NZ605110B2 - Arrays and methods of manufacture - Google Patents

Abstract

Disclosed is a microarray structure including a substrate material layer (2), a continuous three-dimensional (3D) surface layer (3) on the substrate material layer that is capable of functionalisation for use as an array, and an inert material (7). The structure includes accurately defined and functionalisable isolated areas (20) 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. ionalisable isolated areas (20) 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.

Description

OSBNZPR 302858432 ARRAYS AND METHODS OF CTURE 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 y of patterns. The invention also relates to such .

OUND ART There are currently a number of known methods for fabricating arrays. These include ng ques 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 tely define the arrays over a large e 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 detrimentaliy affected. In particular, issues of cost arise with the fabrication of ale 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 cy 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 on tor such as a metal or a semiconductor (the electrode) and an ionically conducting medium (the electrolyte) and which involve on transfer between the electrode and the electrolyte or species in solution. Catalysis concerns the on of a new reaction pathway with a lower activation energy, thereby allowing more reactant molecules to cross the reaction barrier and form reaction products.

VIASOSDSBNZPR 302868432 In a typical ochemical detection process it is, in general, able to employ an array of r electrodes as opposed to a single large electrode. Reasons for this include: 0 the ability to use smaller sample volumes; - application in both in vivo and in vitro measurement; a low depletion rate of target molecules; 0 low background charging due to their reduced surface area; 0 reduced iR drop; and - high current density arising from enhanced mass transport to the electrode surface as a result of convergent diffusion.

Accurately defined arrays would also be valuable for use in: - the is 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, 0 in vitro or in vivo biological sensing such as enzyme-linked assays and the ion of many other biomolecules; - catalysis; - trace metal ring in the environment; . corrosion ring; and 0 energy production and storage devices.

Co-pending PCT application number PCT/2011/000052 also concerns microarray structures.

However, the microarrays as described in PCT/2011/00052 simply include a continuous inert base substrate with onalisable areas isolated by an inert material. The functionalisable areas are not stated to be conductively onnected and the structures do not include at least one uous 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 functionalisabie areas and/or methods of forming such . 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 functionalisabie 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 SD e layer. in a particular embodiment, the invention provides a rray structure including a three dimensional (3D) substrate al layer, a continuous 3D surface layer on the substrate material layer, and an inert al; n the continuous 3D surface layer is capable of functionalisation for use as an array; and 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 iayer and are isolated by the inert material but are interconnected within the structure by the continuous 3D surface layer.

Preferably, the continuous 3D surface layer is electrically conductive. More ably, the 8D surface iayer is a metal.

Aiternatively, the continuous 3D surface iayer is a carbon based material, ing but not limited to carbon fiber, carbon paste, graphite, graphene, giassy carbon, carbon nanotubes and conducting poiymers.

Preferably, the continuous 3D surface iayer is a unitary layer that covers the ate material layer.

Preferabiy, 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 piurality of onalisable areas, wherein each group of functionalisable areas is capable of separate functionalisatlon.

Optionally, the inert al 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 ure 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 des from the inert material such that the functionalisable areas are exposed above the inert material. ably, 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 ion, the intermediate structure including a substrate material layer that includes an accurately defined 3D n 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.

In a further aspect, the invention provides an intermediate structure for use in fabricating an array, wherein the intermediate structure includes a 3D substrate material 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 e of functionalisation for use as an array over at least part of the pattern. ably, substantially all the patterned area is coated with the uous 3D surface layer. 3O Preferably, the substrate material layer is formed from a conductive material or a non— tive inert material which, optionally, is also an insulating material.

Preferably, the n is formed by embossing, casting, stamping, etching, grinding, lithography, pressure forming, vacuum forming, roll g, injection moulding and laser scribing iablation.

Preferably, the substrate material layer is coated with the continuous 3D surface layer by sputtering, evaporation or electroless deposition ques.

Preferably, the continuous 3D surface layer forms a coating layer which is electrically tive. More preferably, the BD g layer is a metal.

Alternatively, the continuous 3D surface layer is a carbon based material, including but not limited to carbon fiber, carbon paste, te, graphene, glassy carbon, carbon nanotubes and conducting polymers.

Optionally, the intermediate structure es 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 30 surface layer, said array formed from an 058NZPR 302868432 intermediate structure of the second aspect of the invention, n a layer of inert material fills the spaces between the tips in the 3D pattern on the surface layer to give an inert material e 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 ing a uous 3D surface layer from which an array having accurately defined and functionalisable ed 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 e of a substrate material; and b. coating at least part of the patterned substrate materiai with a uous 3D surface Iayen Preferably, the pattern is placed on the surface of the substrate material by embossing, casting, stamping, etching, grinding, lithography, re 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 eiectroless deposition techniques.

Preferably, the continuous 3D surface layer covers substantially all of the patterned area of the substrate material.

Alternativety, the continuous 30 e layer is cut into a plurality of isolated uous 30 surface layer segments, wherein the plurality of segments cover substantiaily all of the patterned area of the substrate al.

Optionally, the method includes the step of adding an adhesion layer between the substrate material and the continuous 3D surface layer.

TISSOBOSSNZPR 303747891 Preferably, an inert al is placed on the continuous 3D e 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 ediate 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 onalisable areas which are isolated by the inert material but are interconnected within the structure by the continuous 3D surface layer and are e of functionalisation.

In an embodiment of the fourth aspect, the present invention provides a method for the formation of an array according to the first aspect, the method including the steps of taking a 3D substrate material that includes a continuous SD surface layer and filling individual spaces between tips of the uous 3D surface layer with an inert material to give a surface through, or from, which the tips of the continuous 30 surface layer protrude or are otherwise exposed; wherein the tips define a 3D pattern to a millimetre to nanometer scale; and wherein the tips are isolated by the inert al 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 ntially 2D structure e of functionalisation as an array, said structure including a continuous 3D surface layer, the method including the steps of taking the ediate ure according to the second aspect of the ion and covering the 3D pattern on the intermediate structure 3O with an inert material, removing sufficient of the inert material to only expose the tips of the 3D pattern, wherein the exposed SD tips are isolated by the inert materiai but are interconnected within the structure by the continuous 3D surface layer and are capable of functionalisation.

In a sixth aspect, the present invention provides a further method for the formation of a structure capable of onalisation as an array including a uous 30 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: TlSSOSOfiBNZPR 303747891 a. electroplating the continuous 3D surface layer of the intermediate ure according to the second aspect of the present invention to form a metal layer that covers the tips of the BD pattern on the intermediate structure; b. separating the metal layer and continuous 3D surface layer from 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. lling 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 othenrvise exposed; d. wherein the functionalisable areas are isolated by the inert material but are interconnected within the structure.

In a further aspect, the present ion provides a method for the formation of a structure capable of functionalisation as an array, said structure including a 3D substrate material layer and a continuous 31'.) surface layer on the substrate material; wherein the ure further includes an accurately defined 3D pattern of functionalisable areas in the millimeter to ter scale, the method including the steps of: a. oplating the continuous 3D surface layer to form a metal iayer that covers tips of the 3D pattern; b. separating the metal layer and continuous surface layer from the substrate material to form a metal ve structure which includes a ve of the 3D pattern; 0. backfilling spaces between tips on the metal negative structure with an inert al to give an inert surface through, or from, which the tips protrude or are otherwise exposed; wherein the functionalisable areas are isolated by the inert al but are interconnected within the structure.

Preferably, the metal layer covers at least substantially all of the 3D pattern on the intermediate structure.

TISSOQOSBNZPR 303747891 Optionally, the tops of the tips can be cut away to align with the surface of the inert al to form a 2D surface including functionalisable areas.

In a seventh aspect, the present invention provides an intermediate structure including a uous 3D surface capable of functionalisation for use as an array, wherein the intermediate structure includes an accurately defined 30 pattern at the millimeter to nanometer scale on at least one surface and also includes an inert al between the tips of the 3D n which creates a surface through, or from, which the tips of the SD pattern protrude or are otherwise exposed, the tips of the 3D pattern thus being ed by the inert material and being onnected within the ediate structure by the continuous 30 surface.

DESCRIPTION OF FIGURES Figure 1: 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 e 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.

V|A509058NZPR 302868432 Figure 5: (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; (0) shows a 10 cm disc of sensor chips; and (D) shows a microelectrode array comprising isolated gold tips.

Figure 6: shows the typical types of electrode arrays. (A) shows a microdisk electrode array (could be ordered or random); (B) shows a microband electrode array; (C) shows an inter-digitated 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.

Figure 7: shows, in schematic form, (A) roller embossing of a substrate material (for example, a polymer or glass substrate material); (B) roller embossing of a ate material; and (C) embossing a substrate material using a stamp.

Figure 8: shows the sensor design for er modelling experiments in which two arrays of gold coated tips were precisely aligned over each other.

Figure 9: shows two different geometries. (A) shows droplet ion and (B) shows a chamber filled with electrolyte showing ial distribution and current density vectors (arrows) in 2D and SD .

Figure 10: shows a 3 dimensional rendering of impedance results.

Figure 11: shows impedance for various parameters of space between the 2 electrodes (10 and 20 micron), where r is the radius of the tip.

Figure 12: shows impedance for various parameters of distance.

Figure 13: shows impedance comparison between two distance ries. Red shows fully immersed geometry. Green and blue show the droplet formation with different radius, geometry.

Figure 14: shows Frequency vs. Impedance (Bode plot) for two different geometries, wherein the solid line shows ry 8, and the dotted line shows a droplet with radius 5 um.

VIASDSOSBNZPR 302868432 Figure 15: shows an equivalent t model for the whole system including the interface impedance.

Figure 16: shows model geometry along with domain equations and boundary conditions.

Figure 17: shows impedance st plot) for various double layer lengths of the interface for two different geometries.

Figure 18: shows a Bode plot showing difference in the Real Z (ohms) for two different double layer thickness.

Figure 19: 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 (Ftct) for the two different geometries.

Figure 21: shows change in the impedance due to change in Fiat for the fill geometry.

Figure 22: shows ence between the impedance due to change in Ftct from 1M 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. (8) shows a closer View of the light shining h 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 ment of a SAM via thiol~gold chemistry; (B) shows electrochemical deposition of a conducting polymer ed by coupling to a probe; (C) shows controlled electrochemical deposition of different probes to conducting polymers for multiplexing of capture agents.

Figure 31: shows a schematic diagram for attachment of the 1 micron ed 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 ating working electrode and a counter electrode with a laser scribe between.

Figure 36: shows attachment of P4 onto ovalbumin to form a conjugate.

Figure 37: shows steps in the production of a P4 sensor array.

ED DESCRIPTlON The present invention concerns the pment of arrays of various sizes for use in a variety of applications including sensors, eiectrochemistry and catalysis. In particular, the present invention relates to method for fabricating arrays comprising onalisabie areas at the millimeter to nanometer (inclusive) scale. These functionalisable areas are preferably tive (but may not be) and are isolated at the surface of the array butjoined 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 ion of enzymatic sed 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 g of their complimentary antigen via an associated electrochemical method including the measurement of changes in the resistance between the binding e 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 conduotive, continuous 3D, array surface.

VIA509058NZPR 302868432 The microarrays of the present invention may be said to broadly consist of a substrate material on which functionalisable 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 functionalisable areas which are part of the continuous 3D surface layer and are isolated by the inert material but are onnected within the structure by the continuous 3D surface layer.

As used herein, the “substrate material layer" (herein ed to as substrate material) refers to the base of the microarrays of the present invention. it may be flexible or rigid and is preferabiy planar ranging in thickness from the micrometer to eter scale. As will be known to a skilled person in the art, the ess 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. ore, 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 al layer is inert, it may also act as an ting material. Examples of suitable flexible als for use in the present invention include thermoplastic polyurethane, rubber, silicone rubber, and le epoxy. es of suitabie rigid substrate materials for use in the present invention include glass, PMMA, PC, PS, ceramic, resin, ite 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, “functionalisable areas" should be taken broadly to encompass those parts of the rrays of the present invention which protrude, or are otherwise exposed, h an inert material or are exposed and are therefore capable of being tunctionalised as desired by a user. When the inert material protrudes through the inert materiai, it may be exposed above that material. The functionalisable 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 t invention. However, a person skilled in the art would understand that the functionalisable areas can also form the uppermost surface of a 3D rib like structure formed as part of the substrate al of the microarrays of the present invention.

ViASOSOSSNZPR 302858432 Throughout the specification reference to 30 should be taken to mean a three-dimensional structure, or where required by context, a three dimensional coated structure, wherein, the three-dimensional ure is in the form of a pillar like structure or a rib like structure.

The functionalisable areas preferably range in size from the millimeter to the nanometer scale.

More preferably, the functionalisable areas are between about 10 nm to about 1 micron in size, more preferably between about 200 nm to about 1 micron in size. Likewise, the spaces between individual functionalisable areas can be on the millimeter to ter scale.

In one embodiment of the present ion, the functionalisable areas are accurately defined areas in that they form a defined pattern on the surface of a microarray to the scale d.

This, in turn, allows a user or a computer program to pinpoint specific functionalisable areas on the surface of a microarray and make a desired ement and allows for the functionalisation of only selected functionalisable areas on the surface of a microarray.

Alternatively, the functionalisable 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 n that is tely 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 lly made from silicon or . r, 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 18). 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 3O casting, stamping (Figure 7C), etching, ng, lithography, pressure forming, vacuum forming, roll g (Figures 7A and 7B), injection moulding and laser scribing / ablation. Other suitable methods for forming an accurately d 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 e 10). The coating step forms a continuous single 3D e over the substrate material 2.

OSBNZPR 302868432 As used herein, “continuous 3D surface layer” (herein referred to as continuous 30 surface) refers to the g 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 nuous 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 nuous 3D surface) is laser scribed or otherwise cut using techniques such as lithography to give a plurality of isolated uous 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 onalisabie areas is capable of separate functionalisation.

Preferabiy the coating layer 3 (continuous 3D surface) is formed from an ically tive 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, te, graphene, glassy carbon, carbon nanotubes and conducting rs such as polypyrrole and polythiophene. ation 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 tion. The continuous 3D surface may be used as a seed layer as will be described later herein.

The continuous SD surface (coating layer 3) typically includes an adhesion layer (not shown in Figure 1) to promote adhesion to the ate material 2. This adhesion layer therefore sits n 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 ent of the surface to increase surface roughness, tion 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 onal and structural flexibility over OSBNZPH 802868432 other microarrays known in the art. In particular, the continuous 30 surface may achieve any one of a number of important roles in the present invention. For example, it protects the ying substrate material 2 (Figure 1). It also promotes attachment of binding chemistry at the functionalisable areas of the microarrays of the present ion. When it is formed from a conducting material, it allows electrochemical reactions to occur at the e of the microarray at the functionalisable areas. When also formed from a conducting al, it ensures that the functionalisable areas are tively 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 d in the art. Where the continuous SD e (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 SD e (coating layer 3) has been laser scribed or otherwise cut into ed 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 SD surfaces within the array structure).

Gold, as a choice of coating layer 3 (continuous 3D e), achieves all of these roles. ln 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 tand 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 abovr identified roles of the continuous 3D surface.

As indicated above, gold is the red 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 and harsh al cleaning treatments which in turn ensures that the arrays of the present invention can be used more than once.

VIASOQOSSNZPFI 302868432 As used herein, "inert material" refers to a flexible or rigid material which physically isolates individual functionalisable 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 e (coating layer 3) and thus allowing those areas to be functionalised as desired. In this arrangement, the array remains as a 3D array. atively, 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 lly used where flexibility is not required. The inert material may also be formed from a solid film or a yer 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, CM, and —CH0. The choice of functional group depends on the target s 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 e 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 d by g back the inert material by reactive ion etching. Numerous alternative s 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 l of the inert al from areas to be functionalised (for example, by wiping the tips), spray- coating a dilute coating al onto the g 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 rray of the first aspect of the present invention may be functionalised to be a micro- electrode 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 functionallsable array, ing a continuous 3D surface layer, formed from an intermediate V|A50905BNZPR 302868432 structure 4. Again, dual onalisable areas of the array are separated by a layer of the inert material to give an inert surface through, or from, which the functionalisable areas protrude or are otherwise exposed and the individual functionalisable areas are interconnected by the continuous BD surface. Preferably, the individual functionalisable 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, functionalisable array according to the first aspect of the invention. The intermediate structure is formed from a substrate al layer 2 that includes an accurately d SD pattern to a eter 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). ally, the intermediate ure 4 will include an adhesion layer between the coating layer 8 (continuous 3D surface) and the substrate material 2.

Optionally, the intermediate structure 4 will include an adhesion layer between the coating layer 3 and the substrate material 2.

Figure 10 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 ZB shows a 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 uous sheets thus providing ies 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 eter to the nanometer (inclusive) scale. Thus, in a third , the present invention provides a method for the formation of an ediate structure according to the first aspect of the invention, including a uous 3D surface layer from which an array having accurately defined and functionalisable isolated areas can be formed, the method involving the steps of: a. piacing an accurately defined 3D pattern at the millimeter to nanometer scale on the surface of a substrate material; and VlASOQOSBNZPR 302868432 b. coating at least part of the ned substrate al with a continuous 30 single coating layer.

Preferably, the coating layer 3 covers substantially all of the patterned area of the substrate material to form a continuous SD 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 10) is preferably formed from an inert polymer material. However, as indicated above, there are a number of other suitable le and non-flexible materials which may be used including thermoplastic polyurethane, rubber, silicon rubber, epoxy, PMMA, PC, PS, ceramic, resin and ite materials. Suitable techniques for placing an accurately defined SD pattern at the millimeter to ter scale on the surface of the substrate material are described above and include embossing, g, stamping, etching, grinding, lithography, pressure forming, vacuum forming, roll forming, injection moulding and laser scribing / ablation techniques.

Alternatively, the intermediate ure 4 (as ed in Figure 10) 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 couid then be embossed (or otherwise patterned) with a desired SD pattern. Again, suitable techniques for forming the metal surface with a desired n are as bed above and could include casting, stamping (Figure 7C), etching, grinding, lithography, pressure forming, vacuum forming, roll forming (Figures 7A and 78). 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 ion in one of three ways. in the first method, individual spaces 5 between onalisable areas (depicted in the form of tips) 6 in the 3D pattern on intermediate structure 4 (Figure BC) are filled with an inert material 7. When used in this manner, the inert materiai 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 ure 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 SG shows a diagrammatic top View of the onalisable array of Figure 3F. The functionalisable areas or tips 6 remain connected to each other by the coating layer 3 (continuous 3D surface) of VIASOBOSBNZPR 302868432 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 dual spaces 5 between the tips 6 of the 3D pattern on the intermediate ure 4 with an inert al 7 to give an inert e 8 through which the tips 6 protrude or are otherwise exposed, said tips 6 forming functionalisable areas. Thus an array including a continuous SD 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 e 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 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 g, 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 ZD e, as the onalisable areas or tips 6 do not protrude above the inert materiai 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 Therefore, in a fifth aspect, the present invention es a method for the formation of a 20 microarray structure of the present invention, the method including the steps of taking the intermediate structure 4 and covering the SD pattern on the intermediate structure 4 with an inert materiai 7, and removing ient 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 es an intermediate structure 4 formed from a substrate material 2 (which is inert) and 3D ned to a millimeter to nanometer scale, a coating layer 3 over at least part of the patterned area to form a uous 3D surface, and a OSBNZPR 302863432 layer of inert al 7 which is layered over the uous SD surface and fills spaces 5 between functionalisable 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 BD pattern protrude or are otherwise d. As indicated above, the onalisable areas or tips 6 are isolated by the inert al 7 but are continuously interconnected via the coating layer 3 (continuous 3D e) which is present over at least part of the 30 patterned area (preferably substantially all 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 SD 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 ode(s), a redox eiectrode(s) and g 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 micro- electrode arrays can be configured in a number of ways, two examples of which are shown in Figures 48 and 40. in Figure 4B, the micro—electrode array includes two working electrodes 21 a and 21 b, separated by a counter electrode 22, and a reference electrode 23. in Figure 40, the micro-eiectrode array includes three working electrodes (21a, 21 b and 21c), a counter electrode 22, a reference electrode 23 and a redox ode 24, wherein the counter electrode separates working electrodes 21a and 21 b and the nce electrode 23 and redox electrode 24 together separate working electrodes 21b and 210. The isolated micro-electrode arrays may also be arranged such that each functions as a g electrode 21. in the third method, the coating layer 3 (Figure 30) acts as a seed layer. The intermediate structure 4 is placed into an electroplating bath to electrochemicaliy grow the coating layer 3 (continuous 3D surface). it is therefore preferable that the coating layer 3 (continuous 3D surface) is electrically tive 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 ochemically 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 SD pattern 10 (a negative of the pattern on structure 4 (Figure 3E)). individual spaces 11 between the functionalisabie areas or tips 12 in the negative BD pattern on the metal negative 10 are then backfiiled with an inert material 7 to give a flat surface 8 through, or from, VIASOQOSSNZPR 302858432 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 materiai 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 ing isolated but interconnected functionalisable areas. The onalisable areas or tips 12 are isolated by the inert material 7 and are capable of being functionalised as desired to form functional areas (eg. 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 e layer with an accurately defined 3D pattern of functionalisable areas in the millimeter to nanometer scale, said method including the steps of: a. electroplating the uous 3D surface layer of the intermediate structure 4 to form a metal layer 9 that covers the tips 12 of the 3D pattern rably at least substantially all of the 3D pattern) on the intermediate structure 4; b. 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 tive 30 pattern") on the intermediate structure 4; c. backfilling spaces 11 between tips 12 within the ve 30 pattern on the metal negative structure 10 with an inert material 7 to give an inert surface 8 through, or from, which the tips 12 of the negative 3D n protrude or are otherwise exposed.

Any metal can be ed 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 piating metal.

Figure 3G shows a diagrammatic top view of the functionalisable array formed by the filling of individual spaces 11 between functionalisable areas or tips 12 in the negative pattern on the metal negative 10 (Figure 3H). Thus, the metal ve 10 acts as the substrate material layer of a microarray structure. The present invention may ore 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 SD surface (conductive or non-conductive) and/or an inert al 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 SG.

VIASOSOSBNZPR 802868432 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 t invention provides an economic approach to the creation of such arrays.

As is clear from Figures 3F and 3H and as sed above, the isolated functionalisable areas d by tips 6 and 12) in the array are interconnected below the inert material 7 via a continuous 30 surface (Le. coating layer 3 or metal negative pattern 10). Where the continuous 3D surface is formed from a conductive material (for example, gold), the isolated onalisable areas are conductively interconnected with each other as discussed above and ore act as onnected but isolated conductive islands. Aiso, as indicated above, the continuous 3D surface can be laser scribed or otherwise cut into individuai sections such that individually isolated blocks of functionaiisable 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 sed 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 iunctionalisation of the isolated sites in the micro-electrode array. Micro-electrode arrays can be of a variety of types as shown in Figure 6, inciuding: - microdisk ode arrays, on which the arrays may be arranged in a ordered or random fashion; - microband electro arrays; - digitated lectrode arrays, which may be planar or vertical; — linear microelectrode arrays; and — 3D microelectrode arrays.

When functioning as a micro-electrode array, the continuous SD surface is connected to an electroanalytical device, electrical contact is made with an olytic 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 d” by the micro-electrode array. Capture agents that are ic to the target species can also be appended to the functionalisable areas of the micro-electrode array to aid in this interaction. Individual microelectrode arrays may also be used as counter- ViASOQOSSNZPR 302858432 electrodes 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 e 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 xidant 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 ion 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 kers 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 nductive g material 3 can be employed or the metal negative 10 can be coated with a non-conducting layer.

The isolated onalisable areas (identified at 6/12 in Figure SG) can also be used in a number of other array applications. For e, they may be iunctionalised to act as catalysts in a y of micro reactions, or to act as sensors for various target biomolecules 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 ing 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 uously interconnected by a 30 coating layer 3 within the structure. Preferably, the functionalisable areas are ically conductive.

OSBNZPR 302868432 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 s a surface 8 through which the tips 6 of the SD 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 uous 3D surface.

Reference to “accurately defined” means that the 3D pattern (or ZD pattern) es a known, pre—determined (or calculatable) number of functionalisabie areas in a known n. It is of course possible for the pattern to be randomised. Accuracy also includes the concept that the size and/or position of the tunctionalisable areas are pre-determined and accurately included in the structure.

As indicated above, the microarrays of the present invention are le for use as micro- electrode arrays, atalyst arrays, and sensors for various target ecules or compounds of interest.

When a microarray of the present invention is used as a micro-electrode array (and therefore includes a conductive continuous SD 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 s for attaching the capture agent to an array. Methods of attachment typically include l 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 ding PCT application number PCT/2011/000052, the sure 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 sms.

Electrical connection to a micro-electrode array of the present ion is typically achieved by attaching a clip or pressing a conductor (conductive paste, wire, ) against the part of the electrode which is not in contact with the solution to be passed over the surface of the micro- electrode array.

VIASOSOSBNZPR 302868432 When a microarray of the present invention is used as a microcatalyst array, it is essential that suitabie binding chemistry is first attached to the functionalisabie areas of the array. The attachment of suitable binding chemistry may be achieved in a number of ways inciuding, electrochemical deposition of the binding chemistry where a conductive continuous 3D surface layer is used, and exposure of the functionalisable areas to suitable functional . Suitable functional groups may include a metal catalyst (for example, platinum or palladium), DNA, and conducting polymers such as polypyrrole and iophene. The different surfaces of the microcatalyst arrays so created react with the s in solution to a greater or lesser extent.

The combination of responses allows the solution to be characterised electrochemically.

Examples e One: Computer Modelling of Micro-electrode Sensors A series of computer modelling experiments were carried out on a single functionaiisable area or tip of two individual electrode arrays of the present invention, wherein the two micro- electrode arrays 25a and 25b were preciseiy 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 ments was to calculate the impedance profile for different shapes and sizes of condensed droplets formed in n 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 n two odes is the sum of the impedance at the electrode-electrolyte interface and the impedance of the electrolyte solution. l n order to measure the impedance changes due to changes in the geometry of the , 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 ace 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 t distribution for a frequency range of 1 KHz to 1x106 KHz. The buffer was considered as a solution with conductivity of 0.028/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 odes, and the volume of the electrolyte (as either a t between the tips or as a solution that completely covers the tips).

In summary, the s 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 VlASOQOSBNZPR 302868432 electrolyte at the micron scale. in Figure 9, two different tip geometries are shown. In Figure 9A droplet formation is shown white Figure QB shows a chamber filled with an electrolytic solution. impedance results are shown by the potential bution (Figures 9 and 10). The arrows indicate current density vectors in the 20 and 30 domain.

Figures 11 to 13 show various impedance measurements, white Figure 14 shows frequency vs. impedance for two different geometries.

For bio-sensing applications, the electrodes were assumed to be functionaiized with capture agents and then the impedance was measured before and after the capture. The change in the impedance was primarily due to s 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 nce is illustrated in Figure 15, and the domain equations and boundary conditions are shown in Figure t6.

Variations in double layer capacitance (the ability of a body to store an electric charge) are measured using Non-faradaic ochemical impedance spectroscopy (E18). This involves ting any changes due to redox reactions and measuring the capacitance changes due to s 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 ess (Doll ) (Figures 17, 18 and 19). For all the cases charge transfer resistance (Fict) equates to 1M ohm. The s 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 s the current at the interface, which consequently changes the Rat of the system. The Hot values vary for various ent interfaces. Impedance changes of the system were simulated for various Rot. Results are illustrated in Figures 20, 21 and 22 and show that a droplet provides slightly better ivity at lower frequencies.

The computer modelling experiments showed that the r the dimensions of the tips, and the gaps between the tips, the greater the sensitivity of the sensor array.

OSSNZPH 302868432 The design was also fied to arrange g and counter electrodes side—by-side via inter- digitation ning, thus enabling the different electrodes of an electrochemical set up (for example, working odes, counter electrode and reference electrode) to all be placed on the same micro-electrode array sensor chip. The ochemical interactions of side-by-side electrode sions were modelled and similar s were observed to that of the micro- electrode array set up shown in Figure 8. Therefore, in summary, impedance measurements reiate to the distance between working and counter odes 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 bes the development of microarrays according to the fourth, fifth and sixth s 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 100 microns in height. PMMA (an amorphous polymer) is a preferred substrate material 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 ing steps: 1. Gold coating the substrate to form a continuous 30 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 VIASOSOSBNZPR 302868432 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 thlol 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 15s in 0.5M H2804, and then cycling between 0V to 1.65 V. Figure 26 shows a l 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 g 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 48 and C, Figure 5A and B and Figure SF).

The lasered pattern electrically isolates dual areas of the microarray from each other, resulting in the formation of more than one electrode in a single microarray. 2. ting an inert al 7 between dual tips Three te s were used to deposit the inert material onto the continuous 3D gold e so as to act as an isolation layer n the tips: A. Deposition of a photoresist layer (SU-8) over the entire structure, ed by reactive ion etching to expose the gold tips; 8. 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 100 micron thick layer of SU-8 was applied to a 10 cm wafer of gold coated substrate which had been previously laser scribed into 1 cm inter—digitated sensor chips (Figure SC). 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 358 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 VlASOBOSSNZPH 302868432 tracks of an inter-digitated array in which alternating tracks have ylated polytherthiophene deposited onto the tips. 8. 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(CH2)60H are es of suitable hydroxylated self-assembled monolayer’s for use in the present ion.

The continuous 3D gold e of micro—electrodes were subjected to cyclic metry in Potassium ferricyanide (KaFeCNe) 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 ted to cyclic voltammograms in 5mM Potassium ferricyanide solution with KCl supporting electrolyte (Figure 28). The CV showed gradual disappearance of the ferricyanide peak as the length of time the electrodes were ed in the SAM solution was increased indicating the gradual adsorption of SAM onto the continuous 3D gold surface of the odes.

After a period of 20 minutes the current reached a steady state value showing that the electrodes were ted 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 ferrioyanide solution showed the typical ion oxidation peak at significantly reduced t. This indicated that only the gold on 3O 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.

VIASOSOfiBNZPR 302863432 C. Coating the gotd 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 terricyanide solution showed the typical reduction oxidation, and was ce that the gold tips were ed. 3. Attaching the binding chemistry (-X) onto the tips The binding chemistry (-X, of. Figure 24) was attached to the tips of the SU-8, SAM-OH or the epoxy coated uous 3D gold surface of the substrate. Where SU-8 or an epoxy coat was employed as the inert material 7, ment of the binding chemistry was achieved by electrochemically depositing carboxylated polytherthiophene or ed polyterthiophene onto the tips (Figure 308 and C). White 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 carboxyiic SAM and ng the potential of the different working electrodes as would be apparent to those skilied in the art. Where SAM-OH was employed as the inert material 7, attachment of the g chemistry was achieved by exposing the tips to SAM- COOH (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 ophene (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 g chemistry onto the tips, 1 micron aminated polystyrene beads were covalently attached via the appropriate linker chemistry.

Figure 31 illustrates the process for the amine onalised tips (A). The aminated substrate was exposed to a bi—functional linker solution (6 mg linker/0.5 ml PBS), and shaken at room temperature for 45 minutes. After washing, the substrate was immersed into a on containing a blue HgN-Bead solution (30 pl 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 s including, but not limited to, antibodies, DNA and celis.

As an illustration, the following shows the use of the method to fabricate a sensor for Progesterone (P4). The steps include: 1. Attaching P4 onto ovalbumin to form an ovalbumin P4 conjugate (P4-PEG-OVA 23-atorn tinker) as shown in Figure 36; 2. lmmersing Nl-i2 substituted arrays into a bi-functional linker on (6 mg iinker/O/S ml PBS) and shaking at room temperature for 45 minutes to give an activated array (labelled l in figure 37); 3. Adding P4-PEG-OVA (0.4 ml solution in 0.2 ml PBS) and ng it to react for two hours with shaking to give the Haptan functionalised array (labelled II in figure 37); and 4. Exposing the array to the P4 primary antibody, and then a secondary antibody with attached beads (labelled II! in figure 37). The attachment of the ary antibody bead conjugate allows the sful bonding of the primary antibody to be visuaily confirmed.

VIA509058NZPR 302858432 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 (1 mm) of nickel onto the gold, separating the nickel and r layers and sputter coating the nickel with gold.

Figure 33 shows an SEM of a tmicron 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 couid 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 metai layer can be laser d to isolate groups of tips for selective functionalisation of ed areas. For the production of sensors using electrochemical detection this ability to scribe is an advantage as it allows the electrodes and the g between those electrodes to be d 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), 02 micron tips (1.5 micron at their base and 10 nm 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. e Three: Use of a Single Microarray in Multiplexing Assays Laser scribing was ed 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-eiectrode arrays within a larger array, thus constituting a platform of multiple working electrodes, reference electrodes and r electrodes. This enabled multiplexing on a single sensor chip or array (as ed 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, e oxidase was tethered to ViA50905BNZPR 302868432 working electrode 9 for detection of serum glucose. The tenth working ode was a redox electrode to measure non-adhered bilirubin concentration in solution.

In another example, one working electrode array was functionalised with an RNA ment 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 onalised 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 eter scale.

The ing 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 ion 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 ive or exhaustive sense, that is to say, in the sense of "including, but not limited to".

Tl$509058NZPR 303747891

Claims (15)

1. A microarray structure including a three dimensional (3D) substrate material layer, a continuous 3D surface layer on the substrate material iayer. and an inert al; 5 n the continuous SD surface layer is capable of functionalisation for use as an array; and wherein the structure includes accurately d and functionalisable isolated areas which are millimeter to ter in size; and wherein the functionaiisable areas are part of the continuous 30 surface layer and 10 are isolated by the inert material but are onnected within the structure by the continuous SD e layer.

2. A microarray as claimed in claim 1, wherein the continuous 3D surface layer is formed from an ically 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 rray as claimed in claim 1 or claim 2, wherein the continuous 30 surface 20 layer is cut into a plurality of isolated continuous 3D surface layer segments on the substrate al 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 material 25 is also an insulating material.

6. A microarray as claimed in any one of the previous claims, wherein the microarray ure includes an adhesion layer between the continuous 3D e layer and the substrate material layer.

7. A microarray as claimed in any one of the previous claims, wherein the microarray structure is functionalised as a micro-electrode sensor array and/or a micrccatalyst array. 35

8. 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. T|8509058NZPR 303747891

9. A microarray as claimed in any one of claims 1 to 7, wherein the inert material covers the 3D surface layer to such a degree that the functionalisable areas form a substantially two-dimensional (ZD) surface including exposed onalisable areas.

10. A method for the ion of an array according to any one of claims 1 to 9, the method including the steps of taking a 3D substrate material that includes a continuous 3D surface layer and filling individual spaces between tips of the continuous 3D surface layer with an inert material to give a surface through, or from, which the tips of the continuous 3D e layer protrude or are otherwise exposed; 10 wherein the tips define a 3D pattern to a millimetre to nanometer scale; and wherein the tips are ed by the inert material but are onnected within the structure by the continuous 3D surface layer and are capable of functionalisation.

11. A method as claimed in claim 10, wherein the method es the step of cutting 15 away the tips to align with the surface of the inert material to form a substantially 2D surface including functionalisable areas.

12. A method for the formation of a structure capable of functionalisation as an array, said structure including a 30 substrate material layer and a continuous 3D e 20 layer on the substrate material; wherein the structure further includes an accurately d 3D pattern of functionalisable areas in the millimeter to nanometer scale, the method ing the steps of: a. electroplating the continuous 3D surface layer to form a metal layer that covers 25 tips of the 30 pattern; b. separating the metal layer and continuous surface layer from the substrate material to form a metal negative structure which includes a negative of the 3!) pattern ; c. backfilling spaces between tips on the metal negative structure with an inert 30 material to give an inert surface through, or from, which the tips protrude or are otherwise d; wherein the functionalisable areas are isolated by the inert material but are interconnected within the structure.

13. A method as claimed in claim 12, wherein the metal layer covers substantially all of the 3D pattern. TISSOQOSBNZPR 303747891

14. A method as claimed in claim 12 or claim 13, wherein the method includes the step of g away tips to align with the e of the inert materiai to form a substantially 2D surface including functionalisable areas. 5

15. A microarray structure as d in claim 1, substantially as hereinbefore described with particular reference to any one or more of the Examples and/or