GALVANICALLY FUNCTIONALIZED SENSORS

Abstract

The present invention relates to a biosensor for determining an analyte comprising a substrate, a working electrode comprising an electrically conductive pad in conductive contact with a mediator layer, and an enzyme layer in diffusion-enabling contact with said mediator layer, wherein said mediator layer is an electrodeposited mediator layer, and wherein said mediator layer comprises, in an embodiment consists of, an electrocatalytic agent. The present invention further relates to a method for manufacturing a biosensor, comprising providing a substrate having at least one conductive pad, electrodepositing a mediator layer onto at least part of said conductive pad, wherein said mediator layer comprises, in an embodiment consists of, an electrocatalytic agent, and depositing an enzyme layer onto at least part of said mediator layer. Moreover, the present invention relates to uses and methods related to the biosensor of the present invention.

Claims

1. (canceled)

2. A method for the in vivo electrochemical determination of a glucose analyte comprising: transcutaneously implanting an in vivo biosensor beneath a patient's skin in contact with a bodily fluid comprising glucose, the in vivo biosensor comprising a substrate, a working electrode comprising a first electrically conductive pad, a counter and/or reference electrode comprising a second electrically conductive pad, a mediator layer electrodeposited on and in conductive contact with the working electrode, the mediator layer comprising manganese and/or MnO.sub.2, and an enzyme layer in diffusion-enabling contact with the mediator layer, the enzyme layer comprising a glucose oxidase, the mediator layer and the enzyme layer being separate from each other; closing an electrical circuit comprising the working electrode, the mediator layer, and the counter electrode; applying a voltage to the electrical circuit; measuring the resulting current; and determining the glucose analyte in the bodily fluid from the measured current.

3. The method of claim 2, comprising a method performing continuous monitoring of the glucose analyte.

4. The method of claim 2, wherein the working electrode comprises a multiplicity of electrically conductive pads, of which at least one is in conductive contact with the mediator layer in diffusion-enabling contact with the enzyme layer.

5. The method of claim 2, wherein the biosensor comprises a layer of reference material.

6. The method of claim 5, wherein the reference material comprises silver.

7. The method of claim 2, wherein the mediator layer consists of MnO.sub.2.

8. The method of claim 2, wherein the mediator layer separates the enzyme layer from the working electrode.

9. The method of claim 2, wherein the separate enzyme layer is deposited on the mediator layer.

10. The method of claim 2 in which the biosensor further comprises a diffusion membrane separating the working electrode, the mediator layer, and the counter electrode from the bodily fluid.

11. The method of claim 10 in which the biosensor further comprises an outmost layer comprising a biocompatible layer.

Description

FIGURE LEGENDS

[0095] FIG. 1: Galvanostatic Chronoamperometry of Ag deposition at 250 μA in example 1. x-axis: time t (s); y-axis: potential ΔU (V).

[0096] FIG. 2: Stability of the potential of an electrode produced by electrodeposition of silver, followed by electro-oxidation in chloride-containing solution. Shown is the potential vs. a MnO.sub.2 electrode over time; dashed line: Ag only (before oxidation) solid line: AgCl (after oxidation). x-axis: time t (s); y-axis: potential ΔU (V).

[0097] FIG. 3: Determining deposition voltage for depositing MnO.sub.2 from a MnSO.sub.4-solution; galvanostatic Cyclic Voltammogramm of MnSO.sub.4 at a potential of 350 mV vs. Ag/AgCl/0.15 M Cl.sup.−. x-axis: current I (A); y-axis: potential ΔU (V).

[0098] FIG. 4: Functionality of an electrodeposited MnO.sub.2 layer in catalysis of electrochemical H.sub.2O.sub.2-oxidation. At time points 100 s, 280 s, 430 s, and 550 s, H.sub.2O.sub.2 was added, followed by sporadic mixing (100 s), moderate mixing (280 s), intensive mixing (430 s), and intensive mixing by inverting the whole sensor cell (550 s). x-axis: time t (s); y-axis: current I (A).

[0099] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

EXAMPLE 1: SILVER DEPOSITION ON A SUBSTRATE: REFERENCE ELECTRODE

[0100] A 3-electrode setup was used (working electrode, reference electrode, counter electrode; all having Au-Pads) on an Autolab PGSTAT128N potentiostat/galvanostat. Using galvanostatic chronopotentiometry at −250 μA (FIG. 1), a particularly high rate of deposition of silver from a silver nitrate solution onto a gold electrode pad could be obtained; deposition starts at approximately −120 mV (vs. the Au-pseudo-reference in AgNO3 solution). The deposited shiny silver was visible in the microscope. The amount deposited was determined as: Charge Q=180 s×250 μA=45 mC; amount of substance n=Q/F=(45 E−3 C)/96484 C/mol=460 nmol. Accordingly, silver can be selectively galvanically deposited onto a reference electrode pad.

EXAMPLE 2: POTENTIAL STABILITY AFTER PARTIAL OXIDATION

[0101] To test usability as reference electrode, the Ag-coated reference electrode of Example 1 was tested for potential stability. The electrode initially showed the expected reference potential (approx. 275-325 mV vs. manganese dioxide) without oxidation; however, the potential was not stable. After a short (10 seconds) galvanostatic oxidation at 1 μA in a chloride-ion containing solution, the potential against MnO.sub.2 was essentially constant. The ratio Ag/AgCl was adjusted via the amount of charge applied. In FIG. 2, 45 mC Ag deposition (Example 1) (dashed line) and only 10 μC oxidation to AgCl (solid line) were used, which reduced potential drift drastically.

EXAMPLE 3: MNO.SUB.2 .DEPOSITION ON A SUBSTRATE: WORKING ELECTRODE

[0102] MnO.sub.2 was deposited onto multiple pads of a sensor electrode in a 2-electrode setup (working electrode, reference electrode/counter electrode, sensor reference electrode unused), using a galvanostatic CV from 0 to 15 μA (vs. reference electrode/counter electrode in Mn2+); deposition potential was 3.4 V with a disc voltage reference electrode=counter electrode at 2 V on sensor counter electrode (FIG. 3). Under these conditions, MnO.sub.2 was deposited onto all working electrode pads. Accordingly, galvanically depositing MnO.sub.2 is possible.

EXAMPLE 4: FUNCTIONALITY TESTING (H.SUB.2.O.SUB.2.-OXIDATION)

[0103] Functionality of the electrodeposited MnO.sub.2 in H.sub.2O.sub.2-oxidation and functionality of the Ag/AgCl reference electrode were tested in chronoamperometry at 350 mV vs. sensor reference and addition of H.sub.2O.sub.2. As shown in FIG. 4, the sensor shows an H.sub.2O.sub.2-dependent signal at 350 mV. Notably, the zero current is very low (approx. 50 pA). Moreover, a running-in of zero current is not observed, since the galvanically deposited electrode does not contain ether peroxides from the paste solvent DEGMBE.