U.S. patent application number 12/217087 was filed with the patent office on 2009-12-31 for density modification in arrays of surface-attached nucleic acid molecules.
Invention is credited to Yuan Gao, Wei Wang.
Application Number | 20090325819 12/217087 |
Document ID | / |
Family ID | 41448181 |
Filed Date | 2009-12-31 |
United States Patent
Application |
20090325819 |
Kind Code |
A1 |
Gao; Yuan ; et al. |
December 31, 2009 |
Density modification in arrays of surface-attached nucleic acid
molecules
Abstract
Substrates having nucleic acid polymers attached at varying
surface densities and methods for creating substrates having
nucleic acid polymers attached at varying surface densities are
provided. Methods according to embodiments of the invention are
adapted to the rapid synthesis of arrays of DNA polymers on a
substrate surface. In embodiments of the invention an array of DNA
molecules on a substrate comprises a plurality of DNA polymers
attached to a trifunctional linker such that at least two DNA
polymers are attached to one trifunctional linker that is attached
to the surface of the substrate. By coupling trifunctional linkers
to trifunctional linkers that are attached to a substrate surface,
the density of DNA polymers on a substrate surface is
increased.
Inventors: |
Gao; Yuan; (Mountain View,
CA) ; Wang; Wei; (San Jose, CA) |
Correspondence
Address: |
INTEL CORPORATION;c/o CPA Global
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Family ID: |
41448181 |
Appl. No.: |
12/217087 |
Filed: |
June 30, 2008 |
Current U.S.
Class: |
506/17 ;
506/32 |
Current CPC
Class: |
C12Q 1/6837 20130101;
C40B 50/18 20130101 |
Class at
Publication: |
506/17 ;
506/32 |
International
Class: |
C40B 40/08 20060101
C40B040/08; C40B 50/18 20060101 C40B050/18 |
Claims
1. An array of DNA molecules on a substrate comprising, a substrate
that has a surface wherein the surface comprises regions and the
regions comprise a plurality of DNA polymers attached to the
surface of the substrate, wherein at least one region comprises a
plurality of DNA polymers that are attached to a trifunctional
linker such that at least two DNA polymers are attached to one
trifunctional linker, wherein the one trifunctional linker has
three sites for attachment, and wherein a first of the two DNA
polymers is attached to one of the three sites, a second of the two
DNA polymers is attached to a second of the three sites, and a
third site is attached to the surface of the substrate.
2. The array of claim 1 wherein the array comprises 1,000 to 10,000
regions.
3. The array of claim 1 wherein the array comprises 100 to 1,000
regions.
4. The array of claim 1 wherein a region of the array is less than
100 .mu.m.sup.2.
5. The array of claim 1 wherein the DNA polymers each comprise from
5 to 50 nucleic acids.
6. The array of claim 1 wherein the DNA polymers each comprise from
10 to 30 nucleic acids.
7. The array of claim 1 wherein the DNA polymers are attached to
the trifunctional linker through a spacer group and the spacer
group comprises a polyethylene glycol.
8. An array of DNA molecules on a substrate, comprising a substrate
that has a surface wherein the surface comprises regions and the
regions comprise a plurality of DNA polymers attached to the
surface of the substrate, wherein at least one region comprises a
plurality of DNA polymers that are attached to a first and a second
trifunctional linker such that at least two DNA polymers are
attached to the first trifunctional linker and at least two DNA
polymers are attached to the second trifunctional linker, and the
first and the second trifunctional linker each have three sites for
attachment and a first DNA polymer is attached to one of the three
sites, a second DNA polymer is attached to a second of the three
sites, and a third site is attached to a third trifunctional
linker, and wherein the third trifunctional linker molecule has
three sites for attachment and one of the three sites for
attachment is attached to the first trifunctional linker, a second
of the three sites for attachment is attached to the second
trifunctional linker, and the third site for attachment is attached
to the surface of the substrate.
9. The array of claim 8 wherein array contains 1,000 to 10,000
regions.
10. The array of claim 8 wherein the array contains 100 to 1,000
regions.
11. The array of claim 8 wherein the DNA polymers each comprise
from 5 to 50 nucleic acids.
12. The array of claim 8 wherein the DNA polymers are attached to
the trifunctional linker through a spacer group and the spacer
group comprises a polyethylene glycol.
13. An array of DNA molecules on a substrate, comprising a
substrate that has a surface and the surface comprises regions,
wherein the regions comprise electrodes and wherein at least one
region comprises a plurality of DNA polymers, wherein the
electrodes are connected to a source capable of providing voltage;
and wherein the DNA polymers are attached to a trifunctional linker
such that at least two DNA polymers are attached to one
trifunctional linker and the one trifunctional linker has three
sites for attachment and wherein a first of the two DNA polymer is
attached to one of the three sites, a second of the two DNA
polymers is attached to a second of the three sites, and a third
site is attached to the surface of the substrate.
14. The array of claim 13 wherein the electrodes comprise
platinum.
15. The array of claim 13 wherein the DNA polymers each comprise
from 5 to 50 nucleic acids.
16. The array of claim 13 wherein a feature size of the array is
less than 100 .mu.m.sup.2.
17. The array of claim 13 wherein the DNA polymers are attached to
the trifunctional linker through a spacer group and the spacer
group comprises a polyethylene glycol.
18. An array of DNA molecules on a substrate, comprising a
substrate that has a surface and the surface comprises regions and
at least one region comprises a plurality of DNA polymers, and
wherein the region that comprises a plurality of DNA polymers
comprises a molecule according to the structure: ##STR00001##
wherein when X and Y are different, X and Y are selected from the
group consisting of CH.sub.2, CHR.sup.1, phenyl, O, S, NH, and
NR.sup.2, and in the case where X and Y are the same X and Y are
selected from the group consisting of CH.sub.2, CHR.sup.2, and
phenyl wherein R.sup.1 is selected from the group consisting of
--OH, --CH.sub.3, --CH.sub.2OH, --CH.sub.2CH.sub.3, and
--CH.sub.2CH.sub.2OH and R.sup.2 is selected from the group
consisting of --CH.sub.3, --CH.sub.2CH.sub.3, and
--CH.sub.2CH.sub.2CH.sub.3, n is an integer from 0 to 10, and A is
a DNA polymer, and wherein the molecule is attached to the surface
of the substrate.
19. The array of claim 18 wherein the DNA polymers each comprise
from 5 to 50 nucleic acids.
20. The array of claim 18 wherein n is an integer from 0 to 5.
21. The array of claim 18 wherein the regions comprise
electrodes.
22. The array of claim 18 wherein the regions comprise platinum
metal.
23. The array of claim 18 wherein A comprises a DNA polymer and a
spacer
24. A method for synthesizing a DNA polymer on a substrate
comprising, a) providing a substrate wherein the substrate has a
surface and the surface has attached hydroxyl groups, b) supplying
a solution to the substrate surface comprising a trifunctional
linker molecule wherein the trifunctional linker molecule has three
sites for attachment, and wherein a first and second site for
attachment are protected by protecting groups, under conditions
that allow the trifunctional linker molecule to attach to the
hydroxyl groups attached to the surface, wherein a third site for
attachment is attached to the surface of the substrate and wherein
a second and third site comprise protecting groups, c) removing the
protecting groups attached to the first and second sites for
attachment, d) supplying a solution to the substrate surface
comprising nucleotides having a 5' protecting group under
conditions that allow a first nucleotide to attach to the first
attachment site of the trifunctional linker and a second nucleotide
to attach to the second attachment site of the trifunctional
linker, e) removing the 5' protecting groups, f) supplying a
solution to the substrate surface comprising nucleotides having a
5' protecting group under conditions that allow a third nucleotide
to attach to the first nucleotide and a fourth nucleotide to attach
to the second nucleotide, and repeating e) and f) a plurality of
times.
25. The method of claim 24 wherein the substrate is an array that
comprises 1,000 to 10,000 regions.
26. The method of claim 24 wherein the substrate is an array that
comprises 100 to 1,000 regions.
27. The method of claim 24 wherein the substrate is an array having
regions and the regions comprise electrodes.
28. The method of claim 24 wherein the substrate is an array having
regions and the regions comprise electrodes and wherein the
protecting groups are removed by an electrogenerated acid.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is related to U.S. patent
application Ser. No. 11/646,602, entitled "Method and Apparatus for
Combined Electrochemical Synthesis and Detection of Analytes,"
filed Dec. 28, 2006, now pending, U.S. patent application Ser. No.
11/646,615, entitled "Method and Apparatus for Match Quality
Analysis of Analyte Binding," filed Dec. 28, 2006, now pending, and
U.S. patent application Ser. No. 11/646,600, entitled "Quality
Control Methods for the Manufacture of Polymer Arrays" filed Dec.
28, 2006, now pending, the disclosures of which are considered part
or and are incorporated by reference in the disclosure of this
application.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The embodiments of the present invention relate to arrays of
polymers on the surface of a substrate, and more particularly to
arrays of polymers having varied surface densities, to arrays of
nucleic acid polymers, and to methods of making arrays of nucleic
acid polymers having varied surface densities.
[0004] 2. Background Information
[0005] Microarrays of nucleic acids, peptides, proteins, and
oligosaccharides continue to gain importance as powerful tools for
research and diagnostic applications in the biomedical sciences.
Nucleic acid microarrays, for example, can be used to monitor gene
expression and genetic mutations in a massively parallel manner.
Proteinaceous microarrays provide the ability, for example, to
characterize the molecular progression of disease, research
cellular pathways, and perform high throughput screening in drug
discovery applications. The ability to collect large volumes of
information is an integral part of biomarker discovery and
personalization of medical treatments. Further, other applications
in bioscience, such as for example, the analysis of the proteomic
content of an organism, disease detection, pathogen detection,
environmental protection, food safety, and biodefense are capable
of benefiting from tools that allow rapid multiplexed interrogation
of analyte samples.
[0006] Genetic information in living organisms is contained in the
form of very long nucleic acid molecules such as deoxyribonucleic
acid (DNA) and ribonucleic acid (RNA). Naturally occurring DNA and
RNA molecules are typically composed of repeating chemical building
blocks called nucleotides which are in turn made up of a sugar
(deoxyribose or ribose, respectively), a phosphate group, and one
of five bases, adenine (A), cytosine (C), guanine (G), and thymine
(T) or uracil (U). The human genome, for example, contains
approximately three billion nucleotides of DNA sequence and an
estimated 20,000 to 25,000 genes. DNA sequence information can be
used to determine multiple characteristics of an individual as well
as the presence of and or susceptibility to many common diseases,
such as cancer, cystic fibrosis, and sickle cell anemia.
[0007] As the genomic and proteomic knowledge base expands, so does
the need for methods to collect, understand, and apply biologically
relevant information. The drive towards personalized medicine
magnifies these needs. Methods, such as analyses using microarrays
that allow the use of small volumes of sample for highly
multiplexed analysis of a plurality of components are valuable
tools. Methods that provide for the controllable automated
manufacture of arrays derive value from these same biomedical
detection and analysis goals.
BRIEF DESCRIPTION OF THE FIGURES
[0008] FIG. 1 schematically diagrams a surface chemistry
methodology that allows for the controllable modification of the
density of polymers attached to the surface of a substrate.
[0009] FIG. 2 diagrams an alternate example in which a spacer
molecule has been included in the example shown in FIG. 1.
[0010] FIG. 3A provides further exemplary trifunctional linker
molecules and FIG. 3B provides exemplary spacer molecules.
[0011] FIG. 4 is a simplified diagram of an exposed electrode array
that can be used to synthesize polymers on the electrodes and to
detect molecular recognition events using the array of
electrodes.
[0012] FIG. 5 provides a schematic of the electronic detection of a
molecular recognition event using an array of exposed
electrodes.
[0013] FIG. 6 shows a circuit model for the electronic detection of
a molecular recognition event on an electrode.
[0014] FIG. 7 provides a graph showing the electronic detection of
a molecular recognition event, such as a DNA hybridization event,
using exposed electrodes.
[0015] FIG. 8 diagrams a method for solid-phase nucleic acid
synthesis that can be used to controllably build nucleic acid
molecules having a desired sequence on a solid surface.
DETAILED DESCRIPTION OF THE INVENTION
[0016] Embodiments of the invention are directed to methods for
modifying the density of polymers attached to the surface of a
substrate and to the resulting substrate having polymers attached
to its surface. Methods according to embodiments of the invention
are useful for making arrays of polymers on a substrate. The
ability to control the density of polymers on the surface of a
substrate allows, for example, for enhancing signal to noise ratio
in situations in which the presence of the polymer and or the
binding of a second molecule to the polymer is to be detected.
Increased density provides increased signal for detection which is
especially useful when surface chemistry is detected
electronically. Further, for example, a substrate having regions in
which polymers are provided at different densities allows for the
ability to perform concentration-based assays.
[0017] DNA hybridization is used in micro-array based techniques
for DNA analysis and detection. Embodiments of the invention enable
production of DNA microarrays in which the surface densities of
immobilized DNA probes can be directly compared and optimized on
the same chip for the capture of different target molecules.
Additionally, embodiments of the invention provide for spacing the
DNA probes above the surface of the substrate to reduce steric
constraints imposed by the surface of the substrate and improve
hybridization efficiencies.
[0018] FIG. 1 provides an exemplary scheme for modifying polymer
density on a substrate surface. In this example, the substrate
surface provides hydroxyl groups (--OH) for molecular attachment.
The substrate may be, for example, a glass, a silicon, a metal, or
other surface (or combination thereof) as described more fully
herein. Optionally, the substrate may be modified in order to
provide an available hydroxyl for molecular attachment, according
to well known techniques, such as silanation, oxidation, or
coating, and as described more fully herein. Other protecting
groups, linkers, trifunctional linkers, and arrangements of
building blocks are possible as described more fully herein. In
FIG. 1, a trifunctional molecular building block having two DMT
(dimethoxytrityl) protected hydroxyl groups and a phosphoramidite
functional group is reacted with a surface attached --OH group of
substrate 10. The trifunctional linking molecule has a functional
end for linking to the substrate and two additional functional ends
for attaching desired polymers to the surface of the substrate. The
resulting substrate has an attached building block capable of
providing two hydroxyl groups (--OH) for further molecular
attachment. The linkage between the phosphoramidite and the
DMT-protected hydroxyls is, for example, an alkyl or aromatic
structure, such as a hydrocarbon chain or ring, a sugar structure,
or a phenyl or diphenyl. The DMT protecting group is removed by
adding an organic acid such as trichloroacetic acid or
dichloroacetic acid in methylenedichloride, an inorganic acid, or
an electrogenerated or photogenerated acid (EGA or PGA,
respectively) to provide an --OH group for further molecular
attachment. In this way, surfaces can be created having an
increased number of hydroxyl groups available for further molecular
attachment.
[0019] In an optional further coupling reaction, the DMT protecting
groups are removed from the molecule (using an acid) in FIG. 1, and
two trifunctional linker molecules are coupled to the deprotected
hydroxyl groups creating a structure having four sites for further
molecular coupling that are protected by DMT protecting groups, as
shown in FIG. 2. By the coupling of multiple linker molecules to a
single surface-attachment site, surfaces can be created having an
increased number of hydroxyl groups available for further molecular
attachment. This methodology allows the creation of surfaces having
a controllable number of attachment sites for functional
polymers.
[0020] In FIG. 2, a molecule capable of providing multiple
attachment sites for desired molecules is attached through one
attachment point to substrate 10. Two additional trifunctional
linker molecules are coupled to the first trifunctional linker
molecule that is attached to the surface of the substrate. In this
example, spacer groups are added to the trifuntional linker
molecule and the spacer group is attached to the trifunctional
linker through an optional phosphate group. For optical detection
and fluorescent assays, the spacer molecule can function to reduce
undesired self-quenching or signal reduction that occurs through
the interaction of closely spaced label molecules (interactions
such as, for example, through energy transfer processes that occur
in the place of fluorescent emissions). Spacing a probe molecule
further from the surface of the substrate to which it is attached
can also function to increase the probe molecule's ability to bind
to target analytes by reducing steric constraints imposed by the
surface and neighboring molecules that are themselves attached to
the surface of the substrate.
[0021] FIG. 3A diagrams a trifunctional linker molecule useful in
embodiments of the present invention for increasing and or
controlling the density of polymers attached to a substrate
surface. In the example of FIG. 3A, R.sup.1 represents a protecting
group, such as, for example a DMT group, or another protecting
group as is know in the art or described herein. The functional
group R.sup.1 can be the same group or a different group for the
two instances in which R.sup.1 appears in the molecule of FIG. 3A.
The R.sup.1 group is removed and a DNA polymer is attached or built
onto the deprotected arm of the trifunctional linker. The
functional groups X and Y can be the same or different and can be a
CH.sub.2, a CHR.sup.2, CR.sup.2R.sup.3, an O, an S, a NH, or a
NR.sup.3. In the case where X (or Y) is an O, a NH, or a S, or a
NR.sup.3, then the functional groups X and Y are not the same, and
then Y (or X) is a CH.sub.2, or a CHR.sup.2. The functional group
R.sup.2 can be --OH, --NH.sub.2, --CH.sub.3, --CH.sub.2OH,
--CH.sub.2CH.sub.3, --CH.sub.2CH.sub.2OH, or other alkane or alkene
having from 3 to 7 carbon atoms and from 0 to 4 oxygen atoms. The
functional group R.sup.3 can be --CH.sub.3, --CH.sub.2CH.sub.3, or
other alkane having from 3 to 5 carbon atoms. The value for "n" in
FIG. 3A is from 0 to 10. In alternate embodiments, the X-Y of FIG.
3A is replaced by a phenyl or diphenyl or a sugar having 5 or 6
atom chain length or ring (a pentose, hexose, or heptose molecule).
The phosphoramidite functional group facilitates coupling of the
phosphate group to a surface-attached hydroxyl group.
[0022] FIG. 3B diagrams an exemplary spacer group that can be used
in embodiments of the invention. In the example of FIG. 4A, R.sup.1
represents a protecting group, such as, for example a DMT group, or
another protecting group as is know in the art or described herein.
The value for "n" in FIG. 3B is from 0 to 10. The phosphoramidite
functional group facilitates coupling of the phosphate group to a
surface-attached hydroxyl group.
[0023] In FIGS. 1, 2, and 3A and 3B, the phosphorous of the
phosphate group is shown as attached to a --OCH.sub.2CH.sub.2CN,
however, other configurations are possible. For example, the
--OCH.sub.2CH.sub.2CN could be replaced by --OCHR.sup.1CHR.sup.2CN
or --OCR.sup.1R.sup.2CR.sup.1R.sup.2CN where R.sup.1 and R.sup.2
are --CH.sub.3, --CH.sub.2CH.sub.3, or other alkane having from 3
to 5 carbon atoms. The --OCH.sub.2CH.sub.2CN group is shown here
because it is typically used in solid phase DNA synthesis
procedures.
[0024] Although exemplary structures are diagramed in FIGS. 1 and
2, other molecular arrangements are possible. For example, the
trifunctional linker molecule of FIG. 1 or 2 can be attached to a
substrate surface through a spacer group as described by FIG. 3B or
as described further herein. The trifunctional linker molecule of
FIG. 1 may also have spacer molecules attached to the arms that are
DNA attachment points such that spacer molecules are located
between the DNA polymer and the trifunctional linker group. The
resulting structure having two DNA molecules attached to one
trifunctional linker that is attached to a substrate surface may
optionally contain zero, one, two, or three spacer molecules.
Similarly a structure having two trifunctional linker molecules
attached to a third trifunctional linker that is attached to a
surface (such as in FIG. 2) may optionally comprise one, two,
three, or seven spacer molecules. In the example of three
trifunctional linkers (such as in FIG. 2), the spacer molecules may
be, for example, between the surface of the substrate and the first
trifunctional linker, between the first trifunctional linker and
the second and third trifunctional linkers, and or between the
second and third trifunctional linkers and the DNA polymers. In one
embodiment, the spacer molecule is located between the second (and
third) trifunctional linker and the DMT protecting group (the site
at which a DNA polymer will be attached) as shown in FIG. 2.
[0025] A spacer molecule typically is a molecule inserted into the
growing polymer, inserted between a DNA molecule and a
trifunctional linker, or inserted between the surface of the
substrate and a trifunctional linker molecule that does not
necessarily convey functionality to the a surface-attached probe or
nucleic acid molecule, such as molecular recognition functionality,
but instead elongates the distance between the substrate surface
and the probe functionality to enhance the exposure (steric
availability) of the probe functionality on the surface of the
substrate. A spacer molecule has a chain length of 2 to 30 atoms
and is comprised of C, H, O, S, N, and or P. The spacer molecules
may be, for example, aryl acetylene, ethylene glycol oligomers
containing 2-10 monomer units (PEGs), diamines, diacids, amino
acids, and polypeptides and polymers of amino acid derivatives such
as for example, amino hexanoic acids, and combinations thereof.
Diamines are molecules of the general formula NH.sub.2RNH.sub.2,
where R is a branched or unbranched hydrocarbon (a molecule
composed of carbon and hydrogen) having from 2 to 25 carbon atoms,
wherein one or more carbon atoms may be replaced by oxygen, sulfur,
silicon, and or nitrogen atoms. Examples of diamines include
ethylene diamine and diamino propane. Diacids are molecules of the
general formula R'OOC--R''--COOR', where R' is a branched or
unbranched hydrocarbon having from 2 to 25 carbon atoms, wherein
one or more carbon atoms may be replaced by oxygen, sulfur,
silicon, and or nitrogen atoms, and R' is H or a hydrocarbon having
from 1 to 10 carbon atoms. Typically, the OR' groups are removed
during the reactions to attach the spacer molecule to the substrate
surface and attach the spacer molecule to the nascent polymer and
the resulting linker molecule has the structure --CO--R''--CO--.
Spacer groups may also comprise phosphates, such as in the example
shown in FIG. 3B. Alternatively, the spacers may be the same
molecule type as that being synthesized (i.e., nascent polymers),
such as polynucleotides and oligonucleotides.
[0026] The molecule diagramed in FIGS. 3A and 3B can be attached to
a substrate surface through standard solid phase chemistry used in
solid-phase DNA synthesis processes. The phosphoramidite group is
coupled to an available hydroxyl group attached to a substrate
surface. For example, molecules having a phosphoramidite group can
be dissolved in a 5-ethylthio-1H-tetrazole solution in acetonitrile
and incubated for 30 minutes at room temperature to couple the
molecule to a substrate surface having available --OH groups for
coupling.
[0027] In general, a trifunctional linker molecule is a molecule
that is a branched or bifurcated molecule having three points for
molecular attachment. Typically the molecule is capable of being
attached to the substrate surface through one point and is capable
of attaching to a polymer through two points of attachment. To the
extent that the trifunctional linker has three functional arms it
might also be described as a Y-shaped molecule or molecular
building block, although unlike the Y, the three functional arms
might be of different lengths and the Y-shape is a simplification
that also does not necessarily capture the relative angles of the
arms with respect to one another.
[0028] In general, a molecular chain length describes the number of
atoms connected to each other along the longest chain of
continuously connected atoms in the molecule.
[0029] In general, molecules shown in FIGS. 1, 2, and 3A and 3B,
can be synthesized by transforming the hydroxyl into a
phosphoramidite group using
2-cyanoethyl-N,N-diisopropylchlorophosphoramidite and
diisopropylethylamine in THF (materials are commercially available,
from for example, Sigma Aldrich, St. Louis, Mo.) at -78.degree. C.
to 25.degree. C. Additionally, synthesis methods for trifunctional
linkers can be found in, for example, "Fluorescence Enhancement
through Enzymatic Cleavage of Internally Quenched Dendritic
Peptides: A Sensitive Assay for the AspN Endoproteinase," Angew.
Chem., 41:3233 (2002) and "Dendrimers and Combinatorial
Chemistry--Tools for Fluorescent Enhancement in Protease Assays,"
Tetrahedron, 60:8721 (2004) and references therein.
[0030] An array is an intentionally-created collection of molecules
housed on a solid support in which the identity or source of a
group of molecules is known based on its location on the array. The
molecules housed on the array and within a feature of an array can
be identical to or different from each other. A macroarray
generally contains feature sizes of about 300 .mu.m or larger and
can be imaged by gel and blot scanners. A micro array generally has
feature sizes of less than 300 .mu.m.
[0031] The features, regions, spots, or sectors of an array may
have any convenient shape, for example, circular, square,
rectangular, elliptical, or wedge-shaped. In some embodiments, the
region in which each distinct molecule is synthesized within a
sector is smaller than about 1 mm.sup.2 or less than 0.5 mm.sup.2.
In further embodiments the regions have an area less than about
10,000 .mu.m.sup.2 or less than 2.5 .mu.m.sup.2. Additionally,
multiple copies of a polymer are located within any region. The
number of copies of a polymer can be in the thousands to the
millions within a region. In general, an array can have any number
of features, and the number of features contained in an array may
be selected to address such considerations as, for example,
experimental objectives, information-gathering objectives, and cost
effectiveness. An array could be, for example, a 20.times.20 matrix
having 400 regions, 64.times.32 matrix having 2,048 regions, or a
640.times.320 array having 204,800 regions. Advantageously, the
present invention is not limited to a particular size or
configuration for the array.
[0032] A solid support, support, or substrate is an object having a
rigid or semi-rigid surface or surfaces. In some aspects at least
one surface of a solid support is planar or substantially planar.
The features of an array optionally form synthesis regions that are
for example, wells, depressions, raised regions, pins, or etched
trenches. In embodiments of the invention the substrate comprises a
silicon wafer or a portion of a silicon wafer. A silicon wafer may
also be referred to as a chip or a semiconductor substrate. A wafer
or chip may be fashioned in various shapes and sizes. The chip
could be overlaid or embedded with circuitry for driving
electrodes, sensing voltages, microprocessors, memory functions,
and input/output capabilities. In embodiments of the invention, the
chip comprises at least surface-accessible electrodes and embedded
circuitry for driving the electrodes and sensing voltages. A
substrate may also be comprised of silicon, glass, nylon, plastic
or other polymeric material, silicon nitride, metals, metal oxides,
metal nitrides, or combinations thereof.
[0033] Many substrate materials, such as metals, metal oxides, and
SiO.sub.2, have surface-attached --OH groups that are available for
further reaction and molecular coupling. Further, surfaces that
present --OH groups for molecular coupling can be created on
substrate surfaces, through, for example, creating a thin oxide
layer on a metal (such as through chemical or plasma etching
processes) and depositing a thin layer of SiO.sub.2 onto the
surface. If the substrate surface is SiO.sub.2, the surface has
been coated with SiO.sub.2, the surface is a metal having available
--OH groups, molecules are optionally attached to the sensor
surface through the use of silane linkers (or organo silane
compounds). Silane linkers are molecules that have at least two
different reactive groups bonded to the silane atom of the
molecule: Y--R--Si--(X).sub.2. One of the reactive groups is
capable of bonding to inorganic materials such as glass (SiO.sub.2)
and metals, the X group. These functional groups that are capable
of bonding to inorganic materials are groups such as methoxy,
ethoxy, chlorine, and silanolic hydroxyl groups. The second
functional group is a group such as a vinyl, an epoxy, a methacryl,
an amino, a mercapto, or a carboxylic acid group that is capable of
forming a chemical bond to an organic material, the Y group. The R
group is typically an organic group comprised of from 1 to 10
carbon atoms, such as a straight chain or branched alkane. For
example, a silanating agent, such as hydroxypropyltriethoxysilane
can be vapor deposited or supplied in a solution to the surface to
be silanated. After reaction, the surface presents a --OH group for
further molecular coupling. Metal surfaces such as nickel,
palladium, platinum, titanium dioxide, aluminum oxide, indium tin
oxide, copper, iridium, aluminum, titanium, tungsten, rhodium or
other surface having available hydroxy groups or other similar
surface groups can also be silanated for further attachment of
molecules. A very thin layer of oxide can be created on a metal
surface, for example, by etching the metal surface with an oxygen
plasma or through damascene processes.
[0034] A probe or probe molecule is a small molecule or biomolecule
capable of undergoing a binding or molecular recognition event with
a target molecule. Molecular recognition is a specific interaction
between molecules. Examples of molecular recognition events are
receptor-ligand, antibody-antigen, sugar-lectin, DNA-protein, and
nucleic acid hybridization reactions. A target or target molecule
refers to a small molecule or biomolecule that is specifically
recognized by a probe molecule through a molecular recognition
event. In the case of nucleic acids, a molecular recognition event
occurs when nucleic acids hybridize to complementary nucleic
acids.
[0035] The length chosen for a nucleic acid to be used as a probe
molecule in an assay (such as a hybridization assay) depends on
several factors, including G/C content of the sequence, positioning
of mismatched bases (if any), degree of uniqueness of the sequence
as compared to the population of target nucleotides, the chemical
nature of the polynucleotide (e.g., methylphosphonate backbone and
phosphorothiolate), desired conditions for hybridization reaction
(e.g., temperature and ionic strength of the solution). Typically a
probe molecule will be at least 5 nucleotides and less than 75
nucleotides in length. Preferably the probe is between 24 and 60
nucleotides in length. Embodiments of the invention provide arrays
of nucleic acids that are suitable for use as probe molecules to,
for example, detect the presence of particular nucleic acid
sequences in samples to be analyzed.
[0036] A hybridization reaction is a process in which two
single-stranded polynucleotides bind and form a stable
double-stranded polynucleotide. In a hybridization event
complementary nucleic acid bases pair up, and an adenine (A) pairs
with a cytosine (C), and a guanine (G) pairs with a thymine (T) or
uracil (U) (through, for example, standard Watson-Crick
hydrogen-bonding interactions). Depending on conditions of pH,
temperature, salt concentration, nucleic acids that are not
absolutely complementary are able to hybridize. In general,
substantially complementary nucleic acids refer to nucleic acids
that have 80% or greater complementary base pairing. Highly
complementary nucleic acids refer to nucleic acids having 90% or
greater complementary base pairing. The proportion of the
population of polynucleotides that forms stable hybrids is referred
to as the degree of hybridization. Hybridization refers to the
formation of double stranded species between a probe polynucleotide
and a target nucleic acid wherein the probe preferentially
hybridizes target nucleic acids that are substantially
complementary to the probe nucleic acid and does not hybridize
nucleic acids that are not substantially complementary.
[0037] Methods for preparing, isolating, and manipulating various
forms of nucleic acids are well known. (See for example, Berger and
Kimmel, eds., Guide to Molecular Cloning Techniques, Academic
Press, New York, N.Y. (1987); Sambrook, Fritsch and Maniatis, eds.,
Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Press, Cold Spring Harbor, N.Y. (1989).) Additionally, techniques
for hybridization reactions are well known and a variety of kits
are commercially available for nucleic acid manipulations.
[0038] In general, nucleic acids include polymers of
deoxyribonucleotides (DNA) or ribonucleotides (RNA) and analogs
thereof that are linked together by a phosphodiester bond. A
polynucleotide can be a segment of a genome, a gene or a portion
thereof, a cDNA, or a synthetic polydeoxyribonucleic acid sequence.
Polynucleotides and nucleic acid polymers refer to polymeric forms
of nucleotides and nucleotide analogs that comprise purine and
pyrimidine bases, or other natural, chemically or biochemically
modified, non-natural, or derivatized nucleotide bases, of any
length. Polynucleotide and nucleic acid also refer to non-natural
analogs of nucleic acids, such as peptide nucleic acids (nucleic
acids with peptide backbones), and polyamide polynucleotides. An
oligonucleotide is a polynucleotide having from 2 to 20 nucleotide
monomer units.
[0039] A polynucleotide, including an oligonucleotide, (for
example, a probe or a primer) can contain nucleoside or nucleotide
analogs, or a backbone bond other than a phosphodiester bond. In
general, the nucleotides comprising a polynucleotide are naturally
occurring deoxyribonucleotides, such as adenine, cytosine, guanine
or thymine linked to 2'-deoxyribose, or ribonucleotides such as
adenine, cytosine, guanine or uracil linked to ribose. However, a
polynucleotide or oligonucleotide also can contain nucleotide
analogs, including methylated nucleotides, non-naturally occurring
synthetic nucleotides or modified naturally occurring
nucleotides.
[0040] The covalent bond linking the nucleotides of a
polynucleotide generally is a phosphodiester bond (the
sugar-phosphate backbone). However, the covalent bond also can be
any of a number of other types of bonds, including a thiodiester
bond, an O-methyl phosphate, a phosphorothioate bond, a
peptide-like amide bond or any other bond known to those in the art
as useful for linking nucleotides to produce synthetic
polynucleotides. The incorporation of non-naturally occurring
nucleotide analogs or bonds linking the nucleotides or analogs can
be particularly useful where the polynucleotide is to be exposed to
an environment that can contain nucleolytic activity, since the
modified polynucleotides can be less susceptible to
degradation.
[0041] Electronic detection is the detection of a molecule through
a measurement of voltage, resistance, and or current
characteristics of an electronic sensor in the presence of the
molecules to be detected. Optionally, the electronic signal
measured in the presence of the molecule to be detected is compared
to an electronic signal measured in the absence of the molecules to
be detected.
[0042] A wafer refers to a semiconductor substrate used in the
fabrication of integrated circuits and other microdevices and is
for example a substrate comprised of a silicon crystal. The wafer
serves as a substrate for a micoelectronic device having a large
number of electronic features that is built through the use of nano
and microfabrication techniques such as deposition of various
materials, such as conductors, semiconductors, and insulators,
photolithographic patterning, etching, and ion implantation.
[0043] An array of electrodes can be equipped with circuitry for
individually addressing the electrodes, driving the electrodes at
selected voltages (or set current values corresponding to the
desired voltage), memory for storing voltage current information to
be supplied to the electrodes, memory and microprocessors for
measuring electrode characteristics, differential amplifiers, field
effect transistors (direct and floating gate). Alternatively, one
or more of these functions can be performed by an attached computer
system.
[0044] Electrode arrays are optionally used both to perform
solid-phase synthesis of nucleic acids on the surface of the
electrode and to detect the presence of single and double stranded
nucleic acids on the surface of the electrode. For an electrode
functionalized with a probe nucleic acid molecule exposed to a
solution containing a target nucleic acid molecule, the presence of
double stranded nucleic acids on the surface of the electrode is
indicative of the occurrence of a hybridization reaction.
Electronic detection provides the ability to monitor synthesis and
hybridization reactions in real time without the use of labels.
Since no wash is required to remove unbound labeled analytes,
binding kinetics can be monitored using dynamic measurements at the
solid-solution interface.
[0045] FIG. 4 provides a simplified diagram showing an array of
exposed sensing and reference electrodes 30 and drive electrodes 32
on a substrate 34. Nucleic acid probes (not shown) are attached to
the sensing and or reference electrodes. The reference electrode
may or may not have a similar or different affinity probes
attached. The sensing electrodes optionally have a plurality of
probe molecules attached and the probe molecules attached to one
sensing electrode are different from the probe molecules attached
to a different sensing electrode. Drive electrodes 32 are typically
larger in surface area than the micron or sub-micron scale sensing
and reference electrodes 30. Electronics associate with driving the
electrodes and signal handling (sensing and referencing capacity)
(not shown) are located in the substrate 34. An integrated charge
value from an electrode is converted to a voltage value through a
two stage amplifier. An internal (not exposed) monolithic NMOS or
metal-insulator-metal capacitor is optionally connected to the
amplifier via an internal switch and used as a reference
capacitor.
[0046] Probe molecules can be attached to the surface of an
electronic sensor according to a variety of methods. Additionally,
the electronic sensing surface may be coated with thin layers of
porous materials or with conducting polymers that facilitate the
attachment of probes onto the surface of the sensor.
[0047] Optionally, an integrating charge amplifier is connected to
an electrode (or the electrodes comprising the array) and
configured to detect capacitance changes at the electrode surface.
A differential amplifier (or a differential-input single-ended
output amplifier) is a device that amplifies the difference between
two input signals (-) and (+). Optionally, the integrating charge
amplifier includes a drive circuit that is capable of providing
voltage pulses which can be supplied, for example, as a square,
sine, or sawtooth wave form to a solution-accessible (exposed)
electrode. The integrating charge amplifier optionally also
includes an input that is from an exposed sensing electrode and
another input from a solution-exposed or unexposed reference
electrode.
[0048] A device including one or more integrating charge amplifiers
is preferably configured to measure the integrated charge and
effective capacitance at the analyte-electrode interface. A change
in integrated charge or effective capacitance can then be used to
ascertain whether a hybridization reaction has occurred (i.e.,
whether analytes have bound at the electrode surface or to the
affinity probe attached to the electrode surface). An array of
integrating amplifiers and a corresponding electrode array are
optionally fabricated on the same substrate. The substrate may also
include synthesis and detection drive circuits, logic for
switching, latches, memory, input/output devices.
[0049] FIG. 5 provides a schematic of an exemplary electronic
device capable of detecting a hybridization event between a
surface-attached DNA probe molecule and a target molecule. The
electronic molecular detection device has a substrate 40 that
houses electronics for detecting 42 (as described more fully
herein), exposed sensing electrodes 44, exposed reference electrode
46, and drive electrode 49. The electrodes 44, 46, and 49 are
connected to electronics through physical electrical connections.
In FIG. 5, dotted lines 50 demonstrate resistive and capacitive
paths (virtual capacitive plates 48 are shown) established in the
conductive matrix of buffer solution 52 and insulating affinity
probe/analyte layer 54 on the electrode 44. The probe/analyte layer
54 is not to scale with respect to the electrode size and only one
probe/analyte complex is shown (for simplicity) where many would
typically be attached to an electrode surface. In FIG. 5, the
electronic detection device can be operated in differential
detection mode, in which both reference electrodes 46 and sensing
electrodes 44 have attached affinity probes 54 and 56. The
electronics 42 comprising a differential charge amplifier provide
differential sensing data to an output amplifier and A/D or analog
output.
[0050] For measurements of effective capacitance, the analyte is
preferably provided in a conductive solution that provides a
conductive path between the driving and the integrating electrodes
of the amplifier. A conductive solution comprises for example, an
aqueous solution having an ionic concentration or a conductive gel.
A preferred method for operating a device including one or more
integrating charge amplifiers includes providing a voltage pulse
through the drive electrode to the conductive matrix. This pulse
can be applied to the matrix with respect to an integrating
electrode and the charge is accumulated on the integrating
electrode over a fixed time.
[0051] The measured capacitance is established by the fixed sensing
electrode, the dielectric formed by the attachment chemistry,
attached probe, and bound analyte (if present), and a virtual
parallel plate formed above the sense electrodes by the charge/ion
distribution in the matrix. The measured capacitance is a function
of the electrode area, the dielectric constant, and the distance of
the virtual plate from the sensing electrode. Analytes binding to
the electrode or the attached affinity probe will change the
dielectric constant and or the distance between the virtual plate
and the sensing electrode, thereby changing the effective
capacitance and accumulated charge on the sensing electrode when a
voltage is applied. The area and distance to the drive electrode
are not material since the conductive matrix carries the voltage to
the virtual plate. FIG. 4 provides a theoretical circuit model for
the electronic detection of a hybridization reaction. In FIG. 6,
ssDNA (single stranded DNA) represents the probe attached to the
electrode and dsDNA (double stranded DNA) represents the probe
hybridized to a target analyte. Any capacitance contributed by the
drive electrode is in series with the measured capacitance and is
small owing to the large electrode area.
[0052] Optionally, to compensate for noise that may be present (low
frequency noise, thermal noise, etc.) a calibrating reference pulse
is applied to an internal test capacitor to normalize the response
of the amplifier during each measurement cycle. The output of this
amplifier can then be digitized and post-processed. Post-processing
comprises a software algorithm to remove random noise, slopes, or
other artifacts from the data. Parameters can be determined
experimentally by characterizing the various contributing
parameters, such as electrode size, drive voltage, and
environmental conditions such as temperature and analyte binding
concentration.
[0053] Optionally, individually addressable sensing electrode
arrays of various effective areas are created to increase the
detection range of the amplifier to various concentrations of
target in the solution. A large array of driving electrodes can be
created to allow close coupling of driving voltage to the solution.
Since the drive electrode capacitance is in series with the sense
electrode plus probe or probe/target complex through the solution,
preferably the driving electrode area is larger than the sensing
electrode area to reduce parasitic effects. A system comprised of a
large capacitor in series with a small capacitor is dominated by
the small capacitor. In the case of a large capacitor in series
with a small capacitor, 1/Cseries=1/Cintegrated+1/Cdrive and this
approaches 1/Cintegrated in value as Cdrive gets large.
[0054] The exposed reference electrode allows for common mode noise
rejection by inputting to one input the amplifier signal
representing the same environmental conditions (pH, temperature,
ion concentration, presence or absence of non-binding analytes,
etc.). Alternatively, the reference capacitor can be exposed to air
or covered to establish an absolute reference.
[0055] FIG. 7 provides graphs the operation of an exemplary device
such as the exemplary devices shown in FIGS. 4 and 5 in the
detection of a molecular recognition event at the surface of an
electrode between a bound nucleic acid probe and a complementary
nucleic acid molecule. FIG. 7 shows the measurement of integrated
charge on two electrodes, the electrode having only the probe
attached (trace labeled C.sub.probe) and the electrode having an
attached probe that is hybridized to a complementary nucleic acid
(trace labeled C.sub.hybridized). An integrating charge amplifier
is used to detect capacitance on the functionalized electrode
surface. A dynamic measurement at the solid-solution interface is
obtained by applying a pulse to the functionalized electrode and
integrating the current flow response over time as the capacitor
discharges.
[0056] A pulse of voltage is applied to the solution with respect
to an integrating electrode and charge is accumulated on the
electrode over a fixed time. A calibrating reference pulse can be
applied to the solution through an internal test capacitor to
normalize the response of the amplifier during each measurement
cycle. A two-stage integrating charge amplifier converts measured
charge to voltage.
[0057] The measurement of change in capacitance at the sensing
electrode can be accomplished in the following manner. The change
can be detected with respect to the exposed reference capacitor
when the reference electrode is exposed to the same solution as the
sensing electrode. Optionally, a nucleic acid molecule that has
similar electrical characteristics as the affinity probe attached
to the sensing electrode but that does not bind to a target analyte
in solution is attached to the reference electrode. A change in
integrated charge is measured as binding occurs on the sensing
electrode (binding to the probe attached on the sensing electrode)
and no change is measured on the reference electrode.
Alternatively, two measurements of the same electrode, before and
after analyte binding, can be compared to determine the change in
integrated charge resulting from target analyte binding. In this
example, a measurement of the electrode at a previous time serves
as the reference. Data is optionally gathered and analyzed using a
computer.
[0058] In an additional alternative, the reference electrode can be
configured to take direct capacitance measurements at the sensing
electrode (non-differential mode). The reference electrode is
covered with a dielectric substance (such as, for example, epoxy)
or a dielectric layer (such as, for example, silicon dioxide) or
left exposed to air. The signal from the electrode is compared to
an open circuit thereby establishing an absolute reference for
measurement.
[0059] The solution-accessible (or exposed) electrodes used in
embodiments of the invention are made from metals, combinations of
metals, or other conducting materials. For example, an electrode
may be made from, for example, platinum, palladium, nickel, copper,
iridium, aluminum, titanium, tungsten, gold, rhodium, as well as
alloys of metals, conducting forms of carbon, such as glassy
carbon, reticulated vitreous carbon, basal plane graphite, edge
plane graphite, graphite, indium tin oxide, conducting polymers,
metal doped conducting polymers, conducting ceramics, and
conducting clays. A functionalized electrode is an electrode having
a probe molecule that has a specific affinity for a target molecule
attached to the electrode surface. An unfunctionalized electrode is
an electrode having no probe molecule attached or an attached
molecule that has no specific chemical affinity for a target
molecule to be analyzed.
[0060] Electrodes are connected to sensing and driving circuitry
according to known methods. For example, CMOS (complementary metal
oxide semiconductor) circuitry could be used, magnetic
radiation-addressable switches, direct connections from an
electrode to a bond pad on the perimeter of a semiconductor chip,
and or combinations thereof. Data is optionally gathered and
analyzed using a computer.
[0061] Electrodes are connected to a source capable of providing
voltage and current. For example, electrodes that form an array are
connected to CMOS (complementary metal oxide semiconductor)
switching circuitry, radio frequency (RF) and microwave addressable
devices, light addressable devices, and or metal lines leading to
the perimeter of the array. In embodiments of the invention, CMOS
switching circuitry involves the connection of each of the
electrodes to a CMOS transistor switch and provides the ability to
individually address electrodes comprising an array. The switch is
accessed by sending an electronic address signal down a common bus
to SRAM (static random access memory) circuitry associated with the
electrode. Radio and microwave frequency addressable switches
involve the switching between on and off states through the use of
a microwave or RF radiation. RF and microwave frequency switches
can be tuned to receive a particular frequency or modulation
frequency and switch without the use of array-based switching
logic. Light addressable switches are moved between on and off
positions by light in the ultraviolet through infra red frequency
ranges. An electromagnetic signal can be spatially localized to
provide switching.
[0062] A region of nucleic acid molecules on an electronic
detection device may be created by a variety of methods. For
example, the nucleic acid molecules may be placed into a solution
and spotted onto the surface of the electronic detection device.
Spotting systems that allow a plurality of solutions to be spotted
onto an array in a controlled manner are commercially available,
form for example, Agilent Technologies, Santa Clara, Calif.
[0063] An array of nucleic acids can also be created using in situ
synthesis methods. For example, the synthesis of an array of
nucleic acid polymers on a substrate can be accomplished using
photochemical synthesis methods, photoresist synthesis methods, and
electrochemical synthesis methods. In photochemical synthesis
techniques, protecting groups that prevent polymer growth are
removed photochemically using light energy (electromagnetic
radiation).
[0064] In photoresist synthesis methods, a photoresist is applied
over the array synthesis area and the photoresist is patterned with
electromagnetic radiation to expose areas in which the polymer
chain is to be extended by monomer addition or to protect regions
in which the polymer chain is not to be extended from monomer
addition. In an exemplary synthesis method, photoresists such as
poly(methyl methacrylate) (PMMA) are provided with sulfonium,
polonioum, or halonium salts that generate an acid upon exposure to
UV light. The photo-chemically generated acid deprotects the
protected polymer chain (for example, through the removal of a DMT
group) to allow the addition of a monomer to the growing end of the
unprotected polymer chain. Optionally, the photoresist layer also
includes a photosensitizer, such as a benzophenone, a
thioxanthenone, an anthraquinone, a fluorenone, an acetophenone, or
perylene. In the case of a photosensitizer, the generation of the
protecting group removal reagent may occur through the absorption
of light by a photosensitizer followed by reaction of the
photosensitizer with the protecting group removal reagent precursor
(the molecule capable of generating an acid upon activation),
energy transfer from the photosensitizer to the cleavage reagent
precursor, or a combination of two or more different mechanisms.
After the photoresist is removed, the polymer chain in the
light-exposed regions is available for monomer addition. Through
repeated cycles of photoresist coating, light exposure (optionally
through a mask to pattern the photoresist), and monomer addition,
an array of polymers is built on the surface of a substrate.
[0065] A monomer addition cycle is a series of chemical reactions
that result in the addition (or covalent attachment) of a monomer
to a growing polymer chain or linker molecule. For example, the
following steps typically comprise a common method used to
synthesize a polynucleotide on a solid support (i.e.,
phosphoramidite-based solid phase polynucleotide synthesis).
Referring now to FIG. 8, a first step in the polynucleotide
synthesis is the deprotection of the surface-attached polymer
growth site through removal of the DMT group from, for example, a
5'-protected nucleotide wherein the 5'-hydroxyl is blocked through
the covalent attachment of DMT. The deprotection is accomplished
using a protic acid (for example, a protic acid such as
trichloroacetic acid, dichloroacetic acid, or an electrochemically
generated acid). The substrate optionally is then washed to remove
the cleaved protecting group and other reagents and mobile reaction
products (with, for example, acetonitrile). A molecule, such as a
phosphoramidite nucleotide, optionally activated with tetrazole, is
then coupled to the surface-attached deprotected molecule.
Optionally unreacted surface-attached deprotected molecules are
capped to prevent further participation in subsequent monomer
addition cycles. The trivalent phosphate trimester linkage is
converted to a pentavalent phosphate triester through oxidation
with, for example, iodine, and the pentavalent phosphate triester
is converted to a phosphodiester through reaction with ammonium
hydroxide.
[0066] A protecting group is a chemical functional group that is
designed to block a reactive site in a molecule, but that may be
removed upon exposure to an activator or a deprotecting reagent.
When the protecting group is removed, the reactive site is more
readily available to react and form chemical bonds. A deprotecting
agent is an agent that can remove a protecting group from a
molecule leaving the reactive site available for further chemical
reaction. Deprotecting reagents include, for example, acids, bases,
free radicals, and electromagnetic radiation. Protecting groups can
be bound to a monomer, a polymer, a linker molecule or a monomer,
or polymer, or a linker molecule attached to a solid support to
protect a reactive functionality on the monomer, polymer, or linker
molecule. Hydroxyl groups on phosphoramidites may be protected by
dimethoxytrityl (DMT), which is acid labile (removable). Exocyclic
amine groups on nucleotides, in particular on phosphoramidites, are
preferably protected by dimethylformamidine on the adenosine and
guanosine bases, and isobutyryl on the cytosine bases, both of
which are base labile protecting groups. This protection strategy
is sometimes known as fast oligonucleotide deprotection (FOD).
[0067] Any unreacted deprotected chemical functional groups may be
capped at any point during a synthesis reaction to avoid or to
prevent further bonding at the selected molecule. In general,
capping reagents are agents that prevent further chain growth at
the site of polymer chain formation such as, for example, an acid
anhydride without further reactive functionalities. Capping groups
cap deprotected functional groups by, for example, binding with the
unreacted amino functions to form amides. Capping agents suitable
for use in an embodiment of the invention include: acetic
anhydride, n-acetylimidizole, isopropenyl formate, fluorescamine,
3-nitrophthalic anhydride and 3-sulfoproponic anhydride.
[0068] Electrochemical reagents are reactive species that can be
generated electrochemically at an electrode through an oxidation or
reduction process. Electrochemical reagents can be generated at an
electrode by supplying a minimum voltage that corresponds with the
oxidation or reduction potential of the desired species in
solution. Reagents that are acids (H.sup.+) and bases (such as
OH.sup.-) can be generated electrochemically. Molecules that can be
used to generate an acid electrochemically (EGAs) that can used to
deprotect a growing nucleic acid polymer attached to an electrode
surface (e.g., remove a DMT group) include, for example
hydroquinone that is converted to benzoquinone upon oxidation
thereby releasing two protons (H.sup.+) and a hydroquinone that is
converted to anthraquinone upon oxidation thereby releasing two
protons (H.sup.+) (a non-aqueous system).
[0069] An electrochemical reagent can be generated at a
solution-accessible electrode by applying sufficient electrical
potential to an electrode. The electrochemical reagent is capable
of removing a protecting group from the growing end of a polymer
being synthesized on the electrode. In hydroquinone/benzoquinone
example, the electrochemical reagent produced (H.sup.+) is a
deprotecting agent. In other reactions, the electrochemical reagent
may be an intermediate in the formation of the deprotecting
agent.
[0070] The electrodes of the array optionally are also used to
synthesize polymers. For synthesis the electrodes are used to
create an acidic or basic region around the electrode surface. The
acid or base causes deprotection of the growing polymer chain and
allows monomer addition. Optionally, confinement electrodes of
opposite polarity or floating separate attachment electrodes are
provided to confine the acid or basic region produced and prevent
drift to surrounding electrodes that may not be activated for
synthesis. Further optionally, a set of two latches are provided at
each electrode capable of being activated for polymer synthesis to
allow the electrode to exist in multiple states: driven by a first
voltage, driven by a second voltage, or floated during the
synthesis cycle.
[0071] Voltage sources for the electrodes can be internally
multiplexed from external source(s) through digital control and can
optionally be applied in parallel to a large array of electrodes.
In operation, voltages are applied to a programmed selection of
electrodes in the presence of an acid-generating reagent (EGA) as a
solution containing a monomer is supplied to the exposed
electrodes. The applied voltage creates an acidic region and allows
polymer growth at the selected electrode. Through selection of
electrodes and choice of monomer to supply, polymers of known
desired sequence are synthesized at the electrodes.
EXAMPLES
[0072] Functionalization of a SiO.sub.2 surface with a
trifunctional linker: A silicon dioxide substrate is washed with
piranha solution at room temp for 1 hour, rinsed with de-ionized
water three times, and air dried. A trifunctional linker molecule,
1,1-diDMT-oxygen-3-phosphoramidite propane is dissolved in
acetonitrile and is then added onto the silicon dioxide surface and
allowed to react for 30 min. at room temperature. DMT is deblocked
with trichloroacetic acid room temperature for 20 min. and the
surface is treated with DMT protected polyethyleneglycol
phosphoramide in acetonitrile for 30 min. at room temperature.
[0073] Synthesis of a trifunctional linker: A sugar molecule having
multiple hydroxyl groups, two of which are protected by a DMT
protecting group, one is transformed into phosphoramidite by using
2-cyanoethyl-N,N-diisopropylchlorophosphoramidite,
diisopropylethylamine, THF, -78 to 25.degree. C. The other
hydroxyls are capped with acetic anhydride. The sugar molecule is a
pentose, hexose, or heptose, such as arabinose, ribose, ribulose,
zylose, xylulose, lyxose, allose, altrose, fructose, galactose,
glucose, gulose, idose, mannose, sorbose, talose, tagotose,
sedoheptulose. Reagents are purchased from Sigma-Aldrich, St.
Louis, Mo.
* * * * *