ELECTRODE ASSEMBLY

Abstract

The present invention relates to an electrode assembly (eg a nanoelectrode assembly), to an electrochemical glucose biosensor comprising the electrode assembly and to an apparatus for combating (eg management of) diabetes mellitus which comprises the electrochemical glucose biosensor.

Claims

1. A nanoelectrode assembly having a laminate structure comprising: a first insulating capping layer; a first conducting layer capped by the first insulating capping layer and substantially sandwiched or encapsulated by at least the first insulating capping layer such as to leave exposed only an electrical contact surface; and an array of etched voids extending through at least the first insulating capping layer and the first conducting layer, wherein each void is partly bound by a surface of the first conducting layer which acts as an internal submicron electrode upon or adjacent to which is immobilised a glucose sensitive enzyme, wherein in use a glucose-containing bodily fluid passes into the etched voids for exposure to the immobilised glucose sensitive enzyme.

2. The nanoelectrode assembly as claimed in claim 1, wherein in use, relative mass transfer of glucose and oxygen from the bodily fluid to the immobilised glucose sensitive enzyme is unselective.

3. The nanoelectrode assembly as claimed in claim 1, wherein in use, relative mass transfer of glucose and oxygen from the bodily fluid to the immobilised glucose sensitive enzyme is interventionless.

4. The nanoelectrode assembly as claimed in claim 1, wherein in use, relative mass transfer of glucose and oxygen from the bodily fluid to the immobilised glucose sensitive enzyme is unimpeded.

5. The nanoelectrode assembly as claimed in claim 1, which is free of a glucose-restricting membrane.

6. The nanoelectrode assembly as claimed in claim 1, comprising: a plurality of conducting layers (which may be the same or different) including the first conducting layer and a plurality of insulating capping layers (which may be the same or different) including the first insulating capping layer, wherein the plurality of conducting layers and the plurality of insulating capping layers are alternating in the laminate structure, wherein each conducting layer is sandwiched or encapsulated to leave exposed only an electrical contact surface and the array of etched voids extends through the plurality of insulating capping layers and the plurality of conducting layers, wherein each void is partly bound by a surface of each of the plurality of conducting layers which acts as an internal submicron electrode upon or adjacent to which is immobilised the glucose sensitive enzyme.

7. The nanoelectrode assembly as claimed in claim 1, wherein the thickness (w.sub.n) of the (or each) conducting layer (which may be the same or different) is in the range 0.10 to 75 nm.

8. The nanoelectrode assembly as claimed in claim 1, further comprising: an insulating substrate layer; a second insulating capping layer fabricated on the insulating substrate layer, wherein the first conducting layer is fabricated on the second insulating capping layer and is substantially sandwiched or encapsulated by the first insulating capping layer and the second insulating capping layer such as to leave exposed only an electrical contact surface of the first conducting layer.

9. The nanoelectrode assembly as claimed in claim 1, which is substantially planar.

10. The nanoelectrode assembly as claimed in claim 1, which is cylindrical.

11. The nanoelectrode assembly as claimed in claim 1, wherein the glucose sensitive enzyme is glucose oxidase.

12. An electrochemical glucose biosensor comprising: a nanoelectrode assembly as defined in any preceding claim operable as a working electrode; and a reference electrode and a counter electrode or a combined counter reference electrode.

13. An apparatus for combating diabetes mellitus in a subject comprising: an electrochemical glucose biosensor as defined in claim 12 for continuously measuring the glucose level in the subject; a signal generating device for generating an actuating signal in response to the glucose level exceeding a threshold; and a delivery device for delivering insulin to the subject in response to the actuating signal, wherein in use the electrochemical glucose biosensor, signal generating device and delivery device communicate in a closed loop.

Description

[0089] The present invention will now be described in a non-limitative sense with reference to the accompanying Examples and Figures in which:

[0090] FIG. 1: An illustration of the typical response of an embodiment of the electrochemical glucose biosensor of the invention;

[0091] FIG. 2: A schematic partial cross-section and top view of a first embodiment of the nanoelectrode assembly of the invention;

[0092] FIGS. 3a-b: A top view of two variations of the nanoelectrode assembly of the first embodiment;

[0093] FIG. 4: A response from a 150 μm needle-like sensor;

[0094] FIG. 5: A schematic perspective view of a second embodiment of the nanoelectrode assembly of the invention; and

[0095] FIG. 6: A schematic perspective view of a third embodiment of the nanoelectrode assembly of the invention.

EXAMPLE 1

[0096] A commercial electrode (303D platinum 50 nm nanoband electrode, NanoFlex Ltd (UK)) was used to prepare a glucose oxidase (GOx)-immobilised working electrode mediated by oxygen (as described below) for use in a three electrode electrochemical cell with a saturated calomel electrode (Scientific Laboratory Supplies (UK)) and a 0.5 mm diameter platinum wire counter electrode (Fisher Scientific (UK)).

Conditioning of the Electrode

[0097] The commercial electrode was cleaned by soaking in acetone for 10 minutes, iso-propanol for 10 minutes and 18.2 MΩ deionised water for 10 minutes and then dried under nitrogen.

[0098] The electrode was conditioned electrochemically using cyclic voltammetry. Firstly 50 cm.sup.3 of 0.1 mol dm.sup.−3 citrate buffer was placed in an electrochemical cell and appropriate connections were made with the potentiostat. The electrode was conditioned using parameters detailed in Table 1.

TABLE-US-00001 TABLE 1 Conditioning parameters for citrate buffer Conditioning Solution 0.1 mol dm.sup.−3 citrate buffer (50 cm.sup.3) Start E (V) 0.5 High E (V) 1.7 Low E (V) −1.2 Scans 10 Scan Rate (V s.sup.−1) 0.1 Initial Polarity Positive Step (V) 0.001

[0099] The electrode was then removed from the electrochemical cell and rinsed with copious amounts of 18.2 MΩ deionised water. The electrode was then immersed in 50 cm.sup.3 of 0.05 mol dm.sup.−3 sulfuric acid solution and conditioned using parameters detailed in Table 2.

TABLE-US-00002 TABLE 2 Conditioning parameters for sulfuric acid Conditioning Solution 0.05 mol dm.sup.−3 sulfuric acid (50 cm.sup.3) Start E (V) 0.5 High E (V) 1.0 Low E (V) −1.2 Scans 10 Scan Rate (V s.sup.−1) 0.1 Initial Polarity Positive Step (V) 0.001

[0100] Preparation of the GOx-Immobilised Working Electrode

[0101] The conditioned electrode was placed in a separate glass beaker with the array facing upwards. 2 cm.sup.3 of concentrated sulfuric acid (99.99% purity) was pipetted onto the electrode to cover the entire surface and left for 5 minutes to remove all traces of organics. The electrode was then rinsed in copious amounts of 18.2 MΩ deionised water and dried under nitrogen.

[0102] The electrode was immersed in 50 μmol dm.sup.−3 ethanolic mercaptohexyl amine (MHA) prepared in a glass container. The container was back-filled with dry nitrogen and the cap was sealed and wrapped with parafilm. The electrode was stored in this condition at room temperature (21° C.) for 24 hours in the dark.

[0103] The thiolated electrode was then taken out of the ethanolic MHA solution and rinsed in ethanol for 10-15 seconds using a clean solvent bottle to remove excess thiol. It was then immediately rinsed in 18.2 MΩ deionised water and then dried under dry nitrogen.

[0104] 150 μL of a 5% solution of glutaraldehyde prepared in deionised water was pipetted onto the electrode which was then left to incubate for 45 minutes at room temperature. 40 mg/mL GOx was then made up in 0.01 mol dm.sup.−3 phosphate buffered saline (PBS) and 150 uL of the GOx solution was added onto the electrode. The electrode was left to incubate for 2 hours at room temperature. The GOx solution was then removed and the electrode was rinsed with 0.01 mol dm.sup.−3 PBS (pH 7.0) and left immersed in 0.01 mol dm.sup.−3 PBS until used.

[0105] The working electrode is an example of a first embodiment of the nanoelectrode assembly of the invention with internal submicron electrodes upon each of which is immobilised GOx. The nanoelectrode assembly 30 is illustrated schematically in part cross-section and from the top in FIG. 2 and is a planar laminate structure which has a substantially square (plate-like) profile. The nanoelectrode assembly 30 comprises a conducting layer 33 of platinum (thickness w.sub.1=50 nm) deposited on an insulating capping layer 32 of silicon oxide which is thermally grown on a silicon wafer substrate 34. An insulating capping layer 31 is deposited over the extent of the conducting layer 33 with the exception of one corner 36 which is left exposed to act as an electrical contact for direct and simple connection to an electrochemical measuring device (eg potentiostat). An array of square voids 37 is etched through insulating capping layer 31 and conducting layer 33 and partly through insulating capping layer 32 to an etch depth (d.sub.d) which is short of the substrate 34.

[0106] Performance of the GOx Immobilised Working Electrode

[0107] Table 4 provides details of the reagents and the parameters used for glucose detection.

TABLE-US-00003 TABLE 4 Sample preparation Blank electrolyte 250 cm.sup.3 of 1 × 10−4 mol 1. Weigh out 0.345 g of sodium phosphate dm.sup.−3 ferrocenemethanol monobasic and 0.445 g of sodium phosphate in 0.01 mol dm.sup.−3 dibasic and transfer to a 250 cm.sup.3 volumetric Sodium Phosphate Buffer flask and make up to mark with deionised Solution (PBS) (pH 7.0). water. Mix thoroughly to dissolve (final concentration = 0.01 mol dm.sup.−3 PBS) 2. Weigh out 0.0054 g of ferrocenemethanol and transfer to a separate 250 cm.sup.3 volumetric flask and make up to mark with the 0.01 mol dm.sup.−3 PBS. Sonicate for 30 minutes to dissolve completely. β-D-glucose stock Weigh out 0.3603 g of D-glucose into a 20 cm.sup.3 solution 1 volumetric flask and make up to the 20 cm.sup.3 of 0.1 mol mark with 0.01 mol dm.sup.−3 PBS. Mutarotate dm.sup.−3 β-D-glucose the solution at 4° C. for 24 hours to gain equilibration between α- and β-forms of glucose. β-D-glucose stock Pipette 2 mL of the β-D-glucose stock solution 2 solution 1 into a 20 cm.sup.3 volumetric flask 20 cm.sup.3 of 0.01 mol and make up to the mark with 0.01 mol dm.sup.−3 dm.sup.−3 β-D-glucose PBS. Mutarotate the solution at 4° C. for 24 hours to gain equilibration between α- and β-forms of glucose. Parameters for Cyclic Voltammetry Start E (V) 0.2 High E (V) 0.9 Low E (V) −0.2 Scans 1 Scan Rate (V s.sup.−1) 0.05 Initial Polarity Positive Step (V) 0.001

[0108] Sensor Performance

[0109] The electrochemical cell was filled with 50 cm.sup.3 of the blank electrolyte solution. The solution was used as received (no aeration). A measurement was first taken in the blank using parameters detailed in Table 4. The solution was mixed thoroughly with a magnetic stirrer for 1 minute at 500 rpm and a measurement was taken using parameters in Table 4. Glucose was added sequentially up to a concentration of 60 mmol dm.sup.−3 and the current response was measured.

[0110] It can be seen from FIG. 1 that the electrode responded across the full concentration range over which the enzyme is capable of working. It is therefore apparent that it is possible to use the enzyme in conditions similar to the expected physiological concentrations of oxygen where glucose concentration remains the limiting factor and therefore enables the device to act as a glucose biosensor without the need for the use of glucose-restricting membranes. A limit of detection of 2.6 μmol dm.sup.−3 was calculated using the IUPAC methodology.

[0111] In alternative embodiments shown in FIGS. 3a-b, the profile of the planar laminate structure is substantially rectangular (strip-like) and may incorporate an end projection 40 to facilitate implantation (needle-like—see FIG. 3b). FIG. 4 illustrates the response from a 150 μm needle-like electrochemical glucose biosensor.

EXAMPLE 2

[0112] FIG. 5 illustrates a schematic perspective view of a second embodiment of the nanoelectrode assembly of the invention which is a substantially cylindrical laminate structure. The nanoelectrode assembly 50 comprises a conducting layer 3 deposited on an insulating capping layer 2 which itself is on a hollow cylindrical support 1. An insulating capping layer 4 is deposited over the extent of the conducting layer 3 and incorporates an electrical contact 5 for direct and simple connection to an electrochemical measuring device (eg potentiostat). An array of square voids 7 is etched through insulating capping layer 4 and conducting layer 3 and partly through insulating capping layer 2 to an etch depth which is short of the hollow cylindrical support 1. The array of square voids 7 extends over only a lower portion 10 of the cylindrical laminate structure. The lower portion 10 is selectively implantable into the body to leave exposed an upper portion 11. The hollow cylindrical support 1 defines a receiving bore for gas or fluid delivery.

EXAMPLE 3

[0113] FIG. 6 illustrates a schematic perspective view of a third embodiment of the nanoelectrode assembly of the invention which is a substantially cylindrical laminate structure. The nanoelectrode assembly 60 comprises a conducting layer 63 deposited on an insulating capping layer 62 which itself is on a solid cylindrical support 61. An insulating capping layer 64 is deposited over the extent of the conducting layer 63 and incorporates an electrical contact 65 for direct and simple connection to an electrochemical measuring device (eg potentiostat). An array of square voids 67 is etched through insulating capping layer 64 and conducting layer 63 and partly through insulating capping layer 62 to an etch depth which is short of the solid cylindrical support 61. The array of square voids 67 extends over only a lower portion 80 of the cylindrical laminate structure. The lower portion 80 is selectively implantable into the body to leave exposed an upper portion 81.