Note: Descriptions are shown in the official language in which they were submitted.
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OVERLYING ELECTRODE FOR ELECTROCHEMICAL MICROARRAYS
Technical Field of the Invention
The present invention provides an improved electrochemical-based microarray
device
that is able to increase chip density by moving a counter-electrode out of a
grid pattern of
microarrays and into an overlaying position using thin film electrodes.
Specifically, the
overlaying electrode is preferably a thin film. In those embodiments where the
overlaying
electrode remains during detection, the overlaying electrode does not
interfere with label (often
fluorescent) detection nor does the overlaying electrode have non-specific
binding properties.
Background of the Invention
In the world of microarrays, biological molecules (e.g., oligonucleotides,
polypeptides
and the like) are placed onto surfaces at defined locations for potential
binding with target
samples of nucleotides or receptors. Microarrays are miniaturized arrays of
biomolecules on a
variety of platforms. Much of the initial focus for these microarrays have
been in genomics
with an emphasis of single nucleotide polymorphisms (SNPs) and genomic DNA
detectionlvalidation, gene expression, functional genomics and proteomics
(Wilgenbus and
Lichter, J. Mol. Med. 77:761, 1999; Ashfari et al., Cancer Res. 59:4759, 1999;
Kurian et al., J.
Pathol. 187:267, 1999; Hacia, Nature Genetics 21 suppl.:42, 1999; Hacia et
al., Mol.
Psychiatry 3:483, 1998; and Johnson, Curr. Biol. 26:8171, 1998).
There are, in general, three categories of microarrays (also called "biochips"
and "DNA
Arrays" and "Gene Chips" but this descriptive name has been attempted to be a
trademark): (1)
those spotted onto a solid surface (i.e., usually silicon-based and most often
a glass
microscopic slide) with a computer -controlled printing device, (2)
photolithographic
techniques for in situ oligonucleotide synthesis (see, for example, Fodor U.S.
Patent 5,445,934
and the additional patents that claim priority from this priority document),
(3) electrochemical
in situ synthesis based upon pH based removal of blocking chemical functional
groups (see, for
example, Montgomery U.S. Patent 6,093,302 the disclosure of which is
incorporated by
reference herein and Southern U.S. Patent 5,667,667), and (4) electric field
attraction/repulsion
of fully-formed oligonucleotides (see, for example, Hollis et al., U.S. Patent
5,653,939 and its
duplicate Heller U.S. Patent 5,929,208). Only the first three basic techniques
can form
oligonucleotides in situ, that is, building each oligonucleotide, nucleotide-
by-nucleotide, on the
microarray surface without placing or attracting fully-formed
oligonucleotides.
The electrochemistry platform (Montgomery U.S. Patent 6,093,302) provides a
microarray based upon a semiconductor chip platform having a plurality of
microelectrodes.
This chip design uses Complimentary Metal Oxide Semiconductor (CMOS)
technology to
create high-density arrays of microelectrodes with parallel addressing for
selecting and
controlling individual microelectrodes within the array. The electrodes
activated with current
flow generate electrochemical reagents (particularly acidic protons) to alter
the pH in a small,
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defined "virtual flask" region or volume adjacent to the electrode. The
microarray is coated
with a porous matrix as a reaction layer material. Thickness and porosity of
the material is
carefully controlled and biomolecules are synthesized within volumes of the
porous matrix
whose pH has been altered through controlled diffusion of protons generated
electrochemically
and whose diffusion is limited by diffusion coefficients and the buffering
capacities of
solutions. However, in order to function properly, the microarray biochips
using
electrochemistry means for in situ synthesis has to alternate anodes and
cathodes in the array in
order to generated needed protons (acids) at the anodes so that the protons
and other acidic
electrochemically generated acidic reagents will cause an acid pH shift and
remove a blocking
group from a growing oligomer and are spaced such as to provide a more uniform
electric field
to generate electrochemical reagents. Therefore, only about 50% of the cells
or electrode sites
on a chip can be active sites for creation of biomolecules in situ. Therefore,
there is a need in
the art to increase the site densities of microarray biochips by moving
cathodes out of the array
surface. The present invention was made to meet this need and essentially
double chip site
1 S densities.
Electrodes have been made from a variety of conductive materials including
thin
metallic oxides used as transparent conductors. For example, films of tin
oxide doped with
fluorine or of indium oxide doped with tin (i.e., indium tin oxide) have been
obtained by
standard procedures, such as thermal evaporation, sputtering, or hydrolysis of
metallic
chlorides (spraying), or pyrolysis of organometallic compounds (chemical vapor
deposition)
(Manifacier, Thin Solid Films 90:297-308, 1982). Transparent and conductive
layers of some
metallic oxides, such as tin oxide or indium oxide, have been known for more
than 50 years,
have high stability, hardness and adherence to many substrates. They are
deposited by a
variety of techniques. For example, one technique involves cathode sputtering.
Briefly, every
sputtering process involves the creation of a gas plasma (often in argon) in a
low-pressure
chamber between a cathode, target holder, and the anode which is often used as
the substrate
holder. The discharge was set up by applying a voltage between the anode and
the cathode.
The flow of electrons from the cathode towards the anode ionizes the argon in
the vicinity of
the anode and these positive ions, in turn, bombard the target on the cathode.
By momentum
transfer they then eject particles that are deposited onto the substrate.
Chemical vapor
deposition, for example, involves gases introduced into a chamber where they
react. The
oxidizing agents are usually O2, H20 or even H20z. The tin or indium compounds
may be
evaporated at relatively low temperature (about 100 °C) when
organometallic compounds are
used or at higher temperatures when chlorides are used. In a spraying method,
a solution of
SnCl4 or InCl3 in a mixture of alcohol and water is sprayed onto a heated
substrate. However,
despite the ease of spraying methods, in order to vaporize a solution, a
relatively large
separation of the nozzle and substrate is required. This leads to a relatively
low efficiency and
a high consumption of chemical in comparison with other techniques.
CA 02442975 2003-10-03
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In addition, double-stranded DNA and DNA/RNA and RNA/RNA complexes in a
double helical configuration are stable molecules that require aggressive
conditions in vitro in
order to separate complimentary strands of the nucleic acid. Commonly employed
methods
include, for example, heating the sample to at least 60 °C for about 10
min or use of an alkaline
pH of about 11 or higher. Other methods include the use of helicase enzymes,
such as Rep
protein of E. coli that can catalyze the unwinding of the DNA in an unknown
way, or binding
proteins, such as the binding protein of 32-protein of E. coli phage T4, that
can stabilize the
single stranded form of DNA. The most common method for denaturation is heat
or basic pH
to produce a single stranded form of DNA for subsequent amplification cycles
by common
PCR techniques.
PCR techniques generally require a thermocycler that is able to alternatively
heat and
then cool a sample to denature and allow renaturation for nucleic acid samples
around primer
regions of a DNA backbone. In the case of microarray assays having
oligonucleotide capture
probes, DNA samples for analysis require a significant workup that is often
done with a
1 S reagent kit. The DNA samples are first isolated and then denatured, flowed
by amplification,
such that amplified single-stranded samples are applied to microarrays with
oligonucleotide
capture probes as "content" (meaning the compilation of oligonucleotide
sequences on a
particular microarray). Sample preparation is done in solution test tubes or
microtiter plates.
Once completed, the "prepped" sample is applied to the microarray by a manual
transfer with a
pipette. However, there exists a need in the art to better automate and
facilitate this sample
preparation process by adding features to the microarray so that is also
functions as a sample
preparation biochip. The present invention further addresses this need with a
feature that can
both double site density in electrochemically-based microarrays and provide
resistive heat
control to automate denaturation heating and renaturation cooling of a sample
already applied
to the test microarray. This has resulted in a simplification of handling and
reducing the
hardware required for manufacturing microarrays and for synthesizing oligomer
content.
Summary of the Invention
The present invention provides a blank biochip for synthesis of oligomers at
selected
sites comprising:
(a) a semiconductor chip base having a top surface and a bottom surface, and
having a plurality of cells arranged in a pattern, wherein each cell comprises
an electrode and
switching circuitry able to control current or voltage flow to the electrode;
(b) a porous membrane layer overlaying the top surface of the semiconductor
chip;
(c) a spacing layer overlaying the porous membrane layer so that the porous
membrane acts as a first wall of a fluidics channel, wherein the spacing layer
is capable of
acting as the fluidics channel; and
(d) an overlaying electrode comprising a conductive film layered onto a solid
surface, wherein the conductive film acts as a second wall of the fluidics
channel.
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Preferably, the fluidics channel is in communication with an inlet means and
an outlet
means for fluid flow within the spacing layer. Preferably, the overlaying
electrode acts as a
cathode and one or a plurality of electrodes in the semiconductor chip acts as
anodes so that
when current is flowing, the anodes generate protons to affect pH of a
solution in a regional
volume of the porous matrix defined by the geometry of the electrode as a base
and top of the
volume with walls extending between each two-dimensional structure. Most
preferably, the
electrode is in the form of a circle and the volume defines a cylinder. Most
preferably, the
electrode is in the form of a square and the volume defines a cube.
Preferably, the overlaying
electrode extends to an array covering the entire top surface of the cells in
the semiconductor
chip. Preferably, the overlaying electrode is composed of a conductive film
that is transparent
or translucent to allow passage of at least 50% of electromagnetic radiation
in the visible and
surrounding spectra. Most preferably, the overlaying electrode is composed of
a metallic oxide
doped with tin or aluminum. Most preferably, the overlaying electrode is
composed of a
conductive metal or alloy thereof, platinum, or a metallic oxide selected from
the group
consisting of tin-doped indium oxide (ITO), aluminum-doped zinc oxide (AZO),
and
combinations thereof.
The present invention further provides a microarray having a plurality of
different
oligonucleotides synthesized in situ at selected sites comprising:
(a) a semiconductor chip base having a top surface and a bottom surface, and
having a plurality of cells arranged in a pattern, wherein each cell comprises
an electrode and
switching circuitry able to control current flow to the electrode;
(b) a porous membrane layer overlaying the top surface of the semiconductor
chip;
(c) a spacing layer overlaying the porous membrane layer so that the porous
membrane acts as a first wall of a fluidics channel, wherein the spacing layer
is capable of
acting as the fluidics channel; and
(d) an overlaying electrode comprising a conductive film layered onto a solid
surface, wherein the conductive film acts as a second wall of the fluidics
channel.
Preferably, the fluidics channel is in communication with an inlet means and
an outlet
means for fluid flow within the spacing layer. Preferably, the overlaying
electrode acts as a
cathode and one or a plurality of electrodes in the semiconductor chip acts as
anodes so that
when current is flowing, the anodes generate protons to affect pH of a
solution in a regional
volume of the porous matrix defined by the geometry of the electrode as a base
and top of the
volume with walls extending between each two-dimensional structure. Most
preferably, the
electrode is in the form of a circle and the volume defines a cylinder. Most
preferably, the
electrode is in the form of a square and the volume defines a cube.
Preferably, the overlaying
electrode extends to a uniform covering of the entire top surface of the cells
in the
semiconductor chip. Preferably, the overlaying electrode is composed of a
conductive film
that is transparent or translucent to allow passage of at least 50% of
electromagnetic radiation
in the visible and surrounding spectra. Most preferably, the overlaying
electrode is composed
4
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of a metallic oxide doped with tin or aluminum. Most preferably, the
overlaying electrode is
composed of a metallic oxide selected from the group consisting of tin-doped
indium oxide
(ITO), aluminum-doped zinc oxide (AZO), and combinations thereof.
The present invention further provides a process for controlling temperature
of a
microarray having a plurality of different oligonucleotides capture probes at
selected sites for
affecting denaturation of double stranded oligonucleotides and hybridization
conditions,
comprising:
(a) providing a microarray chip having a plurality of different
oligonucleotide
capture probes, wherein the microarray chip comprises:
(i) a semiconductor chip base having a top surface and a bottom surface,
and having a plurality of cells arranged in a pattern, wherein each cell
comprises an electrode
and switching circuitry able to control current flow to the electrode;
(ii) a porous membrane layer overlaying the top surface of the
semiconductor chip;
(iii) a spacing layer overlaying the porous membrane layer so that the porous
membrane acts as a first wall of a fluidics channel, wherein the spacing layer
is capable of
acting as the fluidics channel; and
(iv) an overlaying electrode comprising a conductive film layered onto a
solid surface, wherein the conductive film acts as a second wall of the
fluidics channel and is
wired to a current or voltage source; and
(b) adding a DNA sample for analysis in a liquid to the spacing layer; and
(b) applying a resistive current across the overlaying electrode to increase
the
temperature of the liquid in the spacing layer to control a hybridization
reaction, whereby
increasing the temperature favors denaturation of double stranded nucleic
acids and lower
temperatures favors renaturation or hybridization of nucleic acids.
Preferably, the fluidics channel is in communication with an inlet means and
an outlet
means for fluid flow within the spacing layer. Preferably, the overlaying
electrode acts as a
cathode and one or a plurality of electrodes in the semiconductor chip acts as
anodes so that
when current is flowing, the anodes generate protons to affect pH of a
solution in a regional
volume of the porous matrix defined by the geometry of the electrode as a base
and top of the
volume with walls extending between each two-dimensional structure. Most
preferably, the
electrode is in the form of a circle and the volume defines a cylinder. Most
preferably, the
electrode is in the form of a square and the volume defines a cube.
Preferably, the overlaying
electrode extends to a uniform covering of the entire top surface of the cells
in the
semiconductor chip. Preferably, the overlaying electrode is composed of a
conductive film
that is transparent or translucent to allow passage of at least 50% of
electromagnetic radiation
in the visible and surrounding spectra. Most preferably, the overlaying
electrode is composed
of a metallic oxide doped with tin or aluminum. Most preferably, the
overlaying electrode is
composed of a a conductive metal, platinum, or a metallic oxide selected from
the group
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consisting of tin-doped indium oxide (ITO), aluminum-doped zinc oxide (AZO),
and
combinations thereof. Preferably, the microarray further comprises a
temperature sensor wired
in a feedback look to the overlaying electrode to regulate temperature.
Brief Description of the Drawings
Figure 1 shows a cross-section schematic of an overlaying cathode over a
microarray
device having a plurality of electrodes arranged in a grid pattern.
Figure 2 shows the results of an expression assay using the inventive
microarray
device, as detailed in Example 1. Specifically, average 35 mer oligonucleotide
capture probes
were synthesized on an electrode-containing microarray device having a common
overlaying
cathode according to the present invention. The overlaying cathode was made
from an ITO
film. The sample used was placental cI2NA using a fluorescent label (Cy5).
Figure 3 shows a patterned overlaying electrode scheme to function as a
resistive
heater.
Detailed Description of the Invention
The present invention provides a blank microarray biochip that is able to
double the
number of sites available for in situ synthesis by electrochemical means in
the same area chip
with the same size of unit cells and electrodes in each unit cell. The present
invention is an
improvement over the original microarray designs for in situ electrochemical
synthesis of
oligomers (e.g., oligonucleotides, polypeptides and small molecules) in that
it moves the
needed counter electrode to an overlaying electrode and away from an
alternative electrode-
counter electrode grid pattern. The net result of this step is to effectively
double the number of
active sites available for electrochemical in situ synthesis on a microarray
grid. A preferred
microarray for in situ synthesis of oligomers is described in United States
Patent 6,093,302, the
disclosure of which is incorporated by reference herein. The '302 Patent shows
a microarray
semiconductor chip having a grid of cells, wherein each cell has an electrode
(Figure 16 of the
'302 Patent shows the electrode in a circular shape) and switching circuitry
such that each
electrode is separately addressable and can be a cathode or an anode, wherein
the anode (that
generates protons and other electrochemical reagents) is often called the
electrode and the
cathode the counter electrode because in situ synthesis based upon acid pH
shifts occurs at the
anode. However, basic shifts with appropriate base-cleavable blocking groups
can occur at a
cathode with an anode being the counter electrode. In addition, Southern U.S.
Patent
5,667,667 shows alternating rows or columns of anodes and cathodes with DNA
synthesis
occurring on an overlaying glass slide or "surface." Therefore, Southern shows
a different
geometry whose elements are located in different places.
Semiconductor chips having microarray structures with separately addressable
electrodes have been fabricated. The density per unit area of chip surface or
the number of
cells that can be packed into a unit area is a function of the ever-decreasing
size of the features
CA 02442975 2003-10-03
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that can be laid down. Therefore, the microarrays having separately
addressable multiple
electrodes for in situ synthesis, preferably electrochemical synthesis, have
been made and
continue to become denser as semiconductor technology allows for the
fabrication of smaller
and smaller feature sizes.
S Making An Overlayin~ Electrode
The overlaying electrode element is made of a conductive thin film layered
onto a solid
substrate. Preferably, the solid substrate is glass or another silica-based
material. Preferred
conductive films are platinum, ITO (tin-doped indium oxide), and AZO (aluminum-
doped zinc
oxide). Only ITO and AZO are also transparent. Preferably, the conductive
material was
coated onto the substrate by standard techniques. A pulsed laser deposition
process for ITO
and AZO was described in Kim et al. (SPIE 3797:290, 1999). Briefly, a KrF
excimer laser
(Lambda Physics LPX 305 at 248 nm and pulse duration of 30 ns) was used to
deposit
conductive films onto glass substrates. The laser was operated at a pulse rate
of 10 Hz and the
laser beam quality was improved by passing it through a spatial filter. The
laser beam was
focused through a 50 cm focal length lens onto a rotating target at a
45° angle of incidence.
The energy density of the laser beam at the target surface was maintained at 2
J/cmz. The
target substrate distance was 4.7 cm. The laser beam was rastered across the
surface of the
target with a computer-controlled mirror while the target was rotated. This
process produced
uniform films over a 1.5 cm by 1.5 cm square surface with a thickness
variation of less than
10%.
ITO targets have been prepared from In203 and Sn02 powders. AZO has been
prepared from Zn0 and A1203 powders. The powders can be mixed in a mixer or
shaker and
pressed into pellets and then scintered.
The overlaying cathode is made, for example with ITO as a film, by depositing
by
electron beam evaporation. A glass substrate (SOmm x 75mm x l.lmm) was coated
on one
side by ITO deposited by electron beam evaporation (Thin Film Technology, Inc.
Buelton,
CA). The thin film has a resistance of between 17 and 20 ohms/square. The
thickness of the
film was about 1700.
In order to form an electrode, TiW and Au metal was deposited sequentially on
the
glass to form electrical contacts to the ITO film. The deposition system was
an MRC Model
822 sputter deposition system (Washington Technology Center Microfabrication
Laboratory,
Seattle, WA). The TiW film was deposited for 3 min at 200W of RF power and 8mT
of argon
to an approximate thickness of 300. The Au film was deposited at 200W of RF
power and
8mT of~rgon for 10 minutes (5 sputter 5 min rest/cool down followed by final 5
min
deposition) to an approximate thickness of 3000. The areas to be free of metal
on the glass
slide were masked prior to deposition using "medium tack" blue dicing tape
(Semiconductor
Equipment Corp. Moorpark, CA). After deposition, the tape was removed by
soaking in a
solvent solution of acetone followed by isopropyl alcohol. The glass was then
cut to a size of
5.1 mm by 26.0 mm to fit over a microarray chip.
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In a preferred embodiment a polymeric coating layer is placed over the thin
film
overlaying electrode. The polymeric coating layer functions to prevent non-
specific bonding
of biologic molecules and target samples to the overlaying electrode to create
"noise" during
analysis of binding of target molecules to specific binding sites on the
microarray. Examples
of polymeric materials are hydrophilic polymers. Preferred hydrophilic
polymers are selected
from the group consisting of PEG (polyethylene glycol having a molecular
weight from about
600 daltons to about 6000 daltons), oligoethylene glycol, polyhydroxyethyl
methacrylate,
polyvinylalcohol, phospholipids, and combinations thereof. Preferred
techniques for
depositing a polymeric layer, preferably a hydrophilic polymeric layer,
include techniques such
as radiation grafting, radio frequency deposition, chemical grafting, co-
polymer adsorption and
chemisorption.
A CMOS (Complementary Metal-Oxide Semiconductor) process was used to fabricate
the electronic component of the microarray device. The assembly of the
microarray biochip
starts by having a die diced, attached and wire bonded onto a PGA (Pin Grid
Array) ceramic
package (Corwil, Inc., San Jose, CA). A porous reaction layer is then
deposited and cured onto
the chip. The inventive overlaying electrode is configured to have the film
placed at a spacing
of from about 2500 microns (0.01 inches) to about 25 microns (0.001 inches)
above the surface
of the porous reaction layer or microarray device. Preferably, the spacing is
approximately
0.004 inches. In this implementation, a spacer film is used to establish the
gap or spacing. For
example, a polyimide film of about 100 pm thickness is used to establish the
spacing. The
spacer film is adhered to the microarray using an epoxy paste, however, care
should be taken to
avoid contact of the epoxy resin with the porous reaction layer so that the
porous reaction layer
is not contaminated. The chip is assembled and then baked to cure the epoxy.
The microarray
chip is now considered a "blank chip" ready for oligomer content to be added.
Finally, a flex
circuit is soldered to form an electrical contact to the overlaying electrode.
The inventive microarray design, having content synthesized in situ, further
allows for
an electric field to be placed across the spacing layer during assay
hybridization. The electric
field will better align the oligonucleotide capture probes previously
synthesized due to a
directional electric field. The oligonucleotide alignment provides for better
access of the single
stranded nucleic acid from the assay sample to the oligonucleotide capture
probes on multiple
sites on the microarray within the porous reaction layer.
Example 1
This example provides the results of a gene expression assay with a known
target
mRNA sample using a higher density microarray having an inventive overlaying
electrode
(cathode) structure wherein the cathode is a film of ITO made as described
herein. Briefly, a
microarray chip having an overlaying electrode was manufactured as described
herein for the
overlaying electrode and as described in Montgomery U.S. Patent 6,093,302 (the
disclosure of
which is incorporated by reference herein) for the CMOS microarray having a
plurality of
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microelectrodes. The microarray was synthesized with oligonucleotide content
with all sites
having the same 15-mer oligonucleotide binding probe (S' TACGCCACCAGCTCC 3').
The
3' end of the capture probe was bound to the chip. The chip, with synthesized
capture probes,
was swelled by dipping in a solution of 6X SSPE and washed. A solution of
oligonucleotide (5
S nM) having a fluorescent probe (Texas Red) (and the sequence 3'
ATGCGGTGGTCGAGG)
was placed over the chip and incubated (for hybridization) at 40 °C for
30 min. The chip was
washed with a buffer solution and then allowed to dry. The surfaced was imaged
using proper
wavelengths of light for excitation and emission for the fluorescent dye. The
image (Figure 3
showed that each cell showed hybridization with the target. Therefore, these
data showed that
the inventive overlaying electrode allowed for proper in situ electrochemical
synthesis of
oligonucleotide capture probes on the surface of a CMOS chip allowing all
cells to be used as
test sites and not just SO% of the cells due to the need for counter-
electrodes.
9
