METHOD FOR PREPARING A WORKING ELECTRODE

Abstract

The present invention relates to a method for the preparation of a working electrode, the method comprising application of a sensing material in several steps. Further, the present invention relates to an analyte sensor comprising the working electrode as well as to the use of the analyte sensor for detecting at least one analyte in a sample.

Claims

1. A method for manufacturing a working electrode of an analyte sensor, the method comprising the steps: a) providing a substrate comprising a first side and a second side, at least one conductive material positioned on the first side of the substrate, b) applying a sensing material to an application area on the first side of the substrate, comprising b1) applying a first layer of a sensing material at least partially onto the conductive material, b2) applying a second layer of the sensing material at least partially onto the first layer of the sensing material, and c) obtaining the working electrode of the analyte sensor on the first side of the substrate, wherein the sensing material comprises at least one enzyme and at least one crosslinker, wherein the first layer of the sensing material is applied in step (b1) and the second layer of the sensing material is applied in step (b2) independently of one another in a wet layer thickness of at most about 70 μm.

2. The method of claim 1 comprising a further step: b3) applying a third layer and optionally at least one further layer of the sensing material at least partially onto the second layer of the sensing material, wherein step (b3) is carried out after step (b2) and before step (c), wherein the third layer and the optional at least one further layer of sensing material is applied in step (b3) independently of one another in a wet layer thickness of at most about 70 μm.

3. The method of claim 1, wherein the at least one conductive material positioned on the first side of the substrate is selected from gold, carbon, carbon paste and any combination thereof.

4. The method of claim 1, wherein the enzyme is a glucose oxidase (GOx).

5. The method of claim 1, wherein the at least one crosslinker is a diglycidyl ether, particularly poly(ethylene glycol) diglycidyl ether (PEG-DGE).

6. The method of claim 1, wherein the sensing material further comprises at least one metal-containing complex.

7. The method of claim 1, wherein at least one of the steps (b1), (b2), and, if present, (b3) is carried out via cannula-coating.

8. The method of claim 7, wherein the speed of the substrate relative to the cannula during at least one of the steps (b1), (b2), and, if present, (b3) is in the range from about 1 mm/s to about 20 mm/s, particularly about 8 mm/s.

9. The method of claim 7, wherein the flow rate of the sensing material during at least one of the steps (b1), (b2), and, if present, (b3) is in the range from 0.02 ml/min to about 0.04 ml/min, particularly about 0.03 ml/min.

10. The method of claim 7, wherein the distance between the cannula and the surface of the first side of the substrate to which the sensing material is applied during at least one of the steps (b1), (b2), and, if present, (b3) is in the range from about 30 to about 50 μm, particularly about 40 μm.

11. The method of claim 1, wherein after at least one of the steps ((b1), (b2), and, if present, (b3) the sensing material is dried and wherein the sensing material has a total dry thickness in the range from about 1 μm to about 10 μm, particularly from about 1 μm to about 6 μm, and more particularly from about 2 μm to about 5 μm.

12. A method for manufacturing an analyte sensor comprising manufacturing a working electrode according to claim 1 and providing at least one further electrode.

13. An analyte sensor comprising: (i) a substrate comprising a first side and a second side, and at least one conductive material positioned on the first side of the substrate, and (ii) a working electrode comprising a sensing material, which at least partially covers the first side of the substrate, wherein the sensing material is applied to an application area on the first side of the substrate, in a manner so that the sensing material is applied at least partially onto the conductive material, and optionally wherein the sensing material is at least partially removed from a first portion of the application area and is preserved on a second portion of the application area, and wherein the sensing material comprises at least one enzyme and at least one crosslinker, wherein the sensing material has a dry total thickness in the range from about 1 μm to about 10 μm, and wherein the dry total thickness of the sensing material is substantially uniform over the application area including the edges of the application area or optionally over the preserved second portion of the application area including the preserved edges of the application area.

14. The analyte sensor of claim 13 comprising at least one further electrode, particularly a combined counter/reference electrode.

15. Use of an analyte sensor of claim 13 for detecting at least one analyte in a sample.

Description

DESCRIPTION OF THE FIGURES

[0225] FIG. 1 shows a schematic depiction of a working electrode comprising a sensing material layer applied in a single step.

[0226] FIG. 2 shows a schematic depiction of a working electrode comprising a sensing material layer applied in two separate steps according to the present invention.

[0227] FIG. 3 shows a topographic measurement of the dry total thickness of a sensing material on a substrate after application of the sensing material in a single step.

[0228] FIG. 4 shows a topographic measurement of the dry total thickness of a sensing material on a substrate after application of the sensing material in three steps according to the present invention.

[0229] FIG. 1 shows an embodiment of a comparative working electrode comprising a sensing material layer prepared in a single application step. An analyte sensor 124 comprises a sensor substrate 114 having a first side 120. The first side 120 comprises at least one conductive material 111 comprising more preferably two materials, e.g. gold and/or carbon. Specifically, the conductive material may comprise a layer of gold 112 and a layer of a further material 110, for example carbon. A sensing material layer 118 applied in a single step onto the conductive material 111 positioned on the first side 120 of the sensor substrate 114 and subsequently dried is shown. The sensing material layer 118 covers at least a portion of the conductive material 111. At the edges 121a and 121b of the sensing material layer 118 a substantial increase in the total dry thickness 125a compared to the total dry thickness 125b in the central section of the sensing material layer 118 is observed.

[0230] FIG. 2 shows an embodiment of a working electrode according to the present invention. As in FIG. 1, an analyte sensor 124 comprises a sensor substrate 114 having a first side 120. The first side 120 comprises at least one conductive material 111 comprising more preferably two materials, e.g. gold and/or carbon. Specifically the conductive material may comprise a gold layer 112 and a layer of a further material 110, for example carbon.

[0231] In contrast to FIG. 1, a sensing material layer 118 applied in two separate steps and dried after each application step onto the conductive material 111 positioned on the first side 120 of the sensor substrate 114 is shown. The sensing material layer 118 covers at least a portion of the conductive material 111. The dried sensing material layer 118 comprises a first dried sensing material layer 118a and a second dried sensing material layer 118b. The first and the second sensing material layers 118a and 118b usually have the same composition. They are applied in wet form by cannula-coating (not shown) each having a wet layer thickness of at most about 70 μm. After drying, a layer of dry sensing material 118 having a substantially uniform dry total thickness typically between about 1 μm and 6 μm, preferably about 2 μm and about 4 μm over the application area is obtained. At the edges 121a and 121b of the sensing material layer 118, the total dry thickness 125a is substantially the same as the total dry thickness 125b in the central section of the sensing material layer 118.

[0232] The analyte sensor 124 is an electrochemical sensor comprising at least one electrode and respective circuitry. More particularly, the analyte sensor 124 is an amperometric electrochemical sensor comprising the at least one working electrode. Typically, the analyte sensor 124 comprises at least one further electrode, particularly a counter electrode and/or a reference electrode and/or a combined counter/reference electrode. The working electrode may be sensitive for the analyte to be measured at a polarization voltage which may be applied between working and reference electrodes and which may be regulated by a potentiostat. A measurement signal may be provided as an electric current between the counter electrode and the working electrode. A separate counter electrode may be absent and a pseudo reference electrode may be present, which may also work as a counter electrode. Thus, an analyte sensor 124 typically may comprise a set of at least two or a set of three electrodes. Specifically, the sensing material 118 is present in the working electrode 122 only.

[0233] The invention is not limited to one of the embodiments described above, but is modifiable in a great variety of ways. Those skilled in the art recognize that the embodiments according to the invention can easily be adapted without departing from the scope of the invention. Thus, simple adaptations are conceivable for the preparation of the analyte sensor. The invention enables the preparation of an analyte with reproducible sensor sensitivity at reduced production costs. Further characteristics, details and advantages of the invention follow from the wording of the claims and from the following description of practical examples based on the drawings.

[0234] The content of all literature references cited in this patent application is hereby included by reference to the respective specific disclosure content and in its entirety.

EXAMPLES

[0235] The following examples serve to illustrate the invention. They must not be interpreted as limiting with regard to the scope of protection.

Example 1: Preparation of a Sensing Material Layer on a Working Electrode in a Single Step

[0236] A sensor substrate based on polyethylene terephthalate and a thin layer of gold was coated with a carbon paste via doctor blading. Suitable Carbon conductive inks are available from Ercon, Inc. (Wareham, MA), E.I. du Pont de Nemours and Co. (Wilmington, DE), Emca-Remex Products (Montgomeryville, PA), or TEKRA, A Division of EIS, Inc (New Berlin, WI). Afterwards, the carbon paste was dried for 12 h at 50° C.

[0237] A layer of sensing material was applied on the sensor substrate by cannula-coating (cannula 1.6 mm (inner diameter), flow rate 0.09 ml/min, speed 8 mm/s, distance between cannula and substrate 100 μm). The sensing material was dried for 10 minutes at 37° C.

[0238] The sensing material comprised 57% by weight of a polymeric transition metal complex (modified poly (vinylpyridine) backbone loaded with poly(biimidizyl) Os complexes covalently coupled through a bidentate linkage), 33% by weight of glucose oxidase and 10% by weight of PEG-DGE (poly(ethylene glycol)-diglycidylether) in each case based on the sum of the percentages by weight of the polymeric transition metal complex, glucose oxidase and PEG-DGE. Water was used as solvent. The total concentration of the polymeric transition metal complex, glucose oxidase and PEG-DGE in water was 50 mg/ml.

[0239] After drying, an increased thickness at the edges of the sensing material layer was found by a topography measurement on the sensor. The thickness of the sensing material layer was 5 to 10 μm at the edges being significantly higher than in the in the center region as shown in FIG. 3.

[0240] The increased thickness at the edges may have negative effects in a laser ablation. In case a layer of about 5 μm is removed by ablation, sensing material remains at the edges and can affect the sensitivity of the sensor.

Example 2: Preparation of a Sensing Material Layer on a Working Electrode in Separate Steps According to the Present Invention

[0241] A sensor substrate coated with gold and carbon paste was prepared as described in Example 1.

[0242] The sensing material of Example 1 was used.

[0243] A layer of sensing material was applied on the sensor substrate by cannula-coating in three separate steps with an intermediate drying time of about 3 min each.

[0244] The sensing material was applied on the sensor substrate by cannula-coating (cannula 1.6 mm (inner diameter), flow rate 0.03 ml/min, speed 8 mm/s, distance between cannula and substrate 30 μm). After each application, the sensing material was dried for 3 min at 22° C.

[0245] FIG. 4 shows a topography measurement on a sensor after application of the sensing material in three separate steps. No increased thickness at the edges of the sensing material was found.

Example 3: Variation of Coating Conditions in the Preparation of a Sensing Material Layer on a Working Electrode in Separate Steps According to the Present Invention

[0246] The uniformity of the coating layer may be improved by the type and amount of crosslinker, the amount of enzyme and the transition metal complex-containing polymer. Particularly, the presence of a crosslinker is advantageous.

[0247] In the experiments of Tables 2 and 3, the sensing material according to Example 1 was used, whereas in the experiments of Table 1, the crosslinker was omitted from the sensing material.

[0248] Table 1 shows the results of coating experiments without crosslinker.

TABLE-US-00001 Wet layer Flow Dimensions thickness Height Height rate Distance Speed width theoretically theoretically measured Edge Crosslinker ml/min [μm] [mm/s] [mm] [μm] [μm] [μm] [μm] — 0.02 30-60 8 1 42 1.5 1 2 — 0.03 30-60 8 1 63 2.2 1 2 — 0.04 30-60 8 1.3 64 2.2 2 3 — 0.05 30-60 8 1.4 74 2.6 2 3 — 0.06 30-60 8 1.5 96 3.4 3 4

[0249] As can be gathered from Table 1, an increased thickness at the edges was observed. Further, a coating over the complete breadth of the cannula was not obtained.

[0250] Table 2 shows the results of coating experiments in the presence of crosslinker PEG-DGE

TABLE-US-00002 Wet layer Flow Dimensions thickness Height Height rate Distance Speed width theoretically theoretically measured Edge Crosslinker ml/min [μm] [mm/s] [mm] [μm] [μm] [μm] [μm] PEGDGE 200 0.02 30-60 8 1 42 1.5 n.a. n.a. PEGDGE 200 0.03 30-60 8 1.3 48 1.7 1 — PEGDGE 200 0.04 30-60 8 1.3 64 2.2 1.5 2 PEGDGE 200 0.05 30-60 8 1.3 80 2.8 1.5 3 PEGDGE 200 0.06 30-60 8 1.3 96 3.4 2 3.5

[0251] When using the crosslinker PEG-DGE 200, an improved spreading of the sensing material was observed, however, not across the complete breadth of the cannula (inner diameter 1.54 mm, outer diameter 1.83 mm). An increased thickness at the edges was only observed when the wet layer thickness was higher than about 40 μm.

[0252] Table 3 shows the results of coating experiments in the presence of crosslinker PEG-DGE 500 10% (w/w) dry.

TABLE-US-00003 Wet layer Flow Dimensions thickness Height Height rate Distance Speed width theoretically theoretically measured Edge Crosslinker ml/min [μm] [mm/s] [mm] [μm] [μm] [μm] [μm] PEGDGE 500 0.01 30-60 8 0.5 42 1.5 — — PEGDGE 500 0.02 30-60 8 1.3 32 1.1 — — PEGDGE 500 0.03 30-60 8 1.8 35 1.2 1 — PEGDGE 500 0.04 30-60 8 1.8 46 1.6 1.5 — PEGDGE 500 0.05 30-60 8 1.7 61 2.1 1.5 2 PEGDGE 500 0.06 30-60 8 1.7 74 2.6 1.5 2.5 PEGDGE 500 0.09 100-130 8 1.6 117 4.1 3 6

[0253] When using the crosslinker PEG-DGE 500, the sensing material was spreading over the complete cannula breadth with a flow rate of at least 0.03 ml/min. An increased thickness at the edges was observed only at a wet layer thickness of more than about 60 μm.