The invention relates to sensors for the detection of target analytes in a solution. More particularly, the invention relates to electrochemical sensors that detect binding of a target analyte to a capture agent on an array.

Background

Many medical, biological and biotechnological applications would benefit from more accurate and reproducible measurement of biological or biochemical processes. However, converting information about a biological event to a detectable electronic signal is challenging due to the complexity of connecting an electronic device directly to a biochemical environment. Electrochemical sensors provide an attractive means to achieve quantitative analysis of the content of a sample due to the direct transduction of a biochemical event to an electronic signal.

Electrochemical sensors are designed to be highly target-specific so as to be able to detect the presence (and preferably the concentration) of the target analyte (TA) in a sample. Electrochemical sensors generally involve using a target-specific binding agent which is immobilised on a sensor substrate. Binding agents may include enzymes, nucleic acids, antibodies, whole cells or receptors.

Although there are many diagnostic sensors in use, they are generally limited to high cost pieces of equipment in a lab environment for operation by skilled operators. In many cases the main limitation in realizing point-of-care testing/sensing devices is the ability to miniaturize the transduction principle and the lack of a cost-effective production method (Grieshaber et al., 2008). The use of cheap, handheld devices for use by the patient or medical professional in the field is limited.

Electrochemical sensing relies on the target analyte (TA) interacting with the sensor surface and influencing / disrupting a measurable property of the electronic pathway. A common approach is depicted in Figure 1 in which a gold electrode is overlaid with a mixed self-assembled monolayer (SAM). The binding agent (X) is attached to the electrode surface via a self-assembled monolayer (SAM)(Figure 1A and IB). A TA / TA-horseradish peroxidase (HRP) mixture is exposed to the binding agent and competitively binds to the binding agent (Fig 1C) in a ratio dependant on the concentration of each component. TMB (3,3', 5,5'-Tetramethylbenzidine) is added (Fig ID) and oxidised by HRP. This results in the subsequent reduction of the TMB on the gold electrodes. Since the reduction of TM B can be measured galvanostatically (i.e. via current across electrodes), this provides a measurement of the concentration of the TA in the sample. HRP and TM B are commonly used in Elisa assays in which the chromogenic (colour producing) TMB is measured optically as opposed to galvanostatically.

Calibration of assays for the detection of target analytes in samples can be complex and prone to inaccuracies due to multiple factors including sample handling, dilution errors and/or interference by the sample matrix (e.g. milk, blood or seawater). In existing assays, considerable effort goes into minimising the impacts of these factors by a skilled technician to ensure that the observed response is accurate, and the comparison against the standards is a true reflection of concentration.

It is an object of the invention to provide an electrochemical sensor and a method of detecting a target analyte that overcomes or ameliorates at least one of the disadvantages of the prior art. Alternatively, it is an object of the invention to provide the public with a useful choice.

Summary of the Invention

In a first aspect, the invention provides a sensor comprising:

a. a support substrate;

b. at least one surface structure protruding from an upper surface of the support substrate, wherein the surface structure includes an electrode layer;

c. a sensing surface on the electrode layer, wherein the sensing surface is adapted to contact a sample containing a target analyte;

d. a binding region on the support substrate, wherein the binding region is separated from the sensing surface;

wherein, in use, a binding agent attached to the binding layer at the binding region is also adapted to contact the sample containing the target analyte.

In one embodiment, the binding layer at the binding region is a planar layer attached to the support substrate.

Preferably the sensor is adapted to detect a detectable response at the sensing surface following binding of the target analyte to a binding agent attached to the binding layer.

Preferably the separation distance between the binding region and the sensing surface is between about lnm and 50mm, and/or the surface structures are separated from each other by about 50 nm to about 2000μιη, and/or the width of the surface structure where it joins the support substrate is between about 20nm to about 5000μιτι, and/or the width of the apex of each surface structure is preferably between about lnm to about 5000 micron.

In another aspect, the invention provides a sensor comprising:

a. a support substrate;

b. at least one surface structure protruding from an upper surface of the support substrate, wherein the surface structure includes an electrode layer;

c. a sensing surface on the electrode layer, wherein the sensing surface is adapted to contact a sample containing a target analyte;

d. a binding region on the support substrate, wherein the binding region is separated from the sensing surface;

wherein, in use, a binding agent attached to the binding layer at the binding region is also adapted to contact the sample containing the target analyte; and wherein the sensor is adapted to detect a detectable response at the sensing surface following binding of the target analyte to a binding agent attached to the binding layer.

In another embodiment, attachment of the binding agent to the binding region is electrostatic, covalent or via a magnetic bead to which the binding agent has been attached.

In another embodiment, the binding layer at the binding region is a functional surface on a magnetic particle wherein the magnetic particle is attached to the support substrate by a magnetic field.

Preferably the sensor includes a magnetic element and the binding layer at the binding region is a functional surface on a magnetic particle wherein the magnetic particle is attached to the support substrate by a magnetic field created by the magnetic element.

In another embodiment the invention provides a method of production of a sensor comprising:

a. providing a support substrate with at least one surface structure protruding from an upper surface of the support substrate;

b. depositing an electrode layer on an upper surface of the at least one surface structure;

c. depositing an inert layer on the electrode layer such that one or more portion of the electrode layer remains exposed to form at least one sensing surface on the electrode layer, wherein the inert layer comprises a material substantially inert to the components of a sample to be analysed;

d. forming a binding region on the support substrate wherein the binding region is adapted to attach at least one binding agent either electrostatically, covalently or magnetically;

wherein the sensing surface is separated from the binding region by a separation distance sufficient, in use, for a detectable response to be detected at the sensing surface following binding of a target analyte to the binding agent.

Preferably the separation distance between the binding region and the sensing surface is between about lnm and 50mm, and/or the surface structures are separated from each other by about 50 nm to about 2000μιη, and/or the width of the surface structure where it joins the support substrate is between about 20nm to about 5000μιη, and/or the width of the apex of each surface structure is preferably between about lnm to about 5000 micron.

Preferably the detectable response comprises an electrochemical detectable response comprising a change in current, voltage, capacitance, resistance, conductance, impedance, magnetic flux or electric field. Preferably the binding of a target analyte to a binding agent results in an electroactive species which mediates the detectable response at the sensing surface. Preferably the binding of a labelled target analyte to a binding agent competes with, displaces, or is displaced by, the binding of an unlabelled target analyte leading to generation of an electroactive species that mediates the detectable response at the sensing surface.

Preferably the support substrate comprises a polymer, silicon or glass. Preferably the support substrate comprises a single layer or multiple layers.

Preferably the at least one surface structure is integral with the support substrate.

Preferably the electrode layer is deposited on an upper surface of the surface structure(s).

Preferably the electrode layer comprises a layer of substantially constant thickness. Preferably the electrode layer covers the surface structure(s) and optionally the support substrate.

Preferably the thickness of the electrode layer is between about lnm and 500nm thick, more preferably between about 40 and 500nm or between about 50 and lOOnm thick. More preferably between about 5nm and 30nm thick, between 50 and lOOnm, between 70 and 400nm, between 900 and 300nm.

Preferably the electrode layers on the upper surface of two or more surface structures are electrically connected within the sensor. The electrical connection may be below the binding layer or inert layer

Preferably the sensing surface is on an upper surface of the electrode layer.

Preferably the sensing surface is on an upper surface of an electrode layer on a surface structure protruding from the support substrate.

Preferably the surface structure comprises a sensing surface defined by the extent of the exposed electrode layer on the support substrate. Preferably the sensing surface is bounded by an inert layer on electrode layer. Preferably the inert layer exists between the surface structures. Preferably the sensing surface is separated from other sensing surfaces by an inert layer. Preferably a sensing surface on one electrode layer is electrically connected to at least one further sensing surface on the same electrode layer. Preferably the electrical connection to the at least one further sensing surface is under the inert layer.

Preferably the sensor comprises a plurality of surface structures each with a sensing surface on the electrode layer.

Preferably a plurality of sensing surfaces are electrically connected via the electrode layer to form a sensing group. In one embodiment, the sensor comprises two or more sensing groups where each sensing group is electrically isolated from other groups.

Preferably the sensing surface is not electrochemically passivated from interacting with an electroactive species. Electrochemical passivation or attenuation of the detectable response may be caused by the binding agent(s), the binding region or a matrix effect induced by the sample.

Preferably the sensing surface comprises a protective coating. Preferably the protective coating comprises a SAM or a protein.

Preferably the protective coating is removable.

Preferably the surface structure(s) protrudes through the inert layer. Preferably the surface structure(s) protrude from the support substrate.

Preferably the surface structure(s) comprises an apex at the top of the surface structure. In some embodiments, the apex is of a surface structure that has an upper portion with a contoured surface and at least one lower portion with a differently contoured surface. In some embodiments, the surface structure or the upper portion thereof is dome-shaped, cone-shaped, pyramid-shaped, papilliform, a ridge or polyhedron-shaped.

Preferably, the ridge has a convex, papilliform, tapered, triangular or polygonal profile along a cross-section along an axis generally parallel to a top surface of the support substrate.

It will be appreciated by those of skill in the art that any surface structure with an apex is likely to be flat when viewed at very high magnification (e.g. atomic or nano-scale). Accordingly, the shapes and measurements provided herein are intended to refer to the overall shape of the surfaces structure rather being precise geometric descriptions.

Preferably the surface structure comprises an upper portion with a convex upper surface. Preferably the surface of the upper portion is tapered to an apex or rounded to an apex.

Preferably a cross-section of the surface structure along a plane orthogonal to a top surface of the support substrate is a triangle, a convex semi-circle or papilliform.

Preferably a cross-section of the surface structure along a plane parallel to a top surface of the support substrate is substantially triangular, substantially circular or substantially square.

Preferably a cross-sectional area of the surface structure diminishes along an axis that is orthogonal to a top surface of the support substrate.

In one embodiment, the surface structures are uniformly arranged on the support substrate. In one embodiment, the surface structures are randomly arranged on the support substrate.

Preferably the surface structures comprise a smooth surface. Preferably the surface structures are regular shapes. Preferably the surface structures have at least one line of symmetry, and preferably 2 lines of symmetry. Preferably all or a plurality of surface structures are substantially identical in shape. Preferably all or a plurality of surface structures are substantially identical in surface area.

In one embodiment, the binding layer is deposited as a planar layer on an upper surface of the inert layer or support substrate. Preferably the binding layer is adjacent to the electrode layer, optionally with an isolating material or gap between the binding layer and the electrode layer.

Preferably the extent of the binding layer defines an aperture through which the sensing surface is exposed to the sample.

Preferably the binding layer is deposited around the surface structures such that an upper portion of the surface structure protrudes with an exposed sensing surface thereon.

Preferably the binding layer comprises a planar surface through which the at least one surface structures

protrude.

Preferably the binding layer comprises a material which binds to a binding agent. Preferably the material has molecular functionality suitable to bind a binding agent such as an antibody. Preferably the binding layer adheres to the top surface of the support substrate. Preferably the binding layer is electrically conductive. Preferably the binding layer is electrically connected to an electrode (or adapted for electrical connection) such as a measurement electrode capable of applying a potential.

Preferably the binding agent comprises an aptamer or antibody specific for a particular target analyte. Preferably the binding agent is selected from the group consisting of antigens, antibodies, antibody fragments, single-chain variable fragments, biotinylated proteins, peptides, nucleic acids (e.g. DNA, ssDNA, mRNA, miRNA, aptamers), avidin, streptavidin, NeutrAvidin, recombinantly expressed proteins containing polyhistidine or glutathione S-transferase, large or small amine-containing molecules, sulfhydryl-containing molecules or proteins expressing glutathione S-transferase (GST), metals and metal salts (such as lead, lead phosphate, chromium, platinum, palladium, iridium, copper etc).

Preferably the binding layer comprises a protective coating (a coating that has negligible binding by the matrix, and renders the recognition portion of the binding agent available for binding).

Where the binding layer is on the surface of an inert layer preferably the binding layer comprises a cross-linked polymer, a photo-resist or a self-assembled mono-layer (SAM). Preferably the cross-linked polymer is a photoresist such as SU-8, AZ40XT, Shipley 3612, polyimide. Preferably the binding layer is deposited by spin-coating, spray-coating, dip-coating, wiping or painting. Preferably the binding layer is deposited on the support substrate by spin coating.

In embodiments where magnetic particles are used as the binding layer, the binding region comprises a region of the support substrate adapted to attach one or more magnetic particles.

Preferably the binding region has at least one magnetic element on, in, or under the support substrate. This magnetic element attracts the magnetic particles to attach them to the binding region.

Preferably the binding region is separated from the sensing surface by a separation distance wherein the separation distance is sufficient for the detectable response to be detected at the sensing surface.

Preferably the separation distance is such that detection of the detectable response at the sensing surface can be made without being attenuated by the binding layer or binding agent(s).

Preferably the separation distance is between about lnm and 50mm. In particular embodiments, the separation distance is between about 30nm and about 5mm, between about lOOnm and about 5000μιη, between about Ιμιη and about ΙΟΟΟμιη or between about 20μιη and about ΙΟΟμιη, about 30nm and about 5000μιη, about lOOnm and about 5mm, about Ιμιη and about ΙΟΟμιη, about 20μιη and about ΙΟΟΟμιη, about 20μιη and about 5000μιη; Ιμιη and about 5mm.

Preferably the particles are attached directly to an upper surface of the support substrate.

Preferably the particles are attached to an inert surface on the support substrate.

Preferably the magnetic particles are separated from the sensing surface by a separation distance as defined above.

Preferably the sensing surface and binding region are housed within a microfluidic system.

Preferably the sensing surface and the binding region are in separate compartments of the microfluidic system.

Preferably the binding region is inert to the components of the sample applied to the sensor.

Preferably the binding region is defined by an area of magnetic field established by a magnetic element on, in or under the support substrate.

In one embodiment, the binding layer comprises a functional surface on one or more magnetic particles attached to the support substrate. Preferably the particles are ferromagnetic or paramagnetic particles. Preferably the attachment of the magnetic particles to the support substrate is by way of magnetic attraction between a magnetic element and the magnetic particles.

Preferably the sensing surface protrudes above the particles such that, in use, the surface structure is exposed to the sample. Preferably the sensing surface protrudes through a layer of particles such that, in use, the surface structure is exposed to the sample.

Preferably the particles are attached to a surface around the surface structures such that an upper portion of the surface structure protrudes with an exposed sensing surface thereon.

Preferably the particles are attached to a surface around the surface structures and cover the surface structure(s) itself. Preferably the magnetic element comprises an element which produces a magnetic

field. Preferably the element comprises an electromagnet or a ferromagnet. Preferably the magnetic element is fixed to the sensor or it is removable.

Preferably the magnetic element is positioned so as to attach the particles around the surface structures such that an upper portion of the surface structure protrudes through the particles with an exposed sensing surface thereon.

Preferably the magnetic element is positioned so as to attach the particles adjacent to the electrode layer, optionally with an isolating material or gap between the particles and the electrode layer.

Preferably the magnetic element is positioned so as to attach the particles to the binding region which is separated from the sensing surface.

Preferably the functional surface on the particle comprises a functional material which binds to a binding agent. Preferably the functional material has molecular functionality suitable to bind a binding agent such as an antibody.

Preferably the magnetic particle comprises a functional surface comprising a carboxylate, aminated, biotinylated, or protein A or G coating. Preferably carboxylated

Preferably the binding layer comprises a protective coating to eliminate non-specific binding.

Preferably a portion of the binding layer is blocked with a blocking agent. Preferably the blocking agent is ethanolamine or a surfactant (e.g. tween), Proteins, OVA, BSA , phosphates

Preferably the binding agent adapted for attachment to the particle comprises an antigen or antibody specific for a particular target analyte. Preferably the binding agent is selected from the group consisting of antigens, antibodies, aptamers, antibody fragments, single-chain variable fragments, biotinylated proteins, peptides, nucleic acids, avidin, streptavidin, NeutrAvidin, recombinantly expressed proteins containing polyhistidine or glutathione S-transferase, large or small amine-containing molecules, sulfhydryl-containing molecules or proteins expressing glutathione S-transferase (GST), metals and metal salts (such as lead, lead phosphate, chromium, platinum, palladium, iridium, copper etc).

In one embodiment, the sensor comprises two or more sensing groups adapted to detect a response from binding of the target analyte to a binding agent.

In a further embodiment, the sensor comprises a measurement electrode electrically connected to one or more sensing surfaces or sensing groups via the electrode layer.

Preferably the measurement electrode is connected to a measuring means which measures a change in one or more of current, impedance, voltage, capacitance, resistance, conductance, magnetic flux or electric field.

Preferably the sensor comprises part of a sensor system comprising a sample container adapted to retain the sample on the sensor surface. Preferably the sensor system further comprises a reference electrode. Preferably the sensor system further comprises a counter electrode. Preferably the counter electrode and the reference electrode contact the sample during detection of the detectable response.

In one embodiment, the sensor is adapted for deployment in a no-flow environment. In another embodiment, the sensor is adapted for deployment in a flow environment.

Preferably the sensor has a single magnetic binding region and single sensing surface.

Preferably the sensor has multiple binding regions to multiple sensing surfaces, multiple binding regions to a single sensing surface.

In a further embodiment, the binding layer or the binding region is adjacent to a first sensing surface on a first electrode layer; and

wherein the first sensing surface is adjacent to a second sensing surface on a second electrode layer; and wherein a first separation distance of the first sensing surface from the binding layer or the binding region is less than a second separation distance of the second sensing surface from the binding layer or the binding region; and

wherein the first and second electrode layers are electrically isolated from one another.

Preferably the sensor is adapted to detect a detectable response at the first and the second sensing surfaces following binding of a target analyte to a binding agent.

Preferably the binding region adjacent to the first sensing surface is defined by an area of magnetic field established by a magnetic element on, in or under the support substrate.

Preferably the sensor further comprises a third, fourth, fifth or further sensing surface respectively situated on a third, fourth, fifth or further electrode layer, wherein each electrode layer is electrically isolated from one another;

wherein the separation distance between the binding layer or the binding region and the respective sensing surface progressively increases as the number of electrode layers increases.

Preferably the sensor is adapted to detect a detectable response at at least the first and the second sensing surfaces following binding of a target analyte to a binding agent.

In one embodiment, the first separation distance (between the sensor surface and the binding region) is between about lnm and 5mm, and the second separation distance is between about lnm and about 5mm. In particular embodiments, the separation distance is between about 30nm and 1mm, between about lOOnm and 500μιη, between about Ιμιη and 200μιη or between about 20μιη and ΙΟΟμιη. The separation distance may be between about 30nm and about 5mm, between about lOOnm and about 5000μιη, between about Ιμιη and about ΙΟΟΟμιη or between about 20μιη and about ΙΟΟμιη, about 30nm and about 5000μιη, about lOOnm and about 5mm, about Ιμιη and about ΙΟΟμιη, about 20μιη and about ΙΟΟΟμιη, about 20μιη and about 5000μιη; Ιμιη and about 5mm.

In one embodiment, a first inter-sensing surface distance between the first sensing surface and the second sensing surface is between about 30nm and 1mm. Subsequent inter-sensing surface distances between adjacent sensing surfaces (each connected to a discrete electrode layer) are preferably between about 30nm and 1mm.

In particular embodiments, the sensor comprises two or more discrete electrode layers each connected to a measurement electrode. Preferably the sensor comprises between 5 and 8 electrode layers.

In one embodiment, the electrode layers are electrically isolated from the binding region or at least one other electrode layer with an isolating material or gap.

In one embodiment of the first aspect, the invention provides a sensor comprising:

a. a support substrate;

b. a plurality of surface structures integral with the support substrate and protruding from an upper surface of the support substrate, wherein each surface structure comprises an electrode layer on an upper surface of the surface structure;

c. a sensing surface on the electrode layer, wherein the sensing surface is adapted to contact a sample;

e. a binding region on the support substrate, wherein the binding region is separated from the sensing surface;

f. a magnetic element on, in or under the support substrate which, in use, attaches to the support substrate, wherein the magnetic particle comprises binding agents attached to a functional surface on the particle, and wherein the binding agents are adapted to contact a sample and bind to a target analyte within the sample.

Preferably, the binding region is defined by an area of magnetic field established by the magnetic element.

Preferably the sensing surface is separated from the binding layer on the magnetic particles. Preferably the sensing surface is separated from the binding layers by a separation distance sufficient for a detectable response to be detected at the sensing surface following binding of the target analyte to a binding agent attached to the binding layer. Preferably the separation distance is between about 30nm and 1mm, between about lOOnm and 500μιη, between about Ιμιη and 200μιη or between about 20μιη and ΙΟΟμιη. The separation distance may be between about 30nm and about 5mm, between about lOOnm and about 5000μιη, between about Ιμιη and about ΙΟΟΟμιη or between about 20μιη and about ΙΟΟμιη, about 30nm and about 5000μιη, about lOOnm and about 5mm, about Ιμιη and about ΙΟΟμιη, about 20μιη and about ΙΟΟΟμιη, about 20μιη and about 5000μιη; Ιμιη and about 5mm.

Preferably the separation distance between the binding region and the sensing surface is between about lnm and 50mm, and/or the surface structures are separated from each other by about 50 nm to about 2000μιη, and/or the width of the surface structure where it joins the support substrate is between about 20nm to about 5000μιη, and/or the width of the apex of each surface structure is preferably between about lnm to about 5000 micron.

WE CLAIMS

1. A sensor comprising:
a. a support substrate;
b. at least one surface structure protruding from an upper surface of the support substrate, wherein the surface structure includes an electrode layer;

c. a sensing surface on the electrode layer, wherein the sensing surface is adapted to contact a sample containing a target analyte;

d. a binding region on the support substrate, wherein the binding region is separated from the sensing surface;

wherein, in use, a binding agent attached to a binding layer at the binding region is also adapted to contact the sample containing the target analyte.

2. A sensor comprising:

a. a support substrate;

b. at least one surface structure protruding from an upper surface of the support substrate, wherein the surface structure includes an electrode layer;

c. a sensing surface on the electrode layer, wherein the sensing surface is adapted to contact a sample containing a target analyte;

d. a binding region on the support substrate, wherein the binding region is separated from the sensing surface;

wherein, in use, a binding agent attached to the binding layer at the binding region is also adapted to contact the sample containing the target analyte; and wherein the sensor is adapted to detect a detectable response at the sensing surface following binding of the target analyte to a binding agent attached to the binding layer.

3. A sensor as claimed in claim 1 or 2 wherein attachment of the binding agent to the binding region is electrostatic, covalent or via a magnetic bead to which the binding agent has been attached.

4. A sensor as claimed in claim 1 or 2 wherein the sensor includes a magnetic element and the binding layer at the binding region is a functional surface on a magnetic particle wherein the magnetic particle is attached to the support substrate by a magnetic field created by the magnetic element.

5. A sensor as claimed in any one of the preceding claims wherein the separation distance between the binding region and the sensing surface is between about lnm and 50mm, and/or the surface structures are separated from each other by about 50 nm to about 2000μιη, and/or the width of the surface structure where it joins the support substrate is between about 20nm to about

5000μιτι, and/or the width of the apex of each surface structure is preferably between about lnm to about 5000 micron.

6. A sensor as claimed in any one of the preceding claims wherein the electrode layer is deposited on an upper surface of the surface structure(s).

7. A sensor as claimed in any one of the preceding claims wherein the binding layer comprises a functional surface on one or more magnetic particles attached to the support substrate.

8. A sensor as claimed in any one of the preceding claims wherein the sensor comprises a measurement electrode electrically connected to one or more sensing surfaces or sensing groups via the electrode layer.

9. A sensor as claimed in any one of the preceding claims wherein the binding layer or the binding region is adjacent to a first sensing surface on a first electrode layer; and

wherein the first sensing surface is adjacent to a second sensing surface on a second electrode layer; and

wherein a first separation distance of the first sensing surface from the binding layer or the binding region is less than a second separation distance of the second sensing surface from the binding layer or the binding region; and wherein the first and second electrode layers are electrically isolated from one another.

10. A sensor as claimed in any one of the preceding claims wherein the support substrate is integral with the surface structure(s).

11. A sensor as claimed in any one of the preceding claims wherein the sensing surface and the binding layer are separated by an inert material or a gap.

12. A method of production of a sensor comprising:

a. providing a support substrate with at least one surface structure protruding from an upper surface of the support substrate;

b. depositing an electrode layer on an upper surface of the at least one surface structure; c. depositing an inert layer on the electrode layer such that one or more portion of the electrode layer remains exposed to form at least one sensing surface on the electrode layer, wherein the inert layer comprises a material substantially inert to the components of a sample to be analysed;

d. forming a binding region on the support substrate wherein the binding region is adapted to attach at least one magnetic particle to the support substrate by positioning a magnetic element or a magnetic element positioning means on, in, or under the support substrate to facilitate the establishment of a magnetic field capable of attracting the magnetic particle;

e. optionally attaching a magnetic particle to the binding region;

f. optionally attaching a binding agent to a functional surface on a binding layer on the magnetic particle;

wherein the sensing surface is separated from the binding region by a separation distance sufficient, in use, for a detectable response to be detected at the sensing surface following binding of a target analyte to the binding agent.

13. A method as claimed in claim 11 wherein the separation distance between the binding region and the sensing surface is between about lnm and 50mm, and/or the surface structures are separated from each other by about 50 nm to about 2000μιη apex to apex, and/or the width of the surface structure where it joins the support substrate is between about 20nm to about 5000μιη, and/or the width of the apex of each surface structure is preferably between about lnm to about 5000 micron.

14. A method as claimed in claim 11 or 12 wherein the at least one surface structure is integral with the support substrate.

15. A method as claimed in any one of claims 11 to 13 wherein the method of production further comprises attaching a magnetic element and/or a magnetic element positioning means to the sensor.

16. A method as claimed in any one of claims 11 to 14 wherein a protective coating is applied to the electrode layer.

17. A method as claimed in any one of claims 1 to 15 wherein the sensing surfaces are formed by deposition of a binding layer or an inert layer on the support substrate or surface structures leaving an upper portion of the surface structures free of binding layer or inert layer thus resulting in the surface of the electrode layer on the upper portion of the surface structure being exposed as the sensing surface.

18. A method as claimed in claim 16 wherein the binding layer or inert layer is deposited adjacent to the electrode layer.

19. A method as claimed in any one of claims 16 to 18 wherein the inert layer is electrically conductive and is connected to at least one electrode capable of applying a potential to the inert layer.

20. A method as claimed in any one of claims 16 to 18 wherein the step of depositing a binding layer on either the electrode layer or the support substrate comprises applying a SAM binding layer.

21. A method of detecting binding of a target analyte in a sample to a binding agent, the method comprising:

a. providing a sensor comprising:

i. a support substrate;

ii. at least one surface structure protruding from an upper surface of the support substrate, wherein the surface structure includes an electrode layer;

iii. a sensing surface on the electrode layer, wherein the sensing surface is adapted to contact the sample;

iv. a binding region on the support substrate, wherein the binding region is also adapted to contact the sample and is separated from the sensing surface and comprises a magnetic field;

b. attaching a magnetic particle to the binding region, wherein the magnetic particle comprises at least one binding agent attached to a binding layer on the magnetic particle;

c. contacting the binding agent and the sensing surface with a sample containing a target analyte;

d. allowing the target analyte to bind to the binding agent to produce a detectable response at the sensing surface;

wherein binding of the target analyte to the binding agent produces an electroactive species which mediates the detectable response at the sensing surface and measuring a change at a measurement electrode electrically connected to the at least one sensing surface;

and wherein the separation distance between the binding region and the sensing surface is between about lnm and 50mm, the surface structures are separated from each other by about 50 nm to about 2000μιη apex to apex, and/or the width of the surface structure where it joins the support substrate is between about 20nm to about 5000μιη, and/or the width of the apex of each surface structure is preferably between about lnm to about 5000 micron.

22. A method of determining the concentration of a target analyte in a sample, the method comprising;

a. providing a sensor comprising:

i. a support substrate comprising a binding region;

ii. at least one surface structure protruding from an upper surface of the support substrate, wherein the surface structure includes an electrode layer;

iii. a sensing surface on the electrode layer, wherein the sensing surface is adapted to contact the sample;

iv. a magnetic element;

v. a binding region on the support substrate, wherein the binding region includes a binding layer and is also adapted to contact the sample and is separated from the sensing surface and comprises a magnetic field created by the magnetic element;

b. attaching at least one binding agent to the binding layer at the binding region;

c. contacting the sensor including the binding agent and at least one sensing surface with a sample containing the target analyte;

d. measuring a change at two or more measurement electrodes, each measurement electrode being electrically connected to an electrode layer and at least one sensing surface, the measurement electrodes being electrically isolated from each other;

e. measuring the change in a detectable response as a function of separation distance from the binding site where binding of the binding agent to the target analyte occurs; f. comparing the change in response with the change in response from a known concentration of a target analyte from a control sample; and

g. determining the concentration of the target analyte in the sample;

wherein binding of the target analyte to the binding agent produces an electroactive species which mediates a detectable response at the sensing surface,

and wherein the separation distance between the binding region and the sensing surface is between about lnm and 50mm, the surface structures are separated from each other by about 50 nm to about 2000μιη apex to apex, and/or the width of the surface structure where it joins the support substrate is between about 20nm to about 5000μιη, and/or the width of the apex of each surface structure is preferably between about lnm to about 5000 micron.

23. A method of detecting binding of a target analyte in a sample to a binding agent, the method comprising:

a. providing a sensor as defined in any one of claims 1 to 11;

b. labelling the target analyte in the sample with a ligand;

c. contacting the sensor with the sample containing a pre-determined amount of the labelled target analyte, and an unlabelled target analyte;

d. applying an electroactive substrate to the sensor, such that a portion of the electroactive substrate is oxidised or reduced by the bound labelled target analyte; wherein binding of the labelled or unlabelled target analyte to the binding agent produces an electroactive species which mediates a detectable response at the sensing surface and

measuring a change at a measurement electrode electrically connected to at least one sensing surface.

24. The method of claim 23 wherein the electroactive substrate is TMB.

25. A method as claimed in claim 23 or 24 wherein the electroactive substrate contacts a ligand labelled target analyte which results in oxidation of the electroactive substrate to yield an electroactive species which elicits a detectable response at the sensing surface by contact with the sensing surface held at a reductive potential.

26. A method as claimed in any one of claims 22 to 25 wherein the binding layer is on a magnetic particle attached to a binding region on the support substrate, wherein the binding region is defined by an area of magnetic field established by a magnetic element on, in or under the support substrate which, in use, attracts and attaches magnetic particles to the support substrate.

27. A method as claimed in any one of claims 22 to 26 wherein the sample is applied by flowing the sample across the surface of the sensor in a microfluidic environment.

28. A method of detecting a target analyte in a sample wherein the method comprises:

a. providing a sensor comprising:

i. a support substrate comprising a binding region;

ii. at least one surface structure protruding from an upper surface of the support substrate, wherein the surface structure includes an electrode layer;

iii. a sensing surface on the electrode layer, wherein the sensing surface is adapted to contact a sample;

iv. a binding region on the support substrate, wherein the binding region is separated from the sensing surface and comprises a magnetic field

b. attaching at least one binding agent to a binding layer;

c. labelling the target analyte with a ligand;

d. contacting the sensor, binding agent and at least one sensing surface with a sample containing a pre-determined amount of the labelled target analyte, and an unlabelled target analyte such that a competitive assay occurs;

e. applying an electroactive substrate, for example TMB in the presence of H202, to the sensor, such that a portion of the electroactive substrate is oxidised or reduced by the bound labelled target analyte;

wherein binding of the labelled or unlabelled target analyte to the binding agent produces an electroactive species which mediates a detectable response at the sensing surface and measuring a change at a measurement electrode electrically connected to the at least one sensing surface.

29. A method as claimed in any one of claims 20 to 28 wherein the method comprises use of a sensor with two or more sensing surfaces on two or more electrode layers at increasing distance from the binding region.

30. A method according to claim 29 wherein the step of measuring a change comprises measuring a change at two or more measurement electrodes, each measurement electrode being electrically connected to an electrode layer and at least one sensing surface, the measurement electrodes being electrically isolated from each other; wherein the method further comprises:

measuring the change in the detectable response as a function of separation distance from the binding site;

comparing the change in the detectable response with the change in response from a known concentration of a target analyte from a control sample; and

determining the concentration of the target analyte in the sample.

31. A method of production of a sensor comprising:

a. providing a support substrate with at least one surface structure protruding from an upper surface of the support substrate;

b. depositing an electrode layer on an upper surface of the at least one surface structure; c. depositing an inert layer on the electrode layer such that one or more portion of the electrode layer remains exposed to form at least one sensing surface on the electrode layer, wherein the inert layer comprises a material substantially inert to the components of a sample to be analysed;

d. forming a binding region on the support substrate wherein the binding region is adapted to attach at least one binding agent electrostatically, covalently or magnetically;

wherein the sensing surface is separated from the binding region by a separation distance sufficient, in use, for a detectable response to be detected at the sensing surface following binding of a target analyte to the binding agent.

32. A method as claimed in claim 31 wherein the surface structures are separated from each other by about 50 nm to about 2000μιη apex to apex, and/or the width of the surface structure where it joins the support substrate is between about 20nm to about 5000μιη, and/or the width of the apex of each surface structure is preferably between about lnm to about 5000 micron.

33. A method as claimed in claim 31 or 32 wherein the at least one surface structure is integral with the support substrate.

34. A method as claimed in any one of claims 31 to 33 wherein a protective coating is applied to the electrode layer.

35. A method as claimed in any one of claims 31 to 34 wherein the sensing surfaces are formed by deposition of a binding layer or an inert layer on the support substrate or surface structures leaving an upper portion of the surface structures free of binding layer or inert layer thus resulting in the surface of the electrode layer on the upper portion of the surface structure being exposed as the sensing surface.

36. A method as claimed in claim 35 wherein the binding layer or inert layer is deposited adjacent to the electrode layer.

37. A method as claimed claim 35 or 36 wherein the inert layer is electrically conductive and is connected to at least one electrode capable of applying a potential to the inert layer.

38. A method as claimed in any one of claims 35 to 37 wherein the step of depositing a binding layer on either the electrode layer or the support substrate comprises applying a SAM binding layer.

39. A method of increasing the accuracy of sensing a target analyte in a sample, the method including the steps of providing a sensor comprising:

a. a support substrate;

b. at least one surface structure protruding from an upper surface of the support substrate, wherein the surface structure includes an electrode layer;

c. a sensing surface on the electrode layer, wherein the sensing surface is adapted to contact the sample;

d. a binding region on the support substrate, wherein the binding region is separated from the sensing surface;

e. contacting the binding agent with the sample containing the target analyte;

f. allowing the target analyte to bind to the binding agent to produce a detectable response at the sensing surface;

g. measuring a change at a measurement electrode electrically connected to the at least one sensing surface;

wherein binding of the target analyte to the binding agent produces an electroactive species which mediates the detectable response at the sensing surface and improves the accuracy of sensing the target analyte in the sample.

40. The method according to claim 39 wherein the separation distance between the binding region and the sensing surface is between about lnm and about 50mm.