Continuous analyte monitoring electrode with crosslinked enzyme

Abstract

The present invention relates to an enzymatic electrode comprising a conductive surface and wherein a conjugate comprising at least one enzyme molecule is covalently bound to the conductive surface. The electrode is suitable for continuous analyte monitoring, particularly for continuous glucose monitoring (CGM) with glucose oxidase (GOD) as enzyme molecule. Further, the invention relates to an electrochemical sensor for measuring the concentration of an analyte, e.g. glucose under in vivo conditions comprising the enzymatic electrode.

Claims

1. An electrode comprising a conductive surface and a conjugate comprising at least one enzyme molecule and at least one conductive nanoparticle covalently bound to each other, wherein the conjugate is covalently bound to the electrode surface via the at least one enzyme molecule and wherein the nanoparticle is not directly covalently bound to the conductive surface.

2. The electrode of claim 1, wherein the at least one enzyme molecule is an H2O2 generating and/or consuming enzyme molecule selected from the group consisting of a glucose oxidase (EC 1.1.3.4), a hexose oxidase (EC 1.1.3.5), an (S)-2 hydroxy acid oxidase (EC 1.1.3.15), a cholesterol oxidase (EC 1.1.3.6), a galactose oxidase (EC 1.1.3.9), an alcohol oxidase (EC 1.1.3.13), an L-glutamate oxidase (EC 1.4.3.11) and an L-aspartate oxidase (EC 1.4.3.16).

3. The electrode of claim 1, wherein the covalent binding of the at least one enzyme molecule to the electrode surface occurs via a sulfur containing functional group selected from the group consisting of a sulfide group and a disulfide group, and/or wherein the covalent binding of the at least one enzyme molecule to a nanoparticle occurs via a sulfur containing functional group selected from the group consisting of a sulfide group and a disulfide group.

4. The electrode of claim 1, wherein the at least one enzyme molecule has been modified to incorporate at least one functional group for covalent binding to the electrode surface and a nanoparticle, and wherein the at least one enzyme molecule has been modified at the amino terminus and/or at an amino side chain group.

5. The electrode of claim 4, wherein the at least one enzyme molecule has been modified by reacting the at least one enzyme with a functionalizing reagent in a molar ratio of enzyme to functionalization reagent of about 1:1 to about 1:10.

6. The electrode of claim 1, wherein the nanoparticles are metal nanoparticles selected from the group consisting of platinum, palladium, iridium, gold and silver nanoparticles.

7. The electrode of claim 1, wherein the nanoparticles have an average size of about 1 to about 100 nm.

8. The electrode of claim 1, wherein the conjugate has an average size of about 10 nm to about 300 nm.

9. The electrode of claim 1, wherein the conjugate does not contain a redox mediator.

10. The electrode of claim 1, wherein the electrode surface is a metal surface.

11. An electrochemical sensor for measuring the concentration of an analyte comprising at least one electrode according to claim 1.

12. The sensor of claim 11, wherein the enzyme molecule is a functionalized glucose oxidase, and the analyte is glucose.

13. The sensor of claim 11 for in vivo or in vitro use.

14. A method of manufacturing an electrode of claim 1, comprising the steps: (a) preparing a conjugate of at least one enzyme molecule and at least one nanoparticle under conditions wherein only the at least one enzyme molecule, but not the nanoparticle in the conjugate has free functional groups for covalently binding to a conductive surface of an electrode, and (b) covalently binding said conjugate to the conductive surface of the electrode, wherein the binding exclusively occurs via free functional groups on the at least one enzyme molecule.

15. A method of measuring an analyte in a tissue and/or a body fluid comprising using the electrode of claim 1.

16. A method of measuring an analyte in a tissue and/or a body fluid comprising using the electrochemical sensor of claim 11.

17. An electrode comprising: a conductive surface; and a conjugate comprising at least one enzyme molecule and at least one nanoparticle covalently bound to each other, wherein the at least one enzyme molecule, and not the nanoparticle, has free functional groups covalently bound to the conductive surface.

Description

(1) Further, the present invention is described in the context of the following figures and examples.

(2) FIG. 1 shows an exemplary embodiment of a working electrode according to the invention.

(3) FIG. 2 shows a non-reducing polyacrylamide gel electrophoresis of reaction mixtures of glucose oxidase (GOD) and Lomant's reagent (DSP) and different molar ratios.

(4) FIG. 3 shows the size distribution of different conjugates of GOD and platinum nanoparticles measured by dynamic light scattering.

(5) FIG. 4 shows the results from three different measurements of a working electrode to which a GOD-Pt nanoparticle conjugate has been bound (A): Current during measurement cycle at three voltages (0.20 V-0.35 V-0.20 V). (B): Average current at three voltages during the measurement cycle.

(6) In FIG. 1, an exemplary embodiment of a working electrode for the measurement of glucose is shown. To an electrode surface, e.g. an Au electrode surface, an enzyme molecule is covalently bound via a sulfur-containing functional group. Further, the enzyme molecule is covalently bound via another sulfur-containing functional group to at least one nanoparticle, e.g. a Pt nanoparticle.

(7) The enzyme molecule is a glucose oxidase which has been modified at surface exposed amino side chain groups with Lomant's reagent, thereby generating sulfur-containing functional groups. Glucose oxidase catalyzes the oxidation of glucose to gluconolactone, thereby generating H.sub.2O.sub.2. A catalytic decomposition of H.sub.2O.sub.2 to H.sub.2O occurs on the nanoparticle, resulting in a flux of electrons (e.sup.−). Thereby, an electric current is generated, the strength of which correlates to the glucose concentration.

(8) The electrode of the present invention uses the naturally occurring mediator system O.sub.2/H.sub.2O.sub.2. A synthetic redox mediator is absent. The generated H.sub.2O.sub.2 is efficiently decomposed to H.sub.2O in the presence of the catalytic nanoparticle.

(9) Due to the covalent coupling of the catalytic nanoparticles and enzyme molecules to the working electrode, a loss of molecules from the enzyme layer can be avoided. Further, no transition resistance between the enzyme layer and the electrical conductor of the working electrode will occur due to the presence of a conductive metal nanoparticle.

(10) Thus, the electrode of the invention and a sensor comprising said electrode provide a simple and efficient construct, which can easily be manufactured by applying a conjugate of nanoparticles and functionalized enzyme to the conductive surface of the electrode.

EXAMPLE 1

Coupling of Platinum Nanoparticles to Glucose Oxidase

(11) Glucose oxidase (GOD) from Aspergillus niger has three cysteine residues. Cysteine residue 164 forms a cysteine bridge to cysteine residue 206 and the third cysteine residue 521 is not exposed to the surface. Thus, the GOD was biochemically modified with functional sulfide, namely with Lomant's reagent DSP (3,3′-dithiodipropionic acid di(N)-hydroxysuccinimide ester) available from Sigma Aldrich. The N-hydroxysuccinimide ester group reacts with surface exposed lysine side chains of GOD, thereby introducing sulfur-containing groups into the enzyme molecule.

(12) Several samples of sulfide-modified GOD with different molar ratios of GOD to Lomant's reagent, were prepared by incubating the compounds in molar ratios of GOD:DSP of 1:1, 1:10, 1:100, and 1:200 for 30 min at room temperature. The reaction products were analyzed by non-reducing polyacrylamide electrophoresis as shown in FIG. 2.

(13) Already at a molar ratio of 1:1, cross-linking of GOD molecules was observed. At a ratio of 1:200, nearly all GOD molecules were cross-linked and high molecular weight aggregates were formed. As a control, GOD without DSP was used.

(14) To all samples from FIG. 2, 1 ml of a nanoplatinum dispersion (0.5 mg/ml; particle size<15 nm) was added and incubated for 12 h overnight at room temperature according to Cao et al. (Biosens. Bioelectron. 26(2010), 87-91) in order to achieve covalent binding of the particles to the sulfide group-modified GOD.

(15) On the next day, the samples were measured by dynamic light scattering (DLS) to determine the size of the cross-linked particles. The results are shown in FIG. 3.

(16) In sample A3 (molar ratio of GOD:DSP=1:100) and sample A4 (molar ratio of GOD:DSP=1:200), aggregates could be detected visually. These large particles have a size above the measuring range of the DLS apparatus of 10 μm and are shown as cut off peak.

(17) Control sample A5 (unmodified GOD) exhibits two discreet peaks. Samples A1 (molar ratio GOD:DSP=1:1) and A2 (molar ratio GOD:DSP=1:10) have an increased particle size compared to control sample A5, which is an indication that conjugates of GOD molecules and Pt nanoparticles have been obtained.

EXAMPLE 2

Electrode Coating

(18) Gold chips QFX301 (LOT Darmstadt) were used as a base for the working electrode. For the subsequent measurement, a sensor set-up with three electrodes, a working electrode, a reference electrode, and a counter electrode (Melinex gold film) was used.

(19) 80 μl of the reaction mixture of sample A2 from Example 1 were pipetted onto the working electrode gold chip and incubated for 10 min at room temperature in order to obtain a covalent coupling of free sulfide groups on the conjugates with the gold surface. After incubation, unbound material was removed by several dippings into phosphate buffered saline (PBS).

EXAMPLE 3

Glucose Measurement

(20) The sensor of Example 2 was tested with a Gamry potentiostat (C3 Analysentechnik München). For this purpose, a chronoamperometric measurement with three voltage levels (200 mV, 350 mV, 200 mV) was performed, at which the resulting current was measured each time for 10 mins. The measurements were conducted in a PBS solution as control and 26 mM glucose. The results are shown in FIG. 4.

(21) At a preset voltage of 0.20 V, no significant difference between the solutions was found. At 0.35 V, an increase in current was found in both solutions, wherein the current in 26 mM glucose was significantly higher compared to PBS (FIG. 4A). This result demonstrates that the coated working electrode is sensitive to glucose. The average electric current during the last five minutes of measurement at the three voltage levels (0.20 V, 0.35 V, 0.20 V) of the measurement cycle is shown in FIG. 4B. The significant difference in the measurement signals of PBS and 26 mM glucose at 0.35 V voltage is clearly observable. A notable sensor drift was observed only in the first minutes of measurement at a given voltage, showing that the sensor has a short calibration time.