
physically insulated. Each micro-electrode can still produce the
strong direct currents necessary for the free field electrophoretic
transport of other. charged DNA molecules to and from the
micro-location surface. Thus, the DNA array device provides
complete electronic control over all aspects of the DNA
hybridization and any other subsequent reactions.
[0158] An example of an electronically controlled hybridization
process is shown in FIG. 9. In this case, each addressable
micro-location has a specific capture sequence (90). A sample
solution containing target DNA (92) is applied to the device. All
the micro-locations are activated and the sample DNA is
concentrated at the micro-locations (FIG. 9(B)). Target DNA
molecules from the dilute solution become highly concentrated at
the micro-locations, allowing very rapid hybridization to the
specific complementary DNA sequences on the surface. Reversal of
the micro-electrode potential repels all un-hybridized DNA from the
micro-locations, while the target DNA remains hybridized (FIG.
9(C)). In similar fashion, reporter probes are hybridized in
subsequent steps to detect hybridized complexes.
[0159] The electronic control of the hybridization process
significantly improves the subsequent detection of the target DNA
molecules by enhancing the overall hybridization efficiency and by
removing non-specific DNA from the micro-location areas. It is
expected that 10,000 to 100,000 copies of target sequences in
un-amplified genomic DNA will be detectable. Hybridization
reactions of this type can be carried out in several minutes or
less, under isothermal conditions well below the Tm of the probes;
and with minimal outside manipulations (i.e., conventional washing
steps are completely eliminated).
[0160] Another common format for DNA hybridization assays involves
having target DNAs immobilized on a surface, and then hybridizing
specific probes to these target DNAs. This format can involve
either the same target DNAs at multiple locations, or different
target DNAs at specific locations. FIG. 10 shows an improved
version of this serial hybridization format. In this case
micro-locations (101-107) are addressed with different capture
DNAs. These are hybridized in a serial fashion with different
sequence specific oligonucleotides (108, 109). The micro-locations
are sequentially biased positive to transport molecules to itself
and then biased negative to transport molecules to the next
micro-location. At the proper electrode potential, the specifically
hybridized DNA probes will remain at that micro-location, while
un-hybridized probes are transported to the next micro-location.
The sequence specific oligonucleotide probes can be labeled with a
suitable reporter group such as a fluorophore.
[0161] The claimed device is able to provide electronic stringency
control. Stringency control is necessary for hybridization
specificity, and is particularly important for resolving one base
mis-matches in point mutations. FIG. 11 shows how electronic
stringency control can be used for one base mis-match analysis.
Electronic stringency control can also be applied to multiple-base
mis-match analysis. In FIG. 11(A) the perfectly matched DNA hybrid
(110) is slightly more stable than mis-matched DNA (112) hybrid. By
biasing the micro-locations negative (FIG. 11(B)) and delivering a
defined amount of electrophoretic power in a given time, it is
possible to denature or remove the mis-matched DNA hybrids while
retaining the perfectly matched DNA hybrids (FIG. 11(C)). FIG. (15)
compares the results for an electronic hybridization process
utilizing electronic stringency control with a conventional
hybridization process. The hybridization involves 15-mer G and A
point mutation probes for the Ras 12 oncogene mutation. The
electronic hybridization result show greatly improved hybridization
efficiency and a very large discrimination ratio for the one base
mis-match over the conventional procedure.
[0162] In a further refinement, the claimed device provides
independent stringency control to each specific hybridization
reaction occurring on the device. In effect each hybridization is a
an independent reaction. With a conventional or passive array
format, it is impossible to achieve optimal stringency for all the
hybridization events which are occurring in the same hybridization
solution. However, the active array devices of this invention are
able to provide different electronic stringency to hybridizations
at different micro-locations, even though they are occurring in the
same bulk hybridization solution. This attribute overcomes the
inherent limitation to conventional matrix or array hybridization
formats, multi-sequencing by hybridization (SBH) formats, and other
plex analyses.
[0163] In addition to improving the specificity (i.e.,
discrimination ratio) and sensitivity for hybridization (such as
single point mutations detection), electronic stringency control
allows oligonucleotides outside the normal size range to be used in
these applications. oligonucleotide sequences ranging from 8-mer to
21-mer are considered acceptable for point mutation detection with
conventional hybridization procedures. In the current practice
using conventional hybridization procedures, oligonucleotides in
the 10-mer to 19-mer are used most frequently in these conventional
procedures which utilize temperature and salt concentration for
stringency control. oligonucleotides shorter than 10-mers have been
found to be not acceptable for multiplex hybridizations; and
sequences shorter than 8-mers are not even considered for use
because of poor hybridization efficiencies. Sequences longer than
21-mers are not used because they have very poor discrimination
ratios between the match and mismatch probes. As the sequence
length goes beyond a 21-mer, the ability to distinguish the
difference in the hybridization signals between the match and
mis-match probes is greatly reduced.
[0164] We have found that hybridizations carried out on APEX
devices with electronic stringency control allows both shorter
(7-mer and shorter) and longer (22-mer and longer) oligonucleotides
to be used with very high discrimination ratios. The use of shorter
oligonucleotide sequences (7-mer and less) has advantages for
sequencing by hybridization (SBH). Shorter length sequences allow
arrays with a smaller number of oligonucleotides (8-mers=65,536,
7-mers=16,384, 6-mers=4,096) to be used for this SBH applications.
The use of longer sequences (22-mer and longer) with electronic
stringency control allows more sensitive and selective point
mutation analysis to be carried out. The use of longer probes
provides higher sensitivity in DNA samples with high complexity,
and also higher overall hybridization efficiencies.
[0165] Electronic hybridization techniques can be used to carry out
in-situ hybridizations. In-situ represent a fundamentally different
hybridization format in that target DNA (or RNA) is not removed
from cells, but detected directly inside them. In-situ
hybridization procedures are generally complex and time consuming,
and the detection of short target sequences (i.e. single point
mutations) is nearly impossible. Electronic controlled in-situ
hybridizations can be carried out on an APEX device that attaches
and processes cells directly on the active surface of the device
(see Example 14 concerning sample preparation techniques). However,
rather than extracting DNA from the cells, the APEX device
electronically hybridizes reporter probes directly to the DNA
within the cells. Electronic stringency control is used to increase
both selectivity and sensitivity by eliminating much of the
non-specific binding and improving overall hybridization
efficiency..
[0166] The ability to provide electronic stringency control to
hybridizations also provides new mechanisms for detecting DNA
hybridization without using a reporter group labeled DNA probe. It
provides a way to carry out a more direct detection of the
hybridization process itself. A fluorescent dye detection process
is shown in FIG. 12 and described in Examples 4 and 6. Direct
detection of DNA hybrids can be achieved by using DNA binding dyes
such as ethidium bromide. The dye binds to both double-stranded and
single-stranded DNA but with a greater affinity for the former. In
FIG. 12(B) positively charged dye (122) is transported to
negatively biased micro-locations. The dye binds to both hybridized
(120) and un-hybridized (121) DNA sequences (FIG. 12(C). By biasing
the micro-locations positive and delivering a defined amount of
power in a given amount of time, the dye molecules bound to
un-hybridized micro-locations is selectively removed. A proper
amount of potential can be applied which does not adversely affect
the DNA hybrids. The hybridized DNAs with associated dye molecules
are then fluorescently detected using associated or integrated
optical systems.
[0167] The following reiterates important advantages the devices of
this invention provide for nucleic acid hybridization reactions and
analysis:
[0168] (1) The rapid transport of dilute target DNA and/or probe
DNA sequences to specific micro-location(s) where hybridization is
to occur. This process can take place in the range of 5 to 120
seconds.
[0169] (2) Concentrating dilute target DNA and/or probe DNA
sequences at specific micro-location(s) where hybridization is to
occur. The concentrating effect can be well over a million fold
(>10.sup.6).
[0170] (3) The rapid removal of non-specifically bound target DNA
sequences from specific micro-location(s) where hybridization has
occurred. This process can take place in the range of 5 to 120
seconds.
[0171] (4) Rapid removal of competing complementary target DNA
sequences from specific micro-location(s) where hybridization has
occurred. This process can take place in the range of 5 to 120
seconds.
[0172] (6) The ability to carry out a large number of independent
hybridization reactions in a matter of minutes.
[0173] (7) The ability to carry out a hybridization process at
isothermal conditions well below the Tm of the probes, and with
minimal outside manipulations or washing steps.
[0174] (8) The use of electronic stringency control (ESC) to remove
partially hybridized DNA sequences.
[0175] (9) The ability to carry out hybridization analysis of
un-amplified genomic target DNA sequences in the 1000 to 100,000
copy range.
[0176] (10) The use of ESC to improve the discrimination ratio
(i.e., resolution) and sensitivity of single base mis-match
hybridizations (point mutations).
[0177] (11) The ability to use single point mutation probes that
are either shorter (7-mer and less) or longer (22-mer or greater)
than those used in conventional hybridization procedures.
[0178] (12) The use of ESC to provide individual stringency control
in matrix hybridizations.
[0179] (13) Improving the detection of hybridization event by
removing non-specific background components.
[0180] (14) The ability to carry out electronic in-situ
hybridization on fixed cells.
[0181] (15) The development of a detection method which eliminates
the need for using covalently labeled reporter probes or target DNA
to detect hybridization.
III(b) REPRODUCTION OF DEVICES
[0182] In addition to separately addressing individual devices with
specific binding entities, it is also possible to produce a master
device, which can copy specific binding entities to other devices.
This represents another method for the production or manufacture of
devices. The process for the replication of devices is shown in
FIG. 13. A master device containing micro-locations which have been
addressed with specific binding sequences is hybridized with
respective complementary DNA sequences (130). These complementary
sequences are activated and thus capable of covalent binding to the
micro-location attachment layer.
[0183] An unaddressed sister device (132) containing an attachment
layer is aligned with the hybridized master device (FIG. 13(B)).
The master device micro-locations are biased negative and the
sister device micro-locations are biased positive. The DNA hybrids
are electronically denatured and are transported to the sister
device, where the activated DNA sequence binds covalently to the
micro-location (FIG. 13(C)). The process can be performed in
parallel or in series, depending on the device geometry so that
crosstalk between the micro-locations is minimized. The hybrids can
be denatured by applying a sufficient negative potential or by
using a positively charged chaotropic agent or denaturant.
III(c) COMPONENT DEVICES AND INTEGRATED APEX SYSTEMS
[0184] A number of separate APEX devices or chips can be combined
to form an integrated APEX System. Because APEX type devices can
carry out many different functions, and reactants can be moved
between devices by free field electrophoresis, integrated systems
can be developed. For example, separate APEX devices or chips
which: (1) selectively bind and lyse cells, (2) electronically
dispense reagents, (3) carry out pre-hybridizations, (4) act as
waste disposal units, (5) provide storage for DNA fragments, and
(5) carry out hybridization analysis can be combined to form a
sample preparation and hybridization analysis system (see Example
14 and FIG. 19). These integrated APEX microelectronic systems are
the equivalent of complete clinical analyzers or programmable
molecular biology laboratories (i.e. laboratories on a chip).
However, they go beyond automation (robotics) or other
microanalytical devices in that they require minimal fluidics or
physical manipulation of samples, reagents, and reactants.
Additional types of integrated APEX systems would include , but are
limited to, those which could carry out in-situ hybridizations,
cell selector and processor systems, and immunodiagnostic
analyzers.
III(d) DETECTION SYSTEM AND REPORTER GROUPS
[0185] In the base of binding reactions involving fluorescent
labelled reporter groups, it is possible to use an epifluorescent
type microscope detection system for the analysis of the binding
reactions on APEX devices. The overall sensitivity of the system
depends on the associated detector component (cooled charged
coupled devices (CCD), intensified charged coupled device (ICCD),
microchannel plate detectors, or photon counting photomultiplier
(PMT) systems). Alternatively, sensitive CCD chip detectors or
avalanche photodiode (APD) detectors can be more directly
associated with the APEX device. These systems would somewhat
reduce the necessity for complex optics. More advanced systems will
involve integrating optoelectronic or electronic detection elements
into the APEX chip. Both optical and direct electronic detection of
DNA is possible with these systems. It is contemplated by this
invention that the most advanced versions will ultimately involve
sandwiching together a microelectronic detector and on board
controller component to the basic APEX chip component. Electronic
and optical (waveguide) connections would be made directly through
the bottom of the APEX component. This strategy solves a number of
problems related to fabrication techniques, materials
compatibilities, and cost effectiveness for making the APEX
component disposable.
[0186] In addition to a variety of fluorescent dyes and reporter
groups which can be used to label DNA probes, target DNAs, or
antibodies; other types of labels or reporter groups can also be
used. These include chemiluminescent labels, non-linear optical
(frequency doubler) materials, biotin/avidin complexes and various
enzymes.
III(e) COMBINATORIAL BIOPOLYMER SYNTHESIS
[0187] The devices of this invention are also capable of carrying
out combinatorial synthesis of biopolymers such as oligonucleotides
and peptides. Such a process allows self-directed synthesis to
occur without the need for any outside direction, influence, or
mechanical movements. Other processes for combinatorial synthesis
require physical masks and complex photolithographic procedures,
microrobotic pipetting systems for reagent delivery, or complicated
physical movement of components to carry out the actual synthesis
at microscopic locations. The combinatorial synthesis disclosed in
this invention allows very large numbers of sequences-to be
synthesized on a device. The basic concept for combinatorial
synthesis involves the use free field electrophoretic transport to
deliver, concentrate, and react monomers, coupling reagents, or
deblocking reagents at specific addressable micro-locations on the
device. The concept capitalizes on the inherent ability of the
device to electronically protect other micro-locations from the
effects of nearby reagents and reactants. Also important to the
concept is the identification of selective steps in these chemical
synthesis processes where one or more of the reactants has either a
net positive or negative charge, or to create such suitable
reagents for these processes.
[0188] One method for combinatorial oligonucleotide synthesis is
shown in FIG. 14. This method begins with a set of selectively
addressable micro-locations (140) whose surfaces have been
derivatized with blocked primary amine (X--NH--) groups (142). The
initial step in the process involves selective deblocking of
micro-locations using a charged deblocking reagent (144). In this
case, the reagent would carry a positive (+) charge. The process is
carried out by applying a negative potential to those
micro-locations being de-blocked, and a positive potential to those
which are to remain protected (FIG. 14(B)). Application of positive
and negative potentials to selective electrodes causes the charged
reagents to be moved from a reagent delivery site and concentrated
at the desired micro-location to be de-blocked, while excluding
reagents from the other micro-locations.
[0189] In the second step, chemical coupling of the first base, in
this case cytosine, to the deblocked micro-locations is carried out
by simply exposing the system to the phosphoramidite reagent (x-C)
(146). The (C) nucleotide couples to de-blocked micro-location
surfaces, but not to any of the blocked electrode surfaces (FIG.
14(C) and (D)). At this point normal phosphoramide chemistry is
carried out until the next de-blocking step.
[0190] At the second de-blocking step (FIG. 14(D)), those electrode
positions which are to be coupled with the next base are made
negative, and those which are to remain protected are made
positive. The system is now exposed to the next base to be coupled,
in this case (x-A) (148), and selective coupling to the de-blocked
micro-location is achieved (FIG. 14(E) and (F)). The coupling and
de-blocking procedures are repeated, until all the different DNA
sequences have been synthesized on each of the addressable
micro-location surfaces.
[0191] The above example represents one possible approach for the
synthesis of nucleic acids. Another approach involves a complete
water soluble DNA synthesis. In this case, charged water soluble
coupling agents, such as 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDCA), is used to carry out oligonucleotide synthesis
with water soluble nucleotide derivatives. This approach would have
significant advantages over present organic solvent based methods
which require extensive blocking of the base moieties. Water
soluble synthesis would be less expensive and eliminate the use of
many toxic substances used in the present organic solvent based
processes. A third approach, again for water soluble synthesis,
involves the use of charged monomers and enzymes.
III(e)(1) Oliqonucleotide Synthesis with Terminal Transferase
[0192] This approach for combinatorial synthesis of
oligonucleotides involves the use of a nucleic acid polymerizing
enzymes. This approach utilizes terminal transferase,
3'-monophosphate esters of 5'-deoxyribonucleotide triphosphates,
and a phosphatase. Terminal transferase is used to couple the
nucleotides. The 3'-phosphate ester serves as a blocking group to
prevent the addition of more than one nucleotide in each coupling
step. A 3'-phosphatase is used to remove the 3'-phosphate ester for
the next coupling step.
[0193] Because all reagents are water soluble and charged, general
APEX techniques can be used for all steps in this combinatorial
synthesis procedure. In this approach, an APEX matrix is, used
which has A, T, G, and C nucleotides linked through their
5'-hydroxyl position to the appropriate number of addressed
micro-locations on the device. The first nucelotides are linked be
standard APEX addressing techniques.
[0194] The first round of coupling reactions is initiated by
biasing positive all those micro-locations which are to be coupled
with an A nucleotide in their second position, and biasing negative
the two electronic reagent dispensers containing terminal
transferase and the 3'-phosphate ester of deoxyadenosine
triphosphate. The reagents are free field electrophoresed to the
appropriate micro-locations and the A nucleotide is coupled by the
terminal transferase to the first nucleotide on the matrix. Because
the nucleotide triphosphates are esterified with a phosphate group
in their 3' positions, terminal transferase adds only one
nucleotide at a time.
[0195] After the nucleotide coupling is complete, the
micro-locations are biased negative and the waste disposal system
is biased positive and the enzyme and spent reagents are removed.
The process is repeated for the first round coupling of G, C, and T
nucleotides until all the micro-locations have been coupled.
[0196] When first complete round of coupling (A,T, G and C) is
complete, all the micro-locations are biased positive and a reagent
dispenser with a 3'-phosphatase enzyme is biased negative. The
3'-phosphatase is free field electrophoresed to the micro-locations
where it hydrolyses the 3'-phosphate ester. The removal of the
phosphate ester leaves the 3'-hydroxyl group ready for the next
round of coupling reactions. The coupling reactions are carried out
until the desired oligonucleotide sequences are complete on the
APEX device.
[0197] In addition to DNA synthesis, a similar process can be
developed for RNA synthesis, peptide synthesis, and other complex
polymers.
III(f) ELECTRONICALLY CONTROLLED MOLECULAR BIOLOGY AND
AMPLIFICATION REACTIONS.
[0198] A variety of molecular biological reactions including linear
and exponential multiplication or amplification of target DNA and
RNA molecules can be carried out with APEX microelectronic devices
and chips.
[0199] Restriction enzyme cleavage reactions and DNA fragment
analysis can be carried out under complete electronic control.
Nucleic acid multiplication or amplification reactions with APEX
devices are distinct from other "DNA Chip" devices which are
basically passive micro-matrix supports for conventional
amplification procedures (PCR, LCR, etc.). New mechanisms for
amplification come directly from the active nature of the APEX
devices. The active device provides unique electronic mechanisms
to: (1) selectively denature DNA hybrids under isothermal reaction
conditions and well below their Tm point (thermal melting
temperature); (2) rapidly transport or move DNA back and forth
between two or more micro-locations; and (3) selectively
concentrate DNA modifying enzymes, such as, but not limited to,
restriction endonucleases, DNA or RNA polymerases, and ligases, at
any desired micro-location on the device. Examples of
electronically controlled molecular biology and amplification
reactions which can be carried out on the APEX devices include: (1)
Electronically Directed Restriction Enzyme Cleavage of ds-DNA
Sequences; (2) Electronic Multiplication of Target DNA By DNA
Polymerases; (3) Electronic Ligation and Multiplication of Target
DNA Sequences By DNA and RNA Ligases; and (4) Electronic
Multiplication of Target DNA By RNA Polymerases.
III(g) ELECTRONIC RESTRICTION FRAGMENT ANALYSIS
[0200] In addition to carrying out restriction enzyme cleavage of
ds-DNA, APEX devices and electronic techniques can be used to
analyze and determine the relative size of DNA fragments. This is
possible when DNA fragments with different lengths can be
hybridized to a common capture sequence on individual
micro-locations. Or when DNA fragments of different lengths can be
hybridized to different capture sequences, all of which have the
same hybridization or binding energy. In these cases, electronic
stringency control can be used to selectively de-hybridize the
different DNA fragments according the length of their un-hybridized
or overhanging sequence. The electrophoretic force on the fragments
with longer overhanging sequences causes them to de-hybridize
before the fragments with shorter overhanging sequences. Thus, if
the fragments are labelled for detection, and addressed to specific
micro-locations, their sizes can be determined by the
electrophoretic potential or power level required to de-hybridize
them from the micro-locations. It may be possible to carry out the
equivalent of an electronic restriction fragment length
polymorphism analysis.
[0201] The invention will now be described in greater detail by
reference to the following non-limiting examples regarding the
making and applications of APEX devices.
[0202] The recipes for buffers, solutions, and media in the
following examples are described in J. Sambrook, E. F. Fritsch, and
T. Maniatis, Molecular Cloning: A Laboratory Manual, 2 Ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New York,
1989.
IV. EXAMPLES
EXAMPLE 1: oligonucleotide Synthesis and Modifications
[0203] Synthetic DNA probes were made using conventional
phosphoramidite chemistry on Applied Biosystems automated DNA
synthesizers. oligomers were designed to contain either a 5'-amino
or a 3'-ribonucleoside terminus. The 5' functionality was
incorporated by using the ABI Aminolink 2 reagent and the 3'
functionality was introduced by initiating synthesis from an RNA
CPG support. The 3'-ribonucleotide terminus can be converted to a
terminal dialdehyde by the periodate oxidation method which can
react with primary amines to form a Schiff's base.
[0204] Reaction conditions were as follows: Dissolve 20-30 O.D.
oligomer in water to a final concentration of 1 OD/.mu.l. Add 1 vdl
of 0.1M sodium acetate, pH 5.2 and 1 vol 0.45M sodium periodate
(made fresh in water). Stir and incubate reaction for at least 2
hours at ambient temperature, in the dark. Load reaction mix onto a
Sephadex G-10 column (pasteur pipette, 0.6.times.5.5 cm)
equilibrated in 0.1M sodium phosphate, pH 7.4. Collect 200 .mu.l
fractions, spot 2 .mu.l aliquot on thin layer chromatography (TLC)
and pool ultra violet (UV) absorbing fractions.
[0205] The following oligomers contain 3'-ribonucleoside termini
(U):
1 ET-12R 5'-GCT AGC CCC TGC TCA TGA GTC TCU CP-1 5'-AAA AAA AAA AAA
AAA AAA AAU AT-A1 5'-CTA CGT GGA CCT GGA GAG GAA GGA GAC TGC CTG U
AT-A2 5'-GAG TTC AGC AAA TTT GGA GU AT-A3 5'-CGT AGA ACT CCT CAT
CTC CU AT-A4 5'- GTC TCC TTC CTC TCC AGU AT-A5 5'- GAT GAG CAG TTC
TAC GTG GU AT-A6 5'- CTG GAG AAG AAG GAG ACU AT-A7 5'- TTC CAC AGA
CTT AGA TTT GAC U AT-A8 5'- TTC CGC AGA TTT AGA AGA TU AT-A9 5'-
TGT TTG CCT GTT CTC AGA CU AT-A10 5'- CAT CGC TGT GAC AAA ACA
TU
[0206] Oligomers containing 5' amine groups were generally reacted
with fluorophores, such as Texas Red (TR, excitation 590 nm,
emission 610 nm). Sulfonyl chlorides are very reactive towards
primary amines forming a stable sulfonamide linkage.
[0207] Texas Red-DNA conjugates were made as follows: Texas Red
sulfonyl chloride (Molecular Probes) was dissolved in dimethyl
formamide (DMF) to a final concentration of 50 mg/ml (80 mM).
Oligomer was dissolved in 0.4M sodium bicarbonate, pH 9.0-9.1, to a
final concentration of 1 O.D./.mu.l (5.4 mM for a 21-mer). In a
micro test tube, 10 .mu.l oligomer and 20 .mu.l Texas Red was
combined. Let reaction proceed in the dark for 1 hour. Quench
reaction with ammonia or hydroxylamine, lyophilize sample and
purify by PAGE (Sambrook et al., 1989, supra).
[0208] The following oligomers contained a 5'-amino termini:
2 ET-21A 5'-Amino-TGC GAG CTG CAG TCA GAC AT ET-10AL 5'-Amino-GAG
AGA CTC ATG AGC AGG ET-11AL 5'-Amino-GGT GCT CAT GAG TCT CTC T-2
5'-Amino-TTT TTT TTT TTT TTT TTT T RC-A1 5'-Amino-CAG GCA GTC TCC
TTC CTC TCC AGG TCC ACG TAG RC-A2 5'-Amino-GTC CAA ATT TGC TGA ACT
C RC-A3 5'-Amino-GGA GAT GAG GAG TTC TAC G RC-A4 5'-Amino-GTG GAG
AGG AAG GAG AC RC-A5 5'-Amino-GCA CGT AGA ACT GCT CAT C RC-A6
5'-Amino-GTC TCC TTC TTC TCC AG RC-A7 5'-Amino-GTC AAA TCT AAG TCT
GTG GAA RC-A8 5'-Amino-ATC TTC TAA ATC TGC GGA A RG-A9 5'-Amino-GTC
TGA GAA CAG GCA AAC A RC-A10 5'-Amino-ATG TTT TGT CAC AGC GAT G
EXAMPLE 2: Electronically Addressable Micro-locations on a
Microfabricated Test Device--Polylysine Method
[0209] Micro-locations were fabricated from microcapillary tubes
(0.2 mm.times.5 mm). The microcapillaries were filled with 18-26%
polyacrylamide containing 0.1-1.0% polylysine and allowed to
polymerize. The excess capillary was scored and removed to prevent
air bubbles from being trapped within the tubes and to standardize
the tube length. Capillaries were mounted in a manner such that
they shared a common upper buffer reservoir and had individual
lower buffer reservoirs. Each lower buffer reservoir contained a
platinum wire electrode.
[0210] The top surface of the microcapillary in the upper reservoir
was considered to be the addressable micro-location. The upper and
lower reservoirs were filled with 0.1 M sodium phosphate, pH 7.4
and pre-run for 10 minutes at 0.05 mA constant using a BioRad
500/1000 power supply. About 2 .mu.l (0.1 O.D.) of periodate
oxidized ET-12R capture sequence was pipetted into the upper
reservoir with the power on and electrophoresed for 2-5 minutes at
constant current. The ET-12R capture sequence becomes concentrated
and immediately covalently bound to the primary amines on the
micro-location surface. The polarity was then reversed so that the
test capillary was now biased negative and electrophoresed an
additional 2-5 minutes. Any remaining un-bound DNA sequences were
repulsed while the covalently attached DNA remained at the
micro-location.
[0211] The upper buffer reservoir was aspirated and rinsed with
buffer. The apparatus was disassembled and a fresh reference test
device was mounted. The reservoir was refilled and fluorescently
labeled complement DNA sequence added, i.e., ET-10AL-TR. The
oligomer was electrophoretically concentrated at the positively
biased test micro-location for 2-5 minutes at 0.05 mA constant
current. The polarity was reversed and unbound complement removed.
The test devices were removed and examine by epifluorescence
microscopy. A negative control for non-specific binding was
performed as described above substituting a non-complementary DNA
sequence ET-21A-TR for ET-10AL-TR.
[0212] The cross-sections of the capillary micro-locations surfaces
were examined under a Jena epifluorescent microscope fitted with a
Hamamatsu ICCD camera imaging system. The fluorescent analysis
results indicated that complement ET-10AL-TR sequence hybridized to
the binding entity/capture sequence and remained hybridized even
when the potential was biased negative. The ET-21A-TR
non-complement sequence was not retained at the test device surface
when the potential was reversed.
EXAMPLE 3: Electronically Addressable Micro-locations on a
Microfabricated Test Device--Succinimidyl Acrylate Method
[0213] This example describes an alternative attachment chemistry
which covalently binds the 5'-terminus of the oligonucleotides.
Capillaries were fabricated as described above except that 1%
succinimidyl acrylate (Molecular Probes) was substitute for
polylysine. The capillaries were made up fresh because the
succinimidyl ester used to react with primary amines is relatively
labile, especially above pH 8.0. The capillaries were mounted as
described above and the reservoirs were filled with 0.1 M sodium
phosphate, pH 7.4. The capillaries were pre-run for 10 minutes at
0.05 mA. About 2 .mu.l ET-10AL (0.1 O.D.), which contains a
5'-amino terminus, was pipetted into the upper reservoir with the
power on and electrophoretic transport carried out for 2-5 minutes.
The polarity was reversed so that the test devices were biased
negative and electrophoresed an additional 2-5 minutes. The
un-bound DNA was repulsed, while the covalently attached DNA
remained at the micro-location.
[0214] The upper buffer reservoir was aspirated and rinsed with
buffer. The reference test device was un-mounted and and a new
reference device mounted. The reservoir was re-filled and the
fluorescent labeled complement oligomer ET-11AL-TR was added and
electrophorese as described above. A negative control for
non-specific binding was performed as described above substituting
a non-complement DNA sequence ET-21A-TR for ET-11AL-TR.
[0215] Fluorescent analysis of each of the test devices showed that
the complement ET-11AL-TR hybridized to the capture sequence
(ET-10AL), and remained hybridized even when the potential was
changed to negative. The non-complementary sequence, ET-21A-TR, was
not retained at the micro-location when the potential was
reversed.
EXAMPLE 4: Electronically Controlled Fluorescent DNA/Dye Detection
Process
[0216] Certain dyes such as ethidium bromide (EB) become highly
fluorescent when bound (intercalated) into double-stranded DNA.
While the fluorescence and binding affinity is greater when bound
into double-stranded DNA; the dye also has some affinity for
single-stranded DNA and produces low level fluorescenece when
bound. The following example shows how an electronically controlled
DNA/Dye detection process can be developed.
[0217] Microcapillary test devices were prepared and hybridized as
described in Example 2 and 3. Ethidium bromide (EB) was added to
the buffer solution (.about.0.05 mM EB final concentration) and the
test devices were biased negative to concentrate EB (positively
charged) at both the hybridized and un-hybridized micro-locations.
The test devices were observed by epifluorescence microscopy at 550
nm excitation and 600 nm emission. Both the hybridized and
un-hybridized micro-locations showed intense red fluorescence from
the concentrated EB.
[0218] The test devices were re-mounted biased positive constant
current at 0.05 mA for 0.03 Volt-Hours, to selectively remove the
EB. Fluorescence at the un-hybridized micro-locations diminished
while the hybridized micro-locations retained a very high level of
EB fluorescence. The results are given below:
3 Capture Target Normalized Signal ET-10AL ET-11AL (Pos.) >200
ET-10AL ET-21A (Neg.) 1
[0219] Fluorescent signal was measured using an ICCD imaging camera
system and represent peak fluorescent intensities. The signal to
noise ratio would be more than 1000 fold if the entire fluorescent
signal area was integrated. This demonstrates a method for
increasing signal to noise ratios and the dynamic range of the DNA
assays using intercalating dyes.
EXAMPLE 5: Electronically Addressable Locations on Metal
Substrates
[0220] Aluminum (Al) and gold (Au) wire (0.25 mm, Aldrich) were
reacted with 10% 3-aminopropyltriethoxysilane (APS) in toluene. The
APS reagent reacts readily with the oxide and/or hydroxyl groups on
the metal surface to form covalent bonds between the oxide and/or
hydroxyl groups and the primary amine groups. No pretreatment of
the aluminum was necessary. The gold wire was subjected to
electrolysis in 5 x SSC solution to form an oxide layer.
Alternatively the metal wire can be oxidized by a perchloric acid
bath.
[0221] The APS reaction was performed as follows: Wires were cut to
3 inches and placed in a glass dish. Toluene was added to
completely cover the wires and the temperature was brought to
50-60.degree. C. on a heat plate. APS was added to a final
concentration of 10%. Mix solution and continue the reaction for 30
minutes. Rinse 3 times with copious volumes of toluene, then rinse
3 times with copious volumes of alcohol and dry in 50.degree. C.
oven.
[0222] The APS treated wire can then be reacted with an aldehyde to
form a Schiff's base. Binding entity ET-12R was periodate oxidized
as described elsewhere in the specification. The electrodes were
placed in a reservoir of degassed water. Power was applied at 0.05
mA constant for about 30 seconds. Activated ET-12R was immediately
added. Power was applied, the liquid was aspirated and fresh water
was added and then aspirated again. The test (biased positive) and
reference electrodes were placed in Hybridization Buffer (HB,
5XSSC, 0.1% SDS) containing fluorescent labeled complement DNA,
ET-10-TR. After 2 minutes the electrodes were washed three times
for one minute each in Wash Buffer (1 x SSC, 0.1% SDS) and observed
by fluorescence (ex. 590 nm, em. 610 nm).
[0223] Results demonstrate that ET-12R was specifically coupled to
the treated metal surfaces. The test electrode was fluorescent
while the reference electrode was not. Non-specific adsorption of
the DNA to the metal was prevented by the presence of SDS in the
hybridization buffer. Attachment to gold substrates by electrolysis
and subsequent APS treatment was effective. Signal obtained was
significantly stronger than observed with non-oxidized gold. More
importantly, this example showed that the metal surfaces could be
chemically functionalized and derivatized with a binding entity and
not become insulated from the solution. The APS method represents
one of many available chemistries to form DNA-metal conjugates.
EXAMPLE 6: Electronically Controlled Fluorescent Dye Detection
Process--Metal Wire
[0224] DNA-aluminum electrode substrates were prepared and
hybridized as described in Example 5. A hybridized and an
un-hybridized DNA-Al electrode were processed with an
un-derivatized Al wire as the reference. Ethidium bromide (EB) was
added to the solution and the test DNA electrodes were biased
negative to attract the dye. The solution was aspirated and fresh
buffer was added. The metal surfaces were examined under the
microscope.
[0225] Remount the device and apply a positive potential for. a
defined volt-hour. The buffer was aspirated, the electrodes were
observed by epifluorescence. This was repeated until there was a
significant difference in fluorescence between the hybridized and
un-hybridized metal surfaces.
4 Capture Target Normalized Signal ET-12R ET-10AL (Pos.) >140
ET-12R None (Neg.) 1
[0226] Fluorescence at the unhybridized metal surfaces diminished
while the hybridized metal surfaces retained fluorescence.
Fluorescent signal was measured using an ICCD camera imaging system
and represent peak fluorescent intensities. The signal to noise
ratio would be >>1000 fold if the entire fluorescent signal
area was integrated. This example demonstrates a method for
increasing signal to noise ratios and thus the dynamic range of the
assay. Similar results were obtained using capillary gel
configuration, suggesting that electrochemical effects do not
significantly affect the performance of the assay.
EXAMPLE 7: Active Programmable Electronic Matrix
(APEX)--Micro-Machine Fabrication
[0227] A radial array of 6 addressable 250 .mu.m capillary
locations was micro-machined from plastic substrate material. The
device has a common upper reservoir and separate lower reservoirs
such that each micro-location is individually addressable. A unique
oligomer sequence binding entity is localized and attached to a
specific micro-locations made from highly crosslinked
polyacrylamide by the methods described previously. The test
micro-location has a positive potential while the other
micro-locations have negative potentials to prevent non-specific
interactions.
[0228] The array is washed and then hybridized with a complementary
fluorescently labeled DNA probe. The array is washed to remove
excess probe and then observed under an epifluorescent microscope.
Only the specifically addressed micro-location are fluorescent. The
process is repeated with another binding entity at another location
and verified by hybridization with a probe labeled with another
fluorescent moiety.
[0229] DNA sequences are specifically located to predetermined
positions with negligible crosstalk with the other locations. This
enables the fabrication of micromatrices with several to hundreds
of unique sequences at predetermined locales.
[0230] To select appropriate plastic substrates of low fluorescent
background, different plastic substrates were tested as to their
fluorescent characteristics at 600 nm. The plastics were tested by
an epifluorescent microscope imaging system and by a fluorometer.
The following table provides the list of substrates and fluorescent
readings obtained from an LS50B fluorometer:
5 Intensity Plastic at 610 nm, 5 sec Substrate int. ABS black 0.140
white 6.811 Polystyrene 7.955 Acrylic clear 0.169 white 51.77
tinted 0.151 black 0.035 transwhite 51.22 UHMW black 0.743 white
Delrin black 1.834 white 61.39 TFE 96.05 Polypropylene white 22.18
natural 25.82 Polycarbonate clear 11.32 tinted 3.103 white 45.31
black 0.156 PVC gray 2.667
[0231] The experiments show that black acrylic, ABS, and
polycarbonate have the lowest fluorescence background levels.
EXAMPLE 8: Active, Proqrammable Electronic Matrix
(APEX)--Microlithographi- c Fabrication
[0232] An 8.times.8 matrix (64 sites) of 50 .mu.m square
micro-locations on a silicon wafer (see FIG. 3) was designed,
fabricated and packaged with a switch box (see Device Fabrication
Section for details). Several materials and process improvements,
as described below, were-made to increase the selectivity and
effectiveness of the APEX DNA chip device.
[0233] 8a) Selection of Topcoat
[0234] The APS (3-aminopropyltriethoxysilane) process involves
reacting the entire surface of the chip. Selectivity of this
initial functionalization process is dependent on the relative
reactivities of the various materials on the chip surface. In order
to reduce functionalization and subsequent DNA attachment to the
areas surrounding the micro-locations, a material that is less
reactive to APS than SiO.sub.2 or metal oxide is needed.
Photoresists and silicon nitride were tested. The different
topcoats were applied to silicon dioxide chips. The chips were
examined by epifluorescence and the then treated with APS followed
by covalent attachment of periodate oxidized poly-A RNA sequences
(Sigma, M 100,000). The chips were hybridized with 200 nM solution
of Texas Red labeled 20-mer (T2-TR) in hybridization buffer, for 5
minutes at 37.degree. C. The chips were washed 3 times in washin
buffer and once in 1 x SSC. The chips were examined by fluorescence
at 590 nm excitation and 610 nm emission.
[0235] Silicon nitride was chosen because it had much less
reactivity to APS relative to silicon dioxide and was not
inherently fluorescent like the photoresist materials tested. Other
methods such as UV burnout of the background areas are also
possible.
[0236] 8b) APEX Physical Characterization
[0237] A finished matrix chip was visually examined using a Probe
Test Station (Micromanipulator Model 6000) fitted with a B & L
microscope and a CCD camera. The chip was tested for continuity
between the test pads and the outer contact pads. This was done by
contacting the pads with the manipulator probe tips which were
connected to a multimeter. Continuity ensures that the pads have
been etched down to the metal surface. The pads were then checked
for stability in electrolytic environments. The metal wires were
rated to handle up to 1 mA under normal dry conditions.
[0238] A drop (1-5 .mu.l) of buffered solution (1 x SSC) was
pipetted onto the 8.times.8 matrix. Surface tension keeps the
liquid in place leaving the outer contact pad area dry. A probe tip
was contacted to a contact pad and another probe tip was contacted
with the liquid. The current was incrementally increasd up to 50 nA
at maximum voltage of 50 V using a HP 6625A power supply and
HP3458A digital multimeter.
[0239] The initial fabrication consisted of the silicon substrate,
a silica dioxide insulating layer, aluminum deposition and
patterning, and a silicon nitride topcoat.
[0240] The second fabrication process included a silicon dioxide
insulating layer between the aluminum metal and silicon nitride
layers. Silicon dioxide and Al have more compatible physical
properties and form a better chemical interface to provide a more
stabile and robust chip than that made by the initial fabrication
process.
[0241] 8c) DNA Attachment
[0242] An 8.times.8 matrix chip was functionalized with APS reagent
as described in Example 5. The chip was then treated with periodate
oxidized poly-A RNA (Sigma, average M 100,000). The chip was washed
in washing buffer (WB) to remove excess and unbound RNA. This
process coated the entire chip with the capture sequence, however
there is a much higher density at the exposed metal surfaces than
at the nitride covered areas. The chip was hybridized with a 200 nM
solution of T2-TR in hybridization buffer (HB) for 5 minutes at
37.degree. C. Then washed 3 times in WB and once in lXSSC for one
minute each at ambient temperature. The chip was examined by
fluorescence at 590 nm excitation and 610 nm emission.
[0243] The opened metal areas were brightly fluorescent and had the
shape of the 50 um square pads (micro-locations). Low fluorescent
intensities and/or irregular borders suggest that some pads were
not completely opened. Additional plasma etch times would be
recommended in these cases.
[0244] 8d) Electronically Controlled Hybridization
[0245] Active hybridization was performed by using a chip from
Example 8c and biasing one specific micro-location positive. This
was done by using the switch box which would also automatically
bias the remaining micro-locations negative or by using an
electrode in the external solution. Three microliters of buffer was
deposited on the matrix pads (micro-locations) only. A current,
.about.1-5 nA, was applied for several seconds and 0.1 pmole of
T2-TR was added to the solution. The liquid was removed and the
chip was dried and examined for Texas Red fluorescence at
excitation 590 nm and emission em.610 nm. only the specific
micro-location biased positive was fluorescent. This experiment was
repeated many times, using other specific micro-locations on the
APEX chip. Additionally, the fluorescence DNA at one micro-location
was electronically de-hybridized and translocated to another
micro-location by biasing the initial location negative and the
destination micro-location positive.
[0246] 8e) Electronically Controlled Addressing and Device
Fabrication
[0247] The 8.times.8 APEX matrix was functionalized with APS as
described previously. The oligonucleotide binding entity CP-1 was
activated by periodate oxidation method. Four micro-locations were
biased positive in the matrix and the remainder were biased
negative. Two microliters of buffer was deposited on the matrix and
a current was applied. The binding entity, CP-1, was added and
electronically concentrate at the designated locations. The liquid
was removed, the chip was rinsed briefly with buffer and two
microliters of buffer was deposited on the chip. Again, current was
applied for several seconds and 0.1 .mu.mole of T2-TR was added.
The liquid was removed after a short time and the entire chip was
washed in WB, 3 times. The chip was dried and examined for
fluorescence.
[0248] Results indicate that the four positively biased
micro-locations were all fluorescent. This example demonstrates the
selective addressing of micro-locations with a specific binding
entity, the localization and covalent coupling of the attachment
sequences to the micro-locations, and the specific hybridization of
complementary target sequences to the derivatized
micro-locations.
[0249] 8f) Genetic Typing APEX Chip
[0250] DNA binding entities with 3'-ribonucleoside termini are
synthesized which are specific for the polymorphisms of HLA gene
dQa. The binding entities are activated by periodate oxidation as
described previously. The reverse complements are synthesized with
5'-amino termini and are conjugated with fluorophores, such as
Texas Red, Rhodamine or Bodipy dyes, as described previously. The
micro-locations are functionalized with primary amines by treatment
with APS, as described previously.
[0251] Several microliters of solution are placed over the
8.times.8 matrix. A specific micro-location is addressed by biasing
that micro-location positive, the periodate oxidized DNA oligomer
is added, at .about.0.1 pmole, and is translocated and covalently
coupled to that location. The polarity is reversed and the un-bound
binding entity molecules are removed. This is repeated for another
binding entity at another addressed micro-location until all the
unique attachment binding entities are bound to the chip.
[0252] The chip is then hybridized to individual fluorescently
labeled complement sequences to determine the specificity of the
coupling reaction as well as to visualize all addressed
micro-locations at once.
[0253] On the same chip which is electronically denatured to remove
complementary oligomelrs (10 minutes at 90.degree. C. in 0.05%
SDS), the addressed micro-locations are hybridized with un-labeled
target DNA or genomic DNA. Detection is via the fluorescent dye
detection assay as described previously in the specification.
[0254] Results will demonstrate that micro-locations are
specifically addressed with unique binding entities. Non-specific
binding to negatively biased micro-locations will be negligible.
The device and associated binding entity chemistry is stable under
denaturation conditions, thus making the addressed and fabricated
device reusable. Electronic methods for denaturing the hybrids
would be to increase the current and/or increase the time it is
applied.
EXAMPLE 9: Electronic Stringency Control
[0255] 9A) Sinqle Point Mutations with 15-mer Ras-12 Probes
[0256] The ability of the device to affect a high level of
electronic stringency control was demonstrated with a Ras-12
oncogene model system using 15-mer probes. A single base pair
mis-match in a DNA duplex causes only a slight instability in the
hybrid pair relative to the matched duplex. This slight instability
causes the mis-matched duplex to denature at a slightly lower Tm
than the matched duplex. When the pairs (match and mis-match) are
both hybridized at optimal stringency for the matched pair, the
mis-matched pair will hybridize with less efficiency. The
hybridization signal from the mis-match will be somewhat less than
the signal from the matched pair. With conventional hybridization
procedures, single point mutation analysis an be carried out with
probes in the 8-mer to 21-mer range. Probes in the 10-mer to 20-mer
range are used most often. When mutation specific probes become
shorter than 8-mers or longer than 20-mers, it becomes extremely
difficult to discriminate the match from the mis-match in any
reliable manner. This is because there is little difference in the
hybridization signals between the match and the mis-matched pairs.
The traditional methods of hybridization stringency control used in
point mutation analysis rely on temperature and salt
concentrations. We have found that stringency control can also be
affected by the electrophoretic potential.
[0257] In the Ras-12 example, 15-mer point mutation specific probes
were electronically hybridized to 30-mer target sequences attached
to the micro-locations on test devices. The polarity at the
micro-locations was biased negative, and the hybrids were subjected
to constant current for a given time, providing a defined power
level which denatures the mis-match without affecting the perfect
match.
[0258] The following sequences were synthesized and tested on a set
of three test structures, each with a 250 .mu.m surface
micro-location. The underlined/bold faced base indicates the
mis-match position.
[0259] The attachment sequences were:
6 Ras-G 5'-GGT GGT GGG CGC CGG CGG TGT GGG CAA GAU-3'-
micro-location Ras-T 5'-GGT GGT GGG CGC CGT CGG TGT GGG CAA GAU-3'-
micro-location
[0260] The-reporter probe sequences (labelled with Texas Red)
were:
7 Ras-1 3'-CC-GCG-GCC-GCC-ACA-C-5'-(TR) Ras-2
3'-CC-GCG-GCA-GCC-ACA-C-5'-(TR) Ras-3
3'-CC-GTG-GCA-GCC-ACA-C-5'-(TR)
[0261] Test devices were fabricated from microcapillary tubes as
described previously in the specification. Attachment sequences
Ras-G and Ras-T were periodate oxidized and covalently bound to the
addressed micro-locations.
[0262] Ras-G micro-locations were then hybridized with Ras-1, Ras-2
or Ras-3. Ras-1 is the perfect match to Ras-G. Ras-2 is a one base
pair mismatch (G-A). Ras-3 is a two base pair mismatch (G-A and
G-T). The G-A mis-match produces the least destabilization to the
DNA duplex, and is therefore the most difficult to distinguish from
the perfect match.
[0263] Conventional hybridization was first carried out and the
micro-locations were examined fluorescently to measure to what
extent complementary sequences were hybridized. The test devices
(microcapillaries) were re-mounted and electronic hybridization was
then carried out. The test devices were all subjected to the same
electronic stringency by biasing them at a negative potential (at
constant current) until the mis-matched hybrids were completely
removed without significantly affecting the perfectly matched
hybrid. The procedure and results are shown below:
[0264] Conventional Hybridization Procedure:
[0265] Hybridize in 5X SSC for 15 minutes at 40.degree. C.
[0266] Wash 3 times in 1X SSC for 5 minutes each 20.degree. C.
[0267] Carry out fluorescent analysis
[0268] Observed signal ratio of perfect match (Ras-G/Ras-1) to 1 bp
mis-match (Ras-G/Ras-2): about 10 to 1
[0269] Electronic Stringency Control (ESC) Procedure:
[0270] Hybridize in 5X SSC for 5 minutes at 20.degree. C.
[0271] "No washing procedure"
[0272] Apply an electronic stringency of 0.15 milliamps (MA) at 150
volts (V) for 4 minutes (20.degree. C.)
[0273] Carry out fluorescent analysis
[0274] Observed signal ratio of perfect match (Ras-G/Ras-1) to 1 bp
mis-match (Ras-G/Ras-2): >100 to 1
[0275] The complete results for all the experiments are shown
graphically in FIG. (15). These results show that it is not only
possible to use electrophoretic potential for stringency control in
DNA hybridization reactions; but also show that ESC provides both
higher hybridization efficiencies and higher discrimination ratios
than conventional hybridization procedures. In addition, ESC can be
applied to each individual micro-location, providing independent
stringency control in the same bulk solution.
[0276] (9B) Single-Point Mutation Analysis using 7-mers and 22-mer
Probes
[0277] Both 7-mer and 22-mer probes, which are well outside the
normal size range commonly used in point mutation analysis, were
prepared to further demonstrate the advantages of electronic
hybridization and ESC. The point mutation specific oligomer probes
listed below can be paired such that resulting hybrids have 0, 1,
or 2 base mis-matches. Complementary oligomer sequences were
coupled to micro-locations and hybridized as described above. The
polarity at the micro-locations was reversed (biased negative) and
the hybrids subjected to constant current for a given time,
providing a defined power level to denature the mis-matches without
removing the perfect match.
[0278] The Ras-G or Ras-GA oligomers (shown below) were attached to
micro-locations and used as target sequences. The series of 22-mer
and 7-mer Ras specific oligomer shown below were labeled with Texas
Red fluorophore as described elsewhere in the specification. The
"underlined and bold faced" bases indicates the mis-matched and/or
potential mis-matched positions:
8 Ras-G 5'-GGT GGT GGG CGC CGG CGG TGT GGG CAA GAU Ras-GA
5'-Amino-GGT GGT GGG CGC CGG CGG TGT GGG CAA GA Ras-22C-TR
(TR)-5'-TGC CCA CAC CGC CGG CGC CCA C Ras-22A-TR (TR)-5'-TGC CCA
CAC CGA CGG CGC CCA C Ras-TA (TR)-5'-TGC CCA CAC CGA CGG T GC CCA C
Ras-7C (TR)-5'-ACA CCG C Ras-7A (TR)-5'-ACA A CG C
[0279] Test devices were fabricated from microcapillary tubes as
described previously in the specification. The oligomer target
sequences Ras-G or Ras-GA were covalently attached to the
micro-locations. One micro-location was then hybridized with the
Texas Red labeled perfect 22-mer complement Ras-22C-TR. A second
micro-location was hybridized with Ras-22A-TR, a 22-mer one base
pair mis-match (G-A); or the Ras-22-TA the 22-mer two base pair
mismatch (G-A and G-T).
[0280] The test devices, as described above in the specification,
were run concurrently in the dual channel mode where both
micro-locations experience the same current or power levels
simultaneously. The test devices were first hybridized by
conventional procedures and the micro-locations examined
fluorescently to determine the amount of complementary sequences
which had hybridized. The test devices were then used to carry out
electronic hybridization to controlled time at constant current
until the mis-matched hybrids were removed without significantly
affecting the perfectly matched hybrids. A Bio-Rad 1000/500 power
supply was typically set to 0.02 to 0.1 mA and the experiments were
run at constant current for 0.02 to 0.04 volt-hours. The device was
disassembled and the test devices were observed by epifluorescence
on a Jena microscope fitted with a silicon intensified CAD camera
(Hamamatsu). The images were processed by a Hamamatsu Argus 10
image processor and recorded by a Sony Video Printer. The
capillaries were re-run when additional electronic stringency was
required.
[0281] Single base pair mis-match discrimination was performed on
the 7-mers as described above. However, due to their lower Tm, the
device was run in a cold box at 4-6.degree. C. rather than at room
temperature.
[0282] Results indicated that electronic hybridization and
stringency control could discriminate single base pair mis-matches
using both 7-mers and 22 mers. The match:mismatch ratios were 100:1
or greater. This signal:noise ratio was generally better than what
was reported by any hybridization methods which use temperature and
ionic strength to control stringency conditions.
[0283] Electronic stringency control was able to distinguish a one
base G-A mismatch from the perfect match eventhough the G-A
mismatch is the most stable mis-match because the G imino proton
can participate in hydrogen bonding with A which can stabilize the
duplex.
[0284] Power dissipation calculations and measurements showed
negligible changes in temperature, demonstrating that the
stringency was not caused by temperature changes at the
micro-locations. Micro-locations which were passively hybridized as
described above (not subjected to a electronic hybridization)
showed no discrimination between match and mis-match demonstrating
that diffusion was not causing the discrimination.
[0285] These examples also demonstrate that each micro-location can
have individual stringency control, and thus overcome a major
obstacle to large scale multiplex hybridization techniques which
are limited to a single common stringency level. It is also
possible to correlated electronic stringency power levels with
thermal melting (Tm) data to generate predictive electronic melting
(Em) curves and equations.
[0286] (9C) Electronic Hybridization in High Genomic Background
[0287] Actual target DNA sequences usually make up only a very
small proportion of the total DNA in a genomic DNA sample. By
concentrating total DNA at a very small location on an APEX device,
this invention increase the efficiency of target hybridizations in
the presence of an excess of heterologous DNA.
[0288] In this example, attachment sequences bearing 5'-amine
groups were attached to test devices containing 22% PAGE, 1%
succinimidyl acrylate. The capillaries were derivatized with either
ET-23AL or ET-11AL capture sequences. The target probe ET-12R was
labelled with Texas Red. ET-12R-TR would hybridize to ET-23AL but
not to ET-11AL capture sequences, the test and the control,
respectively.
[0289] The heterologous genomic DNA, calf thymus DNA (CT DNA,
Sigma), was dissolved to a final concentration of 1 mg/ml water,
sonicated and heated to denature the DNA. Samples were prepared in
0.5X TBE containing 10.sup.10 copies of ET-12R-TR target with 0,
0.1 .mu.g, or 1.0 .mu.g of denatured CT DNA in a final volume of
100 .mu.l. This represented a 0, 1,000, or 10,000 fold excess of CT
DNA relative to target DNA.
[0290] Test devices were pre-run 5 minutes at 0.03 mA in 0.5X TBE
using a Bio-Rad 1000/500 power, supply. The device was set to run
in dual channel mode so that a test and control capillary could be
run at the same time under exactly the same conditions. The sample
was applied (100 .mu.l) and the capillaries were biased with a
positive potential to attract DNA for 5 minutes at 0.03 mA. The
polarity was reversed and the same power was applied to remove all
un-hybridized ET-12R-TR target from the test device surface. The
buffer was aspirated and the test devices were observed by
epifluorescence on a Jena microscope fitted with a silicon
intensified CAD camera (Hamamatsu). The images were processed by a
Hamamatsu Argus 10 image processor and recorded by a Sony Video
Printer.
[0291] There was no difference between the absolute hybridization
signal and the signal/noise ratios in the presence and absence of
0.1 .mu.g CT DNA per 100 .mu.l. The signal intensity was equivalent
and the signal was uniformly distributed across the active
area.
[0292] At the level of 1 .mu.g CT DNA per 100 .mu.l, the signal was
predominantly distributed around the perimeter of the capillary,
suggesting that the capture sequences were blocked or saturated.
This artifact was easily surmounted by oscillating the polarity
during the hybridization step. This would pulse the total DNA
towards and away from the active area, allowing the target to
hybridize more efficiently and uniformly.
[0293] (9D) Passive Hybridization vs. Electronically Controlled
Hybridization
[0294] Electronically controlled hybridization is more efficient
and faster than passive hybridization because of the concentration
effect in the electronically controlled hybridization.
[0295] Microcapillary test devices were made up with ET-23AL and
ET-11AL attachment sequences, as test and control devices
respectively. A hybridization solution containing 1.times.10.sup.10
copies of ET-12R-TR with 1 Ag CT DNA in a total volume of 100 .mu.l
was made up.
[0296] Passive hybridization:
[0297] A set of test and control devices were placed in a small
test tube with 100 .mu.l of hybridization solution at 50.degree.
C., and hybridized for 15 minutes. The samples were then washed 3
times in 1 x SSC, 0.1% SDS, 5 minutes for each wash at 45.degree.
C.
[0298] Electronically controlled hybridization:
[0299] Test devices were mounted and pre-run for 5 minutes at 0.06
mA. The buffer was then aspirated and 100 ul of hybridization
solution was added. The test devices were biased positive for 3
minutes at 0.06 mA. the polarity was then reversed for 30 seconds,
and reverse again so the test devices were once again positive for
additional 3 minutes. The test devices were biased negative for 3
minutes to electronically wash.
[0300] The efficiency and extent of hybridization was significantly
better with the active format than with the passive format.
Absolute signal in the active (electronic) format was more than 100
fold higher than the signal in the passive format. The signal/noise
ratio in the active format was increased 10 fold over the signal in
the passive format. The active hybridization assay was completed in
under 10 minutes with minimal manipulations. The passive format
required--30 minutes with several manipulations of tubes and
buffers.
[0301] Traditional hybridization methods use 2.5 nM probe at 3
times C.sub.ot, for 15 minutes., for 90% completion of the
reaction. At our experimental concentration of 0.17 nM probe, the
passive hybridization reaction kinetics would normally require
.about.4 hrs.
[0302] Active hybridization enables the use 'of lower probe
concentrations which result in lower background. Traditional
methods depend on diffusion and thus must use higher probe
concentrations to drive the reaction kinetics. The active method is
able to concentrate the sample into a very small volume which
results in a very high local probe concentration and subsequently
very fast hybridization reaction kinetics.
EXAMPLE 10: Hybridization with Fluorescent DNA Nano-Structure
[0303] Normally, the overall sensitivity for non-amplification type
hybridization assays is limited by background from the non-specific
binding. This is often a major problem when multiple reporter
groups, or secondary complexes with multiple reporter groups, are
used to label DNA probes. Therefore, the assay detection limit is
often reached well before the actual or intrinsic detection limit
of the reporter label(s) is reached.
[0304] Using electronic controlled hybridization methods, we have
found that highly fluorescent sub-micron or nano-scale beads may be
used with attached DNA probes for ultra-sensitive assays. We have
been able to control the movement of DNA probe-fluorescent
nanostructures using free field electrophoresis. Since electronic
stringency control provides high level discrimination of hybridized
from un-hybridized structures, DNA-probe-fluorescent nanostructures
can significantly increase hybridization sensitivity. Electronic
stringency control allows us to utilize these highly fluorescent
nanostructures or other multiple labeling scenarios for low copy
number (50 to 1000 targets) detection, without amplification being
necessary. To date, this has not been possible with conventional
hybridization methods and procedures.
[0305] Fluorescent nahoparticles, Fluorospheres, were obtained from
Molecular Probes, Inc.. The particles are composed of carboxymethyl
latex spheres loaded with fluorescent dyes, such as Texas Red or
fluorescein. The latex spheres can be obtained with different
functional groups on their surface, such as amine or aldehydes. The
particles are available in sizes from 0.01 to 5 .mu.m in
diameter.
[0306] 1) Characterization of the Fluorescent Nanoparticles
[0307] The nanoparticles, unmodified, amine modified, or aldehyde
modified, have a net positive charge. In an electric field these
particles migrate-towards the negatively biased
micro-locations.
[0308] 2) DNA Attachment Chemistry to the Fluorospheres
[0309] The amine modified particles can be coupled to nucleic acids
bearing terminal aldehyde groups. The latter can be generated by
DNA probes synthesized with a 3'-terminal riboside which is
subsequently oxidized by the periodate method as described
previously in the specification.
[0310] The particles are stored as a 2% suspension in distilled
water. An aliquot of 25 to 50 .mu.l of the 0.02-1.0. .mu.m amine
modified red fluorescent Fluospheres was pelleted and re-suspended
in O.lM sodium phosphate, pH 7.4. An excess of periodate oxidized
poly ribo-A was added to the suspension. The reaction was allowed
to incubate for 90 minutes at room temperature. The particles were
washed and pelleted several times in 1 x SSC, 0.1% SDS (0.15 mM
sodium chloride, 0.015 mM sodium citrate, 0.1% (w/v) sodium
docecyl, sulfate, pH 7.0) to remove unbound and non-specifically
bound poly ribo-A.
[0311] The DNA-fluorospheres in buffered solution were placed in a
direct current electric field. It was observed that 'the
DNA-Fluorospheres migrated towards the positive electrode,
indicating that their net charge was now negative. This is a simple
and convenient method to determine if the DNA coupling reaction was
successful. Traditional hybridization methods would require using a
radiolabeled reporter probe because the intense fluorescence from
the particles would obscure any hybridization signal.
[0312] 3) DNA Attachment to Test Devices
[0313] The test devices were polymerized with highly cross-linked
polyacrylamide, containing 1% succinimidyl acrylate, which can be
subsequently reacted with 5'-amine terminated DNA probes. The
attachment of the capture sequence, oligo-T, was verified by
hybridization with fluorescently labeled complement probe, CP-1-TR.
The test device surfaces were highly fluorescent which indicates
that the surface was derivatized with capture sequences.
[0314] 4) Electronic Hybridization and Detection of
DNA-Fluorospheres
[0315] The hybridization reactions were performed in a structure
which holds 2 microcapillary test devices sharing a common upper
reservoir and independent lower reservoirs. The reactive surfaces
are exposed to the common upper reservoir.
[0316] The test devices were mounted in the structure and pre-run
in 0.5x TBE at 0.05 mA, for 15 minutes. One test device had the T2
complementary attachment sequences, and the other had ET-10AL
non-complementary attachment sequence. One microliter of
DNA-fluospheres at was added to the upper reservoir. The test
devices were biased positive at 0.02 mA, for 5 minutes, to attract
the DNA-Fluorospheres (fluorescent nanoparticles). The test devices
were inspected to determine that the particles were presefnt.on the
surfaces. The polarity was reversed such that the test devices were
now biased negative and the un-hybridized DNA-Fluorospheres should
be repelled.
[0317] There was no discrimination between the test and the control
devices. The particles could not be removed after repeated attempts
regardless of the amount of power applied.
[0318] 5) Passive Hybridization and Detection of
DNA-Fluospheres
[0319] Without being bound by any theory or hypothesis, we believe
that electronic hybridization of the particles, physically embeds
or traps the particles in the surface gel matrix of the test
devices. Thus, DNA-Fluospheres which are passively hybridize to the
attachment sequences on the gel surfaces, should be more easily
removed by electronic de-hybridization.
[0320] New test devices was mounted as described above. A 0.05%
suspension of DNA-Fluorospheres were pipetted into the upper
reservoir and passively hybridized for 5 minutes. The buffer was
aspirated and fresh 1x TBE buffer was added. The test devices were
now biased negative to repel the particles. The test devices was
operated for 5 minutes at 0.02 mA, and then inspected by
fluorescence.
[0321] There was now significant discrimination between the test
and control devices after performing ECS for a total of 10 minutes
at room temperature. The signal was not uniformly distributed
across the test surface, but concentrated in signal pockets. This
may suggest that the availability of the surface attachment
sequences is limited. Improvements can be made using longer spacer
arms with either hydrophobic, hydrophilic, or mixed character. Such
spacers for example can be built using diaminohexane, succinic
anhydride, and a variety of other spacer groups well known in
art.
EXAMPLE 11: Electronically Directed Restriction Enzyme Cleavage of
Specific ds-DNA Sequences
[0322] Two examples are used to demonstrate the ability of APEX
devices to selectively carry out restriction endonuclease cleavage
of ds-DNA sequences. The M13mp 18 (having a Xba I restriction site)
and M13mp8 (not having Xba I restriction site) vectors are used in
these examples. These vectors are commonly used in many cloning and
DNA sequencing procedures.
[0323] The first example demonstrates. (1) the electronic
hybridization of M13mp sequences to specific micro-locations on the
test device, (2) the free field electrophoretic transport of the
Xba I restriction enzyme to the micro-locations, and (3) the
subsequent capture of the cleaved fragments at other
micro-locations. The example also demonstrates the ability of the
device to self-assemble itself with specific binding entities
(oligonucleotide capture sequences, etc.).
[0324] The basic steps in the procedure are shown in FIG. (16).
Four specific micro-locations (ML-1, ML-2, ML-3, and ML-4) which
covalently bind oligonucleotide capture sequences are used in the
procedure. Electronic delivery systems are used to deliver reagents
(oligonucleotides, restriction enzyme, etc.) and for disposal of
reactants.
[0325] The first step involves the transport and covalent
attachment of the M13-1 oligonucleotide capture sequence to ML-1
and ML-2 micro-locations, and the transport and attachment of the
M13-2 oligonucleotide capture sequence to ML-3 and Ml-4
micro-locations. Since nucleic acids are negatively charged at pH
>4, they always move toward the positively charged electrode
when electrophoresed in buffer solutions which range from pH
5-9.
[0326] The second step involves the free field electrophoretic
transport and hybridization of the M13mp18 sequence to the M13-1
capture sequence at ML-1 micro-location, and the M13mp8 sequence to
the M13-1 sequence at the ML-2 micro-location.
[0327] The third step involves the transport of the XbaI
restriction enzyme to the ML-I (M13mp18) micro-location and the
ML-2 (M13mp8) micro-location. The Xba I cleaves the M13mp18 at
ML-1, but not the M13mp8 at ML-2. The cleaved fragments from ML-1
are transported and hybridized to the M13-2 sequence at ML-3. As an
experimental control, free field electrophoresis is carried out
between ML-2 and ML-4. Since the M13mp8 sequence at ML-2 has not
been cleaved, no fragment is detected at ML-4.
[0328] The various M13 attachment and probe sequences used in this
example are prepared as previously described in the specifications.
These sequences are shown below:
9 M13-C1 5'-CCA GTC ACG ACG TTG TAA AAC GAC GGC CAG U M13-C2 5'-GTA
ATC ATG GTC ATA GCT GTT TCC TGT GTG U MP18-40C 5'-GCA TGC CTG CAG
GTC GAC TCT AGA GGA TCC CCG- GGT ACC G MP8-40C 5'-TGC CAA GCT TGG
CTG CAG GTC GAC GGA TCC- CCG GGA ATT C MP18-R1 (TR)-5'-AAA TTG TTA
TCC GCT CAC AAT TGC MP8-R2 Y(F)-5'-ACA CAA CAT ACG AGC CGG AAG
CAT
[0329] Step 1--Attachment of M13 Capture Sequences
[0330] An APEX test device with 200 .mu.m micro-locations of amine
activated highly cross-linked (26%) polyacrylamide surface or
polycarbonate (5-10 nm) porous membrane surface is used for this
procedure.
[0331] The M13-C1 capture sequence is a 31-mer DNA oligonucleotide
containing a 3'-ribonucleotide. The M13-C1 sequence is
complimentary to the 3'-terminal of the M13mp18 and M13mp8
single-stranded (+) vectors. The M13-C1 capture sequence is
designed to hybridize and strongly bind all un-cleaved M13
vectors.
[0332] The M13-C2 sequence is a 31-mer oligonucleotide containing a
3'-ribonucleotide. The M13-C2 is complementary to a portion of the
M13 sequence upstream from the cloning site containing the Xba I
restriction site. The M13-C2 capture sequence is designed to
hybridize and strongly bind the Xba I cleaved M13 fragments.
[0333] The M13-C1 and M13-C2 capture sequences are activated for
coupling to the amine derivatives on the APEX micro-locations by
the paraded oxidation. The 3' ribonucleotide terminus is converted
to a terminal dialdehyde by the paraded oxidation method which can
react with primary amines to form a Schiff's base.
[0334] Reaction conditions are as follows:
[0335] Dissolve 10-20 O.D. of the M13-C1 or M13-C2 oligomer in
water to a final concentration of 1 OD/.mu.l. Add 1 volume of 0.1 M
sodium acetate, pH 5.2 and 1 vol 0.45M sodium paraded (made fresh
in water). Stir and incubate reaction for at least 2 hours at
ambient temperature, in the dark. Load reaction mix onto a Sephadex
G-10 column (pasteur pipette, 0.6.times.5.5 cm) equilibrated in
0.11 M sodium phosphate, pH 7.4. Collect 200 .mu.l fractions, spot
2 .mu.l aliquots on thin layer chromatography (TLC) and pool ultra
violet (UV) absorbing fractions.
[0336] Four top surfaces of the APEX test devices are designated to
be the addressable micro-locations ML-1, ML-2, ML-3, and ML-4.
[0337] M13-C1 is covalently attached to the ML-1 and ML-2
micro-locations by the following procedure:
[0338] The upper and lower reservoirs are filled with 0.1 M sodium
phosphate, pH 7.4 and prerun for 5 minutes at 0.05 mA constant
current, using a BioRad 500/1000 power supply. The tip of an
electronic delivery system containing 0.1 O.D. units of the paraded
oxidized M13-C1 oligonucleotide is placed into the lower reservoir.
The electronic delivery system is a specially modified plastic
pipet tip with a platinum electrode inside. The electronic delivery
system is biased negative (-) and micro-locations ML-1 and ML-2 are
biased positive (+) at 0.1 mA. M13C-1 is electrophorese to ML-1 and
ML-2 for 2 minutes at constant current, where it becomes covalently
bound to the surface. The polarity is reversed, for .about.4
minutes, so that un-reacted M13C-1 is removed from the ML-1 and
ML-2 micro-locations.
[0339] The M13C-2 sequence is attached to the ML-3 and ML-4
micro-locations with the same procedure described above.
[0340] Step 2--Hybridization of M13 Vectors, Complementary
Sequences, and Fluorescent Reporter Probes
[0341] Since restriction endonucleases require double-stranded DNA
for cleavage, the cloning/restriction site segments of the single
stranded M13mp18 (from 6240 to 6280) and M13mp8 (from 6230 to 6270)
must be hybridized with complementary DNA sequences. Electronic
hybridization is used to hybridize a 40-mer complementary fragment
(MP18-40C sequence) to M13mp18 vector on ML-1/M13C-1
micro-location; and to hybridize a 40-mer complementary fragment
(MP8-40C sequence) to the M13mp8 vector on ML-2/M13C-1
micro-location respectively.
[0342] Electronic hybridization is carried out by negatively (-)
biasing an electronic delivery system containing 0.05 O.D. units of
M13mp18, and positively (+) biasing the ML-1/MP13C-1 micro-location
at 0.1 mA for 2 minutes. The polarity is reversed for 4 minutes and
the un-hybridized M13mp18 is removed from the micro-location. The
same procedure is used to electronically hybridize the M13mp8
vector to the ML-1/M13C-1 micro-location.
[0343] The M13mp18 and M13mp8 sequences are then electronically
hybridized with two different fluorescent reporter probes. The
M13mp18 vector on the ML-1/M13C-1 micro-location is electronically
hybridized with a 24-mer Texas Red labelled reporter probe (MP18-R1
sequence), which hybridizes to the 5'-terminal of the
cloning/restriction sites. The M13mp8 vector is electronically
hybridized with a 24-mer Fluorescein labelled reporter probe
(MP8-R2 sequence), which hybridizes to the 5'-terminal of the
cloning/restriction sites.
[0344] Step 3--Restriction Cleavage of the M13mp18 Vector Using the
Xba I Restriction Enzyme
[0345] Depending upon their Isoelectric Point (pI), many proteins
and enzymes can be negatively charged (pH>pI), neutral (pH=pI),
or positively charged (pH<pI) in the pH 5-9 range. A number of
restriction endonucleases have pI's in the 6-7 range. At pH's
greater than the pI, these enzymes will carry a net negative
charge. Therefore, when free field electrophoresis is carried out
in a buffered solution with a pH>7, these enzymes will migrate
to the positively charged micro-location.
[0346] In the case of many DNA modifying enzyme, like restriction
endonuclease, it is always desirable to choose a buffer solution
which provides a pH which balances the optimal enzyme activity with
relatively fast electrophoretic mobility. In some cases it is
possible to have reasonable enzyme actively both above and below
the pI. These enzymes can be moved toward either a positively or
negatively biased micro-location, depending on the chosen pH.
[0347] The Xba I cleavage of the M13mp18 vector at ML-1 is carried
out as follows. The Xba I endonuclease is first free field
electrophoresed to the ML-1/M13mp18 micro-location using an
electronic delivery system. The electronic delivery system,
containing 100 units of Xba 1 in pH 7.6 buffer, is biased negative
and the ML-1/M13mp18 micro-location is biased positive at 0.1 mA
for 2 minutes. The current is then reduced to 0.02 mA for 3
minutes. The electronic delivery system is turned off, while the
ML-1/M13mp18 micro-location is biased negative and the ML-3/M13C-2
micro-location is biased positive at 0.1 mA for 5 minutes. The
ML-3/M13C-2 micro-location is now biased negative and the
electronic delivery system is turned on and biased positive at 0.1
mA for 2 minutes in order to remove Xba 1 and un-hybridized
fragments from the ML-3/M13C-2 micro-location.
[0348] Observation by epifluorescent microscopy shows loss of red
fluorescent signal at the ML-1/M13mp18 micro-location and presence
of red fluorescent signal at the ML-3/M13C-2 micro-locations,
demonstrating Xba 1 cleavage of the M13mp18 vector. The same basic
Xba 1 cleavage procedure is now repeated for the ML-2/M13mp8
micro-location, which serves as a negative control. Since the
M13mp8 vector has no Xba 1 site, cleavage and production of
fragments is not possible. The ML-2/M13mp18 micro-location thus
maintains its green fluorescent signal, and no fluorescent signal
is observed at ML-4/M13C-2 micro-location.
[0349] A second example involves restriction cleavage reactions
being carried out with the restriction enzymes being covalently
attached to addressable micro-locations on the device. In this
case, restriction endonucleases would be derivatized and free field
electrophoresed to addressable micro-locations on an APEX device
where they would become covalently bound. Methods for the
derivatization and covalent attachment of restriction enzymes to
solid supports are known to those skilled in the art. A variety of
different restriction enzymes could be addressed to the APEX
device. Specific cleavage reactions would be carried out by using
free field electrophoresis to concentrate ds-DNA vectors or DNA
samples at the micro-location containing the desired restriction
endonuclease. The ds-DNA would be cleaved and fragments then moved
to other micro-locations on the device. When desired or useful
other DNA modifying enzymes could be coupled to addressable
micro-locations on the APEX device. Also, this example should not
be considered limited to DNA modifying enzymes, in that most other
enzymes could be attached to addressable micro-locations on APEX
devices.
EXAMPLE 12: Electronic Amplification Methods
[0350] In cases of hybridization analysis with very low target
sequence copy number (e.g., HIV, septic blood infections, etc.),
the multiplication or amplification of target DNA sequence would
enable sensitivity to be improved by amplification of purified
target DNA and/or RNA directly on an APEX device. Amplification
would also reduce the requirement for very high yield preparative
steps prior to hybridization analysis.
[0351] APEX amplification protocol provides complete electronic
control of DNA movements, denaturation, and synthesis reactions.
Most importantly DNA hybrids are denatured electronically without
the use of high temperature or the need for thermophilic
polymerases or other thermal stable enzymes.
[0352] As a first example, DNA synthesis can be achieved with high
fidelity using DNA polymerase (Klenow large fragment) and without
the need for thermal cycling. In this example, one DNA strand is
amplified in a way that leaves it covalently bound to a
micro-location. The procedure is carried out in the following
manner: 1) the known target sequence is electronically hybridized
to a capture probe of known sequence on an addressed
micro-location, 2) synthesis of nascent complementary strand DNA
(-) by DNA polymerase primed by the capture probe is carried out,
3) the newly synthesized DNA hybrids are electronically denatured,
4) annealing of target strand DNA to non-elongated capture probe
and annealing of - strand complementary probe to nascent--strand
DNA is carried out, 5) the synthesis of nascent target strand DNA
(+) by DNA polymerase and concomitant synthesis of--strand DNA as
in 2 is carried out, thereby doubling the number of + and - strands
each time these steps are repeated, and 6) size selection of
amplified target is carried out by hybridization to a specially
designed complimentary probe. The complete procedure, shown in FIG.
17, is described in more detail below:
[0353] Step 1) Attachment of Target Sequence to Capture Probe
[0354] Target sequence is electrophoretically transported to a
micro-location (1) containing covalently bound capture probe.
Target sequence can be present in a background of non-target
(genomic) sequence but must be denatured prior to annealing to
capture probe. Target sequence which is initially captured will be
of variable length.
[0355] Step 2) Synthesis of DNA Complementary to Target
[0356] DNA polymerase and dNTP's are electrophoretically
transported to micro-location 1. The capture probe provides a 3'
end for DNA polymerase and the captured target sequence provides
the template. Current sufficient to maintain a concentration of
reagents amenable to synthesis are applied. The current may be
constant or pulsed. These parameters can be manipulated to obtain
differing ranges of lengths of nascent complementary (-)
strand.
[0357] Step 3) Electronic Denaturation of Newly Synthesized
Strands
[0358] Polarity at micro-location 1 is reversed and voltage is
applied to separate-the two strands. The amount of voltage and the
time period of application will be dependent on the length and base
composition of the hybrid DNA complex. These parameters may be
determined empirically or calculated from electronic denaturation
curves.
[0359] Step 4) Annealing of Primers (Capture and complementary
Probes) to DNA Strands
[0360] Oligos need to be annealed to both + and - DNA strands to
provide primer sites for DNA polymerase. For the target or + strand
this is accomplished by electrophoretic transport of + strand to
un-elongated capture probe. This will occur as long as un-elongated
capture probe is in excess to elongated, covalently bound - strand
DNA. Complementary probe is electrophoresed to the micro-location
and binds to covalently bound - strand DNA. Now both + and -
strands have primer bound to them and are templates DNA polymerase
catalyzed synthesis (see figure). Binding of complementary probe
may also occur with noncovalently bound - strand DNA, however these
hybrids will not be electronically denatured and therefore should
have little impact on the overall amplification.
[0361] Step 5) Synthesis of Two New Strands of DNA
[0362] Step 2 is repeated and since both + and - strands are primed
templates, the amount of sequence specific DNA doubles. This
geometric increase in the amount of DNA will occur each time these
steps are repeated.
[0363] Step 6) Size Selection of Amplified Target Sequence
[0364] The nucleotide sequence of the complementary probe will
determine the size and sequence of the amplified target DNA.
Therefore, the amplified DNA can be custom designed to enhance
efficiency in subsequent analysis and/or manipulation.
[0365] Other enzymes can be used in the amplification method of
this invention, including, but not limited to, other DNA
polymerases, T7 or SP6 RNA polymerases, reverse transcriptases, DNA
ligases, and polynucleotide phosphorylases, and combinations of
other nucleic acid modifying enzymes (endonucleases, exonucleases,
etc.).
EXAMPLE 13: Electronic Controller And Data System
[0366] All devices, whether APEX chip or micromachined devices,
will be of the nature of an addressable array of micro-locations
(or macro-locations). A computer control/data collection system has
been designed to provide independent application of electric
potentials to any pads in the array and to measure the resulting
current flowing in the microlocation-electrolyte system. The
computer control/data collection interface provides:
[0367] a) Representation of the array of micro-locations. Higher
level and lower level representations provide views of all
micro-locations, with resolution of blocks of micro-locations at
the highest level view, and with fully resolved blocks of
micro-locations at the lower levels.
[0368] b) Clicking on a micro-location will pops-up a window view
of the micro-location detailing the characterization of the
micro-location, allowing setting of control of the micro-location
with a time sequence of signals of various shape, electric
potential magnitude and sign, etc., display of the control sequence
overlaying that of other micro-locations, etc. The system also
provides display of the data and signals collected for the
micro-location with statistics and comparisons with data form other
micro-locations. Menus provide analysis, documentation and archival
functions for the control design, the actual control signals
observed and the data collected.
[0369] c) The software provides all switching and data collection
through a hardware interface controlled by inputs from the array
control software described in b).
[0370] d) A separate hardware and software system provides image
collection and processing capabilities. This systems images the
array of micro-locations and records flub-rescence signals from DNA
binding interactions at the active micro-locations to provide
readout of the DNA binding experimental results. Image processing
software provides the ability to quantitatively process these
images and extract quantitative assay results. This software is
fully interfaced with the array controller/data collection software
to provide an integrated system that records all the APEX device
control/electrolyte current data and the assay results from imaging
data, analyzes the data to provide reduced results for the assay
along with ancillary information regarding the consistency and
reliability of these results, and archive all the data and
analyses.
[0371] e) An APEX controller will incorporate all of this software
plus a top layer that provides only "DO ASSAY" and "RESULTS"
displays, plus a button to access a) through c) functionality if
necessary, but a) through c) will be collected and archived in all
cases.
[0372] f) The initial version of the controller to be used for
development projects uses a Macintosh Quadra 950 as a host computer
and uses National Instruments boards interfaced with the Quadra 950
to provide the hardware interface described above. These boards
apply the variable potentials to the APEX micro-locations and
measure the resulting current flowing in the electrolyte system.
The National Instruments boards used in this controller are the
High Resolution Multifunction I/O board, NB-MIO-16XL-18, the Analog
Output board, NB-AO-6, the Timing Input/Output board, NB-TIO-10,
the Block Mode DMA and GPIB Interface board, NB-DMA2800, and the
Analog Signal Conditioning Modules boards and Modules for
thermocouples, and other environmental sensors, 5B series.
Connections between the NuBus boards in the Quadra and the APEX
device will be through SCXI 16-Channel SPDT Relay Module boards
housed in an SCXI-1001 Chassis.
EXAMPLE 14: Electronically Controlled Sample Preparation and
Hybridization Analysis--An Integrated APEX System
[0373] Sample preparation usually involves selection of cells;
disruption of cellular material (e.g., lysis), and a series of
separation procedures and affinity reactions. Sample preparation is
important for molecular biologic reactions. For example,
hybridization assay is often limited because one loses significant
amounts of the actual target DNA sequences due to inefficiencies in
the sample preparation process.
[0374] The basic APEX concept for electronic control can be used
for sample preparation in DNA hybridization assays. Electronic
methods will allow sample preparation, cell selection and analysis
to be carried out on an active electronic system of APEX
components. The sample preparation would begin with cell selection
and lysis, and the gross separation of DNA from cellular and
extraneous materials in the sample. The electronic device would
electronically process the sample DNA and move it efficiently
toward the analytical component of the device, while removing the
other materials. The system provides the proper scaling factor for
efficient processing of the target DNA. For human genomic analysis,
electronic sample preparation would include a highly efficient
prehybridization step by which most of the complex non-specific DNA
would be separated from the target DNA.
[0375] An integrated device or complete APEX system with sample
preparation would take a relatively crude sample (blood, sputum,
urine, etc.), and processes it with minimum mechanical manipulation
and fluidics, and then electronically deliver target DNA to the
analytical component of the device. This "active electronic
processing" differs from automation or robotic processing, which
are generally mechanical versions of the manual process and
techniques.
[0376] An integrated APEX System for DNA sample preparation and
analysis can be fabricated using a number of components all based
on the general APEX concept. The components of the system include
(1) an electronic cell selector unit; (2) an electronic reagent
dispenser unit; (3) an electronic waste disposal unit; (4) a crude
DNA selector unit; (5) a secondary DNA'or restriction fragment
selector unit; (6) a DNA fragment storage unit; and (7) the APEX
analytical unit (chip). The integrated APEX system is shown in FIG.
18.
[0377] Such a system can be fabricated on a large silicon wafer.
Alternatively, individual components can be fabricated by
microlithography or micromachining techniques and arranged on
(e.g., plugged into) a specially designed platform unit. The
components of the complete system are designed so their active area
scales to the relative sample size and the amount of materials in
the sample (such as cells). For example, the cell selector active
area generally would be larger than the crude DNA selector active
area, which in turn would be larger than the restriction fragment
selector active area, which would be larger than the APEX
analytical chip active area.
[0378] By way of example, the cell selector "active area" could be
of the order of several cm.sup.2, while the total "active area" for
a 64 micro-location APEX analytical component would be less than
one mm.sup.2. The platform unit is designed to hold all the
component units in a sealed common buffer reservoir. Up to several
hundred microliter of the appropriate sample is added to the system
through a sample addition port near the cell selector component.
The cell selector component is a larger scale APEX device which can
have one or more selected affinities for different cell types.
These affinity selections can be made on the basis of cell surface
charge, haptens, and antigens.
[0379] By way of example, affinity selection for whole blood
samples can be made to select white blood cells (lymphocytes, etc.)
from red blood cells. Highly selective processes could be used to
select fetal cells from material blood sample. It is also possible
to provide affinity selection for infectious microorganisms (yeast,
fungus, bacteria, and virus). While selected cells remain attached
to the cell selector component; all other cells and proteinaceous
materials are transported to the waste disposal unit. At this point
the cells can be lysed by free field electrophoretic transport of
charged detergents, and/or chaotropic agents, and/or appropriate
lytic enzymes and proteinases (lysozyme, proteinase K, pepsin,
etc.) from the electronic reagent dispenser unit to the cells on
the cell selector unit. Appropriate biasing of the electronic waste
disposal system can be used to remove certain lytic waste
materials. The positive biasing of the crude DNA selector unit can
now be used to transport the crude nucleic acid (DNA/RNA) materials
to this component.
[0380] The crude DNA selector is an APEX device which has a general
affinity for DNA. This/affinity can be a positively charged
surface, or a surface which contains a common or repetitive DNA
sequence. For example, an Alu repeat capture sequence would
effectively capture most of the crude DNA extracted from human
cells. A common or generic bacteria or viral sequence could be used
when infectious disease analysis is the objective. In addition to
removing extraneous materials from the DNA; the APEX system is also
designed to reduce the complexity of the sample DNA. This can be
achieved by using restriction enzymes to selectively cleave the DNA
at the crude DNA selector unit. The restriction enzymes are
transported from the reagent dispenser unit. The cleaved
restriction fragments can now be transported from to the secondary
DNA or restriction fragment selector unit by biasing it positive.
This unit is designed to selectively bind large fragments of DNA,
using appropriate capture sequences on its surface.
[0381] At this point, selected DNA fragments can be transported to
the APEX analytical chip for hybridization analysis. It is also
possible to transport DNA fragments to the storage unit or even out
of the system. The examples above represent just some of the
possible scenarios for sample preparation and multiple
hybridization analysis. The binding affinity programmability of
components and flexibility of combining different components and
functions allows a wide variety of procedures to be carried
out.
[0382] While DNA is used as a primary example, the above described
device and method can also be used for the processing and analysis
of target RNA molecules, proteins, polysaccharides, lipids and
other macromolecules.
[0383] All publications referenced are hereby incorporated by
reference herein, including the nucleic acid sequences and amino
acid sequences listed in each publication.
[0384] Other embodiments are within the following claims.
Sequence CWU 1
1
* * * * *