MXPA00002268A - Low volume electrochemical sensor - Google Patents

Abstract

An electrochemical sensor electrode strip that measures analyte concentration in an aqueous sample as small as 2.5 to 2.0 mL is described. Reduction in the minimum sample size is achieved by means of a dielectric coating impregnated into peripheral regions of one or more hydrophilic mesh layers, thereby reducing sample dead volume. The mesh layers are located between an electrode support and a cover layer, which cover layer includes an aperture located upstream from an electrode arrangement.

Description

LOW VOLUME ELECTROCHEMICAL SENSOR FIELD OF THE INVENTION The invention relates to electrochemical sensors, biomedical tests and blood tests.

BACKGROUND OF THE INVENTION Electrochemical assays have been developed to determine the concentration of enzymes or their substrates in complex liquid mixtures. For example, electrochemical sensor strips have been developed for the detection of blood glucose levels. Electrochemical sensor strips generally include an electrochemical cell, in which there is a working electrode and a reference electrode. The potential of the working electrode is normally maintained at a constant value relative to that of the reference electrode. Electrochemical sensor strips are also used in the chemical industry and the food industry, to analyze complex mixtures. Electrochemical sensors are useful in biomedical research, where they can function as invasive probes, and for external testing (ie, blood test obtained by a needle and syringe or a lancet). Normal electrochemical sensors for blood analysis measure the amount of analyte in a blood sample by using a working electrode coated with a layer containing an enzyme and a redox mediator, and a reference electrode. When the electrodes come into contact with a liquid sample containing a species for which the enzyme is catalytically active, the redox mediator transfers electrons in the catalyzed reaction. When a voltage is applied across the electrodes, a response current results from the reduction or oxidation of the redox mediator at the electrodes. The response current is proportional to the concentration of the substrate. Some sensors include a false electrode coated with a layer containing the redox mediator and lacking the enzyme, which improves the precision and accuracy of the measurements. Electrochemical thin-film sensors having electrodes confined to a covered region of the sensor generally have a ventilation system to release air from the confined electrode region upon displacement by the sample. In a two-mesh electrode construction, the sample is twisted along the primary layer and the displaced air is vented from the second mesh layer. See, for example, US Patent no. 5,628, 890. Reducing the volume of the total sample needed to produce an accurate and accurate analyte concentration reading by an electrochemical sensor would intensify the user's convenience. Reducing the volume of the sample is particularly desirable in a blood test because of the pain, disorder and time required to stop the bleeding usually increases as the size of the blood sample increases. Although reducing the sample size is desirable, several restrictions severely limit the ways in which this can be achieved. The restrictions include the following. The volume of the sample should be sufficient to cover the entire electrode area. Reducing the electrode area changes the response current of the electrode, thereby making the electrode strip incompatible with a given meter. Where the sample is not applied directly to an electrochemical cell, the total volume required included the volume needed to cover a sample loading zone, and a path to the electrodes, as well as the electrode area. The sample loading area should be easily visible, even for diabetics with impaired vision.

BRIEF DESCRIPTION OF THE INVENTION The invention features an electrode strip for use in an electrochemical sensor for measuring an analyte in an aqueous sample. For various reasons, it may be desirable to apply a sample at a location on an electrode strip and transport all or part of the sample to an electrode area at a different location. This arrangement requires a portion of the sample to fill a travel path from the sample loading area to the electrode area, that is, a dead sample volume. The invention features an electrode strip with a reduced sample dead volume. This allows the measurement of analyte in a sample as small as 2.0 up to 2.5 μl. The electrode strip includes an electrode holder and an array of electrodes in the holder. The electrode array includes a working electrode and a reference electrode. The working electrode has an upstream end and a downstream end, and the reference electrode is adjacent to the downstream end of the working electrode. Optionally, the electrode array also includes a false electrode. One or more layers of hydrophilic mesh cover the sample loading area and the array of electrodes, the loading area of the sample being adjacent to the upstream end of the working electrode. A cover layer defines an upper limit of a cell volume enclosing the array of electrodes. The cover layer has an opening located above the sample loading area, with no portion of the opening located above the electrode array. A dielectric coating impregnates the peripheral regions of the mesh layers, thereby forming an occluded region of the mesh layers. The occluded region covers a portion of the sample loading area and also defines the lateral limits of the cell volume. The occluded region does not cover any portion of the electrode array. The mesh layers drag the sample from the sample loading area in the area immediately above the electrodes, via a sample flow channel, by which the sample comes into contact with the electrodes. The electrode strip includes one or more layers of hydrophilic mesh. Preferably, the mesh layers have a total thickness between 40 and 200 μm. The mesh layers can be made of an inherently hydrophilic mesh material, or a mesh material coated with a surfactant. Preferably, the mesh material is woven nylon, coated with a surfactant such as FC 1 70C FLUORADMR. Preferably the mesh layers include a woven mesh material having an open area of about 40 to about 45%, a mesh count of about 95 to about 1 15 filaments per centimeter, a filament diameter of about 20 to about 40 μm and a thickness from about 40 to about 60 μm. Preferably, the cover layer is substantially impervious to aqueous liquids. A suitable cover layer is a polyester membrane. Normally, the electrode strip is between 4.5 and 6.5 mm wide. Normally, the opening has a width between 2.5 and 3.5 mm and a length between 2.5 and 3.5 mm. For an electrode strip and aperture of these dimensions, the sample path length (ie, distance from the upstream end of the non-occluded area of the mesh to the downstream end of the non-occluded area), preferably is between 6 mm and 1 0 mm. More preferably, the sample path length is between 7 mm and 9 mm. Preferably, the dielectric coating is a hydrophobic material, such as POLYPLASTM or SERICARD ™. The dielectric coating forms an occluded region in the mesh layers. The occluded region forms a sample flow channel in the sample loading area. Preferably, the width of the sample flow channel is between 4 mm and 0.5 mm. The width can be uniform or non-uniform. Preferably, the sample flow channel is widened in the direction of said array of electrodes, for example, the sample flow channel is V-shaped, preferably, the sample flow channel represents between 10 and 50 % of the mesh layer area within the opening.

Another feature of the invention is a means to identify the target area of the electrode by providing a contrast color within the load area of the sample. The insulating layer may be colored to contrast with the cover layer, the electrode holder or both. This provides a contrast color in the target area where the sample is applied to the strip, which can help the user to correctly apply the sample to the strip. Other features and advantages of the invention will be apparent from the description of the preferred embodiment thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic view of an electrode strip according to an embodiment of the invention. Fig. 2 is a perspective view of the assembled strip of FIG. 1 . Fig. 3 is a graph summarizing test data comparing a conventional electrode strip with an electrode strip having a reduced dead volume. The reference glucose concentration (mM) is on the X axis. The calibrated response (m M) is on the Y axis. Figs. 4A-4F are top views of openings and sample loading areas of low volume electrode strips. Figs. 4a-4F illustrate examples of patterns or geometries of sample flow channel according to the invention.

Figs. 5A and 5B are top views of a preferred embodiment of the invention. In Fig. 5A, the cover layer is present. In Fig. 5B, the cover layer has been removed.

Description of preferred modalities The volume of the sample should be large enough to cover the entire electrode area, including the working electrode, the reference electrode and the false electrode, if present. Incomplete coverage of the total electrode area can cause erroneous measurements. The area of the working electrode and the area of the false electrode must be compatible with the electrical current requirements of the measuring system with which the electrode strip is used. The current response generated by the electrodes and measured by the meter is directly proportional to the area of the working and false electrodes. Changes in the response current caused by changes in the electrode area will make the electrode incompatible with the calibration parameters in a previously compatible measuring system. Electrodes that have thin-layer sensors in an area covered by an electrode strip require a sample loading area, from which the sample travels to the electrode area. This imposes a volume requirement greater than the volume required to cover the electrode area alone. The total volume requirement thus equalizes the volume required to cover the electrode area plus the sample load area plus the sample flow channel area between them.

The application of the appropriate sample for an accurate and reliable operation of an electrochemical sensor system is essential. Consequently, the sample loading area has a size and color to make it easily visible to the user, including diabetic users, who frequently have impaired vision. The size of the application area significantly affects the dead volume of the sample. The thickness of the sample layer between the electrode surface and the electrode strip cover cap is determined by the thickness of the mesh layers used in the strip construction. The electrochemical test reaction can occur in a thinner section of the sample layer, than that required to transport the sample to the electrode area as it is twisted through the mesh. Therefore, an additional restriction of dead volume is associated with the mesh layers. When locating the reference electrode downstream of the working electrode, a circuit is not established until the working elctrode has been completely covered by the sample and the sample has reached the reference electrode. In consecuense, no response is detected if the sample inadequately covers the working electrode. The mesh layer and the dielectric coating can contribute to the movement of the sample towards the working electrode and reference electrode in a uniform manner. The arrangement of electrodes can prevent the sample from reaching the reference electrode until the working electrode is substantially or completely covered. One embodiment of the electrode strip is illustrated in Figs. 1 and 2. Referring to Figs. 1 and 2, the electrode holder 1 is an elongated strip of plastic, such as PVC, polycarbonate or polyester. This supports three printed rails of electrically conductive carbon ink 2. The printed rails define the positions of the reference electrode 4, the working electrode 5, the false electrode 5a, and the electrical contacts 3. The contacts 3 are for insertion in a compatible meter The elongated portions of the conductor rails are each covered with lanes of silver / silver chloride particles 6a, 6b, 6c. The elements 6b and 4 together form the reference electrode. The working area of the working electrode 8 is formed from an ink that includes an enzyme, a meter and a filler. The ink in the work area forms a paste with the sample. The working area of the false electrode 8a is formed from ink which includes a mixture of a mediator and a filler, without enzyme. The respective inks are applied to positions 5 and 5a of the carbon 2 rails as discrete areas of fixed length. Alternatively, the electrode layer 8 may contain a substrate catalytically reactive with an enzyme to be tested. The conductive material in a preferred embodiment includes particulate carbon having the redox mediator adsorbed therein. An electrode printing ink includes a filler, for example, carbon, and an adsorbed redox mediator. The ink for the working electrode also includes an enzyme or a substrate. When the analyte to be measured is blood glucose, the enzyme is preferably glucose oxidase, and the redox mediator is preferably a ferrocene derivative.

The ink can be printed on the screen. The ink may include an enzyme stabilizer, a film-forming polymer, a filler (e.g., carbon), a redox mediator (e.g., ferrocene or a ferrocene derivative), a buffer and an enzyme or a substrate. The printed ink on a false electrode lacks the enzyme or the substrate. A layer of surfactant-coated mesh 10 extends over the array of electrodes. The mesh layer protects the printed components from physical damage and facilitates the wetting of the electrodes by the aqueous sample. Preferably, the mesh layer extends over the path of the complete sample, between and including the sample application area and the electrode array. The mesh can be made of finely woven nylon. Alternatively, any woven or nonwoven material can be used. Preferably, the web is not more than 70 μm thick. Preferably, the mesh has an open area percentage of about 40 to about 45%, a mesh count of about 95 to about 15 per cent, a fiber diameter of about 20 to about 40 pm, and a thickness of about 40. up to about 60 μm. A particularly suitable mesh is the NY64 HC mesh, available from Sefar (formerly ZBF), CH-8803, Ruschlikon, Switzerland. If the mesh material is hydrophobic (eg nylon or polyester), it is coated with a surfactant. If a hydrophilic mesh is used, the surfactant coating may be omitted. The hydrophilicity of the mesh allows the sample to be twisted along the mesh layer to the electrodes. The twisting properties of the mesh can be controlled by changing the type or amount of surfactant of the mesh material. Several surfactants are suitable for coating the mesh material. A preferred surfactant is the fluorochemical surfactant FC 170C FLUORAD ™ (3M, St. Paul, MN). FLUORADMR is a solution of an adduct of fluoroaliphatic oxyethylene, lower polyethylene glycols, 1,4-dioxane and water. The preferred surfactant charge will vary depending on the type of mesh and surfactant used and the sample to be analyzed. It can be determined empirically by observing the flow of the sample through the mesh with different levels of surfactant. If two layers of mesh are used, the second (upper) mesh layer is preferably hydrophilic, but no more hydrophilic than the first (lower) mesh layer. According to this, the first mesh layer may have a higher surfactant charge than the second mesh layer. With respect to the first mesh layer, the surfactant charge suitable for most applications is about 1-5-20 μg / mg mesh (ie, about 1.0 percent w / v). With respect to the second mesh layer, the surfactant charge suitable for most applications is approximately 1 -10 μg / mg mesh. The 1 0 mesh layer is held in place by a dielectric coating 1 1, which impregnates the periphery of the mesh layer. The dielectric coating can be applied by screen printing. Dielectric coating 1 1 does not cover any portion of the electrode array. Preferably, the dielectric coating is hydrophobic, so that it confines the sample efficiently. A preferred hydrophobic dielectric coating is POLYPLAS ™ (Sericol Ltd., Broadstairs, Kent, UK). A more preferred hydrophobic dielectric coating is SERICARD ™ (Sericol). The top layer on the electrode strip is a cover membrane 13, which may be substantially impermeable. A preferred layer is a flexible polyester tape. The cover layer defines an upper limit of the electrochemical cell volume, and in this way, the cover layer determines the maximum depth of the aqueous sample. The cover layer sets the upper limit of the volume of the cell at a predetermined height, which depends on the thickness of the mesh layers. The height of the cell, and in this way the maximum depth of the sample, is selected to ensure a resistance to the suitably high solution. The cover layer has an opening 14 for access of the sample to the underlying mesh layers. The opening 14 is located over the sample loading area, which is adjacent to the upstream end of the working electrode. The opening may be of any suitable size large enough to allow sufficient sample volume to pass through the mesh layer. It should not be so large as to expose some portion of the electrode array. The opening can be formed in the cover layer by any suitable method, for example, die perforation. In Fig. 1, dielectric coating 1 1 forms a V-shaped sample flow channel 30. Dielectric coating 1 1 surrounds the sample path (sample flow channel plus electrode area) 12, and this geometry reduces the total sample volume that needs to be applied to the strip. The V-shape of the flow channel 30 helps direct the sample towards the electrodes. The dielectric coating 1 1 may have a color that contrasts with the color of the cover layer 1 3, the color of the electrode support 1, or both. The color contrast intensifies the visibility of the aperture 14, thereby facilitating the proper application of a sample to the electrode strip. The cover layer 1 3 is attached peripherally to the strip by means of a suitable adhesive. The cover layer 13 is not fixed in the area of the electrode array or the flow channel of the sample. Preferably, the cover layer 1 3 is fixed by means of a hot melt adhesive. The hot melt adhesive normally has a coating weight between 10 and 50 g / m2, preferably 20 to 30 g / m2. Pressure sensitive adhesives or other suitable adhesives can also be used. When using a heat-sensitive dielectric coating, e.g., SERICARD R, heat welding of the cover layer should be performed in a manner that will not damage the dielectric coating. An adhesive is applied so that the dielectric coating 11 is partially sealed to the cover layer 13, 10 mesh layer and electrode support 1. The layers adhere to the electrode support by applying pressure and heat in discrete areas on both sides and each end of the electrode strip. Heat and pressure are not applied to the central portion of the strip, which contains the electrode array. Preferably, a portion of the cover layer is not sealed to the dielectric coating. When a sample is applied to the target area of the electrode in the aperture 14, the sample passes below the cover layer 13 through the mesh layer coated with surfactant 10, towards the electrodes 4, 5 and 5a. Optionally, the upper surface of the cover layer can be coated with a layer of silicone or other hydrophobic coating. This helps guide the sample applied to the hydrophilic mesh layer in the sample loading area, thereby facilitating the application of small volumes. In use, a sensor strip of the invention is connected, via electrode contacts 3, to a measuring device (not shown). A sample is applied to the sample loading area via the opening 14. The sample moves along the sample flow channel 12. The movement of the sample is sufficiently obstructed by the 1 0 mesh layer, so that the sample advantageously forms a uniform front. The air is displaced through the upper portion of the mesh layer 10 to and through the opening 14. The sample completely covers the working electrode 5 before reaching the reference electrode 4. The arrival of the front of the sample in The reference electrode completes the circuit and causes a response to be detected by the measuring device. In some embodiments of the invention, a second mesh is used on the first mesh. The second mesh layer can additionally control the flow of the sample as it travels from the point of application to the electrodes. The second mesh layer can be coated with a surfactant. Preferably, the second mesh layer is hydrophilic, but not more hydrophilic than the first mesh layer. If necessary, the first mesh layer may have a higher surfactant charge than the second mesh layer. Preferably, the second mesh layer is woven, so as to have a regular repeat pattern of mesh fibers perpendicular to, and parallel with, the long axis of the electrode strip. Preferably, the second mesh layer is substantially thicker than the first mesh, with larger diameter mesh fibers and larger openings. The second mesh layer can have a thickness of about 1 00 to 1 000 μm, with a thickness of 1 00 to 1 50 μm being preferred. Preferably, the second mesh has a percentage of open area of about 50 to 55%, a mesh count of about 45 to about 55 filaments per cm, and a filament diameter of about 55 to about 65 μm. One mesh suitable for use as a second mesh layer is the NY151 HC mesh (Sefar, Ruschlikon, Switzerland). With reference to Figs. 4A-4F, the pattern or geometry of the sample flow channel 30 may vary. The sample flow channel 30 is formed by impregnation of a 1 1 hydrophobic dielectric coating on all the mesh layers present. The opening 14 allows access of the sample to the sample flow channel 30, which directs the sample to the electrodes 4, 5, 5a. In the embodiments of the invention shown in Figs. 4A-4F, the opening 14 is 2.35 mm wide by 3.35 mm long, and the total area below the opening 14 is 6.7 mm2. In Figs. 4A-4F, the areas not occluded within the openings are as follows: Fig. 4A, 1 .28 mm2; Fig. 4B, 2.73 mm2; Fig. 4C, 0.76 mm2; Fig. 4D, 2.05 mm2; Fig. 4E, 1.61 mm2; and Fig. 4F, 0.67 mm2. Figs. 5A and 5B show a preferred embodiment of the invention. In Fig. 5A, an oval-shaped opening 14 in the cover layer 1 3 exposes a sample flow channel 30 and a portion of the dielectric coating that forms the sample flow channel 30. In FIG. 5B, the cover layer 13 it has been removed to show the 10 mesh layer and the electrodes 4, 5, 5a. The following examples are intended to be illustrative and not limiting of the invention.

Examples Low volume electrode strips were constructed with a single mesh layer (NY151, 37% open area, 41 / cm mesh count, 1 50 μm thickness) held down with a single layer of dielectric coating (Sericard ™). Another set of electrode strips with two layers of mesh was constructed. The dielectric coating formed a sample flow channel essentially as shown in Fig. 1. Venous blood samples were obtained and divided into aliquots. A known amount of glucose was added to each aliquot to make a series of whole blood samples with a range of glucose concentrations between 90 mg / dl (5 mM) and 820 mg / dl (45 mM). A small volume (3-5 μl) of each aliquot was applied to the sample loading areas of the strips described above, and to control strips, for comparison. The control strips had two mesh layers and did not have a sample flow channel formed by the dielectric coating that occluded part of the mesh layer area. The responses of the glucose strips in the samples were measured using a compatible measuring system. The measured steady state responses for both the sample and control electrodes were plotted against the glucose level. The results are summarized in Table 1. The low volume electrode strips gave a linear glucose response essentially the same as that of the electrode strips of the prior art. Neither reduction in the thickness of the sample by the use of a single mesh layer, nor the use of a sample flow channel materially affected the response.

TABLE 1 Low volume electrode strips, made as described above, were tested using capillary blood (between 5 and 10 μl) of the fingers of about fifty diabetic patients presenting a range of blood glucose values between 4 and 27 mM ( 70 and 500 mg / dl). The calibrated steady state responses given by the electrodes, measured using an appropriate meter (Medisense QIDMR), were compared against those of a reference whole blood value of a standard laboratory reference analyzer (Yellow Springs, Inc.). The results are plotted in Fig. 3. A linear response of the low volume strips over this glucose range was obtained. The variability of response was low, as shown by the small amount of dispersion on the linear regression line. The responses of low volume electrode strips and control strips were compared using blood sample volumes of 10, 5, 4, 3 and 2 μl. Ten replicate samples were applied to each type of electrode strip in each volume. The electrode response and the number of electrodes were measured giving a measurement response for each sample volume. The results are summarized in Tables 2 and 3.

TABLE 2 TABLE 3 Number of strips giving a measurement response The low volume electrode strips continued to give a response at 2.0 μl, while the control strips did not. This demonstrated that the reduced dead volume of the electrode strips of this invention allowed more of the sample to travel to the electrode area and cover the working and reference electrodes. Samples that were too small to complete the working electrode area gave an answer. Other embodiments are within the following claims.

Claims (22)

1. CLAIMS 1 . In an elongated electrode strip for performing an electrochemical glucose measurement in whole blood, in which the electrodes i. used to perform the measurement are covered with a mesh layer extending a distance beyond the electrodes along the length of the strip, and the mesh is covered by a liquid-impermeable layer, in which there is an opening which does not extend over the electrodes, the improvement comprising: a partial occlusion of the mesh that is placed below the opening, said partial occlusion reduces the total volume of blood necessary to carry out the measurement.
2. 2. In the elongated electrode strip of claim 1, further comprising enclosing at least about 50% of the mesh area underlying the opening.
3. 3. The electrode strip of claim 2, wherein a 2.5 microliter whole blood sample provides a sufficient sample volume to reach the electrodes and cause a measurement.
4. 4. An electrode strip for use in an electrochemical sensor for measuring an analyte in an aqueous sample, comprising: an electrode holder; an array of electrodes in said support, comprising a: working electrode and a reference electrode, wherein said working electrode has an upstream end and a downstream end, and I: said reference electrode is adjacent to said end current below said working electrode; a hydrophilic mesh layer extending over a sample loading area and said array of electrodes, said sample loading area being adjacent to said upstream end of said working electrode; a cover layer defining an upper limit of a cell volume comprising said array of electrodes; an opening in said cover layer, located above said opening and defining the limits of said sample loading area, without any portion of said opening located above said array of electrodes; a dielectric coating impregnated in peripheral regions of said mesh layer, thereby forming an occluded region of said mesh layer, said occluded region extending over a portion of said sample loading area, which rests below said aperture and which defines the lateral limits of said cell volume, without any portion of said occluded region extending over said array of electrodes; wherein said mesh layer draws said aqueous sample from said sample loading area towards said array of electrodes, wherein said aqueous sample comes into contact with said working electrode and said reference electrode.
5. 5. The electrode strip of claim 4, wherein said array of electrodes further comprises a false electrode.
6. 6. The electrode strip of claim 4, comprising a mesh layer.
7. 7. The electrode strip of claim 4, comprising two or more mesh layers.
8. The electrode strip of claim 4, wherein the total thickness of said mesh layer is between 40 and 200 μm.
9. 9. The electrode strip of claim 4, wherein said mesh layer comprises an inherently hydrophilic mesh material.
10. The electrode strip of claim 4, wherein said hydrophilic mesh layer comprises a mesh material coated with a surfactant. eleven .
11. The electrode strip of claim 10, wherein said surfactant is FC 1 70C FLUORAMR.
12. 12. The electrode strip of claim 1, wherein said mesh material is woven nylon.
13. The electrode strip of claim 4, wherein said mesh layer comprises a woven mesh material having an open area of about 40 to 455, a mesh count of about 95 to about 1 15 strands per centimeter, a filament diameter of about 20 to 40 μm and a thickness of about 40 to about 60 μm.
14. 14. The electrode strip of claim 4, wherein said cover layer is substantially impervious to aqueous liquids.
15. 15. The electrode strip of claim 14, wherein said cover layer consists essentially of a polyester membrane.
16. 16. The electrode strip of claim 4, wherein said electrode strip is between 4.5 and 6.5 mm wide.
17. 17. The electrode strip of claim 16, wherein said opening has a width between 2.5 and 3.5 mm and a length between 2.5 and 3.5 mm.
18. The electrode strip of claim 4, wherein said dielectric coating is hydrophobic.
19. 19. The electrode strip of claim 4, wherein said occluded region forms a V-shaped sample flow channel in the sample loading area, wherein said V-shaped flow signal is widened in the direction of said arrangement of electrodes.
20. The electrode strip of claim 19, wherein said V-shaped sample flow channel represents between 10 and 50% of the mesh layer area within said opening. twenty-one .
21. The electrode strip of claim 4, wherein said dielectric coating has a color that contrasts with the color of said cover layer.
22. 22. The electrode strip of claim 4, wherein said dielectric coating has a color that contrasts with the color of said cover electrode holder.