Technical Field
The invention relates to the development of two-and three-dimensional microarrays
for use in detection/sensing applications with high sensitivity and selectivity.
In particular the invention relates generally to the detection of compound(s) or target
anaiytes in a sample, particularly but not exclusively to determining whether a nucleic
acid comprising a specific sequence of bases is present within a sample, quantifying
the number of the specific nucleic acid base sequences within a sample, determining
the presence and/or extent of methylation in the promoter region for a particular
gene, and the effects of various factors, including environment, diet or medications,
on nucleic acid methylation.
Background Art
There is increasing need for fast, accurate and cost effective methods of detecting
and identifying various target anaiytes, including but not limited to, molecules or
proteins including antibodies, across a range of industries. In particular, there is
increased demand for tests that can, if desired, be carried out away from standard
laboratory settings by non technical personnel. While sensor technology is a rapidly
expanding area of technology, a number of problems remain which prevent on-site
testing and accurate detection beyond certain sensitivity limits. These problems
include: the complicated nature of the reading instruments and/or sensing processes,
meaning considerable training and expertise is often required; low sensitivity sensing
technology that requires concentrations of the target analyte before detection is
possible; the large size and correspondingly non-portable nature of the sensing
instrumentation, which need to be contained within a laboratory; in the case of micro
organisms the time required for the growth of detectable concentrations of the target
species; the surface areas on which reactions or binding under study can occur and
the correspondingly low surface area availability; carrying out measurements is often
difficult; and finally the requirement for reference standards against which any
measurements obtained can be referenced.
Nucleic acid (NA) detection and base pair determination, and the determination of the
presence or extent of methylation in the promoter region of a gene could all benefit
from improved sensor technology. NA detection and base pair determination
typically requires the use of Polymerase Chain Reaction (PCR), a process used to
amplify a few copies of nucleic acid by several orders of magnitude using enzymatic
replication. The subsequent methodology used to analyse the sample depends on
the quantity of nucleic acid in the sample and the detail required in the analysis.
These methodologies therefore range in complexity from simple electrophoretic gels,
which give sample to sample comparison, to the more complicated mass
spectroscopic techniques, which give details down to the atomic level.
DNA methylation is the covalent addition of a methyl group to the 5-carbon of
Cytosine in a CpG dinucleotide (the region of DNA where a Cytosine nucleotide is
linked to a Guanine nucleotide through a phosphodiester bond). The covalent
addition reaction is catalysed by DNA methyltransferase. DNA methylation affects
cell function by altering gene expression without changing the DNA sequence. The
alterations can also be heritable during cell division.
Transcription of a gene requires the attachment of RNA polymerase (which carries
out the replication process) to a promoter (or epigenetic) region. The promoter
region of a gene contains specific DNA sequences and response elements.
Promoter regions may contain clusters of CpG dinuc!eotides which may be referred
to as CpG islands or regions. If the Cytosine base(s) in one or more CpG
dinucleotides is/are methylated the promoter region is no longer available, preventing
transcription of the gene. Therefore methylated promoter regions cannot be
accessed, while non-methylated promoter regions can be. DNA methylation has
therefore been found to play an important role in both the development and normal
function of organisms and the development of disease, and is consequently the
subject of intense research.
Much research that correlates DNA with various conditions (e.g. diseases,
phenotypes etc) centers around the role that DNA methylation plays in the promoter
regions of the DNA strand. This is because epigenetic alterations have been shown
to be common in cancer and typically involve hypermethylation and hypomethylation
of DNA. Hypermethylation refers to an increase in the extent of methylation of CpG
regions. This in turn results in heritable transcriptional silencing, and where tumour
suppressor genes are silenced, cancerous cells can result. Tumour suppressor
genes provide the code for anti-proliferation signals and proteins responsible for
suppressing mitosis and cell growth. By comparison, hypomethylation refers to a
decrease in methylation of other regions of the genome. Hypomethylation typically
occurs in repetitive DNA that is normally heavily methylated, resulting in increased
transcription and an elevated mutation rate due to the activation of otherwise
silenced gene expression.
DNA methylation changes in cancer, particularly hypermethylation of CpG regions,
has been found to occur relatively early in the development of cancer. Therefore
DNA methylation could act as an important biomarker for the detection of diseases
such as cancer. Furthermore, because the gene sequence remains unchanged
following methylation, individual genes which have been silenced by methylation
remain intact and can therefore be reactivated by small molecule inhibitors of DNA
methyltransferase's.
Recent research has suggested that the methylation of promoter regions can be
affected by a variety of factors, including diet and environmental factors Mass
spectroscopy is typically employed for determining the existence of methylation and
quantifying changes in DNA methylation, however this involves a number of
preparatory steps (for example, the replication of DNA) and is expensive and time
consuming.
Object of the Invention
It is an object of the invention to provide two-and three-dimensional microarrays for
use in detection/sensing applications with high sensitivity and selectivity. It is a
further or alternate object of the invention to provide a method for determining
whether or not a particular molecule(s) or compound(s) is present within a sample
and/or have been modified in any way. It is a further or alternate object of the
invention to provide a method for determining whether a nucleic acid comprising a
specific sequence of bases is present in a sample. It is a further or alternate object
of the invention to enable the detection of a nucleic acid comprising a specific base
sequence without the need for pre-concentration using PCR. It is a further or
alternate object of the invention to provide a useful method to quantify by counting
the number of nucleic acids comprising a specific nucleic acid base sequence within
a sample. It is a further or alternate object to provide a method of determining the
presence and/or extent of methylation in the promoter region of a gene and/or the
effect of factors, such as environment, diet, or medication, on nucleic acid
methylation. It is a further or alternate object of the invention to at least to provide
the public with a useful choice.
Summary of the Invention
In a first aspect the invention provides a method for determining the presence of a
target compound(s) of interest within a sample, the method including the steps of:
a) Providing a microarray including a plurality of defined functionalized areas,
the defined functionalized areas being defined areas having attached sensory
agent(s) capable of attaching to the target compound(s) of interest within the
sample;
b) Contacting the microarray with at least part of the sample; and
c) Determining the presence of a target compound(s) of interest by detection of
a detectable response to the attachment of the target compound(s) to the
sensory agent(s).
Preferably the plurality of defined functionalized areas on the microarray, are defined
areas on a plurality of surface structures to which the sensory agent(s) capable of
giving a detectable sensor response to the target compound(s) of interest within the
sample are attached.
Preferably a signal entity capable of providing a detectable response is attached to
the target compound to be detected in the sample to form a mixed sample, and the
sensor response given by the sensory agent(s) in the defined functionalized areas is
provided by attachment of the signal entity to the sensory agent(s).
Preferably the signal entity is a chemical, biological, or physical entity which is
capable of providing a detectable signal or response.
Preferably the signal entity is a particle and is selected from a coloured microbead, a
fluorescent microbead, a magnetic microbead, or a light blocking microbead.
Preferably the particle is a polymer microbead including but not limited to Polystyrene
beads, PM A beads and PET beads.
Preferably the detectable sensor response is selected from colour, fluorescence,
magnetic or light blocking.
Preferably the detectable sensor response is capable of being read by digital
counting, weight measurements, fluorescence, optical, and/or electrical means.
Preferably the detectable sensor response results in any one or a combination of
quantitative/qualitative, fluorescence, optical or colour metric measurements.
Preferably the detectable sensor response includes visual responses,
spectrophotometric responses, fluorescent techniques, potentiometric or
galvanostatic responses, magnetic light refraction, heat, frequency and digital
responses.
Preferably the detectable signal response is digital.
Preferably the detectable sensor response results in quantitative or qualitative
measurements.
Preferably the sample is a biological sample, including but not limited to, a tissue
sample, a fluid sample, or an oral swab. In one preferred embodiment, the sample is
a biological sample comprising nucleic acid. In another embodiment, the sample is a
biological sample comprising a micro-organism, a peptide or protein, and/or an
antibody.
In a second aspect the invention provides a method for determining the presence of
a target compound(s) of interest within a sample, the method including the steps of:
a) Providing a microarray including a plurality of surface structures on a base
material, the surface structures having attached sensory agent(s), capable of
attaching to the target compound(s) of interest within the sample, on defined
functionalized areas on the tops of the surface structures;
b) Passing at least part of the sample over the array; and
c) Determining the presence of a target compound(s) of interest by detection of a
detectable response to the attachment of the target compound(s) to the sensory
agent(s).
Preferably a signal entity capable of providing a detectable response is attached to
the target compound to be detected in the sample to form a mixed sample, and the
sensor response given by the sensory agent(s) in the defined functionalized areas is
provided by attachment of the signal entity to the sensory agent(s).
Preferably the compound to which the signal entity is attached is a synthesized
complimentary copy of a single strand of a nucleic acid.
Preferably the signal entity is a chemical, biological, or physical entity which is
capable of providing a detectable signal or response.
Preferably the signal entity is a particle and is selected from a coloured microbead, a
fluorescent microbead, a magnetic microbead, or a light blocking microbead.
Preferably the particle is a polymer microbead including but not limited to Polystyrene
beads, PMMA beads and PET beads.
Preferably the detectable sensor response is selected from colour, fluorescence,
magnetic or light blocking.
Preferably the detectable sensor response is capable of being read by digital
counting, weight measurements, fluorescence, optical, and/or electrical means.
Preferably the detectable sensor response results in any one or a combination of
quantitative/qualitative, fluorescence, optical or colour metric measurements.
Preferably the detectable sensor response includes visual responses,
spectrophotometric responses, fluorescent techniques, potentiometric or
galvanostatic responses, magnetic light refraction, heat, frequency and digital
responses.
Preferably the detectable sensor response is digital.
Preferably the detectable sensor response results in quantitative or qualitative
measurements.
Preferably the surface structures are millimeter to nanometer sized surface structures
which are substantially identical and uniformly separated from each other.
Alternatively, the surface structures are randomly ordered on the surface of the
microarray.
Preferably the surface structures take the form of cones or ridges.
Preferably the defined functionalized area of each surface structure is the tip of the
said cones or ridges.
Preferably the tip of the surface structure is between about 1 n and 1000 micron.
Preferably the tip of the surface structure is between about 1 micron and about 20
micron in diameter.
Preferably the tip of the surface structure is between about 5 micron and about 1
micron in diameter.
Preferably the tip of the surface structure is the same size or smaller than the size of
the signal entity.
Preferably the tips of the surface structures are separated from each other by about 5
nm to about 20 micron spacing.
Preferably the tips of the surface structures are separated from each other by about 1
to about 20 micron spacing.
Preferably there are between about 250,000 and about 1 billion tips per cm2.
Preferably there are about 250,000 tips per cm2 at a 10 pm resolution.
Preferably the microarray includes a base material and is formed from a plastics
material, including PMMA, PET and PS or metals such as aluminium or ceramics or
oxides or silicon or a photoresist.
Preferably the microarray is formed from a polymer substrate.
Preferably a patterned layer is attached to or formed onto the underside of the base
material to disperse light across the surface where light is passed through the
microarray for measurement purposes.
Preferably the three dimensional microarray is formed by etching, lithograph
processes, hot embossing, nano-embossing and injection molding or by the
Continuous Forming Technology processes (as described in WO2007/058548).
Preferably the defined areas are functionalized using NH2 or COOH functional
groups.
Preferably one or more single strands of nucleic acid comprising a promoter region or
one more single strands of nucleic acid comprising a specific base sequence of
interest are attached to the NH2 or COOH functional groups.
Preferably a linker group is attached to the NH2 or COOH functional groups.
Preferably the linker group is a covalent linker group.
Preferably the sensory agent is attached to the linker group.
Preferably the sensory agent is attached directly to the NH2 or COOH functional
groups.
Preferably the linker groups are selected from aliphatic compounds, PEG molecules
and polymers, proteins or DNA chains.
Alternatively the sensory agent is attached directly to the tips of the surface
structures through a linker group.
Preferably the sensory agents are biological recognition groups or binding agents.
Preferably the sensory agent/target compound are biological bindings or recognition
groups selected from antibody/antigen, DNA/DNA, DNA/protein, protein/protein,
protein/receptor, cell/protein and cell/DNA binding partners.
Preferably the microarray includes a plurality of sensory agents, each sensory agent
forming a section of the microarray.
Preferably each defined functionalized area includes a plurality of sensory agents.
Preferably the microarray is coated between the defined functionalized areas with an
inert material.
Preferably, before coating, the microarray is treated with a thiol, protein or PEG
material to ensure coating adhesion.
Preferably the inert material is selected from gold or silver or chromium, a polymer or
an oil.
Preferably the inert material employed for coating purposes is a combination of any
two metals selected from gold, silver or chromium.
Preferably the thickness of the inert coating ranges from sub n to mi to mm
measurements.
Preferably the inert material is applied using evaporation, painting, deposition,
sputtering, plasma treatment, spray coating, dip coating, or spin coating.
Preferably the microarray includes a secondary inert coating.
Preferably the secondary inert coating is a thiol compound.
Preferably the sample is a biological sample, including but not limited to, a tissue
sample, a fluid sample, or an oral swab. In one preferred embodiment, the sample is
a biological sample comprising nucleic acid. In another embodiment, the sample is a
biological sample comprising a micro-organism, a peptide or protein, and/or an
antibody.
In a third aspect the invention provides a three-dimensional microarray for use in
determining the presence of a target compound(s) of interest within a sample, the
microarray including:
a) base material including a plurality of surface structures;
b) the plurality of surface structures including sensory agent(s) capable of
attachment to the target compound(s) of interest within the sample, the sensory
agent(s) being included on defined functionalized areas on the tops of each
surface structure.
Preferably a signal entity capable of providing a detectable response is attached to
the target compound to be detected in the sample to form a mixed sample, and the
sensor response given by the sensory agent(s) in the defined functionalized areas is
provided by attachment of the signal entity to the sensory agent(s).
Preferably the detectable sensor response is selected from colour, fluorescence,
magnetic or light blocking.
Preferably the detectable sensor response is capable of being read by digital
counting, weight measurements, fluorescence, optical, and/or electrical means.
Preferably the detectable sensor response results in any one or a combination of
quantitative/qualitative, fluorescence, optical or colour metric measurements.
Preferably the detectable sensor response is digital.
Preferably the detectable sensor response results in quantitative or qualitative
measurements.
Preferably the surface structures are millimeter to nanometer sized surface structures
which are substantially identical and uniformly separated from each other.
Alternatively the surface structures are randomly ordered on the surface of the
microarray.
Preferably the surface structures take the form of cones or ridges.
Preferably the defined functionalized area of each surface structure is the tip of the
said cones or ridges.
Preferably the defined functionalized area on the surface structure is between about
1 nm and 1000 micron.
Preferably the defined functionalized area on the surface structure is between about
1 micron and about 20 micron in diameter.
Preferably the defined functionalized area on the surface structure is between about
5 micron and about 5 micron in diameter.
Preferably the tip of the surface structure is the same size or smaller than the size of
the signal entity.
Preferably the defined functionalized areas on the surface structures are separated
from each other by about 5 nm to about 20 micron spacing.
Preferably the defined functionalized areas on the surface structures are separated
from each other by about 1 to about 20 micron spacing.
Preferably there are between about 250,000 and about 1 billion defined
functionalized areas per cm2.
Preferably there are about 250,000 defined functionalized areas per cm2 at a 10
resolution.
Preferably the microarray includes a base material and is formed from a plastics
material, metal, ceramics or oxides or silicon or a photoresist.
Preferably the plastics material is selected from PMMA, PET and PS.
Preferably the metal is aluminium.
Preferably the microarray is formed from a polymer substrate.
Preferably the base material has a thickness of between about 500 microns and
about 2 mm.
Preferably a patterned layer is attached to or formed onto the underside of the base
material to disperse light across the surface where light is passed through the
microarray for measurement purposes.
Preferably the surface structures are formed by etching, lithographic processes, hot
embossing, nano-embossing, injection molding or by the Continuous Forming
Technology process as described in WO2007/058548.
Preferably the defined areas of the surface structures are functionalized using NH2 or
COOH functional groups.
Preferably one or more single strands of nucleic acid comprising a promoter region or
one more single strands of nucleic acid comprising a specific base sequence of
interest are attached to the NH2 or COOH functional groups.
Preferably a linker group is attached to the H or COOH functional groups.
Preferably the sensory agent is attached to the linker group.
Preferably the sensory agent is attached directly to the NH2 or COOH functional
groups.
Preferably the linker group is a covalent linker group.
Preferably the linker groups are selected from aliphatic compounds, PEG molecules
and polymers, proteins or DNA chains.
Alternatively the sensory agent is attached directly to the defined functionalized areas
through a linker group.
Preferably the sensory agents are biological recognition groups or binding agents.
Preferably the biological bindings or recognition agents form part of antibody/antigen,
DNA/DNA, DNA/protein, protein/protein, protein/receptor, cell/protein and cell/DNA
binding partners.
Preferably the three-dimensional microarray includes a plurality of sensory agent
groupings, each sensory agent grouping forming a section of the microarray.
Preferably the defined functionalized areas include a plurality of attached sensory
agents.
Preferably the three-dimensional microarray is coated between the defined
functionalized areas with an inert material.
Preferably, before coating, the microarray is treated with a thiol, protein or PEG
material to ensure coating adhesion.
Preferably the inert material is selected from gold or silver or chromium, a polymer or
an oil.
Preferably the inert material is a combination of any of gold, silver or chromium.
Preferably the inert material is applied using evaporation, painting, deposition,
sputtering, plasma treatment, spray coating, dip coating, or spin coating.
Preferably the thickness of the coating ranges from sub n to m h to mm
measurements.
Preferably the microarray includes a secondary inert coating.
Preferably the secondary inert coating is a thiol compound.
In a fourth aspect the invention provides a two-dimensional microarray for use in
determining the presence of a target compound(s) of interest within a sample, the
microarray including:
a) a base material including a plurality of defined functionalized areas;
b) the plurality of defined functionalized areas including sensory agent(s) capable
of attaching to the target compound(s) of interest within the sample.
Preferably a signal entity capable of providing a detectable response is attached to
the target compound to be detected in the sample to form a mixed sample, and the
sensor response given by the sensory agent(s) in the defined functionalized areas is
provided by attachment of the signal entity to the sensory agent(s).
Preferably the detectable sensor response is selected from colour, fluorescence,
magnetic or light blocking.
Preferably the detectable sensor response is capable of being read by digital
counting, weight measurements, fluorescence, optical, and/or electrical means.
Preferably the detectable sensor response results in any one or a combination of
quantitative/qualitative, fluorescence, optical or colour metric measurements.
Preferably the detectable sensor response is digital.
Preferably the detectable sensor response results in quantitative or qualitative
measurements.
Preferably the defined functionalized areas are substantially identical in size and
uniformly separated from each other.
Alternatively the defined functionalized areas are randomly placed on the surface of
the microarray.
Preferably each defined functionalized area is between about 1 nm and 1000 micron
in diameter.
Preferably each defined functionalized area is between about 1 micron and about 20
micron in diameter.
Preferably each defined functionalized area is between about 5 micron and about 5
micron in diameter.
Preferably each defined functionalized area is about 10 micron in diameter.
Preferably each defined functionalized area is about 1 micron in diameter.
Preferably each defined functionalized area is about 500 nm in diameter.
Preferably the tip of the surface structure is the same size or smaller than the size of
the signal entity.
Preferably the defined functionalized areas are separated from each other by about 5
nm to about 20 micron spacing.
Preferably the defined functionalized areas are separated from each other by about 1
to about 20 micron spacing.
Preferably the defined functionalized areas are separated from each other by about 5
to about 15 micron spacing.
Preferably the defined functionalized areas are separated from each other by about a
10 micron spacing.
Preferably defined functionalized areas are separated from each other by about a 1
micron spacing.
Preferably the defined functionalized areas are separated from each other by about 5
to about 1000 nm spacing.
Preferably there are between about 250,000 and about 1 billion defined
functionalized areas per cm2.
Preferably there are about 250,000 defined functionalized areas per cm2 at a 10 m h
resolution. ·
Preferably the base material of the two-dimensional microarray is a planar substrate,
a spherical substrate or a tubular substrate.
Preferably the two-dimensional microarray is formed from a plastics material,
including PMMA, PET or PS, or metals such as aluminium, or ceramics or oxides or
silicon or a photoresist or glass.
Preferably the plastics material is selected from PMMA, PET and PS.
Preferably the metal is aluminium.
Preferably the microarray is formed from a polymer substrate.
Preferably the two-dimensional microarray is between about 500 microns and about
2 mm thick.
Preferably a patterned layer is attached to or formed onto the underside of the base
material to disperse light across the surface where light is passed through the
microarray for measurement purposes.
Preferably the defined functionalized areas are formed by layering an inert material
between surface structures of a three-dimensional microarray to form a flat surface
and then removing the top layer of the inert material by etching techniques to expose
defined areas of the base material of the microarray.
Preferably before layering of the inert material, the microarray is treated with a thiol,
protein or PEG material to ensure adhesion of the inert material.
Preferably the inert material is selected from gold or silver or chromium, a polymer or
an oil.
Preferably the inert material is a combination of any of gold, silver or chromium.
Preferably the inert material is applied using evaporation, painting, deposition,
sputtering, plasma treatment, spray coating, dip coating, or spin coating,
Alternatively the defined functionalized areas are formed by lithographic processes,
printing techniques or masking techniques.
Preferably the defined areas are functionalized using NH2 o COOH functional
groups.
Preferably one or more single strands of nucleic acid comprising a promoter region or
one more single strands of nucleic acid comprising a specific base sequence of
interest are attached to the NH2 or COOH functional groups.
Preferably a linker group is attached to the NH2 or COOH functional groups.
Preferably the sensory agent is attached to the linker group.
Preferably the sensory agent is attached directly to the NH2 or COOH functional
groups.
Preferably the linker group is a covalent linker group.
Preferably the linker groups are selected from aliphatic compounds, PEG molecules
and polymers, proteins or DNA chains.
Alternatively the sensory agent is attached directly to the defined functionalized areas
through a linker group.
Preferably the sensory agents are biological recognition groups or binding agents.
Preferably the biological bindings or recognition agents form part of antibody/antigen,
DNA/DNA, DNA/protein, protein/protein, protein/receptor, celi/protein and cell/DNA
binding partners.
Preferably the two-dimensional microarray includes a plurality of sensory agent
groupings, each sensory agent grouping forming a section of the microarray.
Preferably the defined functionalized areas include a plurality of attached sensory
agents.
Preferably the microarray includes a secondary inert coating between the defined
functionalized areas.
Preferably the secondary inert coating is a thiol compound.
Preferably the thickness of the secondary inert coating ranges from sub nm to mih to
mm measurements.
The microarray according to the third and fourth aspects of the invention wherein
a) the areas on the microarray to be formed into the defined functionalized areas
are first covered in a removable blocking material (e.g. wax) and then the
areas between the areas covered in a removable blocking material are coated
in an inert material;
b) the defined functionalized areas are formed by (i) whole or partial removal of
the inert coating to expose the underlying base material to create defined
areas and (ii) functionalization of the areas with sensory agent(s); and
c) the inert coating is removed by friction, abrasion, heat, cutting, electrical
ablation, microtoming, electropolishing, iron milling, laser or etching
techniques.
In a fifth aspect the invention provides a method for determining whether or not a
nucleic acid comprising a specific sequence of bases is present in a sample, the
method comprising;
a) in a sample of nucleic acid, where the nucleic acid is double stranded,
separating it into single strands;
b) combining the single strands of a nucleic acid with a signal entity conjugate to
form a mixed sample;
c) determining whether the nucleic acid comprising a specific sequence of bases
is present by running the mixed sample across the surface of a functionalized
microarray according to the third or fourth aspects of the invention and
counting the number of bound signal entity conjugates.
In a sixth aspect the invention provides a method for determining the extent of
methylation in the promoter region of a gene, the said method comprising: .
a) in a sample of nucleic acid, where the nucleic acid is double stranded,
separating it into single strands;
b) treating the sample of nucleic acid such that non-methylated Cytosine is
converted to Uracil;
c) combining the single strands of nucleic acid with a signal entity conjugate to
form a mixed sample;
d) determining the presence and/or extent of methylation in the promoter regions
by running the mixed sample across the surface of a functionalized
microarray according to the third or fourth aspects of the invention and
counting the number of bound signal entity conjugates.
Preferably the non-methylated Cytosine bases are converted to Uracil by a chemical
conversion using bisulphite.
The methods according to the fifth and sixth aspects of the invention wherein,
a) preferably the single strands of nucleic acid are functionalized before they are
combined with the signal entity conjugate, preferably they are functionalized
with a terminal carboxyl or amino functional group;
b) preferably the microarray is a two- or three-dimensional microarray according
to the third and fourth aspects of the invention, preferably the defined areas of
the microarray are functionalized by the attachment of one or more single
strands of nucleic acid;
c) preferably the signal entity conjugate is formed by attaching a signal entity to
a complimentary compound which is capable of being bound (directly or
indirectly) to the single strands of nucleic acid attached to the defined
functionalized areas of a microarray;
d) preferably the mixed sample is prepared using a suitable buffer at a suitable
pH, preferably the mixed sample is an aqueous solution;
e) preferably the number of bound signal entity conjugates is ascertained by
visual techniques, spectrophotometric techniques, fluorescent techniques,
potentiometric or galvanostatic techniques, magnetic light refraction, heat,
frequency and digital techniques.
In an seventh aspect the invention provides a method for functionalizing a plurality of
defined areas of a microarray for use in the determination of whether or not a
molecule or compound is present within a sample and/or whether or not the molecule
or compound has been modified in any way, the method comprising attaching the
molecule or compound of interest to the defined areas according to the third or fourth
aspects of the present invention and comprising the following additional steps:
a) forming a signal entit conjugate and attaching the conjugate to molecules or
compounds of interest which are attached to the defined functionalized areas;
b) washing off excess signal entity conjugate(s) which have not bound to the
molecules or compounds of interest attached to the defined functionalized
areas;
c) counting the number of bound signal entity conjugates;
d) releasing the bound signal entity conjugates for use in the fifth and sixth
aspects of the present invention, leaving only the molecules or compounds of
interest attached to the defined functionalized areas.
Preferably the microarray is a two- or three-dimensional microarray according to the
third and fourth aspects of the invention, preferably the defined areas of the
microarray are functionalized by the attachment of a molecule(s) or compound(s) of
interest.
Preferably the signal entity conjugate is formed by attaching a signal entity to a
complimentary compound which is capable of being bound (directly or indirectly) to
the molecule of compound of interest attached to the defined functionalized areas of
a microarray.
Preferably the molecule or compound of interest attached to the defined
functionalized area is a single stranded nucleic acid complimentary to a single
stranded nucleic acid forming a part of the signal entity conjugate.
Preferably the molecule or compound of interest attached to the defined
functionalized area is a peptide, antibody, or microorganism (for example, virus
particles or bacteria).
Preferably the excess signal entity conjugate(s) are removed from the surface of the
microarray by washing with a carrier solution.
Preferably the carrier solution is a buffer.
Preferably the number of bound signal entity conjugates is ascertained by visual
techniques, spectrophotometric techniques, fluorescent techniques, potentiometric or
galvanostatic techniques, magnetic light refraction, heat, frequency and digital
techniques.
Preferably the bound signal entity conjugates are released from the defined
functionalized areas in response to a change in their environment, including and not
limited to a change in the pH of the aqueous solution.
Preferably the release of the signal entity conjugates is reversible.
Preferably this reversibility is achieved by changing the pH of the aqueous solution.
Preferably this reversibility allows the microarrays to be stored and used a number of
times.
Preferably the microarrays are stored in a fridge at about 4°C.
In a preferred embodiment of the seventh aspect, the invention provides a method for
functionalizing a plurality of defined areas of a microarray for use in the determination
of whether or not a nucleic acid comprising a specific sequence of bases is present
within a sample and/or in the determination of the presence and/or extent of
methy!ation within the promoter region of a single strand of nucleic acid, the method
comprising attaching a single strand of a nucleic acid to the defined areas according
to the third or fourth aspects of the present invention and comprising the following
additional steps:
a) forming a signal entity conjugate and attaching the conjugate to a nucleic acid
which is attached to the defined functionalized areas;
b) washing off excess signal entity conjugate(s) which have not bound to the
nucleic acid attached to the defined functionalized areas;
c) counting the number of bound signal entity conjugates;
d) releasing the bound signal entity conjugates for use in the fifth and sixth
aspects of the present invention, leaving only the nucleic acid attached to the
defined functionalized areas
Preferably the single strand of a nucleic acid is attached to the defined areas through
a carboxyl or amino group using standard techniques including and not limited to
DCC coupling.
In an eighth aspect, the method as described in the seventh aspect may comprise
attaching a compound to the defined areas of a microarray and may comprise the
following additional steps:
a) providing a complimentary compound and attaching the complimentary
compound to the compound(s) attached to the defined areas;
b) attaching a signal entity to the complimentary compound to form a signal
entity conjugate on the defined areas;
c) counting the number of bound signal entity conjugates;
d) releasing the signal entity conjugates to leave only the compounds attached
to the defined areas.
Preferably the signal entity conjugates are formed by washing signal entities over the
surface structures of the microarray as an aqueous solution.
Preferably the signal entity employed is as described in the first and second aspects
of the present invention.
In a preferred embodiment of the eighth aspect, the method as described in the
seventh aspect of the invention may comprise attaching a single strand of a nucleic
acid to the defined areas of a microarray and may comprise the following additional
steps:
a) synthesizing a complimentary strand of the nucleic acid and attaching the
complimentary strands to the bound nucleic acid;
b) attaching a signal entity to the complimentary strands to form a signal entity
conjugate on the defined areas of the microarray;
c) counting the number of bound signal entity conjugates;
d) releasing the signal entity conjugates to leave only the single strands of
nucleic acid attached to the surface structures.
Preferably the complimentary strands are synthesized using a nucleic acid
synthesizer and functionalized with a terminal carboxyl or amino group and are
bound to single strands of a nucleic acid through complimentary interactions between
carboxyl and amino groups of the nucleic acid strands involved.
In a ninth aspect the invention provides a method for determining the potential and/or
the real effect of environment, diet, or medication on nucleic acid, the method
including the use of the method according to the sixth aspect of the invention to
determine the extent of methylation in the promoter region of the nucleic acid.
Drawing Description
Figure 1: shows, in diagrammatic form, the preparation of a Three-Dimensional
MicroCone Array Substrate;
Figure 2: shows, in diagrammatic form, stylised depictions of surface structures of
the three-dimensional microarrays;
Figure 3: shows a side view of a diagrammatic expression of the creation of a
two-dimensional microarray from a three-dimensional microarray;
Figure 4: shows a top view of a diagrammatic expression of the creation of a twodimensional
microarray from a three-dimensional microarray;
Figure 5: shows, in diagrammatic form, the preparation of a Digital Biosensing -
Single "Sandwich" Assay;
Figure 6: shows, in diagrammatic form, the preparation of a Digital Biosensing -
Multiple "Sandwich" Assay;
Figure 7: shows, in diagrammatic form, an alternative method for functionalizing
the microarrays as described in detail in one embodiment of the
invention;
Figure 8: shows, in diagrammatic form, how the microarrays are functionalized as
described in detail in one embodiment of the invention;
Figure 9: shows, in diagrammatic form, how the microarrays are employed as
detectors when functionalized as depicted in Figure 5;
Figure 10: shows, in diagrammatic form, how the microarrays are employed as
detectors when functionalized as depicted in Figure 7;
Figure 1 : shows digital images of functional three-dimensional microcone arrays.
Detailed Description
The invention concerns the development of two- and three-dimensional microarrays
for use in detection/sensing applications with high sensitivity and selectivity. In
particular the invention relates generally to the detection of compound(s) in a sample
and also to the determination of whether a nucleic acid comprising a specific
sequence of bases is present within a sample, quantifying the number of the specific
nucleic acid base sequences within a sample, determining the presence and/or
extent of methylation in the promoter region for a particular gene, and the effects of
various factors, including environment, diet or medications on nucleic acid
methylation.
The invention in a general sense provides a method for the determining the presence
of a target compound(s) of interest within a sample, the method including the steps
of:
a) Providing a microarray including a plurality of defined functionalized areas,
the defined functionalized areas being defined areas having attached sensory
agent(s) capable of attaching to the target compound(s) of interest within the
sample;
b) Contacting the microarray with at least part of the sample; and
c) Determining the presence of a target compound(s) of interest by detection of
a detectable response to the attachment of the target compound(s) to the
sensory agent(s).
It is particularly preferred that the plurality of defined functionalized areas on the
microarray, are defined areas on a plurality of surface structures.
Therefore, in a more particular sense, the invention provides a method for
determining the presence of a target compound(s) of interest within a sample, the
method including the steps of:
a) Providing a microarray including a plurality of surface structures on a base
material, the surface structures having attached sensory agent(s), capable of
attaching to the target compound(s) of interest within the sample, on defined
functionalized areas on the tips or tops of each surface structure,; and
b) Passing at least part of the sample over the array; and
c) Determining the presence of a target compound(s) of interest by detection of
a detectable response to the attachment of the target compound(s) to the
sensory agents(s).
The invention also provides:
1) A three-dimensional microarray for use in determining the presence of a target
compound(s) of interest within a sample, the microarray including:
a) a base material including a plurality of surface structures;
b) the plurality of surface structures including sensory agent(s) capable of
attachment to the target compound(s) of interest within the sample, the sensory
agent(s) being included on defined functionalized areas on the tops of each
surface structure.; and
2) A two-dimensional microarray for determining the presence of a target
compound(s) of interest within a sample, the microarray including:
a) a base material including a plurality of defined functionalized areas;
b) the plurality of defined functionalized areas including sensory agent(s) capable
of attaching to the target compound(s) of interest within the sample.
As is clear, the two and three dimensional microarrays according to the present
invention can be used in the methods for determining the presence of a target
compound(s) of interest within a sample, referred to previously.
With reference to Figures 1 and 2, the base material of the microarrays 1 according
to the present invention, consists of a flat base 2 ranging in thickness from about 500
microns to about 2 millimeters. For three-dimensional microarrays defined
functionalized areas take the form of surface structures 3 (e.g. "cones" or "ridges")
which protrude (Figure 1, (B), Figure 2, (A)) from the flat base. For two-dimensional
microarrays the defined functionalized areas are flat sensor sites and therefore do
not protrude from the base. The flat base of the two-dimensional microarray can be
a planar substrate, a spherical substrate or a tubular substrate. The tops of the
surface structures 4 and the flat sensor sites form defined functionalized areas on the
microarrays and include sensory agent(s) capable of attaching to the target
compound(s) of interest within the sample. Attachment of the sensory agent(s) to the
target compound(s) results in, or can be made to result in, a detectable response,
thus those defined areas can be "functionalized" to give sensor responses.
Preferably these defined areas 4 are millimeter to nanometer sized areas which are
substantially identical and uniformly separated from each other. Alternatively, these
defined functionalized areas are millimeter to nanometer sized areas which are
randomly ordered on the surface of the microarray.
In a preferred embodiment, the microarrays are three-dimensional microarrays
wherein uniformly spaced cones or ridges form part of the base material to allow for
more accuracy in the end application. The cones or ridges typically range in size
from about 100 n to about 0 mm in diameter at the base and about 1 n to about
1000 micron in diameter at the tip. Alternative tip diameters are available, as would
be known to a skilled person in the art, and include ranges between about 1 micron
to about 20 micron and about 5 micron to about 5 micron. The tips may also be as
small as about 10 micron, about 1 micron or about 500 nm in diameter. The cones or
ridges are tightly packed in a defined pattern. For example cones with a 1 micron tip
size which are separated by a 1 micron spacing from all other tips will produce an
array of 25 million tips per cm2, while a 500 nm tip separated by a 500 nm spacing
from all others will produce an array of 100 million tips per cm2. The cones or ridges
may be separated from each other by about 5 nm to about 20 micron. Preferably,
the cones or ridges are separated from each other by a spacing of about 1 micron to
about 20 micron. Alternative spacings of about 5 micron to about 15 micron or about
10 micron, or about 1 micron, or about 5 nm to about 1000 nm are also available.
Preferably there is anywhere from about 250,000 functionalized tips at a 10 i
resolution to about 100 million functionalized tips per cm2. The defined functionalized
areas (indicated as 4 in Figures 1 and 2) will be present on the tips of the cones or
ridges, thus the sizes of, and separation of, these areas and the resultant numbers of
functionalized defined areas reflects those separations and numbers. Consequently,
the user knows exactly how many functionalized tips are on the surface of the
microarray. The overall size of the microarrays can be altered according to the end
user's needs. This is a major advantage of the present invention. Typically, the size
area of the microarrays range from as small as about 10 mm2 to about 150 mm2.
Size areas outside this range can be achieved. As seen at Figure 2C, following
functionalization of the defined areas, the presence of a target analyte in the sample
can also be detected by attachment of a microbead conjugate 35 to the target
anaiyte, said microbead conjugate 35 being capable of attaching to the sensory
agent(s) to give a sensor response. This is discussed in more detail below.
In another embodiment the microarrays are two-dimensional microarrays (not shown
in Figures 1 and 2 - best seen in Figure 4) wherein the flat defined functionalized
areas range in size from about nm to about 1000 micro in diameter. Preferably, the
flat defined functionalized areas range in size from about 1 micron to about 20
micron, or about 5 micron to about 15 micron. The flat defined functionalized areas
may also be as small as about 10 micron, about 1 micron or about 500 n in
diameter. The defined functionalized areas are tightly packed in a defined pattern.
For example defined functionalized areas with a 1 micro diameter which are
separated by a 1 micro spacing will produce an array of 25 million defined
functionalized areas per cm2. Ideally the defined functionalized areas are separated
from each other by a spacing of about 1 micron to about 20 micron. Alternative
spacings of about 5 micron to about 15 micron or about 10 micron, or about 1 micron,
or about 5 nm to about 1000 nm are also available. Preferably there is anywhere
from about 250,000 defined functionalized areas at a 10 resolution to about 100
million defined functionalized areas per cm2. As a consequence of the uniform
spacing, the user knows exactly how many defined functionalized areas are on the
surface of the microarray. As with the three-dimensional microarray the size of the
two-dimensional microarray can be altered according to the users end needs.
It is important to note that where the defined functionalized areas are randomly
ordered on the surface of the microarray, exact quantification of the number of
defined functionalized areas on the surface of the microarray is not known until after
the user has digitally scanned the surface of the microarray. Further, it is possible
that not all of the defined areas will be functionalized, even though the intention may
be to do so. Reference to functionalization should therefore be seen as reference to
functionalization of substantially all of the defined areas to reflect reality, unless the
context is clearly otherwise.
The base material of the microarrays can be formed from a plastics material,
including PMMA, PET and PS or metals such as aluminium or ceramics or oxides or
silicon or a photoresist (Figure, 1{A)). Unlike the three-dimensional microarray, the
two-dimensional microarrays can also be made out of materials such as glass.
Preferably the base material of the microarray is made from polymer substrates for
cost and processing advantages. Any process capable of manufacturing surface
structures can be employed to manufacture the base material of the threedimensional
microarrays. For example, etching techniques, lithographs processes,
Continuous Forming Technology, hot embossing, nano-embossing and injection
molding can be employed. A preferred process for forming the base material of the
three-dimensional microarrays is Continuous Forming Technology as described in
WO2007/058548. Where this process is employed the base material can be mass
produced easily and at low cost.
The two-dimensional microarrays can be formed by layering an inert material
between individual structures of a three-dimensional microarray to form a flat surface.
The inert material is preferably gold, silver or chromium. Alternatively, the inert
material may be a polymer or an oil. The inert material is introduced using any one
of a number of different coating methods, including and not limited to, evaporation,
painting, deposition, sputtering, plasma treatment, spray coating, dip coating and
spin coating. Etching techniques can then be employed to remove the top of inert
material to create a flat surface on which defined areas of the base material are
exposed and are therefore available to act as defined functionalized areas. This
method of forming two-dimensional microarrays is particularly suitable where the
height of the structures of a three-dimensional microarray is the same or less than
the thickness of the coating layer (sub n to mhi to mm). Removal of the top of the
coating layer will then result in an essentially flat two-dimensional microarray having
defined areas that can be functionalized as desired. Figures 3 and 4 show a
diagrammatic expression (side view and top view, respectively), of the creation of a
two-dimensional microarray from a three-dimensional microarray. As can be seen in
Figure 3, the three dimensional initial structure 5 has a plurality of surface structures
6 on a base 7. The areas 8 between the surface structures 6 are filled in with a
coating layer 9 as seen in Figure 3B. As can also be seen in Figure 3B, the coating
layer 9 also covers the top of the surface structures 6. To form the defined areas to
be functionalized, the top of the coating layer 9 is removed (Figure 3C) as are the
tops of the surface structures 6. This leaves defined areas 0 on the flat surface that
can be functionalized as desired. The microarray is now two-dimensional. With
reference to Figure 4, the top view of the microarray formation of Figure 3 is shown.
Figure 4B shows how the surface structures 6 are covered with the coating layer 9
(Figure 4B) which is then partially removed to leave defined areas 10 (Figure 4C) that
can be functionalized.
Alternative methods of forming two-dimensional microarrays include, an are not
limited to, lithographic, printing or masking techniques. However, alternative
techniques may be used. Where masking techniques are employed, a masking
element is applied to the flat base in a set pattern and both are then coated with an
inert material. The masking element is then removed to leave behind a substrate
with uncoated areas. These uncoated areas are then available to act as defined
functionalized areas.
The surface structures on the three-dimensional microarray (e.g. cones or ridges) or
the flat defined areas on the two-dimensional microarray forming part of the base
material creates a substrate on which a defined functionalized area can be created.
This area is formed through functionalization of the tops (or tips) of the cones or
ridges, or functionalization of the flat areas.
First, a functional group is attached to the tips or flat sensor sites. Functional groups
such as -NH 2 or -COOH are typically employed. However, many other functional
groups can be used, including the likes of aldehydes and thiols. Preferably a linker
group is then bound to the -NH 2 or -COOH functional groups. Many different linker
groups can be employed including, and not limited to, long or short aliphatic chains,
PEG molecules and polymers, protein chains or DNA chains. Various techniques
can be employed to introduce the linker groups including, and not limited to, plasma
treatment and wet chemistry techniques.
Next a sensory agent is immobilised onto (attached to) the linker group. Alternatively
the sensory agent may be attached to the -NH 2 or -COOH functional group without a
linker, but the use of linkers is a preferred option. Sensory agents are selected from
biological recognition groups, or binding agents and include the likes of
antibody/antigen, DNA/DNA, DNA/protein, protein/protein, protein/receptor,
cell/protein and cell/DNA biological pairings. These are selected according to the
nature of the target compound(s)/molecu!e(s) the user wishes to detect. Thus, the
sensory agent(s) must be capable of acting as a biological recognition group or
binding agent (i.e. attaching) to the target compound(s)/molecules(s) of interest.
In the sequence just described covalent linking is employed between each of the
layers, namely the base material, functional groups, linkers and the sensory agent, to
form the attachments. Covalent linking is preferred, but is not necessary. For
example, and where appropriate, non-covalent linking techniques, such as
electrostatic absorption and charge based absorption, can be employed.
Where steric hindrance poses a problem for the attachment of the sensory agent due
to its size, an additional linker may be inserted between the defined functionalized
area surface and the sensory agent. The sensory agents can also be bound directly
to the cone, ridge tips or flat sites, provided a linker, preferably covalent as stated
above, is incorporated into the tips or flat sensor sites of the base material.
It is the addition of a sensory agent which allows the microarrays to act as digital
sensors with quantitative/qualitative, fluorescence, optical or colour metric
measurements resulting. This is because, when a target analyte recognises the
sensory agent, it binds itself to, or becomes attached to, the sensory agent and thus
becomes attached to the microarray. This attachment of the analyte to the sensory
agent on the microarray results in, or can be made to result in, a sensor response
and aflows the number of attached analytes to be 'counted'. Thus, the user
canaccurately ascertain the proportion of target analytes in their sample.
Quantitative/qualitative measurements are preferred in terms of sensor response, as
these can be accurately interpreted by digital means.
However, many other techniques can be also employed for ascertaining the extent of
target analyte attachment including and not limited to weight measurements,
fluorescence, optical, colour metric and/or electrical (potentiometric or galvanostatic)
techniques. Where digital measurements are preferred, these can be made by
commercial MicroScanners, Microscopes and/or digital imaging, with or without the
need to pass light through the sample. Alternative methods could be employed, as
would be known to a person skilled in the art. Where light is passed through the
sample, a patterned layer attached to or formed onto the underside of the base
material (by etching techniques or the like as discussed earlier) may be required to
diffuse light across the surface. Employment of different wavelengths of light may
allow the user to detect different target analytes within a sample and/or the
occurrence of a reaction and/or information about the bound target analyte, e.g. the
recognition of the cell type or viability.
In one embodiment, the present invention provides a potentially useful screening
method for distinguishing target analytes that may be present in a sample based on
their size and/or shape. For example, target analytes such as bacteria, may exhibit a
particular shape and/or size when bound to the sensory agents on the defined
functionalized areas of a microarray according to the invention. The shape and/or
size can be determined by passing light through the microarray and assessing the
number of defined functionalized areas which are blocked by the target analyte. This
will directly indicate size but indirectly indicate shape as the blocked defined areas
will effectively form a shape. It is likely that the target analyte, such as bacteria, will
usually bind to sensory agents on the defined functionalized areas of the microarray
in the same manner. Therefore, target analytes of the same type could potentiallyexhibit
the same rough shape and/or size when bound to sensory agents on the
defined functionalized areas of the microarrays according to the present invention. It
is in this way that the present invention provides a screening method as the user can
potentially create a key based on different shapes and/or sizes of different target
analytes. It is preferable that the sample employed for such a screening application
is dilute as this will better enable the user to distinguish target analytes based on
their shape and/or size.
Where the target analyte cannot be observed directly signal entities with dimensions
of nanometers to millimeters and specific properties depending on the response
required (for example, colour, fluorescence, magnetic, heat, electrical or light
blocking) may be attached to the remaining sensory agents by simple physical
adsorption techniques or by covalent linkage through different coupling chemistry
techniques. Conversely, signal entities can be attached to the bound target analytes
by similar means.
Preferably, the signal entities employed are the same size or larger than the tip of the
surface structure or the defined flat areas (of a two-dimensional microarray). The
signal entities may be of any shape and may be of such a size that they cover a
number of individual defined areas on the microarray surface. Preferably the signal
entities take the form of particles such as coloured, fluorescence, magnetic or light
blocking microbeads. Where coloured, fluorescent or light blocking particles are
employed, the number of particles bound to the microarray can be counted. Where
different shaped signal entities and/or different sized signal entitles are employed, the
response is visual, i.e. the user is able to detect the presence of different target
analytes based on the differentiation in size and/or shape of the signal entities.
Alternative sensor responses include qualitative or quantitative responses such as
weight measurements, fluorescent responses, spectrophotometric responses,
potentiometric or galvanostatic responses, magnetic light refraction, heat and
frequency responses. The signal entities can also be used to form signal entity
conjugates, which are discussed later herein in more detail.
In another embodiment, the present invention provides a means of determining the
nature of the target analyte present. For example, it is well known that a single type
of bacteria can have a number of strains. The user may functionalize the microarray
with a sensory agent that binds to a number of strains, such that when a
functionalized microarray is exposed to a sample, the user will be able to determine
in general terms if a certain bacteria is present within the sample. The user may then
expose the attached bacteria to particles (for example, signal entities) which have
been functionalized to discriminate between the particular strains. This could be
done by washing the microarray having attached target analytes/bacteria with a
composition including a signal entity that will specifically bind to a certain strain.
Where targeted strains are present, the particles will attach and give a sensor
response.
The choice of sensory agent allows the user to manipulate the defined functionalized
areas to sense one or a number of target analytes. For example, different sensory
agents can be attached to the defined functionalized areas such that different
sections of the microarray hold different sensory agents. Furthermore, the close
packing of the defined functionalized areas, and the correspondingly high number of
defined functionalized areas per cm2 as described above, results in a microarray of
high sensitivity. For example and as per above, where one sensory agent is
attached to each defined functionalized area on the tips of the surface structure of a
three-dimensional microarray, a 1 micron tip separated from all others by a 1 micron
spacing will produce an array of 25 million functionalized tips per cm2. Likewise,
where the tip diameter is 500 n , the array will contain 1 billion defined
functionalized areas (i.e. functionalized tips) per cm2. The high number of defined
functionalized areas enables high throughput screening and/or analysis of target
analytes. Addition of one sensory agent per defined functionalized areas s
preferable as this gives rise to a highly sensitive sensor surface and allows for direct
digital counting of the target compounds/analytes that attache to the agent.
Increased sensitivity is achieved by ensuring the defined functionalized areas are the
same size, or smaller, than the target analyte(s) or the attached particle, as defined
above. Where appropriate, a larger cone, ridge tip or flat sensor site, and hence a
larger defined functionalized area, can be employed, allowing for the addition of
multiple sensory agents to the defined functionalized areas. This results in the
formation of a sensor which is similar to a strip test. For example, if the cone tip size
(i.e. defined functionalized area) of a three-dimensional microarray is 200 micron and
the sensory agent is only 1 micron in size, a microarray comprising millions of
individual and identical strip sensors can be formed. While individual counting of
attached compounds/analytes may no longer be possible, the user remains able to
detect the presence of targets in their sample and may potentially be able to carry out
real time detection experiments. The high sensitivity -of the microarray sensor
surface also potentially allows for single molecule detection, particularly where the
defined functionalized areas can be made smaller than the mih level.
Further increased sensitivity of a three-dimensional microarray is achieved by coating
the base material of the microarray with an inert material to minimize non-specific
binding, allowing for more accurate measurements such as digital counting. This can
best be seen with reference to Figures 1 and 2. Figure B shows the surface
structures 3 and the base 2 in an initial uncoated form. Figure 1C then shows the
surface of the microarray, as a whole, having been coated with a layer 1 of a
suitable inert material. Figure 1D then shows the creation of defined areas 4 for
functionalization that have been created by removal of the coating layer 1 from the
top of the surface structures 3 as well as a part of the surface structures 3
themselves. The side view of Figure 1D shows how this creates a pattern of defined
areas 4. This can also be seen depicted in Figure 2. To achieve this, the inert
material is introduced to the base material surface using any one of a number of
different coating methods, including and not limited to, evaporation, painting,
deposition, sputtering, plasma treatment, spray coating, dip coating and spin coating.
As indicated earlier, where the areas between the individual structures of a
microarray are completely filled with an inert material, a two-dimensional microarray
may be formed.
The inert material itself can be almost anything that results in a non-reactive surface,
including metals such as gold or silver or chromium, or polymers, paints and oils.
The inert material can also consist of a combination of metals used together. The
thickness of the inert coating can range from sub nm to m to mm depending on the
use to which the microarray is to be put. Where adhesion of the inert material is a
problem, the base material can be treated, prior to coating the inert material, with the
likes of thiol-based compounds, proteins or PEG molecules/polymers. In the case of
a gold coating and where non specific binding remains a problem, a secondary
coating of the likes of a thiol can be applied to further reduce the non-specific
binding.
Once the base material is coated, it is preferably treated to remove small specific
areas of coating, allowing for functionalization of defined areas of the microarray (as
described above and represented in Figure 2, (C)) and thus creating its
corresponding sensor capabilities.
Preferably, in the case of a three-dimensional microarray, it is the tip of the cone or
ridge structure (or other like structure) from which any coating is removed (Figure 1
(D), Figure 2, (B)). In the case of a two-dimensional microarray, etching techniques
may be used to form the flat defined areas. Preferably the coating is completely
removed from these areas to expose the underlying base material. Alternatively, the
coating may be only partially removed from these areas such that a thin coating of
the inert material remains. Surprisingly, the inventors have found that the presence
of a very thin coating does not substantially inhibit functiona!ization of the defined
areas.
Various methods can be employed to remove the coating from the tips of the cones
or the flat defined areas (i.e. potential sensor sites). However, the method employed
should preferably be able to remove an accurate size and depth of inert coating from
the tips. For this reason friction, abrasion, heat, cutting, electrical ablation,
microtoming, electropolishing, iron milling, laser and etching techniques are
commonly employed where the microarray is a three-dimensional microarray.
Conversely, masking techniques can also be employed to ensure any inert coating
only ends up on the sides and valleys (and not the tips) of the base material of the
three-dimensional microarray. Using this technique, the cones or ridges could be
masked or protected by dipping the base material into a wax solution upside down or
the die surface used to create the surface structures (which may have an aperture at
the tip) could be used as a mask. The masking compound or die surface is then
removed if it does not have an aperture at the tip to allow for functionalization.
Masking or etching techniques or lithographic or printing techniques can be
employed to remove the coating from the defined areas of a two-dimensional
microarray.
Gold is the preferred inert coating material. However, where this is employed a
secondary thiol coating should preferably be used to further minimise non-specific
binding. Evaporation techniques provide an ideal method of applying the gold
coating to the base material.
Gold-covered three-dimensional microarray sensors can be employed for digital
biosensing of "competition" or "inhibition" assays, particularly where small molecule
compounds (or analytes) such as antibiotics, steroid hormones, drug residues and
the likes of small protein or DNA samples are the desired target compounds.
For example, and with reference to Figure 5, gold-covered three-dimensional
microarrays 20 have been employed in a "competition assay" of antibody/milk
antibiotics or Ab/Ag pairs. After removal of the gold coating 2 1 from the cone tips 22
(Figure 5, (A)) they are functionalized with an a/rt/-antibiotics antibody Y, 25, in a
borate buffer (pH 8.5) which is immobilised onto the surface of the microarray (Figure
5, (B)). The microarray is then left overnight before being exposed to a 2% OVA
solution in PBS to prevent or eliminate non-specific binding that has taken place on
the remaining goid-coated surface. The microarray 20 (Figure 5(B)) is now ready to
use as a sensor.
A solution of milk containing a known milk protein 23 is then passed over the
microarray. This known milk protein is the target analyte which antibody Y, 25,
should bind to. At low concentrations of the protein, few bindings are observed
(Figure 5, (C-1)). Conversely, at high concentrations a number of bindings are
observed (Figure 5, (C-2)).
The microarray of Figure 5, (C-1) and (C-2) is then exposed to coloured
microbead/anti-protein antibody conjugates 24, which bind, or attach, to the surfacebound
milk protein 23 via different epitope (Figure 5, (D-1) and (D-2)). The
microbead 24 acts as a signal entity that provides a detectable signal. Where there
are few analytes 23 bound, attachment of a number of microbead/antibody
conjugates 24 is observed and vice versa. Digital assay of the microarray is then
carried out (Figure 5, (E-1) and (E-2)). This involves counting either the number of
coloured microbead/antibody conjugates or the number of empty cone tips directly.
The concentration of the milk protein is proportional to the number of coloured
microbeads (sandwich assay), but inversely proportional to the number of empty
cone tips. Thus, a high proportion of coloured beads will indicate a high
concentration of target analyte, and vice versa.
Where an "inhibition" assay format is employed, it is the small analyte molecule (such
as a milk antibiotic as the target molecule) which is immobilised onto the surface of
the microarray. Sensor surfaces in inhibition assay formats can be regenerated
many times for multiple measurements, while competition assays are generally used
only once as disposable sensors.
The above methodology can be repeated in standard form, whereby the
functionalized microarray, is exposed to a solution containing a target analyte. These
bind to the sensory agents on recognition and when light is passed through the
microarray, the defined functionalized areas which have sensory agents bound to
target analytes will effectively be blocked by the target analyte allowing for digital
counting of the "darkened" areas.
Gold-covered three-dimensional microarray sensors can also be employed in
multiple "sandwich" assay digital biosensing formats where the user wishes to detect
large target compounds or analytes such as proteins, virus cells, and cells. Such
analytes typically have multiple attachment sites (termed epidopes) for sensory
agents.
For example, and with reference to Figure 6, three different antibodies 25, 26, 27 (in
areas X, Y and Z) are either immobilised in a set position onto the gold-free and
functionalized cone tip 22 surfaces of the microarray 20 (Figure 6, (B-1)), or are
immobilised randomly onto the cone tip 22 surfaces (Figure 6, (B-2)).
The microarrays 20 are then exposed to 2 % OVA in PBS to block any non-specific
binding on the gold surface 21. Three different large analytes 28, 29, 30 are then
exposed to the sensor surface. These bind to their respective antibodies 25, 26, 27
(Figure 6, (C-1) and (C-2)).
Next, three differently coloured microbead/antibody conjugates 31, 32, 33 bind to the
three different large analytes 28, 29, 30 separately (sandwich assay) to form
multicoloured arrays on the surface of the sensor (Figure 6, (D-1) and (D-2)).
The sample concentrations of the three analytes 28, 29, 30 can then be ascertained
by counting each of the three different coloured microbeads 31, 32, 33 separately or
by carrying out digital, weight, fluorescence or electronic measurements.
Concentrations of the analytes 28, 29, 30 are directly proportional to the number of
coloured microbeads 31, 32, 33 on the sensor surface (Figure 6, (E-1) and (E-2)),
such that a sandwich assay format is generally more sensitive than a competition or
inhibition assay format. Such multiple analyses format can also be applied to
"competition" or "inhibition" assays for digital biosensing small molecular analytes.
In a preferred embodiment of the present invention two- and three-dimensional
microarrays can be employed for determining whether or not molecules or
compounds of interest are present in a sample and/or have been modified in any
way. The molecules or compounds may be of any biological or chemical nature and
include, but are not limited to, nucleic acids, peptides or proteins, micro-organisms
(including but not limited to prions, viruses, bacteria), antibodies or any type of small
chemical molecule, for example. Examples of the types of modification for which the
invention could be used to detect include, but are not limited to, structural changes,
substitutions, post transcriptional and post translational modifications (including
glycosy!ation), and methylation of nucleic acids.
The invention may be of use for a number of applications, including diagnostic and
forensic applications, for example to identify the presence or absence of a specific
nucleic acid sequence (including mutations in a nucleic acid sequence which may
signal disease), or a methylation pattern that may be associated with disease.
The description hereinafter focuses on the analysis of a target compound being
nucleic acid molecules. However, i should be appreciated that the general
methodology described is applicable to other molecules or other target compounds
as mentioned above. Therefore, a skilled person will recognise that the invention has
many uses.
To determine whether or not a molecule or compound is present within a sample
and/or whether the molecule or compound has been modified in any way, the sample
of interest is first combined with a signal entity conjugate (the formation of which is
discussed in more depth below) to form a mixed sample. The mixed sample is then
run across the surface of a two- or three-dimensional microarray and the number of
bound signal entity conjugates are counted. Preferably, the mixed sample is an
aqueous solution and is prepared using a suitable buffer, for example PBS, at a
suitable pH. Preferably the pH is between about 4.0 to about 9.0, more preferably
between about 7.0 to about 7.5. Preferably, the signal entity conjugates only bind to
the molecule or compound of interest within the mixed sample to form signal entity
conjugate complexes.
Therefore the present invention provides a method of determining whether or not one
or more nucleic acids (for example, a DNA or RNA strand), comprising a specific
sequence of bases is present in a sample. Likewise the invention provides a method
for determining the presence and/or extent of methylation of a nucleic acid,
preferably methylation within or around the promoter of a gene (herein referred to as
the promoter region). A gene comprising a methylated promoter region is
unavailable for transcription purposes.
The method for determining whether or not one or more nucleic acids comprising a
specific sequence of bases is present in a sample the following preparatory steps
must be carried out. First, a sample of nucleic acid is collected. The nucleic acid
may be obtained from any appropriate biological material, including but not restricted
to a tissue sample, fluid sample or an oral swab. Where the nucleic acid is double
stranded it is separated into single strands using known techniques. Second, the
single strands are combined with a signal entity conjugate (the formation of which is
described below) to form an aqueous mixed sample. The mixed sample is run
across the surface of a single (if the assay is for a single base sequence) or multi- (if
the assay is for more than one base sequence) functionalized two- or threedimensional
microarray described above.
The method for determining the presence and/or extent of methylation of a nucleic
acid also forms an aspect of the present invention and comprises the following
preparatory steps. First, a sample of nucleic acid is collected from an appropriate
biological material as described above for the first preferred embodiment of the first
aspect of the invention. Where the nucleic acid is double stranded it is separated
into single strands using known techniques. Second, the promoter regions within the
single strands of nucleic acid are treated such that all non-methylated Cytosine
bases present are converted to Uracil. Preferably the conversion of non-methylated
Cytosine to Uracil is achieved by a chemical conversion whereby the non-methylated
Cytosine bases are sulfonated and deaminated upon reaction with bisulphite (Nucleic
Acids Research, (1996) Vol 24, No. 24, pp 5064-5066). It is known that bisulphite
will only react with non-methylated Cytosine bases. Therefore any methylated
Cytosine bases present within the promoter region remain unchanged. Third, the
treated single strands of nucleic acid are combined with a signal entity conjugate (the
formation of which is described below) to form an aqueous mixed sample. Following
completion of these steps, determination of whether the Cytosine bases have been
converted and have therefore not been methylated is carried out using a single (if the
assay is for a single promoter type) or multi- (if the assay is for more than one
promoter type) functionalized two- or three-dimensional microarray described above.
With reference to Figures 7 to 10 the following key can be used:
Promoter region or base sequence of interest (P1):
Signal entity:
O
Signal entity conjugate:
Signal entity conjugate/nucleic acid complex:
Nucleic acid containing P1:
For the purpose of the present invention as it relates to the detection of nucleic acids
(and with reference to Figure 7), the defined areas of two- and three-dimensional
microarrays, respectively, can be functionalized according to the following
methodology (preferably the remainder of the surface of the microarray, other than
the defined areas, will have been coated with an inert material):
a) one or more single strands of a nucleic acid comprising a promoter region or
specific base sequence of interest is bound to the defined areas (Figure 7,
(B));
b) a signal entity conjugate is formed and then washed over the defined areas of
the microarray allowing for attachment of the conjugate to the bound nucleic
acid (Figure 7, (C));
c) excess signal entity conjugates which have not bound to the nucleic acid are
washed off allowing the user to ascertain or count the number of signal entity
conjugates bound to the defined areas of the microarray; and
d) the bound signal entity conjugates are released leaving only the nucleic
acid(s) of step (a) bound to the defined areas of the microarray (Figure 7, (D);
e) The areas are now defined functionalized areas including a known amount
and type of sensory agent (the nucleic acid).
A plurality of nucleic acids comprising promoter regions or other base sequences of
interest can be bound to the defined areas through carboxyl or amino groups using
standard techniques. Dicyclohexylcarbodiimide (DCC) coupling provides an example
of a suitable technique for this purpose. Other coupling techniques may be used,
including the use of linker molecules, as described earlier. Likewise, each defined
area may be functionalized by only one nucleic acid comprising a promoter region or
other nucleic acid sequence of interest.
Broad methodology for functionalizing the microarray and forming a signal entity
conjugate where molecules or compounds other than nucleic acid are of interest are
broadly described above in relation to the detection of target analytes. Skilled
persons will readily appreciate appropriate compounds and conditions for attaching
such compounds or molecules to the flat sensor sites, surface structures and signal
entities, having regard to the nature of the compounds of interest.
The signal entity conjugate is preferably formed by attaching a signal entity to a
synthesized complimentary copy of the nucleic acid comprising a promoter region or
other base sequence of interest. The complimentary copies of nucleic acids are
preferably synthesized using a nucleic acid synthesizer, and are functionalized with a
terminal functional group to which the signal entity attaches. Alternatively, the
complimentary copies are obtained using recombinant techniques. Examples of
suitable terminal functional groups include carboxyl or amino groups.
Dicyclohexylcarbodiimide (DCC) coupling provides an example of a suitable standard
technique for coupling the complimentary copy to the signal entity.
The signal entity employed may be any chemical, biological or physical entity or
particle which is capable of providing a detectable signal or response. Examples of
suitable chemical entities include, but are not limited to, small chemical molecules,
fluorophores, chemiluminescent tags, or polymers. Polymeric forming chemical
reactions or a series of reactions to form a known entity that can be attached and will
provide a signal entity can also be employed. Examples of suitable biological entities
include, but are not limited to, bacteria, viruses, or the stacking of biological material
(for example, the stacking of multiple strands of DNA, or chlorophyll). Examples of
suitable physical entities include, but are not limited to, the likes of particles or
microbeads. In a preferred embodiment, the signal entity is a physical entity such as
coloured microbeads, fluorescent microbeads, magnetic microbeads or light-blocking
microbeads. Microbeads formed from polymer materials are preferably employed as
they can be readily obtained in a variety of forms, (including coloured, magnetic, and
fluorescent), and functionalities (for example, carboxyfated or aminated). Examples
of suitable polymer microbeads include Polystyrene beads, PMMA beads and PET
beads.
Preferably the particles or beads have a dimension of nanometers to millimeters such
that the defined areas of the microarray are the same size or smaller than the signal
entity. This will allow for a preferred 1:1 ratio between the defined functionalized
areas and the particles, irrespective of the number of nucleic acids comprising
promoter regions or base sequences bound to the defined functionalized areas, and
this will in turn aid in the precise quantification of the number of bound signal entity
conjugates used in later sensing applications. The precise quantification also allows
the user to ascertain whether there are any non-activated defined areas.
Alternatively, the particles or beads may be significantly larger in size than the
defined areas of the microarray and may be of any shape.
The number of bound signal entity conjugates is preferably ascertained using the
techniques described above in relation to the detecting of target analytes. These
techniques include but are not limited to visual (optical) techniques, fluorescent
techniques, electrical techniques, magnetic light refraction techniques, heat and
frequency responses and digital techniques. Examples of other suitable techniques
include, but are not limited to spectrophotometry techniques. The signal entity
employed should therefore preferably have specific properties, dependant on the
technique employed. For the purposes of this embodiment of the invention and as
indicated above, the preferred technique and response is digital, allowing the user to
count the number of particles bound.
The signal entity conjugates are preferably washed over the defined functionalized
areas of a microarray as an aqueous solution and preferably attach to bound nucleic
acids comprising a promoter region or base sequence through a carboxyl or amino
group using standard techniques to form a non-covalent double helix. Excess signal
entity conjugates are then removed from the surface sites by washing with the carrier
solution (typically a buffer) before quantification of the number of bound signal entity
conjugates takes place. Detection and quantification of the bound signal entity
conjugates can occur by any appropriate technique having regard to the nature of the
signal entity to be used. By way of example, if the microarray is mono-functionalized
then counting would preferably be achieved by employing light-blocking microbeads
such that when light is passed through the microarray the number of bound
microbeads can be ascertained by counting the number of defined functionalized
areas appear either illuminated or blocked. If the sensor is multi-functionalized then
counting would preferably be achieved by employing coloured microbeads, allowing
for determination of the numbers of each different colour present on the microarray.
The bound signal entity conjugates are then released by a change in the pH of the
aqueous solution, and are subsequently employed in the analysis of nucleic acid
molecules as indicated above and described below.
An alternative method provided by the present invention for functionalizing the
defined areas of a two- or three-dimensional microarray, with reference to Figure 8,
comprises the following steps:
a) one or more single strands of a nucleic acid comprising a target promoter
region or other base sequence of interest is bound to the flat sensor sites or
surface structures, i.e. the defined areas of a microarray, through a carboxyl
or amino group using standard techniques, including but not limited to DCC
coupling (Figure 8, (A));
b) a complimentary strand of the nucleic acid comprising a promoter region or
other base sequence of interest (herein defined as the complimentary strand)
is synthesized and attached to the bound nucleic acid through complimentary
interactions between the carboxyl or amino groups of the nucleic acid strands
involved (Figure 8, (B));
c) a signal entity is attached to the complimentary strands to form a signal entity
conjugate (Figure 8, (C));
d) the number of bound signal entity conjugates are counted; and
e) the signal entity conjugates are released for use in analysis of nucleic acid
molecules, leaving only the nucleic acid(s) bound to the flat sensor sites or
surface structures of the microarray (Figure 8, (D)).
As with the previously described method of functionalization, the complimentary
strand may be synthesized using a nucleic acid synthesizer (or produced via
recombinant techniques, for example) and functionalized with a terminal carboxyl or
amino functional group. Likewise, the preferred signal entity is as described above.
Preferably, the signal entity conjugate is formed by washing the signal entity over the
surface structures of the microarray as an aqueous solution. The signal entities then
bind to the functionalized complimentary strands through a carboxyl or amino group,
using standard techniques, which include but are not limited to DCC coupling. As for
the previously described method of functionalization, the number of bound signal
entity conjugates is counted.
Again, functionalizing the microarray and forming a signal entity conjugate in
accordance with this alternative method of the present invention where molecules or
compounds other than nucleic acid are of interest are broadly described above in
relation to the detection of target analytes and skilled persons will readily appreciate
appropriate compounds and conditions to be used.
It is preferable that the final step in each of the above functionalization methods,
involving the release of the signal entity conjugate, is reversible. This may provide a
number of benefits. First, as stated above, the released signal entity conjugates may
then be used in the analysis of nucleic acid molecules. Second, it is the release of
the signal entity conjugates that allows the microarrays to act as detectors. That is,
the inventors have found that functionalizing the defined areas of a two- or threedimensional
microarray with attached nucleic acids comprising a promoter region or
base sequence of interest allows for either the subsequent analysis of the presence
and/or extent of methyiation within a promoter region of a gene or the detection of
specific base sequences. Furthermore, reversibility of the final step aids in the
quantification of the number of signal entity conjugates used during the sensor
application.
In one embodiment of the present invention, the use of the microarrays as sensors
involves running a mixed aqueous sample, containing single strands of nucleic acid
from a sample to be analyzed and the signal entity conjugates, which have been
released from the array (as described previously), across the microarray (Figure 9).
Preferably, and as indicated above, the mixed sample is formed by combining the
released signal entity conjugates with the nucleic acid sample in a suitable buffer and
at a suitable pH (preferably the pH is 7.0). Preferably, the nucleic acid sample
contains the promoter region or other nucleic base sequence of interest.
n an alternative embodiment, only unreacted conjugates are run across the
microarray (Figure 10). For example, the sample is combined with the signal entity
conjugates, signal entity conjugates which do not bind to the sample are separated
and run across the microarray.
Within the mixed sample, it is preferable that the signal entity conjugates bind only to
base sequences of interest or to methylated Cytosine bases within the promoter
regions of interest as they will not have been converted to Uracil during treatment
with bisulphite. This selectivity is achieved through interactions between
complimentary base pairs. Binding of the signal entity conjugates to the single
strands of nucleic acid results in the formation of signal entity conjugate/nucleic acid
complexes within the mixed samples. When the functionalized microarray is
subsequently exposed to the mixed sample, only those signal entity conjugates
which are unbound are available for binding to the functionalized defined areas on
the microarray. The user is then able to count the number of signal entity conjugates
that bind to the defined areas using the techniques described above. Alternatively,
as indicated above, the mixed sample is separated into its component parts, namely
the complexes and the free signal entity conjugates, and only the free signal entity
conjugates are then run across the defined areas of the microarray. Preferably, the
binding of the signal entity conjugates to the functionalized flat sensor sites or
surface structures is reversible as this allows the microarrays to be used repeatedly
and stored, and preferably this reversibility is achieved by changing the pH of the
aqueous solution. Preferably, the microarrays functionalized according to the above
methods are stored at about 4°C.
A decrease in the number of un-reacted signal entity conjugates, when compared to
the known number of signal entity conjugates as ascertained from the
functionalization of the microarray, will signify that a base sequence of interest is
present. Likewise, a decrease in the number of un-reacted signal entity conjugates
will signify that the promoter region of the gene was methylated to some extent. No
change in binding will indicate that either the base sequence of interest was not
present or that the promoter region of the gene was not methylated.
It is the qualitative and quantitative nature of the functionalized microarray which
therefore allows detection of specific nucleotide sequences or the presence and/or
extent of methylation to be determined. The qualitative and quantitative nature of the
functionalized microarray also allows the user to determine whether the promoter
region for a specific gene is available for transcription. The ability to determine the
presence and/or extent of methylation also allows the user to determine the
influences of various factors, including but not limited to, environmental factors, diet
or medication.
The ability to accurately count the number of signal entity conjugates bound or
attached to the surface of the microarray is an important aspect of the present
invention because it allows for direct quantification of the number of nucleic acid
strands present within a sample. For example, if a promoter region of interest in the
sample occurs only once and is methylated then the number of signal entity
conjugates bound to the microarray will be equal to the number of nucleic acid
strands in the sample. Surprisingly, the inventors have also found that due to the
sensitivity of the process the invention obviates the need for amplification of the
nucleic acids in a sample using techniques such as PCR, although for certain
applications one may still choose to employ an amplification technique. This is a
significant aspect of the present invention, as the replication or amplification of
nucleic acids is time consuming and can result in a number of errors. Eliminating the
need for replication restricts the possibility of errors occurring. Furthermore, the
method itself allows the user to determine the concentration of DNA within a sample.
As indicated previously, and using the techniques described previously, increased
sensitivity of the defined functionalized areas of the microarray (i.e. flat sites on a
two-dimensional microarray or the tips/tops of surface structures on a threedimensional
microarray) is achieved by coating the base material of the threedimensional
microarray with an inert material. This minimises non-specific binding
and allows for more accurate measurements such as digital counting (best seen at
Figure 1, (C) and (0)).
Finally because microarrays are employed as the sensor platform, this invention also
allows for simultaneous assaying of nucleic acids comprising promoter regions, other
regions of a nucleic acid, genes and/or genomes (or one or more other compounds).
Multiple assays could be carried out using microarrays whose surfaces have either
been functionalized in a uniform or random manner with a variety of the appropriate
complimentary genes or genomes.
In summary, as a result of being able to attach sensory agents or nucleic acid
Strands either directly to sensory agents on the surface of a two- or threedimensional
microarray, or indirectly through linker groups, the detection of single
molecules and the analysis of nucleic acid molecules can potentially be achieved
with high sensitivity due to the large number of individual flat sensor sites or surface
structures per cm2. Coating the base material of the two- or three-dimensional
microarray with an inert material can be preferable to ensure a high degree of
sensitivity is achieved and to eliminate non specific binding. Preferably gold is used
as the inert coating material.
Examples
Example 1: Immobilisation of bead conjugates on the surface of a threedimensional
microarray.
Following the process shown in the diagrammatic depiction in Figure 5, and with
reference to Figure 1, the following experiment was carried out using coloured bead
conjugates as the target analyte:
A three-dimensional microcone array 20, manufactured from a PMMA polymer
substrate and coated in gold 2 1 was employed (Figure 1, (A)). The cone tips 22
measured 100 m in diameter. Following removal of gold from the cone tips 22 by
abrasion an -NH 2 functional group was attached to the exposed PMMA polymer
surface at the defined area formed by the exposed cone tips 22. Thus a defined
fuctionalized area at exposed cone tips 22 is formed. This was achieved by exposing
the surface of the microarray to 1 to 20% ethylenedamine in DMSO for 5 to 20
minutes. The surface was then washed with IPA and dried under N2 gas. The
surface was then exposed to 2 % GA in sodium carbonate buffer (pH 9.2) for two
hours with shaking at room temperature. This was followed by a water wash.
Subsequent attachment of a linker group was achieved by exposing the surface of
the microarray to 2 % 1,6-hexyldiamine in sodium carbonate buffer (pH 9.2) for two
hours with shaking at room temperature, followed by water washes. The NH2-
functionalized surface was then attached by micro-PS-C0 2H beads (8 microns in
diameter) in Borate buffer (pH 8.5) after beads activation with EDC and NHS in MES
buffer (pH 6.8) (Figure 5). In future, such NH2-functionalized sensing surface can be
used for attachment of a sensory agent (for example, an antibody, DNA, etc). For
example, a tip surface NH2 group can join a protein NH2 group through a GA linker.
This was achieved by loading the sensory agent in a borate buffer (pH 8.5) onto the
surface of the microarray and leaving it overnight. A PBS buffer wash was then
carried out. The surface was then exposed to microbead conjugates 36 (pictorially
represented as 35 in Figure 2, (C), and shown in Figure 11, (B) and (C)) comprising
an appropriate binding partner for the sensory agent (for example, antigens, DNA,
etc) to test the functional working of the three-dimensional microarray. Digital
measurements were carried out to ascertain the extent of binding of the microbead
conjugates 36. To achieve this, light was passed through the sample. Where light
was blocked, microbead conjugates were attached and their number was able to be
digitally counted using a commercial MicroScanner, Microscope or/and Digital image.
Figure 11, (D) shows a diagrammatic representation of a digital result showing the
blocked areas.
Examples 2 and 3: Schemes 1 and 2 in the Examples below show schematic
representations of the process described in Examples 2 and 3, respectively. Steps
(1) to (4) of Scheme 1 are microarray preparation steps as discussed earlier and are
applicable to both examples 2 and 3. A key for components A to G in both Schemes
is provided in Scheme 1.
Example 2: Sandwich assay for large protein molecules
Scheme 1, steps (5) to (7) show a sandwich assay for rat IgG. The threedimensional
microarray employed was 100 m in diameter and was formed from
PM A substrates. After removing gold from the cone tips and introducing a -NH 2
functional group to each cone tip, the cone tips were further reacted with
glutaraldehyde in PBS buffer (pH 7.4) to bind a -CHO functional group to the
functionalized cone tips. A solution of commercial ani/-rat IgG (secondary antibody)
(R 1 8, Sigma) in PBS was then reacted with the cone tips overnight to immobilize
the anti-rat IgG onto the tips. After washing the microarray with PBS, a solution of
commercial proteins (Rat IgG) (P1922, Sigma) in PBS and in various concentrations,
are reacted with the anti-rat IgG on the tips at 40 °C for 1 hour. Finally, after a PBS
wash of the microarray, a suspension of anti-rat IgG coated microparticles was
further reacted with the substrates at 40 °C for 1 hour to attach the microbeads to the
cone tips. After PBS washing and gently drying the substrates, the concentration of
the protein analyte (Rat IgG) was proportional to the numbers of microbeads
attached on the tips, which were then able to be digitally counted using a commercial
MicroScanner, Microscope and/or Digital image.
Scheme 1: Sandwich assay for large proteins
4)
Glutaraldehyde in PBS
(5)
Anti-rat IgG immobilization
7)
A: PMMA; B: Chromium; C: Gold; D: Anti-rat IgG (secondary antibody); E: Antiprogesterone
(P4) antibody (mAb) (primary antibody), also as a protein analyte (Rat
IgG); F: Microbeads; G: Progesterone (P4).
Example 3: Inhibition assay for small steroid molecules
Scheme 1, steps (5), (8) and (9) shows an inhibition assay for small molecules.
Following introduction of -CHO functional groups to the cone tips of threedimensional
microcone array (100 m in diameter) PMMA substrates, a solution of a
Progesterone-PEG-NH 2 (P4-PEG-NH2) , prepared according to a literature method (J.
S. Mitchell et al, Analytical Biochemistry, 2005, 343:125-135), in PBS (pH 7.4) was
reacted with the cone tips overnight for immobilization of steroid progesterone (P4) on
the microcone tips. After washing the substrates with PBS, a fixed amount of the
commercial monoclonal anf/-progesterone antibody (mAb, primary antibody) (P1922,
Sigma) was mixed with various standard progesterone (P ) solutions in PBS (pH 7.4)
at 40 °C for 1 hour. The resulting mixture was then bound to the Progesterone (P )
attached to the tips. This reaction was carried out at 40 °C for 1 hour. Finally, after a
PBS wash of the substrates, a suspension of anti-rat IgG (secondary antibody)
coated microparticles was further reacted with the substrates at 40 °C for 1 hour to
attach the microbeads to the cone tips. After PBS washing and gently drying the
substrates, the concentration of the steroid (progesterone) was reverse proportional
to the numbers of microbeads attached on the tips, which were then able to be
digitally counted using a commercial MicroScanner, Microscope and/or Digital image.
Scheme 2: Inhibition assay for small molecules
5)
Progesterone-PEG-NH immobilization
Surface regeneration 1). Rat IgG (analyte);
2). Anti-rat IgG/Microbeads
Example 4: DNA hybridizations
Scheme 3, shown below, is a schematic representation of the process described in
Example 4. Scheme 2 provides a alternative route from step (5) shown in schemes
1 and 2 via new steps (10) to (12). The key provided for Scheme 1 is relevant to
Scheme 3.
Scheme 3: DNA hybridization
DNA immobilization 3'- H2N-(CH2)6- CCT AAT AAC AAT - 5'
on microarray tips
After -CHO functionalization of the three-dimensional microcone tips, a H20 wash
and N2 drying, a solution of synthetic amine-attached 12-base oligonucleotides of a
given sequence [H2N-(CH2)6-CCTAATAACAAT] in phosphate buffer (pH 7.0) was
immobilized on the microcone tips overnight, followed by washings with 5XSSC, H20
and N2 drying.
Prior to hybridization, glutaraldehyde-activated substrates were treated with Na2BH .
The first hybridization was carried out as follows: 12-base DNA immobilized
microcone tips were hybridized with a synthetic target DNA (27-base, 5'-
GGATTATTGTTAAATATTGATAAGGAT-3') in a PBS hybridization buffer (8 x PBS,
pH7.0) for 24 hours, followed by 2 x SSC washes. The second hybridization was
performed by a further reaction of the hybridized microcone tips with DNA attached
micropartic!es [15-base, 3'-TTATAACTATTCCTA-(CH 2)6-NHCO-Microparticle] in the
same hybridization buffer (8 x PBS) for 5 to 24 hours. Finally, the microparticlesattached
substrates were washed with 8 x PBN buffer (0.3M NaN03 and PBS,
pH7.0) several times. The quantification of target DNA (27-base) was also carried out
by digitally counting using a commercial MicroScanner, Microscope and/or Digital
image. The concentration of the target DNA was proportional to the microparticles on
the microcone tips of the substrate.
Because microarrays are employed as the sensor platform, this invention also allows
for simultaneous assaying of nucleic acids comprising promoter regions, other
regions of a nucleic acid, genes and/or genomes (or one or more other compounds).
Multiple assays could be carried out using microarrays whose surfaces have either
been functionalized in a uniform or random manner with a variety of the appropriate
complementary genes or genomes.
As discussed previously, sensory agents, which specifically bind to the target analyte
the user wishes to detect, are usually employed. To prevent non-specific binding to
the coated surface the microarray is exposed to 2 % OVA in PBS for 2 hours at room
temperature with shaking. This is followed by a PBS wash. The microarray is then
ready to use.
Because microarrays are employed as the sensor platform, this invention also allows
for simultaneous assaying of nucleic acids comprising promoter regions, other
regions of a nucleic acid, genes and/or genomes (or one or more other compounds).
Multiple assays could be carried out using microarrays whose surfaces have either
been functionalized in a uniform or random manner with a variety of the appropriate
complimentary genes or genomes.
The foregoing describes the invention including preferred forms thereof.
Modifications and alterations that would be readily apparent to the skilled person are
intended to be included within the spirit and scope of the invention described.
Unless the context clearly requires otherwise, throughout the description and the
claims, the words "comprise", "comprising", and the like, are to be construed in an
inclusive sense as opposed to an exclusive or exhaustive sense, that is to say, in the
sense of "including, but not limited to."
The reference to any prior art in the specification is not, and should not be taken as,
an acknowledgement or any form of suggesting that the prior art forms part of the
common general knowledge in any country in the World.

Claims
A method for determining the presence of a target compound(s) of interest
within a sample, the method including the steps of:
a) Providing a microarray including a plurality of defined functionalized
areas, the defined functionalized areas being defined areas having
attached sensory agent(s) capable of attaching to the target compound(s)
of interest within the sample;
b) Contacting the microarray with at least part of the sample; and
c) Determining the presence of a target compound(s) of interest by detection
of a detectable response to the attachment of the target compound(s) to
the sensory agent(s).
The method of claim 1 wherein the plurality of defined functionalized areas on
the microarray, are defined areas on the tops of a plurality of surface
structures.
The method of claim 1 or 2 wherein the target compound(s) is selected from a
micro-organism, a peptide or protein, and/or an antibody.
The method of any one of claims 1 to 3 wherein the sensory agent(s) and
target compound(s) are, together, biological recognition groups or binding
agents.
The method of any one of claims 1 to 4 wherein sensory agent(s) and target
compound(s) are, together, biological bindings or recognition groups selected
from antibody/antigen, DNA/DNA, DNA/protein, protein/protein,
protein/receptor, ce!l/protein and cell/DNA binding partners.
The method of any one of the previous claims wherein the sample is a tissue
sample, a fluid sample, or an oral swab.
A method for determining the presence of a target compound(s) of interest
within a sample, the method including the steps of:
a) Providing a microarray including a plurality of surface structures on a base
material, the surface structures having attached sensory agent(s),
capable of attaching to the target compound(s) of interest within the
sample, on defined functionalized areas on the tops of the surface
structures;
b) Passing at least part of the sample over the array; and
c) Determining the presence of a target compound(s) of interest by detection
of a detectable response to the attachment of the target compound(s) to
the sensory agent(s).
8. The method of claim 7 wherein the sensory agents and target compounds
are, together, biological recognition groups or binding agents.
9. The method of claim 7 or claim 8 wherein sensory agents and target
compounds are, together, biological bindings or recognition groups selected
from antibody/antigen, DNA/DNA, DNA/protein, protein/protein,
protein/receptor, cell/protein and cell/DNA binding partners.
10. The method of any one of claims 2 to 9 wherein the tops of each surface
structure includes a defined functionalized area.
1 . The method of any one of claims 1 to 10 wherein the microarray includes a
plurality of different sensory agents.
12. The method of any one of claims 1 to 11 wherein each defined functionalized
area includes a plurality of sensory agents.
13. The method of any one of claims 1 to 12 wherein a signal entity capable of
providing a detectable response is attached to the target compound.
14. The method of claim 13 wherein the signal entity is attached to the target
compound in the sample prior to step (b), or wherein the signal entity is
attached to the target compound between steps (b) and (c).
15. The method of claim 13 or 14 wherein the signal entity is a chemical,
biological, or physical entity which is capable of providing a detectable signal
or response.
6 The method of claim 15 wherein the signal entity is a particle and is selected
from a coloured microbead, a fluorescent microbead, a magnetic microbead,
or a light blocking microbead.
17. The method of claim 1 wherein the signal entity is a polymer microbead.
18. The method of claim 17 wherein the polymer microbead is selected from
polystyrene beads, PMMA beads and PET beads.
19. The method of any one of claims 13 to 15 wherein the signal entity includes a
synthesized complimentary copy of a single strand of a nucleic acid.
20. The method of any one of claims 1 to 19 wherein the detectable response is
selected from colour, fluorescence, light blocking, visual responses,
spectrophotometric responses, potentiometric or galvanostatic responses,
magnetic light refraction, heat, frequency and digital responses.
21. The method of any one of claims 1 to 20 wherein the detectable response is
capable of being read by digital counting, weight measurements,
fluorescence, optical, and/or electrical means.
22. The method of any one of claims 1 to 19 wherein the detectable response
results in any one or a combination of quantitative/qualitative, fluorescence,
optical or colourmetric measurements.
23. The method of any one of claims 1 to 22 wherein the defined functional area
is between about 1 nm and 1000 micron in diameter.
24. The method of any one of claims 1 to 23 wherein the defined functional area
is the same size or smaller than the size of the signal entity or the target
compound.
25. The method of any one of claims 1 to 24 wherein the defined functional areas
are separated from each other by about 5 nm to about 20 micron spacing.
26. The method of any one of claims 1 to 25 wherein there are between about
250,000 and about 1 billion defined functional areas per cm2 of the
microarray.
27. The method of any one of claims 1 to 26 wherein the microarray is formed
from a plastics material, a metal, a ceramic, an oxide, silicon or a photoresist.
28. The method of claims 27 wherein the plastics material is selected from
PMMA, PET and PS; and the metal is aluminium.
29. The method of any one of claims 1 to 26 wherein a patterned layer is
attached to or formed onto the underside of the microarray to disperse light
across the surface where light is passed through the microarray for
measurement purposes.
30. The method of any one of claims 1 to 29 wherein the microarray is formed by
etching, lithograph processes, hot embossing, nano-embossing and injection
molding or by a Continuous Forming Technology process.
31. The method of any one of claims 1 to 30 wherein the defined areas are
functionalized using NH2 or COOH functional groups.
32. The method of claim 3 1 wherein a linker group is attached to the NH2 or
COOH functional groups.
33. The method of claim 32 wherein the linker group is a covalent linker group.
34. The method of claim 32 or 33 wherein the sensory agent is attached to the
linker group.
35. The method of claim 3 1 wherein the sensory agent is attached to the NH2 or
COOH functional groups.
36. The method of claims 32 to 34 wherein the linker groups are selected from
aliphatic compounds, PEG molecules and polymers, proteins or DNA chains.
37. The method of claims 1 to 29 wherein the sensory agent is attached directly
to the tips of the surface structures through a linker group.
38. The method of any one of claims 1 to 37 wherein the microarray is coated
between the defined functionalized areas with an inert material.
39. The method of claim 38 wherein the inert material is selected from gold or
silver or chromium, a polymer, or an oil.
40. The method of claim 38 wherein the inert material is a combination of any two
metafs selected from gold, silver or chromium.
4 . The method of claims 38 to 40 wherein the inert material includes a layer of a
thiol, protein and/or PEG material to ensure coating adhesion.
42. The method of claims 38 to 4 1 wherein the microarray includes a secondary
inert coating.
43. The method of any one of claims 7 to 42 wherein the sample is a biological
sample.
44. The method of claim 43 wherein the biological sample is a tissue sample, a
fluid sample, or an oral swab.
45. The method of any one of claims 7 to 44 wherein the target compound is a
nucleic acid.
46. The method of any one of claims 7 to 44 wherein the target compound is a
biological sample comprising a micro-organism, a peptide or protein, and/or
an antibody.
47. A three-dimensional microarray for use in determining the presence of a
target compound(s) of interest within a sample, the microarray including:
a) a base material including a plurality of surface structures;
b) the plurality of surface structures including sensory agent(s) capable of
attachment to the target compound(s) of interest within the sample, the
sensory agent(s) being included on defined functionalized areas on the
tops of each surface structure.
48. The three-dimensional microarray of claim 47 wherein the surface structures
are millimeter to nanometer sized surface structures.
49. The three-dimensional microarray of claim 47 or 48 wherein the surface
structures are randomly ordered on the surface of the microarray or which are
substantially identical and uniformly separated from each other.
50. The three-dimensional microarray of any one of claims 47 to 49 wherein the
surface structures take the form of cones or ridges.
51. The three-dimensional microarray of any one of claims 47 to 50 wherein the
defined functionalized area is between about 1 n and 1000 micron in
diameter.
52. The three-dimensional microarray of claim 5 1 wherein the defined
functionalized area on the surface structure is between about 1 micron and
about 20 micron in diameter.
53. The three-dimensional microarray of claim 52 wherein the defined
functionalized area on the surface structure is between about 5 micron and
about 1 micron in diameter.
54. The three-dimensional microarray of any one of claims 47 to 53 wherein the
defined functionalized area is the same size or smaller than the size of the
target compound.
55. The three-dimensional microarray of any one of claims 47 to 54 wherein the
defined functionalized areas on the surface structures are separated from
each other by about 5 nm to about 20 micron spacing.
56. The three-dimensional microarray of claim 55 wherein the defined
functionalized areas on the surface structures are separated from each other
by about 1 to about 20 micron spacing.
57. The three-dimensional microarray of any one of claims 47 to 56 wherein there
are between about 250,000 and about 1 billion defined functionalized areas
per cm2.
58. The three-dimensional microarray of claim 57 wherein there are about
250,000 defined functionalized areas per cm2 at a 10 m resolution.
59. The three-dimensional microarray of any one of claims 47 to 58 wherein the
microarray is formed from a plastics material, a metal, a ceramic, an oxide,
silicon or a photoresist.
60. The three-dimensional microarray of claim 59 wherein the plastics material is
selected from PMMA, PET and PS; and the metal is aluminium.
6 . The three-dimensional microarray of any one of claims 47 to 58 wherein the
microarray is formed from a polymer substrate.
62. The three-dimensional microarray of any one of claims 47 to 60 wherein the
base material has a thickness of between about 500 microns and about 2
mm.
63. The three-dimensional microarray of any one of claims 47 to 62 wherein a
patterned layer is attached to or formed onto the underside of the base
material to disperse light across the surface where light is passed through the
microarray for measurement purposes.
64. The three-dimensional microarray of any one of claims 47 to 63 wherein the
surface structures are formed by etching, lithographic processes, hot
embossing, nano-embossing, injection molding or by the Continuous Forming
Technology process as described in WO2007/058548.
65. The three-dimensional microarray of any one of claims 47 to 64 wherein the
defined areas are functionalized by NH2 or COOH functional groups.
66. The three-dimensional microarray of any one of claims 47 to 64 wherein the
sensory groups are one or more single strands of nucleic acid comprising a
promoter region or one more single strands of nucleic acid comprising a
specific base sequence of interest.
67. The three-dimensional microarray of claim 65 wherein the sensory groups are
one or more single strands of nucleic acid comprising a promoter region or
one more single strands of nucleic acid comprising a specific base sequence
of interest are attached to the NH2 or COOH functional groups
68. The three-dimensional microarray of claim 65 wherein a linker group is
attached to the NH or COOH functional groups.
69. The three-dimensional microarray of claim 68 wherein the sensory agent is
attached to the linker group.
70. The three-dimensional microarray of claim 65 wherein a sensory agent is
attached to the NH2 or COOH functional groups.
7 . The three-dimensional microarray of claim 68 or 69 wherein the linker group
is a covalent linker group.
72. The three-dimensional microarray of claim 68, 69, or 7 1 wherein the linker
groups are selected from aliphatic compounds, PEG molecules and polymers,
proteins or DNA chains.
73. The three-dimensional microarray of any one of claims 47 to 64 wherein the
sensory agent is attached directly to the defined functionalized areas through
a linker group.
74. The three-dimensional microarray of any one of claims 47 to 64 wherein the
sensory agents form part of biological recognition groups or binding agents.
75. The three-dimensional microarray of any one of claims 47 to 64 wherein the
sensory agents are selected to attach to the target compounds as part of
antibody/antigen, DNA/DNA, DNA/protein, protein/protein, protein/receptor,
cell/protein and cell/DNA binding partners.
76. The three-dimensional microarray of any one of claims 47 to 75 wherein the
three-dimensional microarray includes a plurality of sensory agent groupings,
each sensory agent grouping forming a section of the microarray.
77. The three-dimensional microarray of any one of claims 47 to 75 wherein the
defined functionalized areas include a plurality of attached sensory agents.
78. The three-dimensional microarray of any one of claims 47 to 77 wherein the
three-dimensional microarray is coated between the defined functionalized
areas with an inert material.
79. The three-dimensional microarray of claim 78 wherein the microarray includes
a layer of a thiol, protein or PEG material beneath the inert coating.
80. The three-dimensional microarray of claim 78 or 79 wherein the inert material
is selected from gold or silver or chromium, a polymer or an oil.
8 . The three-dimensional microarray of claim 78 or 79 wherein the inert material
is a combination of any of gold, silver or chromium.
82. The three-dimensional microarray of any one of claims 78 to 8 1 wherein the
inert material is applied using evaporation, painting, deposition, sputtering,
plasma treatment, spray coating, dip coating, or spin coating.
83. The three-dimensional microarray of any one of claims 78 to 82 wherein the
microarray includes a secondary inert coating.
84. The three-dimensional microarray of claim 83 wherein the secondary inert
coating is a thiol compound.
85. A two-dimensional microarray for use in determining the presence of a target
compound(s) of interest within a sample, the microarray including:
a) a base material including a plurality of defined functionalized areas;
b) the plurality of defined functionalized areas including sensory agent(s)
capable of attaching to the target compound(s) of interest within the
sample.
86. The two-dimensional microarray of claim 85 wherein the defined
functionalized areas are substantially identical in size and uniformly separated
from each other or are randomly placed on the surface of the microarray .
87. The two-dimensional microarray of claim 85 or 86 wherein each defined
functionalized area is between about 1 nm and 1000 micron in diameter.
88. The two-dimensional microarray of any one of claims 85 to 87 wherein the
defined functionalized areas are the same size or smaller than the size of the
target compound.
89. The two-dimensional microarray of any one of claims 85 to 88 wherein the
defined functionalized areas are separated from each other by about 1 to
about 20 micron spacing.
90. The two-dimensional microarray of any one of claims 85 to 89 wherein there
are between about 250,000 and about 1 billion defined functionalized areas
per cm2.
91. The two-dimensional microarray of any one of claims 85 to 90 wherein the
microarray is formed from a polymer substrate; a plastics material, including
P MA, PET or PS; or metals such as aluminium; or ceramics or oxides or
silicon or a photoresist or glass.
92. The two-dimensional microarray of any one of claims 85 to 9 1 wherein the
microarray is between about 500 microns and about 2 mm thick.
93. The two-dimensional microarray of any one of claims 85 to 92 wherein a
patterned layer is attached to or formed onto the underside of the base
material to disperse light across the surface where light is passed through the
microarray for measurement purposes.
94. The two-dimensional microarray of any one of claims 85 to 93 wherein the
microarray is coated between the defined functionalized areas with an inert
material.
95. The two-dimensional microarray of any one of claims 85 to 94 wherein the
defined areas are functionalized using NH2 or COOH functional groups, or
one or more single strands of nucleic acid comprising a promoter region or
one more single strands of nucleic acid comprising a specific base sequence
of interest.
96. The two-dimensional microarray of any one of claims 85 to 95 wherein a
linker group is attached to the sensory agent.
97. The two-dimensional microarray of claim 96 wherein the linker group is
selected from aliphatic compounds, PEG molecules and polymers, proteins or
DNA chains.
98. The two-dimensional microarray of any one of claims 85 to 97 wherein the
sensory agents form part of a biological recognition group or binding agent.
99. The two-dimensional microarray of any one of claims 85 to 98 wherein
sensory agents form part of antibody/antigen, DNA/DNA, DNA/protein,
protein/protein, protein/receptor, cell/protein and cell/DNA binding partners.
100. The two-dimensional microarray of any one of claims 85 to 99 wherein the
two-dimensional microarray includes a plurality of sensory agent groupings,
each sensory agent grouping forming a section of the microarray.
101. The two-dimensional microarray of any one of claims 85 to 100 wherein the
defined functionalized areas include a plurality of attached sensory agents.
102. A method of preparing a two dimensional microarray according to claim 85,
the method including the steps of forming the defined functionalized areas by
layering an inert material between and over the surface structures of a threedimensional
microarray according to claim 47 and then removing sufficient of
the inert material and of the top of the surface structures to expose defined
areas of the surface structures within the inert material.
103. A method for determining whether or not a nucleic acid comprising a specific
sequence of bases is present in a sample, the method comprising:
a) in a sample of nucleic acid, where the nucleic acid is double stranded,
separating it into single strands;
b) combining the single strands of a nucleic acid with a signal entity
conjugate to form a mixed sample;
c) determining whether the nucleic acid comprising a specific sequence of
bases is present by running the mixed sample across the surface of a
functionalized microarray according to claim 47 or 85; and
d) counting the number of bound signal entity conjugates.
104. A method for determining the extent of methylation in the promoter region of a
gene, the said method comprising:
a) in a sample of nucleic acid, where the nucleic acid is double stranded,
separating it into single strands;
b) treating the sample of nucleic acid such that non-methylated Cytosine is
converted to Uracil;
c) combining the single strands of nucleic acid with a signal entity conjugate
to form a mixed sample;
d) determining the presence and/or extent of methylation in the promoter
regions by running the mixed sample across the surface of a
functionalized microarray according to claim 47 or 85; and
e) counting the number of bound signal entity conjugates.
105. The method of claim 104 wherein the non-methylated Cytosine bases are
converted to Uracil by a chemical conversion using bisulphite.
106. The method according to claims 103, 104, or 105 wherein, the number of
bound signal entity conjugates is ascertained by visual techniques,
spectrophotometric techniques, fluorescent techniques, potentiometric or
galvanostatic techniques, magnetic light refraction, heat, frequency and digital
techniques.