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US005916156A [ii] Patent Number:  Date of Patent:
 ELECTROCHEMICAL SENSORS HAVING
IMPROVED SELECTIVITY AND ENHANCED
 Inventors: Karlheinz Hildenbrand, Krefeld;
Hans-Ulrich Siegmund, Leverkusen, both of Germany
 Assignee: Bayer Aktiengesellschaft, Leverkusen, Germany
 Appl. No.: 08/798,387
 Filed: Feb. 7, 1997
 Foreign Application Priority Data
Feb. 15, 1996 [DE] Germany 196 05 583
 Int. CI.6 COIN 27/327; C12Q 1/00
 U.S. CI 600/347; 204/403; 422/82.03;
 Field of Search 600/345, 347,
600/348, 355, 361; 204/403; 422/82.02, 82.03, 90; 436/73, 74, 95
 References Cited
U.S. PATENT DOCUMENTS
5,066,372 11/1991 Weetall 204/403
5,124,128 6/1992 Hildenbrand et al. .
5,130,009 7/1992 Marsoner et al. .
5,385,846 1/1995 Kuhn et al 204/403
5,525,511 6/1996 D'Costa 204/403
ELECTROCHEMICAL SENSORS HAVING IMPROVED SELECTIVITY AND ENHANCED SENSITIVITY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention describes electrochemical sensors, preferably electrochemical biosensors. In addition, a method for fabricating electrochemical, preferably amperometric biosensors for the diagnostics of bodily fluids is described.
2. Description of Related Art
The use of amperometric biosensors, particularly in blood sugar diagnostics, has formed part of the prior art for some years.
Such products are described, for example, in U.S. Pat. No. 4,545,382, in EP 127 958, EP 351 891 and EP Appl. 0 47 1 15 986. The corresponding test systems are commercially available under the product names MediSense®, ExacTex® and Glucocard®. They permit a simple blood glucose diagnosis under home-user conditions.
Particular significance has been gained by the amperometric biosensors containing glucose oxidase as a receptor component. As described in detail in Anal. Chem. 1990, 62, 1111 to 1117, the reaction of glucose with glucose oxidase produces an amount of hydrogen peroxide which is proportional to the sugar concentration.
Since, however, the anodic oxidation H202—»02+2H++ 2e~ requires a relatively high cell voltage (approximately 600 mV), the analysis of whole blood may entail undesirable interference problems. This is because, at the above- 3Q mentioned voltage, certain blood components such as ascorbic acid likewise react, resulting in false positives.
Consequently, with a view to improving the selectivity of amperometric sensors, the idea of mediators has been developed. Frequently used mediators in the case of so-called 35 second-generation biosensors are, for example, ferrocenes or potassium hexacyanoferrate K3Fe(CN)6. The amperometric blood glucose determination in this case proceeds according to the following reaction scheme:
(1) glucose+GO(FAD)^gluconolactone+GO(FADH2) 40
(2) GO(FADH2)+Fe(III)(CN)63-^Fe(II)(CN)64-+GO (FAD)+H+
The amperometric blood glucose determination is therefore confined, as far as measurements are concerned, to the 45 anodic oxidation described under (3), which proceeds at a potential of +360 mV. Such mediator-modified biosensors thus have enhanced selectivity.
With a view to reproducible results, the 02-controlled side reaction GO (FADH2)+02^G0 (FAD)+H202 must be pre- 50 vented as far as possible.
The design of a suitable testing means includes, in addition to the necessary detection reagents, for example glucose oxidase and potassium hexacyanoferrate, at least two electrodes (working electrode and reference electrode), which 55 must be in contact with one another via an electrolyte bridge.
Possible electrode materials according to the prior art are noble metals such as palladium, platinum, gold, silver and copper, or graphite, the anode (working electrode) and cathode (reference electrode) optionally being fabricated 60 from different materials or from the same material and optionally having surfaces of equal or different size.
The test procedure in the case of the commercially available systems is confined, as far as the patient is concerned, to feeding in the liquid sample (whole blood), the 65 analysis value being displayed digitally within at least one minute.
The actual course of the reaction, however, which involves oxidation of the analyte (glucose) and reduction of the mediator, is controlled in such a way, in terms of measurement, that the following steps are observed:
a) Blood is fed in and reaction proceeds according to (1) to (2).
b) After a certain reaction time of approximately 5 to 30 sec is observed, a constant voltage of approximately 400 mV is applied and the anodic oxidation described in (3) takes place.
c) After a short delay time the current is measured. Analytic evaluation takes place within the range of
diffusion-controlled limiting currents, the so-called Cottrell equation
n°F°A^D°C equation (A)
Of) = ——
n=Number of electrons involved in the electrode reaction
C=Concentration of the analyte
A=Area of the working electrode
D=Thickness of the diffusion boundary layer at the work-
If these conditions are to be met, the oxidized form of the redox mediator (K3Fe(CN)6) at the counter electrode must significantly exceed the concentration of the reduced mediator form (K4Fe(CN)6) at the working electrode.
Testing means which allow for separate application— involving, if required, fixing by immobilization—of the mediator system, for example potassium hexacyanoferrate (K3Fe(CN)6), to the counter electrode and of the enzymatic receptor (GO) to the working electrode should correspondingly provide an advantage.
Test systems containing separated reagent zones may also be advantageous with a view to long-term stability of the enzymatic reagent system.
A number of various publications list further desirable characteristics for electrochemical biosensors, which may contribute to an optimization, of the overall system.
Some important ones are listed below: Further enhancement of the selectivity
Europ. Pat. Spec. 0 276 782 describes enzyme electrodes containing albumin layers cross-linked by glutardialdehyde, which, owing to their permeability, protect the working electrode against electroactive interfering components, particularly against proteins having a higher molecular weight.
The use of synthetic membranes to exclude the erythrocytes in the case of electrochemical cells is described in Europ. Pat. Appl. 0 289 265.
WO 94/27140 describes electrochemical sensors provided with erythrocyte exclusion membranes which contain mobile erythrocyte agglutinants.
Europ. Pat. Appl. 0 546 536 describes a system comprising a bipartite working electrode consisting of an enzymefree and an enzyme-containing field, the former detecting oxidizable interfering components which cannot be reacted enzymatically, such as ascorbic acid. The corrected actual blood glucose level is then determined by means of calculation from the measurements of individual potentials.
Nankai et al. describe, in WO 86/07 642, a three-electrode system which, in addition to working electrode and reference electrode, also contains a comparison electrode which compensates for the dependence of cell voltage on the cell current. 5 Increase in the sensor sensitivity
The enhancement of the sensitivity by enlarging the electrode surface areas in line with equation (A) is described in EP 0 385 964.
Improved handleability 10
Nankai et al. describe, in Eur. Appl. 0 471 986, the fabrication of an amperometric blood glucose test system containing expendable sensors, said system being distinguished by particularly good handleability. The expendable sensor plugged into the amperometric analyser is made to 15 touch, by the sensor tip, the drop of blood to be analysed. Via a microcapillary (capillary flow system) whole blood is conveyed into the sensor's working chamber (working electrode and reference electrode plus detection reagents). In the process, the detection reagents (GO/K3Fe(CN)6) dissolve in 20 the liquid (blood) to be analysed, and the previously quoted detection reaction proceeds. If both electrodes are wetted with blood—a precondition for troublefree operability—the reduced resistance value automatically causes the analyser to start. The instrument can therefore be operated without 25 any control buttons. With a view to extracting blood without undue pain, the amount of blood required is kept as low as possible and the volume of the microcapillary system is therefore restricted to approximately 5 /A. From the reaction chamber defined by the microcapillary conductor tracks 30 lead, via the extended sensor section, to the plug-in contacts, any contamination of important functional components in the analyser thus being precluded.
The fabrication of the blood glucose biosensors quoted customarily makes use of a screen printing technology 35 method.
The printing process of the electrode part (transducer) employs commercially available screen printing pastes, for example based on graphite or silver (Acheson), which are printed onto substrate materials such as ceramic or plastic 40 sheets. This requires a number of successive printing and drying steps (conductor tracks, working electrodes, reference electrodes, dielectric layers).
The screen printing pastes which, with a view to workability, contain a number of different additives such as 45 antifoaming agents, thixotropic agents and detergents, often exhibit significant deficiencies in terms of reproducibility.
Frequently, the screen-printed electrode surfaces still have to be activated by plasma treatment. This is because, owing to the high, relatively hydrophobic binder fraction, the 50 surfaces tend to be hydrophobic, poorly wetted and have a markedly reduced conductivity compared with the pure conductor material, for example graphite or silver.
Further drawbacks of the plasma treatment such as ageing or generatinguesirable redox-active surface groups must be 55 taken into account. Fabrication of the electrode part is followed by application of the detection reagent formulation, for example glucose oxidase (GO) and potassium hexacyanoferrate in the case of blood glucose detection. This requires each individual sensor working surface to 60 be doped individually, either the screen printing technology method or the laborious method of micropipetting being employed.
In a third procedure, the microcapillary system is finally applied by bonding appropriately preformed sheets which, if 65 required, have to be provided with hydrophilic layers with a view to good wettability.
Overall this is therefore a relatively complicated fabrication process.
SUMMARY OF THE INVENTION
Surprisingly, a method for fabricating electrochemical sensors has now been found, which is significantly simpler in terms of fabrication and is more reliable in terms of reproducibility.
In particular, the method according to the invention permits the combination, in one system, of those characteristics aimed for and described in the text, which should result in an improved product. In the prior art, said combined and integral profiles of characteristics have not yet been achieved.
Thus an enhancement of the sensitivity is possible in a simple manner by enlarging the reagent matrix area, without a significant increase in the sample volumes (e.g. drops of blood), as in the case of the conventional system, being required.
An increase in selectivity is possible by integrating porous separating layers. The fact is that, as described hereinafter in more detail, it is possible to integrate selectivity-enhancing separation processes in different sensor layers, for example porous reference electrode, membranes as a reagent matrix and possibly via membrane coating of the working electrode.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in further detail with reference to the drawing wherein:
FIG. 1 is an illustration of an amperometric testing device according to the present invention.
DETAILED DESCRIPTION OF THE PREFERED
FIGS. 1A and IB are intended to illustrate the sensor systems according to the invention:
Onto a graphite sheet (2) which is fixed to a base sheet (1) a reagent matrix (membrane) (3) having an area in the range of a few mm2 is applied. On top of this a strip of a graphite web (5) is fastened by means of a perforated double-sided adhesive tape (4). Finally, a perforated sheet (6) is bonded on as a top covering. The sample volume required can be defined by integrating a liquid stop zone (10) in the graphite web, for example behind the reagent membrane (3).
Contact with a potentiostat is established at (7) to the graphite web layer (reference electrode, cathode) and at (8) to the graphite sheet (working electrode, anode). The sample can be fed in via the front edge (9) of the graphite web, entailing—as described in the examples—liquid being transported in the direction of the reagent matrix.
The components employed or possible for the individual functional layers are described below in more detail: a) Working electrode
Preferably use is made of graphite sheets which are available under the brand name Sigraflex® from SOL Carbon Group.
The important characteristics for this intended purpose are:
electric resistivity: 8 to 10 Q fim parallel to the layer 600
to 650 Q fim perpendicular to the layer layer thickness: 0.25 to 1.00 mm purity: >99.85%
With a view to increasing the reaction selectivity it is possible, as will be described later in the examples, for the