ARRAYS AND METHODS OF MANUFACTURE
TECHNICAL FIELD
The invention relates to the development of a 3D ("three dimensional") surface which can be
modified to form an array of isolated but interconnected functionalisable areas for use in a
variety of array applications, in particular microelectrode sensor arrays and microcatalyst arrays.
In particular, the method allows for the fabrication of arrays which include isolated but
conductively interconnected surface areas which can be arranged in a variety of patterns. The
invention also relates to such arrays.
BACKGROUND ART
There are currently a number of known methods for fabricating arrays. These include printing
techniques such as screen printing or ink jet printing, lithographic techniques whereby the array
is etched onto a surface, photolithography, direct electrodeposition (deposition of wires),
patterning of carbon nanotube / nanofiber arrays and assembly techniques, for example, wires
set in an epoxy resin. However, these known methods have a number of limitations. In
particular, they are cumbersome to carry out and it is difficult to accurately define the arrays
over a large surface area and on the millimeter to nanometer scale. Thus, the resolution of the
arrays produced is often poor due largely to that lack of definition. The inability to accurately
place sensor sites on such arrays causes problems as qualitative and quantitative measurement
is detrimentally affected. In particular, issues of cost arise with the fabrication of nanoscale
arrays as, while they can be made, control over definition and cost remain problems which
cannot be easily overcome. Economy of scale is a particular issue.
The fabrication of arrays on the millimeter to nanometer scale, particularly on the micrometer to
nanometer scale over large surface areas having improved accuracy of definition would be
particularly valuable in the areas of sensing, electrochemistry and catalysis. Electrochemistry is
the branch of chemistry that deals with the use of spontaneous chemical reactions to produce
electricity, and the use of electricity to bring about non-spontaneous chemical change. In
particular, it is the study of aqueous chemical reactions which occur at the interface of an
electron conductor such as a metal or a semiconductor (the electrode) and an ionically
conducting medium (the electrolyte) and which involve electron transfer between the electrode
and the electrolyte or species in solution. Catalysis concerns the creation of a new reaction
pathway with a lower activation energy, thereby allowing more reactant molecules to cross the
reaction barrier and form reaction products.
In a typical electrochemical detection process it is, in general, preferable to employ an array of
smaller electrodes as opposed to a single large electrode. Reasons for this include;
the ability to use smaller sample volumes;
application in both in vivo and in vitro measurement;
low depletion rate of target molecules;
low background charging due to their reduced surface area;
reduced IR drop; and
high current density arising from enhanced mass transport to the electrode surface
result of convergent diffusion.
Accurately defined arrays would also be valuable for use in:
· the analysis of fluids (e.g. biological: blood, urine, milk and non-biological: waste water
streams, beverages);
• integration with living, biological systems into lab-on-a-chip devices,
• in vitro or in vivo biological sensing such as enzyme-linked assays and the detection of
many other biomolecules;
catalysis;
• trace metal monitoring in the environment;
• corrosion monitoring; and
energy production and storage devices.
Co-pending PCT application number PCT/201 1/000052 also concerns microarray structures.
However, the microarrays as described in PCT/201 1/00052 simply include a continuous inert
base substrate with functionalisable areas isolated by an inert material. The functionalisable
areas are not stated to be conductively interconnected and the structures do not include at least
one continuous interconnected layer, separate to the base substrate material and inert material,
that allows for improved functional and structural flexibility of the microarrays formed.
It is therefore an object of the present invention to provide arrays including isolated but
conductively interconnected functionalisable areas and/or methods of forming such arrays. It is
a further or alternative object of the present invention to at least provide the public with a useful
choice.
SUMMARY OF THE INVENTION
In a first aspect, the present invention provides a microarray structure including a substrate
material layer, a continuous 3D ("three-dimensional") surface layer on the substrate material
layer that is capable of functionalisation for use as an array, and an inert material;
wherein the structure includes accurately defined and functionalisable isolated areas
which are millimeter to nanometer in size; and
wherein the functionalisable areas are part of the continuous 3D surface layer and are
isolated by the inert material but which are interconnected within the structure by the
continuous 3D surface layer.
Preferably, the continuous 3D surface layer is electrically conductive. More preferably, the 3D
surface layer is a metal.
Alternatively, the continuous 3D surface layer is a carbon based material, including but not
limited to carbon fiber, carbon paste, graphite, graphene, glassy carbon, carbon nanotubes and
conducting polymers.
Preferably, the continuous 3D surface layer is a unitary layer that covers the substrate material
layer.
Preferably, the continuous 3D surface layer is cut into a plurality of isolated continuous 3D
surface layer segments on the substrate material layer, each segment including a plurality of
functionalisable areas, wherein each group of functionalisable areas is capable of separate
functionalisation.
Optionally, the inert material is also an insulating material.
Optionally, the substrate material layer is formed from a conductive material or a non-conductive
inert material which, optionally, is also an insulating material.
Optionally, the structure includes an adhesion layer between the continuous 3D surface layer
and the substrate material layer.
Preferably, the microarray structure is functionalised to be a micro-electrode sensor array and/or
a micro-catalyst array.
Preferably, the continuous 3D surface layer protrudes from the inert material such that the
functionalisable areas are exposed above the inert material.
Preferably, the inert material and the functionalisable areas form a 2D ("two-dimensional")
surface including functionalisable areas.
In a second aspect, the present invention provides an intermediate structure for use in
fabricating an array according to the first aspect of the invention, the intermediate structure
including a substrate material layer that includes an accurately defined 3D pattern to a
millimeter to nanometer scale, and a continuous 3D surface layer on the substrate material layer
that is capable of functionalisation for use as an array over at least part of the pattern.
Preferably, substantially all the patterned area is coated with the continuous 3D surface layer.
Preferably, the substrate material layer is formed from a conductive material or a nonconductive
inert material which, optionally, is also an insulating material.
Preferably, the pattern is formed by embossing, casting, stamping, etching, grinding,
lithography, pressure forming, vacuum forming, roll forming, injection moulding and laser
scribing / ablation.
Preferably, the substrate material layer is coated with the continuous 3D surface layer by
sputtering, evaporation or electroless deposition techniques.
Preferably, the continuous 3D surface layer forms a coating layer which is electrically
conductive. More preferably, the 3D coating layer is a metal.
Alternatively, the continuous 3D surface layer is a carbon based material, including but not
limited to carbon fiber, carbon paste, graphite, graphene, glassy carbon, carbon nanotubes and
conducting polymers.
Optionally, the intermediate structure includes an adhesion layer between the continuous 3D
surface and the substrate material.
In one embodiment of the first aspect, the present invention provides an accurately defined and
functionalisable array including a continuous 3D surface layer, said array formed from an
intermediate structure of the second aspect of the invention, wherein a layer of inert material fills
the spaces between the tips in the 3D pattern on the surface layer to give an inert material
surface through, or from, which the tips of the 3D pattern protrude or are otherwise exposed;
and wherein the tips are isolated by the inert material but are conductively interconnected via
the continuous 3D surface layer between the inert material surface and the substrate material
layer.
Optionally, the inert material surface is also an insulating layer.
In a third aspect, the present invention provides a method for the formation of an intermediate
structure according to the second aspect of the invention including a continuous 3D surface
layer from which an array having accurately defined and functionalisable isolated areas can be
formed, the method involving the steps of:
a. placing an accurately defined 3D pattern at the millimeter to nanometer scale on the
surface of a substrate material; and
b. coating at least part of the patterned substrate material with a continuous 3D surface
layer.
Preferably, the pattern is placed on the surface of the substrate material by embossing, casting,
stamping, etching, grinding, lithography, pressure forming, vacuum forming, roll forming,
injection moulding and laser scribing / ablation.
Preferably, the substrate material is coated with the continuous 3D surface layer by sputtering,
evaporation or electroless deposition techniques.
Preferably, the continuous 3D surface layer covers substantially all of the patterned area of the
substrate material.
Alternatively, the continuous 3D surface layer is cut into a plurality of isolated continuous 3D
surface layer segments, wherein the plurality of segments cover substantially all of the
patterned area of the substrate material.
Optionally, the method includes the step of adding an adhesion layer between the substrate
material and the continuous 3D surface layer.
Preferably, an inert materia! is placed on the continuous 3D surface to form a structure
according to the first aspect of the invention.
In a fourth aspect, the present invention provides a method for the formation of a structure
capable of functionalisation as an array according to the first aspect of the invention, the method
including the steps of taking the intermediate structure according to the second aspect of the
invention and filling individual spaces between the tips of the 3D pattern on the intermediate
structure with an inert material to give a surface through, or from, which the tips of the 3D
pattern protrude or are otherwise exposed; wherein the tips form functionalisable areas which
are isolated by the inert material but are interconnected within the structure by the continuous
3D surface layer and are capable of functionalisation.
Optionally, the tops of the tips can be cut away to align with the surface of the inert material to
form a 2D surface including functionalisable areas. Optionally, a portion of the inert material is
also removed.
In a fifth aspect, the present invention provides a method for the formation of a 2D structure
capable of functionalisation as an array, said structure including a continuous 3D surface, the
method including the steps of taking the intermediate structure according to the second aspect
of the invention and covering the 3D pattern on the intermediate structure with an inert material,
removing sufficient of the inert filler material to only expose the tips of the 3D pattern, wherein
the exposed 3D tips are isolated by the inert material but are interconnected within the structure
by the continuous 3D surface and are capable of functionalisation.
In a sixth aspect, the present invention provides a further method for the formation of a structure
capable of functionalisation as an array including a continuous 3D surface layer, the structure
having an accurately defined 3D pattern of functionalisable areas in the millimeter to nanometer
scale, the method including the steps of:
electroplating the continuous 3D surface layer of the intermediate structure according to
the second aspect of the present invention to form a metal layer that covers the tips of
the 3D pattern on the intermediate structure;
separating the metal layer and the substrate material of the intermediate structure to
form a metal negative structure which includes a negative of the 3D pattern (the
"negative 3D pattern") on the intermediate structure;
c. backfilling spaces between tips within the negative 3D pattern on the metal negative
structure with an inert material to give an inert surface through, or from, which the tips of
the negative 3D pattern protrude or are otherwise exposed;
d. wherein the functionalisable areas are isolated by the inert material but are
interconnected within the structure.
Preferably, the metal layer covers at least substantially all of the 3D pattern on the intermediate
structure.
Optionally, the tops of the tips can be cut away to align with the surface of the inert material to
form a 2D surface including functionalisable areas.
In a seventh aspect, the present invention provides an intermediate structure including a
continuous 3D surface capable of functionalisation for use as an array, wherein the intermediate
structure includes an accurately defined 3D pattern at the millimeter to nanometer scale on at
least one surface and also includes an inert materia! between the tips of the 3D pattern which
creates a surface through, or from, which the tips of the 3D pattern protrude or are otherwise
exposed, the tips of the 3D pattern thus being isolated by the inert material and being
interconnected within the intermediate structure by the continuous 3D surface.
DESCRIPTION OF FIGURES
Figure : shows, in schematic form, the process for preparing a coated and patterned
structure 4 of the present invention.
Figure 2: (A) shows a 50 micron gold coated structure; (B) shows a 10 micron gold coated
structure.
Figure 3: shows, in schematic form, the process for converting a coated and patterned
structure into an array of the present invention.
Figure 4: (A) shows, in schematic form, the use of a laser to scribe lines in the coating
layer in between the tips to produce four isolated micro-electrode arrays (IV) on
the same sensor chip (A, B, C and D). The four isolated micro-electrode arrays
can be configured in a number of ways as shown in (B) and (C), each of which
show a cross-section of a micro-electrode array spanning across electrically
isolated rows of tips.
(A) shows a schematic of the laser pattern for an inter-digitated array; (B) shows
an example of the laser patterning on a 2 cm x 2 cm gold coated sensor chip; (C)
shows a 10 cm disc of sensor chips; and (D) shows a microelectrode array
comprising isolated gold tips.
shows the typical types of micro-electrode arrays. (A) shows a microdisk
electrode array (could be ordered or random); (B) shows a microband electrode
array; (C) shows an inter-digitated micro-electrode array (planar and vertical); (D)
shows a linear micro-electrode array; (E) shows a 3D micro-electrode array; and
(F) shows electrically isolated individual tips with electrical connections from each
tip.
shows, in schematic form, (A) roller embossing of a substrate material (for
example, a polymer or glass substrate material); (B) roller embossing of a
substrate material; and (C) embossing a substrate material using a stamp.
shows the sensor design for computer modelling experiments in which two arrays
of gold coated tips were precisely aligned over each other.
shows two different geometries. (A) shows droplet formation and (B) shows a
chamber filled with electrolyte showing potential distribution and current density
vectors (arrows) in 2D and 3D domain.
shows a 3 dimensional rendering of impedance results.
shows impedance for various parameters of space between the 2 electrodes (10
and 20 micron), where r is the radius of the tip.
shows impedance for various parameters of distance.
shows impedance comparison between two distance geometries. Red shows
fully immersed geometry. Green and blue show the droplet formation with
different radius, geometry.
shows Frequency vs. Impedance (Bode plot) for two different geometries,
wherein the solid line shows geometry B, and the dotted line shows a droplet with
radius 5 urn.
Figure 15: shows an equivalent circuit model for the whole system including the interface
impedance.
Figure 6: shows model geometry along with domain equations and boundary conditions.
Figure 7: shows impedance (Nyquist plot) for various double layer lengths of the interface
for two different geometries.
Figure 8 : shows a Bode p ot showing difference in the Real Z (ohms) for two different
double layer thickness.
Figure 9: shows difference between the impedance due to change in double layer from
50nm to 5 nm for the two geometries.
Figure 20: shows the change in the impedance due to change in charge transfer resistance
(Ret) for the two different geometries.
Figure 2 1: shows change in the impedance due to change in Ret for the fill geometry.
Figure 22: shows difference between the impedance due to change in Ret from ohm to
100K for the two geometries.
Figure 23: shows a picture of 40micron tips in PMMA.
Figure 24: shows the process for sensor fabrication.
Figure 25: shows the process for sensor fabrication
Figure 26: shows electrochemical cleaning of gold electrodes in sulphuric acid.
Figure 27: (A) shows a laser scribed interdigitated array. (B) shows a closer view of the
light shining through the 5 micron laser scribed lines.
Figure 28: shows SAM adsorption on an Au electrode over time.
Figure 29: shows 40 micron tips coated with epoxy.
Figure 30: (A) shows chemical attachment of a SAM via thiol-gold chemistry; (B) shows
electrochemical deposition of a conducting polymer followed by coupling to a
probe; (C) shows controlled electrochemical deposition of different probes to
conducting polymers for multiplexing of capture agents.
Figure 3 : shows a schematic diagram for attachment of the 1 micron animated blue
polystyrene beads to the tips of a sensor array.
Figure 32: shows beads covalently attached to the tips of a sensor array.
Figure 33: shows a gold coated nickel 1 micron sensor array.
Figure 34: shows potential distribution and current flow between inter-digitated electrode
tips.
Figure 35: shows inter-digitated tracks of an alternating working electrode and a counter
electrode with a laser scribe between.
DETAILED DESCRIPTION
The present invention concerns the development of arrays of various sizes for use in a variety of
applications including sensors, electrochemistry and catalysis. In particular, the present
invention relates to method for fabricating arrays comprising functionalisable areas at the
millimeter to nanometer (inclusive) scale. These functionalisable areas are preferably
conductive (but may not be) and are isolated at the surface of the array but joined below the
material used to isolate them. They may be of any shape or size and can be functionalised to
create sensor or catalytic sites (amongst other options) for a multitude of applications.
Examples of applications include the detection of enzymatic catalysed reduction or oxidation
reactions (e.g. glucose oxidase), the direct detection of oxidisable species within a solution (e.g.
metals, metal oxides, organic species), the detection of antibodies, DNA, cells or small
molecules where an appropriate haptan has been attached to the array surface, and detecting
and binding of their complimentary antigen via an associated electrochemical method including
the measurement of changes in the resistance between the binding surface and a counter
electrode or an electrochemical reaction. In each instance, concentration of the target analyte is
related to the level of current passed through the conductive, continuous 3D, array surface.
The microarrays of the present invention may be said to broadly consist of a substrate material
on which functionatisable areas are formed. Thus, in a first aspect, the present invention
provides a microarray structure, including a substrate material layer, a continuous 3D surface
layer on the substrate layer that is capable of functionalisation for use as an array and an inert
material. The structure includes functionaiisable areas which are part of the continuous 3D
surface layer and are isolated by the inert material but are interconnected within the structure by
the continuous 3D surface layer.
As used herein, the "substrate material layer" (herein referred to as substrate material) refers to
the base of the microarrays of the present invention. It may be flexible or rigid and is preferably
planar ranging in thickness from the micrometer to millimeter scale. As will be known to a
skilled person in the art, the thickness of the substrate material is primarily governed by the
thickness required to ensure proper handling. Where required, the substrate material should
also be optically transparent. Therefore, preferably, the substrate material is between about 50
micron to about 2 mm thick, or between about 500 micron to about 2 mm thick, or between
about 50 micron to about 100 micron thick. Preferably, the substrate material is a polymer
material. Alternatively, the substrate material may be a conducting material or an inert, non
conducting material. Where the substrate material layer is inert, it may also act as an insulating
material. Examples of suitable flexible materials for use in the present invention include
thermoplastic polyurethane, rubber, silicone rubber, and flexible epoxy. Examples of suitable
rigid substrate materials for use in the present invention include glass, PMMA, PC, PS, ceramic,
resin, composite materials and rigid epoxy. The substrate material may also be formed from a
metal such as gold, silver, nickel or the like, as discussed in more detail below.
As used herein, "functionaiisable areas" should be taken broadly to encompass those parts of
the microarrays of the present invention which protrude, or are otherwise exposed, through an
inert material or are exposed and are therefore capable of being functionalised as desired by a
user. When the inert material protrudes through the inert material, it may be exposed above
that material. The functionaiisable areas can be in any shape as desired by the user and
preferably form the uppermost surface or tip of a three-dimensional (3D) pillar like structure
(nanometer to millimeter size) formed as part of the substrate material of the microarrays of the
present invention. However, a person skilled in the art would understand that the
functionaiisable areas can also form the uppermost surface of a 3D rib like structure formed as
part of the substrate material of the microarrays of the present invention.
Throughout the specification reference to 3D should be taken to mean a three-dimensional
structure, or where required by context, a three dimensional coated structure, wherein, the
three-dimensional structure is in the form of a pillar like structure or a rib like structure.
The functionaiisable areas preferably range in size from the millimeter to the nanometer scale.
More preferably, the functionaiisable areas are between about 0 n to about 1 micron in size,
more preferably between about 200 nm to about 1 micron in size. Likewise, the spaces
between individual functionaiisable areas can be on the millimeter to nanometer scale.
In one embodiment of the present invention, the functionaiisable areas are accurately defined
areas in that they form a defined pattern on the surface of a microarray to the scale desired.
This, in turn, allows a user or a computer program to pinpoint specific functionaiisable areas on
the surface of a microarray and make a desired measurement and allows for the
functionalisation of only selected functionaiisable areas on the surface of a microarray.
Alternatively, the functionaiisable areas are randomly arranged on the surface of a microarray of
the present invention.
Figure 1 shows, diagrammatically, the use of embossing techniques to shape the surface of the
substrate material 2 into a desired 3D pattern that is accurately defined to the scale desired.
Figure 1 shows the use of a stamp to achieve this. First a stamp 1 is formed to the negative of
the desired pattern (Figure 1A). This pattern is shown in Figure 1 as being of repeating
triangles, however, this could be replaced by other options as desired by the user. The pattern
does not have to be uniform. The embossing creates tips 6 that extend from the surface of the
substrate material 2 and, therefore, also creates the desired spaces between those tips 6. The
stamp 1 is typically made from silicon or nickel. However, it can be formed from any suitable
material that is capable of use in this manner. The stamp 1 is then used to emboss the
substrate material 2 with the desired pattern (Figure 1B). As will be apparent, embossing
techniques are well known and a number of other options may be available for use to create an
appropriate and accurately defined pattern in a desired substrate material. These could include
casting, stamping (Figure 70), etching, grinding, lithography, pressure forming, vacuum forming,
roll forming (Figures 7A and 7B), injection moulding and laser scribing / ablation. Other suitable
methods for forming an accurately defined pattern to the millimeter/nanometer scale would be
known to those skilled in the art.
The 3D patterned substrate material 2 is then pulled away from the stamp 1 and is coated with a
coating layer 3 to form a 3D coated and patterned structure 4 (Figure C). The coating step
forms a continuous single 3D surface over the substrate material 2.
As used herein, "continuous 3D surface layer" (herein referred to as continuous 3D surface)
refers to the coating layer 3 which can be formed from an electrically conductive material or a
carbon-based material and which can be fabricated in large, continuous sheets over the
polymer substrate material 2. Thus, the continuous 3D surface (coating layer 3) is separate
from the substrate material 2 and will effectively be between the substrate material 2 and the
inert material 7 (best seen in Figure 3). The coating layer 3 (continuous 3D surface) is
preferably between about 1 nm to about 5 micron thick, more preferably between about 3 nm to
about 100 nm thick, more preferably about 5 nm to about 100 nm, more preferably between
about 5 nm to about 50 nm thick. Preferably, the coating layer 3 (continuous 3D surface) is a
unitary layer that covers the substrate material 2. Alternatively, the coating layer 3 (continuous
3D surface) is laser scribed or otherwise cut using techniques such as lithography to give a
plurality of isolated continuous 3D surface layer segments on the substrate material 2. Each
isolated continuous 3D surface layer segment includes a plurality of functionalisable areas so
that the surface of the microarray includes a plurality of groups of functionalisable areas (Figure
6F). Preferably, each group of functionalisable areas is capable of separate functionalisation.
Preferably the coating layer 3 (continuous 3D surface) is formed from an electrically conductive
material, preferably it is formed from a metal. Suitable metals for use as a coating layer 3 in the
present invention include gold, platinum, silver, nickel and copper amongst others.
Alternatively, the coating layer 3 is formed from a carbon-based material, preferably from the
likes of carbon fiber, carbon paste, graphite, graphene, glassy carbon, carbon nanotubes and
conducting polymers such as polypyrrole and polythiophene.
Application of the continuous 3D surface (coating layer 3) can be achieved by a number of
methods, including but not limited to sputtering, evaporation or electroless deposition. The
continuous 3D surface may be used as a seed layer as will be described later herein.
The continuous 3D surface (coating layer 3) typically includes an adhesion layer (not shown in
Figure 1) to promote adhesion to the substrate material 2. This adhesion layer therefore sits
between the continuous 3D surface layer and the substrate material. Suitable adhesion
materials for use in forming an adhesion layer would be known to those skilled in the art.
Options would include plasma treatment of the surface to increase surface roughness,
deposition of a thin layer (nanometers) of chromium or vanadium, and plasma deposited or
covalently bound thiols or amines to enhance adhesion.
The inventors have found that inclusion of the continuous 3D surface in the structure of the
microarrays of the present invention allows for improved functional and structural flexibility over
other microarrays known in the art. In particular, the continuous 3D surface may achieve any
one of a number of important roles in the present invention. For example, it protects the
underlying substrate material 2 (Figure 1). It also promotes attachment of binding chemistry at
the functionalisable areas of the microarrays of the present invention. When it is formed from a
conducting material, it allows electrochemical reactions to occur at the surface of the microarray
at the functionalisable areas. When also formed from a conducting material, it ensures that the
functionalisable areas are conductively interconnected with each other. As used herein,
"conductively interconnected" refers to electrical communication of the isolated functionalisable
areas of an array with each other and with an electroanalytical device such as a voltage meter,
a potentiostat, a galvanostat, an impedance analyser and any other device capable of
measuring current as would be known to those skilled in the art. Where the continuous 3D
surface (coating layer 3) is a unitary layer covering the substrate material, it may be connected
to an electroanalytical device at only one point. Alternatively, where the continuous 3D surface
(coating layer 3) has been laser scribed or otherwise cut into isolated continuous 3D surface
layer segments, each segment may not necessarily be interconnected with other segments in
the wider array structure and each may be connected to an electroanalytical device to give
individual electrodes within the array. Figure 6F shows such an arrangement. Reference to a
"continuous 3D surface" in this context is intended to include such options (i.e. there may be a
plurality of continuous 3D surfaces within the array structure).
Gold, as a choice of coating layer 3 (continuous 3D surface), achieves all of these roles. In
some embodiments of the present invention, the coating layer 3 may also need to be
transparent. Again, gold is capable of being transparent. A person skilled in the art will readily
understand that other conductive materials (for example, silver, platinum and conducting
polymers such as polypyrrole and polythiophene) will also be capable of achieving the above
identified roles. Likewise, non-conductive materials (for example, graphene and carbon
nanotubes) will at least be capable of achieving the majority of the above identified roles of the
continuous 3D surface.
As indicated above, gold is the preferred coating material for use as a continuous 3D surface in
the present invention because it is highly conductive (and therefore capable of acting as an
electrode), is inert, forms a strong covalent bond with sulphur, is easy to deposit on the
substrate material, has a well known chemistry and it is readily available. It is also able to
withstand harsh chemical cleaning treatments which in turn ensures that the arrays of the
present invention can be used more than once.
As used herein, "inert material" refers to a flexible or rigid material which physically isolates
individual functionalisab!e areas from each other. Thus, the inert material forms an "inert
surface" through, or from, which the functionalisable areas protrude or are otherwise exposed,
therefore exposing isolated areas of the continuous 3D surface (coating layer 3) and thus
allowing those areas to be functionaiised as desired. In this arrangement, the array remains as
a 3D array. Alternatively, the functionalisable areas do not protrude but align with the inert
material surface to form a 2D (two-dimensional, i.e. flat) surface including functionalisable
areas.
Suitable inert materials for use in the present invention include, but are not limited to, epoxy,
spray-coatable materials such as paint, silicon dioxide, or photoresist materials such as SU-8.
Epoxy or photoresist materials are typically used where flexibility is not required. The inert
material may also be formed from a solid film or a monolayer of thiol terminated molecules, or a
self-assembled monolayer (SAM) which are well known in the laser field. SAM's include an
alkyl chain which is usually terminated by an -SH functional group at one end but may also be
terminated by a variety of other functional groups, including but not limited to, -CH3, -OH, -
COOH, -NH2, -CN, and -CHO. The choice of functional group depends on the target species to
be bound to the microarrays of the present invention. The inert material may also act as an
insulating material, and may also be seen to be a filler material or an isolation layer.
Depending on the inert material to be used in the present invention, its application may involve
spin-coating the coating layer 3 of the microarray to a known thickness. Where this method of
application is employed, the inert material is then cross-linked under ultra-violet light and
individual functional areas are exposed by etching back the inert material by reactive ion
etching. Numerous alternative methods for applying the inert layer would be known to those
skilled in the art and include, but are not limited to, spray-coating followed by physical removal
of the inert material from areas to be functionaiised (for example, by wiping the tips), spraycoating
a dilute coating material onto the coating layer 3 which upon application will flow off the
tips and into the valleys of the 3D array, and dip coating a SAM monolayer followed by physical
removal of the SAM on the tips.
The microarray of the first aspect of the present invention may be functionaiised to be a microelectrode
sensor (as indicated above). It may also function as a microcatalyst array. Further
discussion on the potential uses of the arrays of the present invention are discussed below.
In one embodiment of the first aspect, the present invention provides an accurately defined and
functionalisable array, including a continuous 3D surface layer, formed from an intermediate
structure 4. Again, individual functionalisabie areas of the array are separated by a layer of the
inert material to give an inert surface through, or from, which the functionalisabie areas protrude
or are otherwise exposed and the individual functionalisabie areas are interconnected by the
continuous 3D surface. Preferably, the individual functionalisabie areas are conductively
interconnected by the continuous 3D surface. The intermediate structure forms the base of the
array.
Thus, in a second aspect, the present invention provides an intermediate structure 4 for use in
fabricating an accurately defined, functionalisabie array according to the first aspect of the
invention. The intermediate structure is formed from a substrate material layer 2 that includes
an accurately defined 3D pattern to a millimeter to nanometer scale. All or part of the 3D
pattern is coated with a coating layer 3 to form a continuous 3D surface layer on the substrate
material layer 2 that is capable of functionalisation for use as an array. It is preferred that
substantially all the patterned area is covered with the coating layer 3 (continuous 3D surface).
Optionally, the intermediate structure 4 will include an adhesion layer between the coating layer
3 (continuous 3D surface) and the substrate material 2.
Optionally, the intermediate structure 4 wi l include an adhesion layer between the coating layer
3 and the substrate material 2.
Figure C shows an intermediate structure 4 of the second aspect of the present invention.
Figure 2A shows a 50 micron gold coated patterned substrate material while Figure 2B shows a
10 micro gold coated patterned substrate material according to the present invention. Both are
"intermediate" structures 4.
The intermediate structure 4 can be fabricated separately to the arrays in large continuous
sheets thus providing economies of scale to the user. These large continuous sheets of coated
and patterned material include accurately defined 3D patterns (of any desired type - lines, arcs,
random) on the millimeter to the nanometer (inclusive) scale. Thus, in a third aspect, the
present invention provides a method for the formation of an intermediate structure according to
the first aspect of the invention, including a continuous 3D surface layer from which an array
having accurately defined and functionalisabie isolated areas can be formed, the method
involving the steps of:
a. placing an accurately defined 3D pattern at the millimeter to nanometer scale on the
surface of a substrate material; and
b. coating at least part of the patterned substrate materia! with a continuous 3D single
coating layer.
Preferably, the coating layer 3 covers substantially all of the patterned area of the substrate
material to form a continuous 3D surface layer.
Optionally, the method includes the step of adding an adhesion layer between the substrate
material 2 and the coating layer 3.
The substrate material of the intermediate structure 4 (as depicted in Figure 1C) is preferably
formed from an inert polymer material. However, as indicated above, there are a number of
other suitable flexible and non-flexible materials which may be used including thermoplastic
polyurethane, rubber, silicon rubber, epoxy, PMMA, PC, PS, ceramic, resin and composite
materials. Suitable techniques for placing an accurately defined 3D pattern at the millimeter to
nanometer scale on the surface of the substrate material are described above and include
embossing, casting, stamping, etching, grinding, lithography, pressure forming, vacuum forming,
roll forming, injection moulding and laser scribing / ablation techniques.
Alternatively, the intermediate structure 4 (as depicted in Figure 1C) could be formed from a
single layer of metal such as gold, silver, nickel or the like, depending on shape, size and cost
restraints. The metal surface could then be embossed (or otherwise patterned) with a desired
3D pattern. Again, suitable techniques for forming the metal surface with a desired pattern are
as described above and could include casting, stamping (Figure 7C), etching, grinding,
lithography, pressure forming, vacuum forming, roll forming (Figures 7A and 7B). Other suitable
methods would be known to those skilled in the art.
The intermediate structure 4 of the second aspect can be used to form arrays of the present
invention in one of three ways. In the first method, individual spaces 5 between functionalisable
areas (depicted in the form of tips) 6 in the 3D pattern on intermediate structure 4 (Figure 3C)
are filled with an inert material 7. When used in this manner, the inert material essentially acts
as a filler material or an isolation layer (Figure 3F) to give an inert surface 8 through which the
functionalisable areas or tips 6 of the intermediate structure 4 protrude or are exposed. Figure
3F depicts the use of a solid film as the inert material 7. The functionalisable areas or tips 6 are
thus isolated from each other and are capable of being functionalised as desired. Thus, once
functionalised, they become functional areas (e.g. sensor sites) in an array form. Figure 3G
shows a diagrammatic top view of the functionalisable array of Figure 3F. The functionalisable
areas or tips 6 remain connected to each other by the coating layer 3 (continuous 3D surface) of
the intermediate structure 4. However, not all of the coating layer 3 of intermediate structure 4
needs to be covered by the inert material 7. Those areas of the coating layer 3 which are not
covered are then available for use in making electrical connection to the tips 6.
Therefore, in a fourth aspect, the present invention provides a method for the formation of a
structure capable of functionalisation as an array according to the first aspect of the invention,
the method including the steps of taking the intermediate structure 4 according to the second
aspect of the invention and filling individual spaces 5 between the tips 6 of the 3D pattern on the
intermediate structure 4 with an inert material 7 to give an inert surface 8 through which the tips
6 protrude or are otherwise exposed, said tips 6 forming functionalisable areas. Thus an array
including a continuous 3D surface layer with isolated but interconnected, preferably conductively
interconnected, functionalisable areas in an accurately defined pattern is formed.
Optionally, where the functionalisable areas protrude, the tips 6 of the protruding areas can be
cut away to align with the surface 8 of the inert material 7 to form a 2D surface including isolated
but interconnected functionalisable areas. Optionally, a portion of the inert material is also
removed.
Alternatively, in the second method the inert material 7 can be added in sufficient amount to
cover the functionalisable areas or tips 6 of the 3D pattern on the intermediate structure 4. The
inert material 7 is then partially removed (by etching, abrasion, chemical or plasma techniques)
to expose the functionalisable areas or tips 6 of the 3D pattern on the intermediate structure 4.
This method provides a means of obtaining a 2D surface, as the functionalisable areas or tips 6
do not protrude above the inert material 7. The exposed functionalisable areas or tips 6 are
isolated from each other by the inert material 7 but remain interconnected, preferably
conductively interconnected, within the structure by coating layer 3 of the intermediate structure
4.
Therefore, in a fifth aspect, the present invention provides a method for the formation of a 2D
microarray structure of the present invention, the method including the steps of taking the
intermediate structure 4 and covering the 3D pattern on the intermediate structure 4 with an
inert material 7 , and removing sufficient of the inert material 7 to only expose the
functionalisable areas or tips 6 of the 3D pattern.
The array formed by either of the above methods includes an intermediate structure 4 formed
from a substrate material 2 (which is inert) and 3D patterned to a millimeter to nanometer scale,
a coating layer 3 over at least part of the patterned area to form a continuous 3D surface, and a
layer of inert material 7 which is layered over the continuous 3D surface and fills spaces 5
between functiona!isable areas or tips 6 in the 3D pattern on the intermediate structure 4 to give
an inert surface 8 through, or from, which the functionalisable areas or tips 6 of the 3D pattern
protrude or are otherwise exposed. As indicated above, the functionalisable areas or tips 6 are
isolated by the inert material 7 but are continuously interconnected via the coating layer 3
(continuous 3D surface) which is present over at least part of the 3D patterned area (preferably
substantially a l of the patterned area).
As shown in Figure 4A, electrically isolated groups of arrays can also be formed using the
above methods in combination with a process of laser scribing, wherein the coating layer 3
(continuous 3D surface) between individual functionalisable areas or tips 6 is etched out (as
depicted by 20 in Figure 4B and C, Figure 5A and B and Figure 6F). This allows a single sensor
chip to have individually addressable areas which could include a counter electrode(s), a
reference electrode(s), a redox electrode(s) and working electrode(s). There is no restriction to
the shape of the lasered lines. However, it is preferable that the width of the lasered lines is
between about 1 to about 100 micron. The individually addressable areas or isolated microelectrode
arrays can be configured in a number of ways, two examples of which are shown in
Figures 4B and 4C. In Figure 4B, the micro-electrode array includes two working electrodes
21a and 2 b, separated by a counter electrode 22, and a reference electrode 23. In Figure 4C,
the micro-electrode array includes three working electrodes (21a, 21b and 2 c), a counter
electrode 22, a reference electrode 23 and a redox electrode 24, wherein the counter electrode
separates working electrodes 21a and 21b and the reference electrode 23 and redox electrode
24 together separate working electrodes 2 b and 21c. The isolated micro-electrode arrays may
also be arranged such that each functions as a working electrode 21.
In the third method, the coating layer 3 (Figure 3C) acts as a seed layer. The intermediate
structure 4 is placed into an electroplating bath to electrochemically grow the coating layer 3
(continuous 3D surface). It is therefore preferable that the coating layer 3 (continuous 3D
surface) is electrically conductive and the metal employed is capable of being electrochemically
deposited onto the coating layer 3 (continuous 3D surface) where this method is employed. The
coating layer 3 (continuous 3D surface) is electrochemically grown to form a metal layer 9 that
at least substantially covers the functionalisable areas or tips 6 of the intermediate structure 4
(Figure 3D). The metal layer 9, which includes, and therefore incorporates, the coating layer 3
of intermediate structure 4, is then separated from the remainder of the structure 4 to give a
metal negative 3D pattern 0 (a negative of the pattern on structure 4 (Figure 3E)). Individual
spaces 1 between the functionalisable areas or tips 2 in the negative 3D pattern on the metal
negative 0 are then backfilled with an inert material 7 to give a flat surface 8 through, or from,
which the tips 12 of the metal negative 10 protrude or are otherwise exposed (Figures 3H and
3G). Again, when used in this manner, the inert material 7 essentially acts as a filler material or
an isolation layer. Also, where the tips 12 protrude from the inert material 7, they may be cut
away to align with the surface 8 of the inert material 7 to form a 2D surface including isolated
but interconnected functionalisable areas. The functionalisable areas or tips 12 are isolated by
the inert material 7 and are capable of being functionalised as desired to form functional areas
(e.g. sensor or catalytic sites) in an array form.
Thus, in a sixth aspect, the present invention a method for the formation of an array which
includes a continuous 3D surface layer with an accurately defined 3D pattern of functionalisable
areas in the millimeter to nanometer scale, said method including the steps of:
electroplating the continuous 3D surface layer of the intermediate structure 4 to form a
metal layer 9 that covers the tips 2 of the 3D pattern (preferably at least substantially all
of the 3D pattern) on the intermediate structure 4;
separating the metal layer 9 and the substrate material 2 of the intermediate structure 4
to form a metal negative structure 10 which includes a negative of the 3D pattern
("negative 3D pattern") on the intermediate structure 4;
backfilling spaces 11 between tips 1 within the negative 3D pattern on the metal
negative structure 0 with an inert material 7 to give an inert surface 8 through, or from,
which the tips 2 of the negative 3D pattern protrude or are otherwise exposed.
Any metal can be employed in the electroplating step to form the metal layer 9. The use of
Nickel is preferred as it is a hard and ductile metal and is commonly used as a plating metal.
Figure 3G shows a diagrammatic top view of the functionalisable array formed by the filling of
individual spaces 1 between functionalisable areas or tips 12 in the negative pattern on the
metal negative 10 (Figure 3H). Thus, the metal negative 10 acts as the substrate material layer
of a microarray structure. The present invention may therefore extend to a functionalisable
array when formed by the method of the sixth aspect of the present invention.
Therefore, the present invention also provides a microarray with a conductive base which is
capable of being coated with a continuous 3D surface (conductive or non-conductive) and/or an
inert material 7 to form isolated functionalisable areas.
As will be appreciated, the methods described above for using the intermediate structure 4
(Figure 3C) result in an array having the same top view shown in Figure 3G.
The ability to create accurately defined arrays at the millimeter to nanometer scale has been an
issue in the array field for some time. The small sizes at issue, particularly in the nanometer
scale, present particular problems when seeking to obtain accurate quantitative and/or
qualitative analyses. The present invention provides an economic approach to the creation of
such arrays.
As is clear from Figures 3F and 3H and as discussed above, the isolated functionalisable areas
(formed by tips 6 and 12) in the array are interconnected below the inert material 7 via a
continuous 3D surface (i.e. coating layer 3 or metal negative pattern 10). Where the continuous
3D surface is formed from a conductive material (for example, gold), the isolated
functionalisable areas are conductively interconnected with each other as discussed above and
therefore act as interconnected but isolated conductive islands. Also, as indicated above, the
continuous 3D surface can be laser scribed or otherwise cut into individual sections such that
individually isolated blocks of functionalisable areas are formed within a wider array structure.
The use of a conductive material allows the arrays formed by the methods of the present
invention to be functionalised to form micro-electrode arrays as discussed above. Therefore,
the entire array can act as a single micro-electrode. Alternatively, the array can include multiple
individual electrodes where the continuous 3D surface has been cut into isolated blocks, The
interconnection also allows efficiencies of charge functionalisation of the isolated sites in the
micro-electrode array. Micro-electrode arrays can be of a variety of types as shown in Figure 6,
including:
- microdisk electrode arrays, on which the arrays may be arranged in a ordered or random
fashion;
- microband electro arrays;
- inter-digitated microelectrode arrays, which may be planar or vertical;
- linear microelectrode arrays; and
- 3D microelectrode arrays.
When functioning as a micro-electrode array, the continuous 3D surface is connected to an
electroanalytical device, electrical contact is made with an electrolytic solution and current is
allowed to flow through the solution. Target species in the electrolytic solution bind to the
functionalised areas of the microarray and therefore aid or impede current flow. In this way, the
target species are "sensed" by the micro-electrode array. Capture agents that are specific to
the target species can also be appended to the functionalisable areas of the micro-electrode
array to aid in this interaction. Individual micro-electrode arrays may also be used as counterelectrodes
to each other, whereby a current is passed between individual functionalised areas
on each and the current is measured.
The electrical communication achieved by use of a conducting continuous 3D surface (coating
layer 3) also allows the arrays of the present invention to provide insight into the redox
environment of a sample passing over the surface of the array. For example, the arrays can be
used to ascertain whether the redox environment of the sample is oxidative or reductive
(therefore allowing for the establishment of the likes of anti-oxidant response elements), or
whether there are peroxides present or radicals present.
The use of techniques such as laser scribing or lithography to cut the continuous 3D surface
(coating layer 3) into individual isolated blocks or areas also imparts on the microarrays of the
present invention the ability to function as multiplexing arrays, wherein simultaneous testing or
measurement of multiple analytes or biomarkers can be conducted (Figure 6F). Such a system
could be used to detect known biomarkers relevant to a specific disease, organ or system. This
also allows the user to isolate a known number of sensor sites for different purposes.
Where the user wishes to create non-conducting (or otherwise non-functionalised) isolated
areas, a non-conductive coating material 3 can be employed or the metal negative 0 can be
coated with a non-conducting layer.
The isolated functionalisable areas (identified at 6/ in Figure 3G) can also be used in a
number of other array applications. For example, they may be functionalised to act as catalysts
in a variety of micro reactions, or to act as sensors for various target biomo!ecules or
compounds of interest. Other suitable uses will be known to those skilled in the art. The means
to functionalise the areas would also be well known to a skilled person once in possession of
this invention.
Thus the present invention provides a structure including a continuous 3D surface which is
capable of functionalisation for use as an array, the structure including accurately defined and
functionalisable isolated areas which are millimeter to nanometer in size. The functionalisable
areas are isolated by an inert material 7 (which may also act as an insulator) but are
continuously interconnected within the structure. Preferably, the functionalisable areas are
continuously interconnected by a 3D coating layer 3 within the structure. Preferably, the
functionalisable areas are electrically conductive.
The present invention also provides an intermediate structure 4 including a continuous 3D
surface capable of functionalisation for use as an array, wherein the intermediate structure 4
includes an accurately defined 3D pattern at the millimeter to nanometer scale on at least one
surface. The intermediate structure 4 also includes an inert material 7 between the tips 6 of the
3D pattern which creates a surface 8 through which the tips 6 of the 3D pattern protrudes or are
otherwise exposed, the tips 6 of the 3D pattern thus being isolated by the inert material 7 and
being interconnected within the intermediate structure 4 by the continuous 3D surface.
Reference to "accurately defined" means that the 3D pattern (or 2D pattern) includes a known,
pre-determined (or calculatable) number of functionalisable areas in a known pattern. It is of
course possible for the pattern to be randomised. Accuracy also includes the concept that the
size and/or position of the functionalisable areas are pre-determined and accurately included in
the structure.
As indicated above, the microarrays of the present invention are suitable for use as microelectrode
arrays, microcatalyst arrays, and sensors for various target biomolecules or
compounds of interest.
When a microarray of the present invention is used as a micro-electrode array (and therefore
includes a conductive continuous 3D surface layer), the array would be functionalised by
attaching a capture agent that is specific for the target analyte. Examples of suitable capture
agents include small molecules, antibodies, and single stranded DNA. Other capture agents
would be known to those skilled in the art. There are numerous methods for attaching the
capture agent to an array. Methods of attachment typically include initial attachment of a linker
molecule with a terminal carboxyl or amino group, onto which the capture agent is bound using
standard binding methods, as would be known to those skilled in the art and are also discussed
in related co-pending PCT application number PCT/201 1/000052, the disclosure of which is
included by way of reference. Suitable applications for the use of a micro-electrode array of the
present invention include detection of small molecule biomarkers, proteins, DNA/RNA and
organisms.
Electrical connection to a micro-electrode array of the present invention is typically achieved by
attaching a clip or pressing a conductor (conductive paste, wire, ribbon) against the part of the
electrode which is not in contact with the solution to be passed over the surface of the microelectrode
array.
When a microarray of the present invention is used as a microcatalyst array, it is essential that
suitable binding chemistry is first attached to the functionalisable areas of the array. The
attachment of suitable binding chemistry may be achieved in a number of ways including,
electrochemical deposition of the binding chemistry where a conductive continuous 3D surface
layer is used, and exposure of the functionalisable areas to suitable functional groups. Suitable
functional groups may include a metal catalyst (for example, platinum or palladium), DNA, and
conducting polymers such as polypyrrole and polythiophene. The different surfaces of the
microcatalyst arrays so created react with the species in solution to a greater or iesser extent.
The combination of responses allows the solution to be characterised electrochemical!y.
Examples
Example One: Computer Modelling of Micro-electrode Sensors
A series of computer modelling experiments were carried out on a single functionalisable area
or tip of two individual micro-electrode arrays of the present invention, wherein the two microelectrode
arrays 25a and 25b were precisely aligned over each other as shown in Figure 8.
Thus, each micro-electrode array 25a and 25b was acting as a counter-electrode to the other.
The aim of these experiments was to calculate the impedance profile for different shapes and
sizes of condensed droplets formed in between the tips. This was dependant on the
concentration of the buffer solution and the size and shape of the droplet formed between the
electrodes. The total impedance between two electrodes is the sum of the impedance at the
electrode-electrolyte interface and the impedance of the electrolyte solution. In order to
measure the impedance changes due to changes in the geometry of the system, the total
impedance of the system was simulated by solving the modified Laplace's equation for two
different geometries. The interface impedances were assumed to remain constant for different
geometries and the interface reactions were not considered in the model.
An AC potential of 1 V was applied between the upper and lower electrodes and total
impedance was calculated from the current distribution for a frequency range of 1 KHz to 1x106
KHz. The buffer was considered as a solution with conductivity of 0.02S/m and a relative
permittivity of 80. The results are illustrated in Figures 9 to 14 which model three parameters
including the distance between the electrodes, the area of the electrodes, and the volume of the
electrolyte (as either a droplet between the tips or as a solution that completely covers the tips).
In summary, the results showed that sensitivity is inversely related to both the distance between
the electrodes and the electrode area, but not significantly affected by the volume of the
electrolyte at the micron scale. In Figure 9, two different tip geometries are shown. In Figure
9A droplet formation is shown while Figure 9B shows a chamber filled with an electrolytic
solution. Impedance results are shown by the potential distribution (Figures 9 and 10). The
arrows indicate current density vectors in the 2D and 3D domain.
Figures 1 to 13 show various impedance measurements, while Figure 14 shows frequency vs.
impedance for two different geometries.
For bio-sensing applications, the electrodes were assumed to be functionalized with capture
agents and then the impedance was measured before and after the capture. The change in the
impedance was primarily due to changes at the electrode interface. The equivalent circuit
model of the interface can be given by the Randle's circuit (neglecting the Warburg element due
to diffusion of ions at the interface). The total equivalent circuit of the system with the interface
and the solution impedance is illustrated in Figure 15, and the domain equations and boundary
conditions are shown in Figure .
Variations in double layer capacitance (the ability of a body to store an electric charge) are
measured using Non-faradaic electrochemical impedance spectroscopy (EIS). This involves
neglecting any changes due to redox reactions and measuring the capacitance changes due to
changes in the double layer thickness. In order to determine the total impedance change of the
system due to changes in double layer thickness, the model was simulated for various double
layer thickness (Ddl) (Figures 17, 18 and 9). For all the cases charge transfer resistance (Ret)
equates to 1 ohm. The results indicated that the droplet is only slightly more sensitive than
using a completely submerged sensor tip.
The second way to detect the changes at the interface is by measuring the redox reaction at the
interface. When there is a change in the interface due to biological capture agents, the rate at
which the redox reaction takes place changes. This changes the current at the interface, which
consequently changes the Ret of the system. The Ret values vary for various different
interfaces. Impedance changes of the system were simulated for various Ret. Results are
illustrated in Figures 20, 2 1 and 22 and show that a droplet provides slightly better sensitivity at
lower frequencies.
The computer modelling experiments showed that the smaller the dimensions of the tips, and
the gaps between the tips, the greater the sensitivity of the sensor array.
The design was also simplified to arrange working and counter electrodes side-by-side via interdigitation
patterning, thus enabling the different electrodes of an electrochemical set up (for
example, working electrodes, counter electrode and reference electrode) to all be placed on the
same micro-electrode array sensor chip. The electrochemical interactions of side-by-side
electrode protrusions were modelled and similar results were observed to that of the microelectrode
array set up shown in Figure 8. Therefore, in summary, impedance measurements
relate to the distance between working and counter electrodes and the diameter of the working
electrode (Figure 34).
Example Two: Formation of Array Substrates, Intermediate Structures and Materials
The above description describes different approaches to the formation of arrays of the present
invention having isolated functionalisable areas or tips. In Figure 3F a thin layer of an inert
material 7 has been deposited over a continuous 3D metal surface to sit between individual tips
of a 3D patterned substrate material 2. In Figure 3H a thick layer of metal 9 has been deposited
over and onto the continuous 3D surface of a substrate material 2. However, in both,
functionalisation occurs predominately at the tips of the 3D pattern of the microarray.
The following describes the development of microarrays according to the fourth, fifth and sixth
aspects of the present invention.
Development of Arrays According to the Fourth and Fifth Aspects:
Figure 23 shows a photograph of 40 micron tips embossed into PMMA and which are evenly
spaced at 100 micron intervals and are 00 microns in height. PMMA (an amorphous polymer)
is a preferred substrate materia! for use in the present invention as it is easily processed and
gives highly defined three-dimensional substrate surfaces.
The process for fabricating the sensor (Figure 24) includes the following steps:
1. Gold coating the substrate to form a continuous 3D gold surface (or coating layer 3);
2. Depositing an inert material 7 between individual tips;
3. Attaching binding chemistry (-X) onto just the tips; and
4. Attaching Haptan species onto the tips.
1. Gold coated polymer substrate fabrication and cleaning
A thin layer of chromium (2 to 3 mm in thickness) was deposited onto the PMMA substrate to
act as an adhesion layer. Vanadium can also be used in place of chromium as an adhesion
layer. Likewise, amine and thiol chemicals are known to promote adhesion of gold to a surface.
Gold was then sputtered onto the PMMA substrate to give the electrode with a continuous 3D
gold surface depicted in Figure 25. The electrode was cleaned electrochemically by holding it at
1.65V vs. Ag/AgCl for 5s in 0.5M H S0 , and then cycling between OV to .65 V. Figure 26
shows a typical CV, and a stable gold oxidation and reduction peak at 1.15 V and 0.9V
respectively, thus providing support that gold had been deposited onto the PMMA substrate.
The gold coating layer 3 so formed was between about 7 to about 40 nm thick. Gold tracks
were then defined into the gold by laser scribing. An example of inter-digitated tracks 20 is
shown in Figure 27 (and is depicted as 20 in Figures 4B and C, Figure 5A and B and Figure 6F).
The lasered pattern electrically isolates individual areas of the microarray from each other,
resulting in the formation of more than one electrode in a single microarray.
2. Depositing an inert material 7 between individual tips
Three separate methods were used to deposit the inert material onto the continuous 3D gold
surface so as to act as an isolation layer between the tips:
A. Deposition of a photoresist layer (SU-8) over the entire structure, followed by reactive
ion etching to expose the gold tips;
B. Deposition of a hydroxylated self-assembled monolayer (SAM-OH) over the entire
structure and physical removal of the tip region by rubbing; and
C. Coating the gold coated substrate with a paint layer of suitable viscosity so as to run off
the tips before cross-linking. This resulted in the valleys between the tip being filled and
the gold surface on the tips left exposed.
All three methods resulted in the gold tips protruding out of the inert material.
A. Deposition of a SU-8 Photoresist Layer
A 00 micron thick layer of SU-8 was applied to a 0 cm wafer of gold coated substrate which
had been previously laser scribed into 1 cm inter-digitated sensor chips (Figure 5C). The SU-8
was then cross-linked under ultra violet light and controllably etched by reactive ion etching.
This was found to give very good control of the thickness of the SU-8 polymer layer and also
very clean gold tips. Figure 35B shows two adjacent tips. The tip shown on the right hand side
has bare gold, while the tip shown on the left hand side has had a carboxylated
polyterthiophene layer electrochemically deposited onto the gold. Figure 35A shows adjacent
tracks of an inter-digitated array in which alternating tracks have carboxylated poiytherthiophene
deposited onto the tips.
B. Deposition of a Self-Assembled Monolayer (SAM)
Self-assembled monolayer's (SAM) are well known in the art and typically form spontaneously
on a substrate by chemisorption of "head groups" onto the substrate followed by slow
organisation of "tail groups".
Terthiophene substituted with an alkyl alcohol and HS(CH )6OH are examples of suitable
hydroxylated self-assembled monolayer's for use in the present invention.
The continuous 3D gold surface of micro-electrodes were subjected to cyclic voltammetry in
Potassium ferricyanide (K3FeCN ) solution. The CV showed a standard ferricyanide oxidation
and reduction peak at 0.35 V and 0.15 V respectively. The electrodes were then immersed in
solution containing 5mM SAM-OH in 1:1 ethanol/water solution. The electrodes were then
taken out periodically and washed with water and subjected to cyclic voltammograms in 5mM
Potassium ferricyanide solution with KCI supporting electrolyte (Figure 28). The CV showed
gradual disappearance of the ferricyanide peak as the length of time the electrodes were
immersed in the SAM solution was increased indicating the gradual adsorption of SAM onto the
continuous 3D gold surface of the electrodes.
After a period of 20 minutes the current reached a steady state value showing that the
electrodes were saturated with SAM. The CV of the electrode after 20 minutes of SAM
adsorption had similar characteristics as the CV of the electrode after 2 hours of SAM
adsorption.
Physical removal of the SAM-OH by rubbing the tips on a glass microscope slide resulted in the
gold on the tips being exposed. CV in 5mM Potassium ferricyanide solution showed the typical
reduction oxidation peak at significantly reduced current. This indicated that only the gold on
the tips was exposed.
The SAMs can be removed from the gold-coated substrate, allowing the substrate to be freshly
coated with a new SAM. Thus, the arrays of the present invention can be used a number of
times without degradation of the array.
C. Coating the gold coated substrate with an epoxy coat
Figure 29 shows the gold coated substrate material with a layer of epoxy in the valleys between
the tips. The consistency of the epoxy layer provided sufficient time for the epoxy to run off the
gold tips prior to cross-linking.
CV in 5mM Potassium ferricyanide solution showed the typical reduction oxidation, and was
evidence that the gold tips were uncoated.
3. Attaching the binding chemistry (-X) onto the tips
The binding chemistry (-X, c.f. Figure 24) was attached to the tips of the SU-8, SAM-OH or the
epoxy coated continuous 3D gold surface of the substrate. Where SU-8 or an epoxy coat was
employed as the inert material 7, attachment of the binding chemistry was achieved by
electrochemically depositing carboxylated polytherthiophene or animated polyterthiophene onto
the tips (Figure 30B and C). While Figure 30C shows the controlled electrochemical deposition
of different probes to conducting polymers for multiplexing of capture agents, this can also be
achieved using a carboxylic SAM and altering the potential of the different working electrodes as
would be apparent to those skilled in the art. Where SAM-OH was employed as the inert
material 7, attachment of the binding chemistry was achieved by exposing the tips to SAMCOOH
(Figure 30A). In each case, this resulted in the attachment of either a -COOH or -NH2
group at the end of each of the tips. Use of terthiophene (or pyrrole) substituted with a carboxyl
terminated side chain also allows the binding group to be added selectively at a defined tip as it
can be electrochemically polymerised on those tips.
To test the selectivity of the process for attaching the binding chemistry onto the tips, 1 micron
aminated polystyrene beads were covalently attached via the appropriate linker chemistry.
Figure 3 1 illustrates the process for the amine functionalised tips (A). The aminated substrate
was exposed to a bi-functional linker solution (6 g linker/0.5 ml PBS), and shaken at room
temperature for 45 minutes. After washing, the substrate was immersed into a solution
containing a blue H2N-Bead solution (30 mI beads suspension in 0.5 ml PBS), and shaken at
room temperature for 1 h . Attachment of the blue beads onto the array of tips (A) and onto a
single tip (B) is shown in Figure 32. Visual or electrochemical (for example resistance, CV or
impedance) techniques can be used to detect what is bound to the arrays.
4. Attaching the Haptan Species onto the tips
Once confirmation that the attachment chemistry had been bound to the tips of the array,
standard linker chemistry could be used to attach a variety of haptans including, but not limited
to, antibodies, DNA and cells.
As an illustration, the following shows the use of the method to fabricate a sensor for
Progesterone (P4). The steps include:
. Attaching P4 onto ovalbumin to form a conjugate;
Ovalbumin P4 conjugate
Immersing NH2 substituted arrays into a bi-functional linker solution (6 mg linker/0/5 ml
PBS) and shaking at room temperature for 45 minutes to give an activated array (I);
Adding P4-PEG-OVA (0.4 ml solution in 0.2 ml PBS) and allowing it to react for two
hours with shaking to give the Haptan functionalised array (I ) ; and
Exposing the array to the P4 primary antibody, and then a secondary antibody with
attached beads (III). The attachment of the secondary antibody bead conjugate allows
the successful bonding of the primary antibody to be visually confirmed.
ll
Development of Arrays According to the Sixth Aspect:
Arrays according to the sixth aspect of the present invention were prepared by sputtering a thin
layer (7nm) of gold onto a polymer substrate, electroplating a thick layer (1mm) of nickel onto
the gold, separating the nickel and polymer layers and sputter coating the nickel with gold.
Figure 33 shows an SE of a 1micron gold coated nickel array.
The process provided a substrate which had a gold surface similar to that of the arrays formed
according to the fourth and fifth aspects of the present invention and as shown in Figure 25, and
which could be coated in an identical manner with an inert material.
The arrays according to the sixth aspect of the present invention have the advantage of being
more robust due to the thickness of the metal base. Also, the metal layer can be laser scribed
to isolate groups of tips for selective functionalisation of isolated areas. For the production of
sensors using electrochemical detection this ability to scribe is an advantage as it allows the
electrodes and the spacing between those electrodes to be defined on a single chip and down
to the accuracy of the laser. This approach is widely used in the fabrication of a wide range of
electrochemical sensors including those for monitoring glucoses for diabetes, and dramatically
simplifies the mass-production.
Using the above described process, arrays including 3 micron tips (25 micron at their base), 0.2
micron tips ( 1.5 micron at their base and 0 n tips (160 nm at their base) have also been
produced. It has been found that the smaller tip size is favoured with respect to sensitivity of the
arrays.
Example Three: Use of a Single Microarray in Multiplexing Assays
Laser scribing was utilized to isolate individual micro-electrodes of an array to form an
electrochemical version of the typical DNA or RNA microarray (Figures 5, 27 and 35). Groups
of micro-electrodes were also isolated to form smaller micro-electrode arrays within a larger
array, thus constituting a platform of multiple working electrodes, reference electrodes and
counter electrodes. This enabled multiplexing on a single sensor chip or array (as depicted in
Figure 6F).
For example, one sensor chip design was functionalised with different capture agents on each
of eight working electrodes to constitute a liver panel on one chip. The antibodies used had
affinity for ALT, AST, ALP, GGT, LDH, Hep A, Hep B x-antigen, and full length Hep C E2 protein
on working electrodes 1 to 8, respectively. An enzyme, glucose oxidase was tethered to
working electrode 9 for detection of serum glucose. The tenth working electrode was a redox
electrode to measure non-adhered bilirubin concentration in solution.
In another example, one working electrode array was functionalised with an RNA complement
for the srm gene messenger RNA, a second working electrode was functionalised with 3'UTR of
srm for targeting microRNA detection, and a third working electrode was functionalised with an
antibody raised against the srm gene product spermadine synthase.
The above describes the formation of arrays on a substrate material, including functionalisable
areas that are accurately defined in desired patterns and/or shapes at a milli- to nano-meter
scale.
The foregoing describes the invention including preferred forms thereof. Modifications and
alterations as would be readily apparent to a person skilled in this particular art 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".
CLAIMS
. A microarray structure including a substrate material layer, a continuous threedimensional
(3D) surface layer on the substrate material layer that is capable of
functionalisation for use as an array, and an inert material;
wherein the structure includes accurately defined and functionalisable isolated areas
which are millimeter to nanometer in size; and
wherein the functionalisable areas are part of the continuous 3D surface layer and
are isolated by the inert material but which are interconnected within the structure by
the continuous 3D surface layer.
2. A microarray as claimed in claim 1, wherein the continuous 3D surface layer is
formed from an electrically conductive material or a carbon based material.
3. A microarray as claimed in claim 1 or claim 2, wherein the continuous 3D surface
layer is a unitary layer that covers the substrate material layer.
4. A microarray as claimed in claim 1 or claim 2, wherein the continuous 3D surface
layer is cut into a plurality of isolated continuous 3D surface layer segments on the
substrate material layer, each segment including a plurality of functionalisable areas,
wherein each group of functionalisable areas is capable of separate
functionalisation.
5. A microarray as claimed in any one of the previous claims, wherein the inert materia!
is also an insulating material.
6. A microarray as claimed in any one of the previous claims, wherein the substrate
material layer is formed from a conductive material or a non-conductive, inert
material.
7. A microarray as claimed in any one of the previous claims, wherein the microarray
structure includes an adhesion layer between the continuous 3D surface layer and
the substrate material layer.
8. A microarray as claimed in any one of the previous claims, wherein the microarray
structure is capable of being functiona!ised as a micro-electrode sensor array and/or
a microcatalyst array.
9. A microarray as claimed in any one of the previous claims, wherein the continuous
3D surface layer protrudes from the inert material such that the functionalisable
areas are exposed above the inert material.
10. A microarray as claimed in any one of claims 1 to 8, wherein the inert material and
the functionalisable areas form a two-dimensional (2D) surface including exposed
functionalisable areas.
11. An intermediate structure for use in fabricating an array according to claim 1,
wherein the intermediate structure includes a substrate material layer that includes
an accurately defined 3D pattern to a millimeter to nanometer scale, and a
continuous 3D surface layer on the substrate material layer that is capable of
functionalisation for use as an array over at least part of the pattern.
12. An intermediate structure as claimed in claim 11, wherein substantially all the
patterned area is coated with the continuous 3D surface layer.
13. An intermediate structure as claimed in claim 1 or claim 12, wherein the substrate
material layer is conductive or non-conductive.
14. An intermediate structure as claimed in any one of claims to 13, wherein the
continuous 3D surface layer is formed from an electrically conductive material or a
carbon based material.
15. An intermediate structure as claimed in any one of claims to 14, wherein the
intermediate structure includes an adhesion layer between the continuous 3D
surface layer and the substrate material.
6. A method for the formation of an intermediate structure according to claim 11,
wherein the method includes the steps of:
a. placing an accurately defined 3D pattern at the millimeter to nanometer scale on
the surface of a substrate material; and
b. coating at least part of the patterned substrate material with a continuous 3D
surface layer.
A method as claimed in claim 16, wherein the pattern is placed on the surface of the
substrate material by embossing, casting, stamping, etching, grinding, lithography,
pressure forming, vacuum forming, roll forming, injection moulding and laser scribing
/ ablation.
A method as claimed in claim 6 or claim 17, wherein the continuous 3D surface
layer is applied to the substrate material by sputtering, evaporation or e!ectro!ess
deposition techniques.
A method as claimed in any one of claims 6 to 18, wherein the continuous 3D
surface layer covers substantially all of the patterned area of the substrate material.
A method as claimed in any one of claims 16 to 18, wherein the continuous 3D
surface layer is cut into a plurality of isolated continuous 3D surface layer segments,
said plurality of segments covering substantially all of the patterned area of the
substrate material.
A method as claimed in any one of claims 16 to 20, wherein the method includes the
step of adding an adhesion layer between the substrate material and the continuous
3D surface layer.
A method for the formation of a structure capable of functionalisation as an array
according to claim 1, the method including the steps of taking the intermediate
structure as claimed in any one of claims 1 to 15 and filling individual spaces
between the tips of the 3D pattern on the intermediate structure with an inert material
to give a surface through, or from, which the tips of the 3D pattern protrude or are
otherwise exposed; wherein the tips form functionalisable areas which are isolated
by the inert material but are interconnected within the structure by the continuous 3D
surface layer and are capable of functionalisation.
A method as claimed in claim 22, wherein the tops of the tips can be cut away to
align with the surface of the inert material to form a 2D surface including
functionalisable areas.
24. A method for the formation of a 2D structure capable of functionalisation as an array,
said structure including a continuous 3D surface and having an accurately defined
3D pattern of functionalisable isolated areas in the millimeter to nanometer scale, the
method including the steps of taking the intermediate structure according to any one
of claims to 15 and covering the 3D pattern on the intermediate structure with an
inert material, removing sufficient of the inert filler material to expose the tips of the
3D pattern, wherein the exposed 3D tips are isolated by the inert material but are
interconnected within the structure by the continuous 3D surface and are capable of
functionalisation.
25. A method for the formation of a structure capable of functionalisation as an array,
said structure including a continuous 3D surface layer and having an accurately
defined 3D pattern of functionalisable areas in the millimeter to nanometer scale, the
method including the steps of:
a. electroplating the continuous 3D surface layer of the intermediate structure
according to any one of claims to 15 to form a metal layer that covers the tips
of the 3D pattern on the intermediate structure;
b. separating the metal layer and the substrate material of the intermediate
structure to form a metal negative structure which includes a negative of the 3D
pattern (the "negative 3D pattern") on the intermediate structure;
c. backfilling spaces between tips within the negative 3D pattern on the metal
negative structure with an inert material to give an inert surface through, or from,
which the tips of the negative 3D pattern protrude or are otherwise exposed;
wherein the functionalisable areas are isolated by the inert material but are
interconnected within the structure.
26. A method as claimed in claim 25, wherein the metal layer covers at least
substantially all of the 3D pattern on the intermediate structure.
27. A method as claimed in claim 25 or claim 26, wherein the tops of the tips can be cut
away to align with the surface of the inert material to form a 2D surface including
functionalisable areas.
28. An intermediate structure including a continuous 3D surface capable of
functionalisation for use as an array, wherein the intermediate structure includes an
accurately defined 3D pattern at the millimeter to nanometer scale on at least one
surface and also includes an inert materia! between the tips of the 3D pattern which
creates a surface through, or from, which the tips of the 3D pattern protrude or are
otherwise exposed, the tips of the 3D pattern thus being isolated by the inert material
and being interconnected within the intermediate structure by the continuous 3D
surface.