Small volume in vitro analyte sensor

The present invention relates to analytical sensors for detecting biological analytes in small volumes of samples and methods of making and using the sensors.

Analytical sensors are useful in the fields of chemistry and medicine for measuring the presence and concentration of biological analytes. Such sensors are needed, for example, to monitor glucose and lactate in the context of critical treatment in diabetic patients.

Currently available techniques are for measuring biological analytes in relatively large sample volumes, for example, 3 microliters or more of blood or other biofluid is usually required. These liquid samples are taken from the patient, for example using a needle or syringe, or a portion of the skin, such as a fingertip, is incised with a lancet and "squeezed" from that site to obtain a useful sample volume. Collected from the patient. These procedures are inconvenient and often painful for the patient, especially when samples are frequently needed. For example, less painful sampling methods such as incisions in the arm or thigh with low nerve terminal density are known. However, the blood sample obtained by incising a preferred area of the body is generally submicroliter. This is because there are not so many near-surface capillaries in these areas. Therefore, it is desirable to develop a blood analyzer sensor that allows accurate and sensitive analysis of the concentration of the analyte in a small volume of sample, is relatively painless, and is easy to use. It is also very useful.

<p> It is also desirable to develop a method for manufacturing small volume electrochemical sensors that can reduce the error caused by the size of the sensor or sample.</p>

<p>The sensors of the present invention provide a method for detecting and quantifying an analyte in a submicroliter sample. The present invention generally includes methods and sensors for analyzing an analyte in a small volume sample, for example, by coulometry, amperometry, and / or potentiometry. The sensor of the present invention preferably utilizes a non-exudative or diffusive redox mediator. The sensor further comprises a sample chamber that holds the sample in electrolytic contact with the working electrode. In many examples, the sensor also contains a non-exudative or diffusible second electron transfer agent.</p><p> In a preferred embodiment, the working electrode faces the counter electrode and forms a measurement area in the sample chamber between the two electrodes, which measurement area is about 1 μL or less, preferably about 0.5 μL or less, more preferably. Is set to a size that can accommodate a sample of about 0.32 μL or less, more preferably about 0.25 μL or less, and most preferably about 0.1 μL or less.</p><p> In one embodiment of the invention, a working electrode and a counter electrode are in a sensor configured for insertion into an electronic meter, and if the sensor is properly inserted into the meter, it is electrically with the electronic meter. A contact conductive insertion monitor is formed. The conductive insertion monitor is configured and placed in close proximity to the electrical circuit when the sensor is properly inserted into the electronic connector.</p><p> In another embodiment of the present invention, the sensor is formed with a plurality of contact portions, each contact portion including a contact pad which is a contact area with an electronic meter. The plurality of contact portions and contact pads are arranged on a substrate having a length and width, and each contact pad has a contact pad width measured in parallel with the width of the substrate. The total contact pad width is greater than the board width. In a preferred embodiment, six contact pads make six electrical connections on a sensor with a width approximately corresponding to the width of the four contact pads. For example, one working electrode, three counter electrodes (eg, one counter electrode and two indicator electrodes), and two insertion trace connections each have one contact pad. Connections are made to each of these six contact pads, which are the same width as the working electrode and three counter electrode contact pads.</p><p> The present invention also includes an electrical connector that provides electrical contact between a sensor and an electrical meter or other device. The electrical connector comprises a plurality of contact structures, each of which has a proximal contact end for electrical connection with a sensor contact and a distal end for electrical connection with an electrical device. .. In one embodiment, the plurality of first contact structures extend longitudinally parallel from the distal end to the proximal end. In addition, one or more second contact structures extend longitudinally near the first contact structure, from the distal end to the proximal end of the first contact structure, extending in the longitudinal center of the connector. It is inclined toward the line. It is then in contact with the sensor via the proximal contact end.</p><p> Preferably, the electrical connector comprises at least two second contact structures that extend longitudinally through the proximal end of the first contact structure and incline towards the longitudinal centerline of the connector. Behind the sloping or curved portion, the proximal contact end of the second contact structure in one embodiment is electrically connected to a single conductive surface for the sensor, such as a conductive insertion monitor. Has been made. In another aspect, the first contact structure may be configured and arranged to contact one or more working electrodes and / or counter electrodes of the sensor, and the second contact structure may be one or more. It is configured and arranged to contact the conductive insertion monitor of the.</p><p> The sensor of the present invention can be configured for either side filling or top filling. Further, in some embodiments, the sensor may be part of an integrated sampling and analysis instrument. The integrated sampling and analysis material measuring device includes the sensor and the skin perforating member so that the device can be used to perforate the user's skin and allow a liquid sample such as blood to flow out and be collected by the sensor. May be provided. In at least some embodiments, the liquid sample can be collected without moving the integrated sampling and analyzer measuring device.</p><p> In one embodiment, the sensor is connected to an electrical device to provide a processor coupled with the sensor. The processor is configured and arranged to be able to measure continuous current values during the electrolysis of the sample in the sample chamber. The processor measures the peak current value from a continuous current value. After the current value decreases below the threshold fraction of the peak current value, the slope value is measured from the current value, and the logarithmic value of the current over time is expressed as a linear function. The processor determines the extrapolation gradient from the gradient value. From the extrapolation gradient and the measured current value, the processor measures the amount of charge required to electrolyze the sample, and from that amount of charge measures the concentration of the analyte in the sample.</p><p> As mentioned above, one method of making a sensor is to form at least one working electrode on the first substrate and at least one opposed or opposed / reference electrode on the second substrate. Including forming a pole. The spacer layer is arranged on either the first substrate or the second substrate. The spacer layer defines the chamber from which the sample is extracted and held when the sensor is complete. The redox mediator and / or the second electron transfer agent can be placed on the first or second substrate in the area that will be exposed in the sample chamber when the sensor is completed. The first and second substrates are then aligned and spaced by spacers from the sample chamber to provide access to at least one working electrode and at least one counter electrode or counter / reference electrode. In some embodiments, the first and second substrates form part of a single sheet or continuous material web. The present invention includes particularly effective and reliable methods for making these sensors.</p><p> One such effective and reliable method is to provide an adhesive having first and second surfaces covered with first and second release liners, and then a second release liner. It includes finely cutting out the first release liner and the adhesive without penetrating. These cuts define one or more sample chamber areas. A part of the first release liner is removed to expose a part of the surface of the first adhesive. Thereby, the rest of the first release liner is left above the sample chamber area. This exposed first adhesive surface is applied to a first substrate on which one or more conductive traces are arranged. To expose the surface of the second sticky material, the second release liner is removed together with the sticky material and the first release liner in the sample chamber region. Then, the surface of the second adhesive is applied to a second substrate having one or more conductive traces. By this method, a sensor having a sample chamber corresponding to one of the sample chamber regions is formed.</p><p> These and various other features that characterize the invention are specifically pointed out by the accompanying claims. In order to gain a better understanding of the present invention, its advantages, and the objects obtained by its use, reference should be made to the drawings and accompanying specification illustrating and illustrating preferred embodiments of the present invention.</p>

For each drawing, the same reference numerals and letters indicate the corresponding structures in each drawing. (Detailed description of preferred embodiments) The terms used herein are defined by the following definitions.

An "air-oxidizable mediator" is a redox mediator that is oxidized by air, preferably when at least 90% of the mediators are stored in the air, either solid or liquid, for example. , 1 month or less, preferably 1 week or less, more preferably 1 day or less.

"Amperometry" includes stationary amperometry, chronoamperometry, and Cottrell type measurements. A "biofluid" is any body fluid that can be measured by an analyte, such as blood (including whole blood and cell-free components such as plasma and saliva), interstitial fluid, dermal fluid, and sweat. , Tears, urine and saliva.

"Coolometry" is the measurement of the charge that passes or is believed to pass, either directly on the electrode or during complete or near-complete electrolysis of the analyte via one or more electron transfer agents. Charges are measured by measuring the charge that moves during partial or near-complete electrolysis of the analyte, or often by multiple weight measurements of decay current and elapsed time during electrolysis. .. The decay current is due to the concentration of the species being electrolyzed. It occurs as a result of the decrease.

A "counter electrode" is an electrode that is paired with a working electrode and that carries an electrochemical current that is the same size as the current flowing through the working electrode and has the opposite sign. Represent. Unless otherwise stated that "counter electrode" does not include a reference electrode or counter / reference electrode, the term "counter electrode" includes counter electrodes that also function as reference electrodes (ie, counter / reference electrodes). It shall be.

An "effective diffusion coefficient" is a diffusion coefficient that characterizes the transport of a substance, such as an analyte, enzyme, or redox mediator, in volume between electrodes of an electrochemical cell. In at least some examples, the cell volume may be occupied by more than one medium (eg, sample fluid and polymer membrane). Diffusion of material through each medium may occur at different rates. The effective diffusion coefficient corresponds to the diffusion rate through the volumes of the plurality of media, and is generally different from the diffusion coefficient of the substance in the cell filled only with the sample solution.

An "electrochemical sensor" is an apparatus configured to detect the presence of an analyte and / or measure the concentration of an analyte via an electrochemical oxidation and reduction reaction. This reaction is converted into an electrical signal that correlates with the amount or concentration of the analyte.

"Electrolysis" is the electrooxidation or reduction of a compound, either directly at the electrode or via one or more electron transfer agents (eg, redox mediators and / or enzymes). The term "facing electrode" refers to an arrangement of a working electrode and a counter electrode such that the working surface of the working electrode is arranged so as to be substantially opposed to the surface of the counter electrode. In at least some examples, the distance between the working electrode and the counter electrode is shorter than the width of the working surface of the working electrode.

The compound is "immobilized" on the surface if it is encapsulated or chemically bonded to the surface. An "indicator electrode" is an electrode that detects that the sample chamber and / or measurement area is partially or completely filled.

A "layer" is one or more layers. A "measurement area" is, in the present specification, an area of a sample chamber formed sized to accommodate only the portion of the sample intended to be examined in the analyte assay.

A "non-diffusible", "non-exudative" or "non-releasing" compound is a compound that does not substantially diffuse from the working surface of the working electrode during the analyte assay. The "opposite / reference electrode potential" is the half cell potential of the reference electrode or counter / reference electrode of the cell when the solution in the cell is a 0.1 M NaCl solution at pH 7.

A "redox mediator" is an electron transfer agent that transports electrons between an analyte and a working electrode, either directly or via a second electron transfer agent. The "reference electrode" includes a reference electrode that also functions as a counter electrode (ie, counter electrode / reference electrode), unless it is stated that the "reference electrode" does not include a counter / reference electrode. And.

A "second electron transfer agent" is a molecule that carries electrons between a redox mediator and an analyte. "Surface in sample chamber" means working electrode, counter electrode, counter / reference electrode, reference electrode Includes the indicator electrode, each surface of the spacer, or any other surface coupled to the sample chamber.

A "working electrode" is an electrode in which the analyte is electrooxidized or electroreduced with or without the action of a redox mediator. The "working surface" is the portion of the working electrode that is coated with a non-exudative redox mediator and exposed to the sample, or if the redox mediator is diffusive, the "working surface" is the sample. It is an exposed part of the working electrode.

The small volume in vitro analyzer sensor of the present invention has a volume of about 1 μL or less, preferably about 0.5 μL or less, more preferably 0.32 μL or less, more preferably 0.25 μL or less, and most preferably 0.1 μL or less in a sample. It is designed to measure the concentration of the part of the analyte.

The analyte of interest is generally provided as a solution or biological fluid, such as blood or serum. In general, with reference to FIGS. 1 and 2, the small volume in vitro electrochemical sensor 20 of the present invention typically has a working electrode 22 on a first substrate 32 and a counter electrode (opposite) on a second substrate 34. / Reference electrode) 24, and sample chamber 26. The sample chamber 26 is configured such that when the sample is fed into the chamber, the sample is in electrolytic contact with the working electrode 22, the counter electrode 24 and all possible reference or support electrodes. This allows current to flow between the electrodes, causing electrolysis (electrooxidation or reduction) of the analyte. The spacer 33 is arranged between the first substrate 32 and the second substrate 34, forms a space between the electrodes 22 and 24, and forms a sample chamber 26 for accommodating the sample to be evaluated.

(Working electrode) The working electrode 22 can be formed of a molded carbon fiber composite or is composed of an inert non-conductive base such as polyester with a suitable conductive layer deposited on it. You can also do it. The conductive layer generally has a relatively low electrical resistance and is generally electrochemically inert in the potential range of the sensor in operation. Suitable conductive layers include gold, carbon, platinum, ruthenium dioxide, palladium, and, for example, ECCOCOAT CT5079-3 carbon-filled conductive epoxy coatings (WR Grace Company, Woburn, MA). ), As well as other non-corrosive materials known to those skilled in the art. The electrodes (eg, the conductive layer) are deposited on the surface of the Inactive Material by methods such as vapor deposition or printing. The electrodes are preferably printed on a substrate.

The inert non-conductive substrate is also referred to as a substrate, a substrate, or the like. This substrate is generally an electrically non-conductive material, such as any insulating material that cannot carry an electric charge or current. Examples of substances that can be used as the base material of the sensor of the present invention include polyester, polyethylene (both high density and low density), polyethylene terephthalate, polycarbonate, vinyls and the like. The substrate can be treated with a primer or other such coating to improve contact with the electrodes on it.

A tab 23'may be provided at the end of the working electrode 22 to facilitate the connection of the electrode to an external electronic device (not shown) such as a voltage source or current measuring device. A contact pad 23 connected to the working electrode 22, eg, an extension from the working electrode, can be placed on the tab 23'.

To prevent electrochemical reactions from occurring in the uncoated portion of the working electrode mediator, if a non-leaching mediator is used, a dielectric or other insulating material should be applied to the area of the redox mediator on the electrode. It can be placed above, below, or around. Suitable dielectric materials include non-conductive organic polymers such as wax and polyethylene. The dielectric may also cover a portion of the redox mediator on the electrode. The portion coated with the redox mediator does not contact the sample and therefore does not become part of the working surface of the electrode.

(Chemical properties of sensing) In addition to the working electrode 22, a sensing chemical that analyzes the analyte is provided in the sample chamber 26. The sensing chemical preferably comprises a redox mediator and a second electron transfer agent, but in some cases only one or the other may be used. The redox mediator and the second electron transfer agent can be individually diffusible or non-exudative (ie, non-diffusible), eg, either or both are diffusible or non-exudative. May be good. The placement of the sensitive chemical components depends on whether each component is diffusive or non-exudative. For example, non-exudative and / or diffusive components generally form a sensing layer on the working electrode. Alternatively, one or more diffusible components may be placed on any surface in the sample chamber prior to sample introduction. As another example, one or more diffusible components may be placed in the sample prior to introduction of the sample into the sensor.

When the redox mediator is non-exudative, the non-exudative redox mediator is generally placed on the working electrode 22 as a sensing layer. In an embodiment having a redox mediator and a second electron transfer agent, if both the redox mediator and the second electron transfer agent are non-exudative, both of these non-exudative components act as a working electrode as a sensing layer. 22 Placed on top.

For example, if the second electron transfer agent is diffusive and the redox mediator is non-exudative, then at least the redox mediator is placed on the working electrode 22 as a sensing layer. The diffusible second electron transfer agent need not be placed on the sensing layer of the working electrode and may be placed on any surface of the sample chamber, including within the redox mediator sensing layer, or in the sample. It can also be placed. If the redox mediator is diffusible, the redox mediator may be placed on any surface of the sample chamber or may be placed in the sample. If both the redox mediator and the second electron transfer agent are diffusive, the diffusible components can be placed on either surface of the sample chamber individually or together, and / or in the sample. (Ie, each diffusible component need not be placed on the same surface of the sample chamber or placed in the sample).

The redox mediator, whether diffusible or non-leaching, causes an electric current to flow between the working electrode 22 and the analyte, and the electricity of the molecule, which is not suitable for a direct electrochemical reaction on the electrode. Enables chemical analysis. The mediator functions as an electron transfer agent between the electrode and the analyte.

Analyzants that can be investigated include, for example, glucose, acetylcholine, amylases, bilirubin, cholesterol, chorionic villous gland stimulating hormone, creatine kinase (eg, CK-MB), creatine, DNA, fructothamine, glucose, glutamine, growth hormone. Species, hormones, ketones, lactate, peroxide, prostate-specific antigens, prothrombin, RNA, thyroid-stimulating hormone, and troponin. For example, analysis of drugs or pharmaceuticals such as antibiotics (eg, gentamicin, vancomycin, etc.), digitoxy, digoxin, substance of abuse, theophylline, and warfarin. The concentration of objects can also be measured. Assays suitable for measuring DNA and / or RNA concentrations are disclosed in US Patent Application Nos. 09 / 138,888 and 09 / 145,776 and are also described in PCT application PCT / US99 / 14460.

(Redox mediator) Any organic or organometallic redox species can be used as a redox mediator, but one suitable redox mediator is a transition metal compound or complex. Suitable transition metal compounds or complexes include compounds or complexes of osmium, ruthenium, iron and cobalt. In these complexes, the transition metal is coordinated to one or more ligands. The ligand is generally a monodentate, bidentate, tridentate, or quaternary ligand. The most preferred ligands are heterocyclic nitrogen compounds such as, for example, pyridine and / or imidazole derivatives. The polydentate ligand may include multiple pyridine and / or imidazole rings. Alternatively, for example, a metallocene derivative such as ferrocene can be used. As an example of a mediator, [Os (4- (N- (6-aminohexyl) aminobipyridine) (1,1'-dimethyl-2,2'-biimidazole)₂) Cl₃Can be mentioned.

Redox mediators are diffusible redox mediators, or non-exudative redox mediators such as non-exudative redox polymers. For more information on redox mediators, see, for example, US Patent Application No. 09 / 295,962 (filed April 21, 1999) and PCT International Publication No. WO 98/35225.

(Second electron transport chain) In a preferred embodiment of the invention, the sensor comprises a redox mediator and a second electron transfer agent that allows electron transfer to or from the redox mediator and analyte. The second electron transfer agent may be diffusible or non-leaching (eg, incorporated into a redox polymer or coordinated, covalently, or ionic). An example of a suitable second electron transfer agent is an enzyme that catalyzes the reaction of the analyte. For example, if the analyte is glucose, glucose oxidase, or glucose dehydrogenase such as pyrroloquinoline quinone glucose dehydrogenase (PQQ) is used. If the analyte is lactate, lactate oxidase plays this role. Other enzymes can be used for other analytes.

Counter electrode As shown in FIGS. 1 and 2, the counter electrode 24 can be configured in the same manner as the working electrode 22. The counter electrode 24 may be a counter / reference electrode. Alternatively, another reference electrode may be provided in contact with the sample chamber. Suitable materials used for the counter / reference electrode or reference electrode include, for example, Ag / AgCl or Ag / AgBr printed on a non-conductive substrate, or silver chloride on a silver metal substrate. The counter electrode may be made of the same material and method as used for the construction of the working electrode 22, but a different material and method may be used. The counter electrode or counter / reference electrode is preferably printed on an insulating substrate. A tab 25'with a contact pad 25 may also be provided for easy connection to external electronic devices (not shown) such as coulometers, potentiostats, or other measuring devices.

Optionally, a non-conductive filler material, such as a non-conductive ink, can be formed in the vicinity of the counter electrode or between a large number of counter electrodes to provide a flat surface in the sample chamber along the path of movement of the sample solution. .. The non-conductive filler material forms a smooth surface to facilitate sample chamber filling by capillary action, and / or air bubbles are trapped near the counter electrode. It is preferable to reduce the possibility of This non-conductive material may be colored or uncolored. It may also be formed on the substrate by printing or other methods. The non-conductive material can be deposited before or after the formation of the counter electrode. In one embodiment, non-conductive ink is used to fill between multiple 12.3 micrometer (0.5 mil) thick counter electrodes. In another embodiment, non-conductive ink is used to fill between a plurality of 6.4 micrometer (0.25 mil) thick counter electrodes. Filler inks are generally not required for thicknesses less than about 6.4 micrometers. Also, depending on the sensor design, filler ink may not be required for the counter electrode with a thickness of 6.4 micrometers.

(Electrode configuration) In one embodiment of the present invention, the working electrode 22 and the counter electrode 24 are arranged to face each other and face each other to form a pair of facing electrodes, as shown in FIGS. 1 and 2. In this preferred configuration, the sample chamber 26 is generally located between these two electrodes. In this facing electrode configuration, the electrodes are separated from each other with a distance of about 0.2 mm or less (that is, at least a part of the working electrode is separated from a part of the facing electrode with a distance of 200 μm or less). ), Preferably separated by a distance of 100 μm or less, most preferably 50 μm or less.

The electrodes do not have to face each other directly and may be slightly offset. Moreover, the two electrodes do not have to be the same size. It is preferred that the working electrode 22 extends in the width direction of the sensor 20 and that the counter electrode 24 is part or all of its width. Either the working electrode 22 or the counter electrode 24 may include one or more electrodes, as shown in FIG. 4 (shown as counter electrodes 324,344) and FIG. 5B (shown as counter electrodes 424,441,442). Good. The counter electrode 24 can also be formed in a comb-teeth shape. Other configurations of both counter electrode and working electrode are also included within the scope of the invention. However, in this special embodiment, it is preferable that the distance between at least a part of the working electrode and the part where the counter electrode is located does not exceed the above-mentioned specific range.

In another embodiment of the invention, the working electrode and counter electrode are coplanar. In this case, the sample chamber is in contact with both electrodes and is coupled by a non-conductive inert substrate or substrate on the opposite side of the electrodes. Suitable materials for the inert substrate include non-conductive materials such as polyester.

Other configurations of the sensor of the present invention are also possible. For example, it is possible to form two electrodes on a surface that is angled to each other. One example of such a configuration is having electrodes on a right-angled surface. Another possible configuration is to have the electrodes on a curved surface, such as inside a tube. For example, the working electrode and the counter electrode can be arranged so as to face each other on the opposite side of the tube. This is another example of opposing electrode pairs. Alternatively, the electrodes can be placed adjacent to each other on the wall of the tube (eg, one placed on the top surface of the other, or side by side). In all configurations, the two electrodes must be configured so that they do not come into direct electrical contact with each other to avoid short circuits in the electrochemical sensor.

Returning to FIGS. 1 and 2, electrodes 22 and 24 extend from the sample chamber 26 to the other end of the sensor 20 as electrode extensions called traces. Each trace is equipped with contact pads 23, 25 to provide electrical connectivity to meters and other devices capable of aggregating data and other measurements, as described below. Preferably, each conductive pad 23,25 is located on a tab 23', 25'extending from each non-conductive substrate 32,34. According to one embodiment, the tab has one or more contact pads located on it. According to the second embodiment, using a single contact pad, Contact with one or more electrodes can be provided. That is, a large number of electrodes are combined and connected via one contact pad.

As shown in FIGS. 1 and 2, when the electrodes are opposed to each other, a spacer 33 can be used to keep the electrodes apart. The spacer 33 is clearly shown in FIG. The spacers are generally selected from pressure sensitive adhesives, polyesters, Mylar®, Kevlar®, or other strong and thin polymeric films, or chemically inert. It is composed of an inert non-conductive material such as a thin polymer film such as Teflon (trademark) film. In addition to preventing contact between the electrodes, the spacer 33 also functions as part of the boundary of the sample chamber 26. Other spacers include a layer of adhesive and double-sided adhesive tape (eg, a carrier film with adhesive on opposite sides of the film). The adhesive may be applied to the polymeric material, for example by coating, to provide the spacer 33.

(Sample chamber) The sample chamber 26 is generally defined by a combination of electrodes 22, 24, substrates 32, 34, and spacer 33, as shown in FIGS. The measurement area is the area of the sample chamber contained within the sample chamber and containing only the portion of the sample analyzed in the analyte assay. In the embodiment of the invention shown in FIGS. 1 and 2, the sample chamber 26 is the space between the two electrodes 22, 24 and their non-conductive substrates 32, 34 coupled by the spacer 33. In this embodiment, the sample chamber preferably has a volume of about 1 μL or less, more preferably about 0.32 μL or less, and most preferably about 0.25 μL or less.

In the embodiment of the present invention shown in FIGS. 1 and 2, the measurement area has a volume substantially equal to the volume of the sample chamber. In a preferred embodiment, the measurement range occupies 80% of the sample chamber, in the more preferred embodiment 90%, and in the most preferred embodiment approximately 100%.

In another embodiment of the invention shown in FIG. 3, the sensor 220 comprises a working electrode 222 on the non-conductive substrate 232, a counter electrode 224 on the non-conductive substrate 234, and a spacer 233 between them. .. A contact pad 223 extends from the working electrode 222. Similarly, the contact pad 225 extends from the counter electrode 224. The sample chamber 226 (defined by substrates 232, 234 and spacer 233) extends in the length direction of the sensor 220 and has a much larger space than the area in close proximity to the electrodes 222, 224. In this embodiment, it is possible to provide a plurality of electrodes in contact with one or more sample chambers. In this embodiment, the measurement area corresponding to the region containing the portion of the sample to be analyzed is the working surface of the working electrode 222 and the portion of the sample chamber 226 coupled to the counter electrode 224. In this embodiment, the measurement range is set to accommodate a sample volume of preferably about 1 μL or less, more preferably about 0.32 μL or less, particularly preferably about 0.25 μL or less, and most preferably 0.1 μL or less. To.

In both embodiments described above, the thickness of the sample chamber and measurement area is generally the thickness of spacers 33,233 (eg, in FIGS. 2 and 3 the distance between the electrodes, or if the electrodes are coplanar, the electrodes. Corresponds to the distance between the and the inert substrate). The spacer may be, for example, an adhesive or a double-sided adhesive tape or film. Examples of useful adhesives include urethanes, acrylates, acrylics, latexes, rubbers and other known adhesives. This thickness is preferably small so that more samples are in contact with the electrode surface for a given sample volume in order to facilitate rapid electrolysis of the analyte. In addition, in a thin sample chamber, the measurement time On the other hand, the longer diffusion time reduces the error caused by the diffusion of the analyte from the rest of the sample chamber into the measurement area during the analyte assay. Generally, the thickness of the sample chamber is about 50 to about 200 mm.

(Absorbent) The sample chamber may be empty before the sample is placed in the chamber. Alternatively, in some embodiments, the sample chamber is an absorbent material for absorbing and retaining the liquid sample during the measurement process (shown as adsorbent 50 in FIG. 3). Suitable absorbents include cellulose derivatives such as polyester, nylon, cellulose, and nitrocellulose. The absorbent facilitates the absorption of small volume samples by supplementing or preferably replacing the capillary action of the sample chamber. In addition to or as an alternative, part or all of the walls of the sample chamber are coated with a surfactant with the intention of reducing the surface tension of the liquid sample and increasing the flow of liquid in the sample chamber. You may. Examples of surfactants that can be used are available under the trade name Zonyl FSO from DuPont, Wilmington and DE (Dupont of Wilmington).

Samples can be transported to the sample chamber or measurement area using methods other than the absorption action of the absorbent material. Examples of such means of transport include not only the absorbing action of the absorber, but also the application of pressure to the sample to push the sample into the sample chamber, a pump or other vacuum forming method to pull the sample into the sample chamber. Vacuum formation in the sample chamber and capillary action due to the surface tension of the sample with the wall of the thin sample chamber can be mentioned.

All sensor assemblies are tightly joined to ensure that the sample is in contact with the electrodes and that the sample chamber and measurement area maintain the same volume. This is an important consideration in the analysis of samples by coulometry, where measurements at a defined sample volume are required.

(Another sensor design) Figures 4-12 show different sensor designs for both tip filling and side filling. According to FIG. 4, the sensor 320 includes a working electrode 322, a counter electrode 324, a second counter electrode 344 (which may have a filling instruction function, as shown below), and at least one of the sensors 320. It comprises a sample chamber 326 extending along the section and may optionally include an absorber 350.

Figures 5A-5C show three layers that, when assembled, provide a sensor with a preferred configuration. FIG. 5A includes a working electrode 422 on a substrate 432. The working electrode 422 comprises a trace 453 extending from the sample chamber 426 to the tab 423'with the contact pad 423 above it. Contact pad 423 connects the sensor to a meter or other measuring device. FIG. 5B (shown with FIG. 5A inverted and the electrode side facing up) has a counter electrode 424 on the substrate 434. It also includes a first indicator electrode 441 and a second indicator electrode 442. The counter electrode 424 comprises a trace 454, the first indicator electrode 441 comprises a trace 451 and the second indicator electrode 442 comprises a trace 452. It ends with contact pads 425,443,444 on tab 425', respectively. Spacer 433 in FIG. 5C defines the sample chamber 426 and provides space between the electrodes when the two electrodes 432,434 are installed facing each other on opposite sides.

Figures 6A-6C show three layers that serve as sensors when assembled. In FIG. 6A, the working electrode 502 is formed on the first substrate 500. The working electrode 502 is an external Includes contact pad 503 for connecting to electronic devices. The contact pad 503 is connected to the working electrode 502 by a trace 552. As shown in FIG. 6B, a layer of sticky material or a spacer 504, such as double-sided tape, defines channel 506 and forms the sample chamber of the sensor. As shown in FIG. 6C (the figure showing FIG. 1A inverted and the electrode side facing up), two facing (or facing / reference) electrodes 510 and 512 are formed on the second substrate 508. As described below, the arrangement of the large number of counter electrodes also fulfills the filling instruction function by using the counter electrode 512. Each counter electrode 510,512 has a contact area or pads 511,513 for connecting to an external electronic device. These contact pads 511 and 513 are connected to the counter electrode by traces 551 and 553. The second substrate 508 is inverted so that the working electrode 502 and the two counter electrodes 510, 512 face each other in the region of channel 506 and are placed on the first substrate 500 with the spacer 504 in between.

In some examples, the surface area of the counter electrode 510 closest to the inlet 514 of channel 506 (FIG. 6B) in the sample chamber is at least 2 times, and at least 5 or 10 times that of the other counter electrode 512. It may be. A non-exudative or diffusible redox mediator and / or second electron transfer agent may be provided on either the first or second substrate 500, 508 in the region corresponding to channel 506, as described above. it can.

The working electrode and counter electrode can be formed to cover the entire channel region (except for the small space between the two counter electrodes). In this embodiment, the sample chamber and the measurement area are efficiently the same and have the same volume. In another embodiment, the measurement area occupies, for example, 80% or 90% of the volume of the sample chamber. It will be appreciated that similar sensors can be made with one counter electrode or three or more counter electrodes. It will also be appreciated that the sensor may be provided with a large number of working electrodes.

Figures 7A, 7B, and 7C show the placement of side-filled sensors. FIG. 7A shows a first substrate 520 with a working electrode 522. FIG. 7B shows a spacer 524 defining channel 526. FIG. 7C (inverted view of FIGS. 7A and 7B) shows a second substrate 528 with three facing (or facing / reference) electrodes 530, 532, 534. This arrangement of a large number of counter electrodes can provide a filling instruction function as described below. Indentations 536, 538, i.e., recessed or recessed portions, can also be formed from the end of the opening to either channel 526 to facilitate drawing of liquid into the sensor. This configuration aids in the filling of channels (ie, sample chambers) by suction or capillarity. This configuration can also reduce the potential for the user to accidentally block the channel for sample collection by pressing the tip of the sensor against the skin along the edge.

Figures 8A, 8B, and 8C show another example of the placement of side-filled sensors. FIG. 8A shows a first substrate 540 with a working electrode 542. FIG. 8B shows a spacer 544 defining channel 546. FIG. 8C (inverted view of FIGS. 8A and 8B) shows a second substrate 548 with three facing (or facing / reference) electrodes 550, 552, 554.

Figures 9A, 9B, and 9C show another example of the placement of the top-filled sensor. FIG. 9A shows a first substrate 560 with a working electrode 562. FIG. 9B shows a spacer 564 defining channel 566. FIG. 9C (inverted view of FIGS. 9A and 9B) shows a second thin film substrate 568 with two facing (or facing / reference) electrodes 570, 572. To. The arrangement of this large number of counter electrodes is a fill indicator function, as described below. Can be provided. Vent 574 (shown as a shaded area in FIG. 9C) is formed through the second substrate. In the illustrated embodiment, the vent 574 penetrates only the counter electrode and optionally the substrate 568 with the spacer 564. In this embodiment, the vents may be formed, for example, by die-cutting a part of the substrate. This die cut can also remove a portion of at least one counter electrode, but in sufficient quantity for contact with the sample within the channel and for electrical connection with the contact at the other end of the sensor. The counter electrode should be left behind. In another embodiment, the vents 574 may be formed to penetrate all layers or penetrate the first substrate and not the second substrate.

Another embodiment with different shapes is shown in Figures 10A, 10B, and 10C. As shown in FIG. 10A, this sensor includes a first substrate 579 with at least one working electrode 580. As shown in FIG. 10B, the sensor also has a spacer 581 with channel 582 formed within the spacer 581. As shown in FIG. 10C (inverted view of FIGS. 10A and 10B), the sensor further includes a second substrate 583 with two counter electrodes 584, 585. The vent opening 586 is generally cut through all layers and extends from the side of the sensor. In some embodiments, the vent opening and the sensor anterior 587 are simultaneously cut at a reproducible distance between the vent opening and the sensor anterior 587, allowing the length of the channel 582 and the working electrode 580 to be reproducible. .. To facilitate the introduction of fluid into the sensor, a dent 588, a recessed or dented portion, may be formed in the filling opening of channel 582.

11A, 11B, and 11C show another example of the placement of side-filled sensors. FIG. 11A shows a first substrate 640 with a working electrode 642. FIG. 11B shows a spacer 644 defining channel 646. FIG. 11C (inverted view of FIGS. 11A and 11B) shows a second substrate 648 with three facing (or facing / reference) electrodes 650, 652, 654. The arrangement of the large number of counter electrodes can provide a filling instruction function as described later. The length of channel 646 is generally defined by two parallel cuts along the sides 656, 658 of the sensor.

Due to the vertical sides, these sensors (and also shown in Figures 1, 2 and 5) can be made in close proximity to each other, as shown in Figure 13A. is there. By positioning it in this way during production, waste of material can be reduced. Another optional fabrication advantage in the proximity sensor fabrication method is that the redox mediator and / or second electron transfer agent strips these continuous streams along the rows or columns of adjacent sensors. As a result, it can be steadily placed in the channel. This allows these components to be placed more efficiently and without waste than other methods such as placing the redox mediator and / or the second electron transfer agent individually within the individual channels.

Figures 12A, 12B, and 12C show different sensor configurations. As shown in FIG. 12A, the sensor has a first substrate 600 with at least one working electrode 602. As shown in FIG. 12B, the sensor also has a spacer 604, and channel 606 is formed within the spacer 604. As shown in FIG. 12C (inverted views of FIGS. 12A and 12B), the sensor further has a second substrate 608 with two counter electrodes 610, 612. This large number of counter electrode arrangements can also provide a filling instruction function, as will be described later. The sensor may also include an indicator that informs the user which side of the sensor should be adjacent to the sample, such as slot 614 or extension 616 from the sensor body. Surface printing can be applied simultaneously or as an alternative to the sensor acting as an indicator. This is a sample specific It is especially important if accurate readings of the sensor can be obtained only when introduced from the side.

(Multi-electrode sensor and its calibration) Multi-electrode sensors can be used for a variety of reasons. For example, multiple electrodes may be used to test different analytes with a single sample. One embodiment of the multi-electrode has one or more sample chambers, each of which has one or more working electrodes, each defining a different measurement area. If the redox mediator is non-exudative, one or more of the working electrodes may be equipped with the appropriate reagents for testing the first analyte, eg, the appropriate enzymes, and one or more of the remaining working electrodes. A suitable reagent may be provided for testing the second analyte. For example, a multi-electrode sensor may include one or more working electrodes for measuring glucose concentration with glucose oxidase and one or more working electrodes for measuring lactate concentration with lactate in the sensing layer. is there.

The multi-electrode can also be used to improve the reading accuracy of the analysis results. Measurements obtained from each working electrode (all working electrodes detect the same analyte) can be averaged and otherwise combined for a more reliable reading. In some cases, when the difference from the mean exceeds the threshold limit, the measurement could be excluded. This threshold limit can be determined based on statistical parameters such as, for example, the standard deviation of the mean measurements. The average value may be calculated again after omitting the exclusion value. In addition to the use of multi-electrode sensors to improve accuracy, a large number of measurements are obtained for each electrode and averaged to improve accuracy. This technique can also be used for a single electrode sensor for increased accuracy.

An example of a multi-electrode sensor that can be used to accurately measure the volume of the measurement range of a pair of electrodes and is also useful for reducing noise is shown here. In this example, one of the working electrodes is made by a non-exudative redox mediator and a non-exudative second electron transfer agent (eg, an enzyme). Another working electrode is equipped with a non-exudative redox mediator, but not a second electron transfer agent. The optional third working electrode is neither equipped with a redox mediator coupled to the electrode nor a second electron transfer agent. Similar configurations can be formed using a diffusive redox mediator and / or a diffusible second electron transfer agent. However, the diffusible component is not always arranged on the working electrode. Preferably, there is sufficient distance between the electrode pairs and the redox mediator and / or enzyme does not substantially diffuse between the electrodes within the measurement period (the time from the time the same sample is introduced into the sample chamber to the end of the measurement). ..

Sensor errors due to the redox mediator present in a non-uniformly oxidized state prior to sample introduction can be measured by simultaneously electrolyzing the sample in each measurement range close to the working electrode and counter electrode. At the first working electrode, the analyte is electrolyzed to give a sample signal. At the second working electrode, the analyte is not electrolyzed (assuming a second electron transfer agent is required) because the second electron transfer agent is absent. However, electrolysis and / of redox mediators in an oxidized state (ie, some of the redox centers are in a reduced state and some are in an oxidized state) that were mixed prior to sample introduction. Alternatively, the charge moves (and current flows) due to the reciprocation of the diffusive redox mediator between the working electrode and the counter electrode. The small charge that passes through this second working electrode is subtracted from the charge that passes between the first electrodes to substantially eliminate and / or diffuse the error due to the oxidation state of the redox mediator. The background current caused by the sex redox mediator can be eliminated. This process reduces errors related to capacitive charging and Faraday currents, as well as errors related to other electrolyzed interfering substances such as ascorbic acid salt, urate, and acetaminophen. To.

The thickness of the sample chamber can be determined by measuring the capacitance between the electrode and the corresponding counter electrode, preferably in the absence of any liquid. The capacitance of a pair of electrodes depends on the surface area of the electrodes, the spacing between the electrodes, and the permittivity of the material between the plates. In general, the capacitance of this electrode configuration is a few picofarads because the permittivity of air is invariant (or, if there is a liquid between the electrodes and the counter electrode, the permittivity of most biofluids will be. If it's about 75, it's about 100-1000 picofarads). Therefore, since the surface area of the electrode is known, the thickness of the measurement range can be measured within the range of about 1 to 5% by measuring the capacitance of the electrode pair.

In other electrode configurations, these methods (ie, capacitance measurements and coulometric measurements in the absence of critical components) are used to make inaccuracies regarding interfering substances and sample volume to be measured. Background noise and errors caused by knowledge can be reduced. Protocols involving one or more working and counter electrodes and one or more measurements described above can be developed, which is within the scope of the present invention. For example, only one electrode pair is required for capacitance measurement, but additional electrodes may be used for convenience.

(Filling indicator) When using a sample chamber filled with 1 μL or less of liquid, it is often desirable to be able to confirm that the sample chamber is filled. 6A-6C show an example of a sensor having a filling indicator structure. In particular, FIG. 6A shows the first substrate 500 on which the working electrode 502 is printed. For example, a spacer 504 (FIG. 6B), such as a layer of adhesive or double-sided adhesive tape, is formed above the first substrate 500 and the working electrode 502, and a channel 506 formed within the layer provides a sample chamber. As shown in FIG. 6C (the figure in which FIGS. 6A and 6B are inverted and the electrode side is turned up), two counter electrodes 510 and 512 are printed on the second substrate 508. Preferably, the counter electrode 510 closest to the inlet 514 of channel 506 has a surface area of at least twice, preferably at least 5 or 10 times that of the other counter electrode 512 in the sample chamber.

When the sensor is filled with liquid, it can be signaled that the sensor is or is nearly filled by observing the signal between the second counter electrode 512 and the working electrode 502. When the liquid reaches the second counter electrode 512, the signal from that counter electrode should change. Suitable signals for observation include, for example, the voltage, current, resistance, impedance, or capacitance between the second counter electrode 512 and the working electrode 502. Alternatively, observe the sensor after filling to see if the value of the signal (eg, voltage, current, resistance, impedance, or capacitance) has reached a value that indicates that filling of the sample chamber is complete. Can be done.

In an alternative embodiment, the counter electrode or working electrode may be divided into two or more portions and signals from each portion may be observed to confirm that the sensor has been filled. In one example, the working electrode is opposed to the counter electrode and the indicator electrode. In another example, the counter electrode, the working electrode, and the indicator electrode are not opposed to each other and are arranged, for example, side by side. Generally, the indicator electrode is on the downstream side of the sample inlet as compared with the working electrode and the counter electrode.

For side-filled sensors such as the sensors shown in FIGS. 5, 7, 8 and 11, the indicator electrode may be located on either side of the major counter electrode. As a result, the user can fill the sample chamber from either the left or right side with the indicator electrode arranged on the upstream side. This 3-electrode configuration is not always necessary. Side filling type sensor is also , A single indicator electrode, and preferably an indication as to which side should be brought into contact with the sample solution.

Alternatively or additionally, two indicator electrodes are used in combination with one counter / reference electrode to prevent incomplete filling of the sample chamber at the start and completion of filling of the sample chamber. Detect time. The two indicator electrodes are optionally held at different potentials than the counter / reference electrodes. The start and completion of filling the sample chamber is indicated by the flow of current between the reference electrode and the counter / reference electrode.

In another example, the potentials of the opposite / reference electrodes may be equal. When the potentials of all three counter / reference electrodes are equal, eg 0 volt, the liquid causes electrical contact between the working electrode and the first counter / reference electrode when filling of the measurement range is initiated. The reaction of the analyte with the enzyme and mediator causes a current to flow between the first counter / reference electrode. When the liquid reaches the third counter / reference electrode, another current can be measured to indicate that the measurement range has been filled, as in the case of the first counter / reference electrode. Once the measurement area has been filled, the three opposing / reference electrodes may be optionally shorted together, or these signals may be added or otherwise combined.

The indicator electrode can be used to improve the accuracy of the analyte measurement. The indicator electrode may act as a working electrode or as a counter electrode or counter / reference electrode. In the embodiments shown in FIGS. 6A-6C, the indicator electrode 512 functions as a second counter electrode or counter / reference electrode with respect to the working electrode 502. The measured values obtained from the indicator electrode and the working electrode are combined (eg, added or averaged) with the measured values obtained from the first counter electrode or the counter / reference electrode / working electrode to obtain a more accurate measured value. be able to. In one embodiment, the indicator electrode may act as a second working electrode along with a counter electrode or a counter / reference electrode. In another embodiment, the indicator electrode may act as a second working electrode along with a second counter electrode or counter / reference electrode. In yet another embodiment, the indicator electrode may act as a second counter electrode or counter / reference electrode with the second working electrode.

The sensor or sensor reader can include a sign (eg, a visual sign or an auditory signal) that is activated in response to an indicator electrode to inform the user that the measurement area has been filled. The sensor or sensor reading device may be configured such that the indicator electrode begins reading after indicating that the filling of the measurement area is complete with or without warning to the user. The reading may be initiated, for example, by initiating monitoring of the signal generated at the working electrode by applying a potential between the working electrode and the counter electrode.

(General method for manufacturing sensors) 13A to 13B describe one embodiment showing the arrangement of the sensors displayed in FIGS. 5A to 5C. This method can also be used for the placement of various other sensors described above. The sensor 420 is formed when the three layers of FIGS. 5A-5C are combined.

In FIGS. 13A and 13B, a substrate 1000, such as a plastic substrate, is moving in the direction of the arrow. The substrate 1000 may be a separate sheet or a continuous roll on the web. A large number of sensors 420 may form a section 1022 having a working electrode 422 (FIG. 5A) on it, and a section 1024 having a counter electrode 424 and a section 1024 having indicator electrodes 441,442 (FIG. 5B) on the substrate 1000. it can. These action, facing, and indicator electrodes are electrically connected to their corresponding traces and contact pads. Generally, the working electrode section 1022 is made on one side of the substrate 1000. , Counter electrode section 1024 is made on the other half of the substrate 1000. In some embodiments, the substrate 1000 can be cut and bent to integrally form sections 1022,1024 with the sensor. In some embodiments, as shown in FIG. 13A, separate working electrode sections 1022 are placed in close proximity to and adjacent to each other on substrate 1000 to reduce material waste. Similarly, separate counter electrode sections 1024 can be formed in close proximity to or adjacent to each other. In other embodiments, separate working electrode sections 1022 (and similarly counter electrode sections 1024) may be spaced apart, as shown in FIG. 13B. The rest of the process is described for the fabrication of multiple sensors, but can also be easily modified to form individual sensors.

The carbon or other electrode material (eg, a metal such as gold or platinum) can also be formed on the substrate 1000 to provide a working electrode 422 for each sensor. The carbon and other electrode materials can be deposited by a variety of methods, including printing with carbon or metal inks, vapor deposition, and other methods. Printing can be performed by screen printing, gravure roll printing, transfer printing, or other known printing methods. The trace 453 and the contact pad 423 are preferably applied together with the working electrode 422, but may be applied in the next step.

Similar to the working electrode 422, counter electrodes 424,441,422 (shown in FIG. 5B) are formed on the substrate 1000. The counter electrode is formed by supplying carbon or other conductive electrode material onto the substrate. In one embodiment, the material used for the counter electrode is Ag / AgCl ink. The counter electrode material may be deposited by a variety of methods, including printing and vapor deposition. Printing may be performed by screen printing, gravure printing, transfer printing, or other known printing method. It is preferred to apply traces 454, 451 and 452 and contact pads 425, 443, 444 together with counter electrodes 424, 441, 442. However, it can also be applied in a later step.

It is preferable to manufacture a large number of sensors 420 at the same time. That is, working electrodes for multiple sensors, including their traces and contact pads, are made (eg, printed) on a polymer sheet or web. And at the same time or later, counter electrodes for multiple sensors and their traces and contact pads are made (eg, printed). The working electrode and the counter electrode may later be formed on separate substrates so that the electrodes are opposed to each other so as to face each other. Further, in order to facilitate the alignment of the substrate, the working electrode is formed on the first half of the substrate sheet or the web, and the counter electrode is formed on the sheet or the web of the second half of the substrate. Alternatively, the web may be folded and the working electrode and the counter electrode may be overlapped so as to face each other.

To provide the sample chamber 426, spacer 433 is made above at least one of the substrate / working electrode and substrate / counter electrode. The spacer 433 is preferably a layer of adhesive or an adhesive spacer such as a double-sided adhesive tape (eg, a polymer carrier film having an adhesive arranged on opposite surfaces). Suitable spacer materials include urethane, acrylate, polyacrylate, latex, rubber and the like.

A channel that results in a sample chamber is formed within the spacer 433 by cutting out a portion of the sticky spacer, placing two sticky pieces, or placing them in close proximity with a gap. The adhesive can also be printed. Alternatively, it may be arranged on the substrate according to a pattern that defines the channel area. The adhesive spacer may optionally be provided with one or more release liners prior to incorporation into the sensor. The adhesive is cut (eg, die-cut or slit) and before placing the spacer on the substrate It is also possible to remove the sticky portion corresponding to the channel.

In one method of the invention, the sticky material comprises a first and second release liner, as shown in FIGS. 14A-14G. Prior to applying the adhesive to the first substrate / working electrode, or substrate / counter electrode, the adhesive penetrates at least one, preferably all, of the first release liner and the underlying adhesive. The second release liner can be "kiss-cut" without penetrating to form one or more sections. One of the sections will eventually become the sample chamber section. The first release liner is removed from the sticky material over its length, but left on the sample chamber section. The exposed adhesive is then applied to the first substrate / working electrode or substrate / counter electrode. When removing the second release liner, the adhesive located in the sample chamber section is also removed, leaving an opening in the channel or adhesive spacer. A second substrate / working electrode or substrate / counter electrode is then applied to the adhesive to form a layered structure of the sensor.

Figures 14A-14G show in detail the process of the adhesive layer by "kiss cut". The adhesive layer that finally becomes the spacer 433 is preferably obtained from an adhesive structure that includes the adhesive layer 1030 between the first release layer 1020 and the second release layer 1040. The blade (eg, knife or mold blade) cuts out at least one part of the first release liner 1020 and the adhesive layer 1030, but does not cut out the second release liner 1040, as shown in FIGS. 14B-14G. Form liner sections 1020a, 1020b and sticky sections 1030a, 1030b. The space occupied by the 1030b is the area that results in the sample chamber of the sensor and is referred to as the "sample chamber area". With a cutting configuration as shown in FIG. 14G, one release liner section 1030a can be removed, leaving the release liner section 1030b. The liner can provide a separate strip that is cut and extends over the length of the liner. This then requires the separate removal of the liner strips.

The release liner section 1030a is removed to provide the adhesive composition shown in FIG. 14C. The exposed sticky material is positioned above the substrate 1050 and adheres firmly to the substrate 1050 as shown in FIG. 14D. Adhesive section 1030a, which does not include a release liner, adheres substrate 1050. As shown in FIG. 14E, the second release liner 1040 is removed and the cut and unbonded section of the adhesive 1030b and the first liner 1020b are pulled with it. This results in a sticky region 1030a that provides spacer layer sections 443a, 443b with a sample chamber 426 in between, as shown in FIG. 14F. After that, the second substrate is positioned on the adhesive layer and laminated on the first substrate via the spacer 433. The thickness of the spacer 433 generally determines the distance between the working electrode and the counter electrode, which affects the volume of the sample chamber 426. This thickness does not vary by 5% for individual sensors and / or one of the individual sensors in the vat. It is preferable that it is full. The integrity of the sample chamber wall is preserved by using the "kiss cut" method to create a layered sensor structure. This provides a more reliable and reproducible sample chamber volume.

A non-exudative or diffusible redox mediator and / or second electron transfer agent is placed on the substrate, at least in the area of the sample chamber. If either or both of these components are non-exudative, the components are preferably placed on a working electrode. If either or both of these components are diffusible, the components can be placed on any surface of the substrate in the channel region. The redox mediator and / or the second electron transfer agent can be placed individually or together on the substrate before or after placement of the spacers. The redox mediator and / or the second electron transfer agent may be, for example, screen printing, inkjet printing, spraying, painting. It can be placed by a variety of methods, including alignment and / or stripping along rows or columns of adjacent electrodes. Other components may also be placed individually or with a redox mediator and / or a second electron transfer agent. Examples of these components include surfactants, polymers, polymer films, preservatives, binders, buffers, and crosslinkers.

After arranging the spacer, redox mediator, second electron transfer agent, sensory layer, etc., the first and second substrates (which have a working electrode and a counter electrode on it) are arranged so as to face each other. To form a sensor. The substrate surfaces are joined by the adhesive material of the spacer. After joining the faces together, the individual sensors can be removed from the web-like sensor by a variety of methods, including, for example, die cutting, slitting, or other methods of cutting off excess parts of the substrate material and separating the individual sensors. cut out. In some embodiments, methods such as cutting and slitting are used in combination. Alternatively, an individual sensor portion is first cut out from the substrate, and then the two portions are joined by an adhesive method such as using a spacer adhesive to form a sensor.

Cut out the sensor from the rest of the board with the sides of the sensor vertical and / or cut out the sensor from other parts by slitting the board parallel to, for example, the direction in which the gang arbor blade system is used. be able to. The edge of the sensor can define the edge of the sample chamber and / or the edge of the measurement area. Accurate control of the distance between cuts can often reduce variability in sample chamber volume. In other examples, these cuts are preferably parallel to each other. This is because parallel cutting is generally easy to make.

(Connecting the sensor to an electrical device) In general, with reference to FIGS. 16A and 16B, 17A and 17B, and 18A and 18B, the combined sensor of FIGS. 5A-5C (referred to as sensor 1420) is generally an electrical connector 1500. Connect with a meter or other electrical device. The connector is configured to be coupled and connected with contact pads 423,425,443,444 at the end of the sensor 1420. Sensor meters typically include a potentiostat or other component to provide potential and / or current to the electrodes of the sensor. The sensor reader may also include a processor (eg, microprocessor or hardware) for measuring the concentration of the analyte from the sensor signal. The sensor meter also includes a display or a port for connecting the display to the sensor. From the sensor signal, said display comprises a sensor signal and / or, for example, an analyte concentration, a rate of change in the analyte concentration, and / or an excess of the threshold analyte concentration (eg, indicated as hypoglycemia or hyperglycemia) Display the measured results.

Examples of suitable connectors are shown in FIGS. 16A and 16B, 17A and 17B, and 18A and 18B. The connector 1500 (used to connect the sensor to a meter or other electrical device) typically consists of two parts, a top 1510 and a bottom 1520 (see Figure 16B). Positioned and secured between the top 1510 and bottom 1520 are the contact leads 1423,1425,1443,1444, which provide an electrical connection between the sensor 1420 and the meter. These leads 1423,1425,1443,1444 connect to the proximal end of physical contact with contact pads 423, 425, 443, 444 (Fig. 5A and Fig. 5B, respectively) and to all mounted meters. Has a reed. The end of the sensor 1420 with the contact pad slides into the connector 1500 and fits into the connector 1500 by placing the sensor 1420 in the slide area 1530. The contact structure of the connector 1500 is the correct pad and electrical of the sensor so that the working electrode and counter electrode are properly coupled to the meter. It is important to make contact with each other.

One optional feature is the insertion monitor 1450, shown in FIGS. 16A and 16B, which are top views of the sensor before it is inserted into the connector 1500. The insert monitor is a conductive strip across the width of the sensor. This conductive insertion monitor is located on a conductive substrate and has a conductive pad for electrical contact with the connector. The insertion monitor is configured and located near the electrical circuit between the two contact structures 1451 and 1452 when the sensor is properly inserted into the connector. Proper insertion into the connector 1500 means that the sensor strip 1420 is inserted with the right side up, the correct end of the strip is inserted into the connector, and the contact pad of the electrode on the strip and its. This means that the strips are inserted into the connector far enough to ensure a secure electrical connection between the contact structures of the corresponding connectors. When all electrode pads are not in proper contact with the contact structure of connector 1500, it is preferable that there is no closing circuit. The insertion monitor may be in a form other than a strip across the width of the sensor. For example, other designs include individual dots, grid patterns. Alternatively, it may have stylistic features such as words and letters.

Since this insertion monitor 1450 is not at the end with the contact area of the electrodes, the insertion monitor 1450 does not require additional width space on the sensor. The width of the contact pads 443,425,444,423 is defined as the width at which the leads are arranged to result in an electrical connection. Generally, the contact width is the width of the exposed contact area. In one embodiment, six contact lead structures on the connector (ie, 1451, 1443, 1425, 1444, 1423, 1452) are associated with the sensor 1420 and four contact pads (ie, 443, 425, 444, 423). It can contact the sensor 1420 with the same width. The concept of having contact points on a sensor that occupies a width greater than or equal to the width of the sensor may be used for any number of contact points. It can be used with or without an insert monitor.

As a special case, the four leads 1443,1425,1444,1423 come into contact with the contact pads 443,425,444,423. If the width of each lead and / or contact pad is 1 mm, then a sensor with a width of at least 4 mm is required for contact. Additional leads, such as leads for the insertion monitor 1450 (ie, contact leads 1451, 1452), are contacted by extending leads 1451, 1452 along the sides of leads 1443 and 1423, and then leads 1443,1425, Tilt towards the center of strip 1420 behind the point where 1444,1423 contacts strip 1420. The insertion monitor strip leads 1451 and 1452 cross the side edges of the sensor 1420 and come into contact with the sensor. Therefore, no additional sensor width is required.

The contact structures are parallel and do not overlap. They run longitudinally from the distal end to the proximal end. Lead structures 1443,1425,1444,1423 end at their proximal ends, while lead structures 1451,1452 pass longitudinally through the proximal ends of lead structures 1443,1425,1444,1423. Passing through the proximal end, the lead structures 1452,1452 tilt towards the center of the sensor strip.

The insertion monitor can also be used to encode the information on the test strip. The encoded information can be, for example, calibration information for a production lot or for a special strip thereof. Such calibration information may be associated with the sensitivity of the strip, or the y-interception of its calibration curve. In one embodiment, the insertion monitor comprises two or more contact pads for connection to the meter. Two or more contact packs Do are connected by a conductive material such as conductive ink. The resistance of the conductive path between two or more contact pads is associated with the coded information. As an example of discontinuous calibration values, resistance values in a given range can correspond to one calibration setting value. And the resistance values in different ranges can correspond to different calibration settings. As an example of continuous calibration values, the calibration set value corresponds to a continuous function of resistance. Suitable conductive paths between contact pads are shown in Figures 19A-19L.

In FIGS. 19A-19F, a number of conductive paths with different resistances are printed on the strip. Path resistance can be altered to be uncharged by cutting or cutting some or all conductive paths. In FIGS. 19G-19L, the resistance is controlled by the width or length of the conductive path. Although this encoded information is preferably provided to the insertion monitor, it should be recognized that the function of the insertion monitor and the coding of the information can also be done individually using separate conductive traces on the strip. ..

In another embodiment to ensure proper insertion of the sensor into the meter, the meter has a raised area to prevent or prevent the sensor from being inserted in the wrong direction, as shown in FIG. Alternatively, bumps may be included. To ensure proper insertion of the sensor 1100 into the connector 1110, the connector 1110 may include a raised area 1140 that prevents or prevents the sensor from being inserted in the wrong direction. For example, the width w2 of the contact area of the second substrate 1130 can be wider than the width w1 of the contact area of the first substrate 1120. In this example, the raised region 1140 is arranged such that the first substrate 1120 slides the sensor 1100 into the connector so that it is adjacent to the surface 1150 protruding from the raised region 1140. However, the second substrate 1130 will prevent or prevent the raised region 1140 from adjoining the protruding surface 1150. An object other than the raised area can also be used to guide the user to insert the sensor correctly into the meter.

(Integrated sampling and analysis material measuring device) Analytical material measuring devices constructed according to the principles of the present invention generally include a sensor combined with a sampling device to provide an integrated sampling and measuring device. Sampling devices typically include a skin perforator, such as a lancet, that can be injected into the patient's skin to allow blood to flow, for example. In a preferred embodiment, the integrated sample acquisition and analyzer measuring device comprises an incision device that holds the lancet and measuring strip. The incision device is preferably active cocking. By requiring the user to cock the device prior to use, the risk of inadvertently triggering the lancet is reduced. It would also be preferable for the incision device to be able to adjust the depth at which the user inserts the lancet into the skin. Such devices are already available in Boehringer Mannheim and Palco. It can be purchased from companies such as. This feature allows the user to adjust the incision device according to different skin thicknesses, skin durability, or pain sensitivities depending on the body part or the user.

Larger samples of body fluids, such as blood and interstitial fluid, generally appear when pressure is applied to the area around the skin that has been perforated. A sampling device with a protruding ring as shown in FIG. 21 may be used. Ring 1200 surrounds the incision site and applies higher pressure to remove a larger sample from the wound. The lancet emerges from the center 1220 of the ring 1200. Ring 1200 has a surface area 1210 that pushes the skin. Therefore, it increases the tension on the skin and often raises the skin. For tighter, wider skin, a larger volume is obtained from the wound. Ring 1200 provides a larger surface area 1210, tighter skin, The pain experienced by the patient during the incision is reduced. In addition, the tendency to bruise is generally reduced.

In one embodiment, the incision instrument and meter are integrated into a single device. In order to operate the device, the user inserts a disposable cartridge containing a measurement sensor and an incision device into the integrated device, cocks the incision device, presses the incision device against the skin to operate, and reads the measurement results. All you have to do is take it. Such an integrated incision and test reader simplifies the user's test process and minimizes fluid handling.

(Sensor and meter operation) The electrochemical sensor of the present invention can be operated with or without an electric potential applied to the electrodes. In one embodiment, the electrochemical reaction occurs spontaneously and it is not necessary to apply an electric potential between the working electrode and the counter electrode. In another embodiment, a potential is applied between the working electrode and the counter electrode. However, the potential may or may not be constant. The magnitude of the potential required depends on the redox mediator used. The potential at which the electrodes are naturally balanced, or the potential at which an external bias is applied, and the potential at which the analyte is electrolyzed are generally large enough to complete or nearly complete the electrochemical reaction. However, it is preferable that the size does not cause oxidation to the extent that it significantly induces an electrochemical reaction of interfering substances such as urate, ascorbate, and acetaminophen that affect the signal to be measured. For non-exudative redox mediators, the potential is generally about -350 mV to about + 400 mV with respect to the standard caromel electrode (SCE). Preferably, the potential of the redox mediator is more negative than about +100 mV, more preferably negative than 0 mV, and most preferably negative than about -150 mV with respect to SCE.

When applying an external potential, the potential may be applied either before or after the sample is placed in the sample chamber. If the measurement area occupies only a part of the sample chamber, an electric potential is applied after the sample is completely placed in the sample chamber to prevent electrolysis of the sample passing through the measurement area when the sample chamber is filled. Is preferable. Alternatively, if the measurement area occupies most or all of the sample chamber, the potential can be optionally applied either before or during filling of the sample chamber without compromising assay accuracy. When a potential is applied and a sample is present in the measurement range, a current flows between the working electrode and the counter electrode. The current is due, at least in part, to the electrolysis of the analyte in the sample. This electrochemical reaction occurs via a redox mediator and an optionally used second electron transfer agent. For many biomolecules B, the process is represented by the following reaction equation.

<maths num="1"><img file="JP4885508B2_D0001.tif" /></maths>

<maths num="2"><img file="JP4885508B2_D0002.tif" /></maths>

Biochemical B is oxidized to C by redox mediator A in the presence of the appropriate enzyme. Next, the redox mediator A is oxidized at the electrode. Electrons are collected on the electrodes and the resulting current is measured. The measured current may also include background current generated by the measured background charge, at least in part, due to the reciprocation of the diffusible redox mediator between the working electrode and the counter electrode. is there. This background current can be minimized or clarified as described above. It is described in the PCT application PCT / US99 / 23425.

Various techniques can be used to measure the concentration of the analyte in the sample. These techniques include, for example, coulometry, amperometry, and potencyometry. In order to measure the concentration of an analyte (eg, glucose) in a sample by coulometry, the charge passed during the electrolysis of the analyte, or the charge attempted to pass between the working electrode and the counter electrode is measured. Knowing the charge and the volume of the sample chamber, the concentration of the electrolyzed analyte in the sample can be calculated. This charge can be calculated by several methods.

For example, the charge can be measured directly. This can be done using a coulometer or by a known coulometric method. In general, the charge is measured while completely or almost completely electrolyzing the analyte.

As another example, the charge is electrolyzed current i with respect to time t._tCan be measured from. Continuous current (i_x, I_{x + 1}, I_{x + 2}...) for continuous time (t)_x, T_{x + 1}, T_{x + 2}...) to measure. The currents can then be integrated (eg, integrated using a known numerical method) to add charge. In some examples, the current can be measured throughout the electrolysis. In another example, the current is extrapolated after a certain period of time, and the charge is measured using the combination of the measured current and the extrapolated current.

Extrapolation of current utilizes a projected relationship between current and time for at least a portion of the period of electrolysis. In general, when the system is diffusion limited, the natural logarithm of the current is linear with the passage of time. This is generally sensor-controlled. Occurs after Hirojo Ya. Figure 15A is like glucose in the sensor already described. This is an example of a graph showing the relationship between current and time for electrolyzing an analysis object. FIG. 15B is a graph showing the relationship between the natural logarithm of current and time for the same data. While the sensor is in equilibrium, the sample is completely sunned, as shown in Figures 15A and 15B. After filling the pull chamber, the current is increased to the peak current value. Peak currents generally occur while the system is kinetic-restricted rather than diffuse. Generally, the current then begins to decrease, but in some cases it may increase further before diffusion is restricted. Finally, the current value falls into the region shown in Figure 15B. There, electricity There is a linear relationship between the natural logarithm of the flow and time. It is preferred to extrapolate using the non-linear or preferably linear estimation method (eg, linear least squares method) required to electrolyze the rest of the analyte. In Figure 15B, the extrapolation area is tangled. It is shown and the extrapolation is shown by the solid line.

For this discussion, the referenced current value is the absolute value of the actual current. Therefore, the sign of the current measurement is ignored. For example, the peak current deviates most positively or negatively from zero current.

The relationship between the natural logarithm of current and time is linear while the system is diffusion limited. The charge required to completely electrolyze the analyte remaining in the sample can be reliably estimated from the gradient of this straight line. Ensure that the system is in a spread-restricted range before making an estimate You have to be careful as it is. One way to ensure that the system is in diffusion limited state is the peak current i_peakObserve the current value until (P) is achieved. The current value is the threshold i where the current is usually a fraction of the peak current._threashContinue observing the current value until it drops below. For example, the threshold is one-half, one-third, or one-fourth of the peak current (eg, i)._threash= j * i_peak, Where j is, for example, 0.5, 0.33 or 0.25). The threshold is selected based on the characteristics of the sensor. Thereby, the system has high reliability when it becomes a diffusion limit.

Many other methods can be used to ensure that the system is in a spread-restricted state. For example, i_peakAfter that, you will be able to observe the current value until it reaches the inflection point and the second derivative of the natural logarithm of the current turns positive. In this respect, the system is generally in a diffusion restricted state.

After reaching the threshold, two or more measured current values (eg, m)_x= (log (i)_x)-(log (i)_xy)) / (t_x-t_xy), However, m_xIs time t_xIt is the gradient at the time of i_xIs time t_xIt is the current at the time of i_xyIs time t_xT before_xyThe gradient is measured using the natural logarithm of). The gradient can be measured using the natural logarithm of any two current values (eg, one current value and the other current values measured immediately after), but the gradient is a number of consecutive currents. It is preferred to measure using the natural logarithm of the values or current values at least about 10 (ie y = 10), preferably at least about 30 (ie y = 30) apart. This process can reduce the effect of random noise in the system. Optionally, for confirmation, the continuity of multiple gradient values (eg, 3 or 5 or 10 gradient values) can be observed. For example, the system can look for 10 monotonically decreasing gradient values before accepting the gradient values for use in the estimation.

The measured gradient values are used to extrapolate the charge required to completely electrolyze the analyte remaining in the sample. That is, the current is extrapolated to zero current. A known extrapolation method (eg, linear extrapolation) can be used. By adding the measured charge to the extrapolated charge, the total charge required to electrolyze the analyte is determined.

Current values (or other measurements) can also be used to monitor the operation of sensors and meters. For example, the ratio of current values can be checked to see if the measurement failed (eg, the sensor was pulled out of the measuring device). The range of acceptable ratios will generally depend on the type and component of the sensor, the speed at which measurements are made, and the noise level that is acceptable. As an example, the range of ratios accepted for approximation measurements would be, for example, 2: 1 to 1: 2.

What has been said above focuses on the extrapolation of the charge required to completely electrolyze the analyte in the sample, using the gradient of the relationship between the natural logarithm of the current and time under diffusion limiting conditions. ing. Those skilled in the art will recognize that this gradient is related to the effective diffusivity and can be used to extrapolate other linear or non-linear mathematical functions related to the effective diffusivity.

Extrapolation of current measurement and measurement of the charge and concentration of the analyte to pass or pass through and other functions by the processor with or without a storage medium, software, hardware or its It can be done by combination. According to another embodiment, this same process is accomplished using configured discontinuous or semi-programmable hardware, eg, using a hardware description language such as Verilog. In yet another embodiment, this process uses at least one look-up table arrange me with the data stored therein. Performed using a processor with nt), the complete or partial result of the above equation is expressed based on the settings of a given input data.

The current measurement can be performed by various methods. For example, the current can be measured by directly measuring the current using a known technique or device. Another method of measuring current includes discharging a known amount of charge to the sample (by electrolysis of the analyte) and measuring the time required for the discharge. The current is then calculated as an index of charge and discharge time. As an example, a capacitor can be charged by a circuit in the meter, then coupled to a working electrode or counter electrode and discharged by electrolysis of the analyte. The time to discharge to the threshold level can be measured, for example, using a clock circuit that is part of the meter's processing circuit. By using a clock circuit, time can be measured very accurately. This is advantageous over designs where current or charge must be measured directly and these analog measurements must be converted to a processable digital display using an expensive A / D (analog-to-digital) converter. Is.

The accuracy of charge and time measurements that provide the current value affects the accuracy of the final analyte concentration measurement. Although it is desirable to use a high quality and high precision measuring device, the capacitors used to store the charge can be high quality and expensive capacitors with exactly known capacitance. Alternatively, accurate charge measurement can be performed by using a calibration resistor in parallel with the capacitor. During the calibration period, the capacitor is discharged through a resistor, and the current passing through the resistor or the time constant of discharge can be measured. It can then be used to measure the charge stored in the capacitor. High quality and highly accurate resistors are generally less expensive than similar precision capacitors and will provide similar accuracy in charge measurements.

Analytical material (C) regardless of how it is measured_A) Concentration is calculated using the charge Q by the following equation (when the redox mediator is non-exudative): C_A= Q / nFV (3a) However, n represents the electron equivalent required to electrolyze the analyte, F represents the Faraday constant (approximately 96,500 coulombs / equivalent), and V represents the volume of the sample in the measurement range. .. When a diffusible mediator is used, the concentration of the analyte can be obtained from the following formula.

C_A= (Q_tot-Q_back) / NFV (3b) However, Q_totRepresents the total amount of charge transferred during the measurement, Q_backRepresents the amount of charge that is transferred by the reciprocation of the diffusible mediator between the working electrode and the counter electrode, regardless of the analyte. At least in some cases, the sensor is configured such that the background charge does not exceed five times the magnitude of the charge generated by the electrolysis of an amount of the analyte. Preferably, the background signal does not exceed 200%, 100%, 50%, 25%, 10%, or 5% of the charge generated by the electrolysis of the analyte.

An example of a method for measuring the ratio of the hackground signal to the signal generated by the electrolysis of the analyte is described below. If the reciprocation of the redox mediator is not neutralized by the applied potential, the charge generated by that reciprocation is expressed by the following equation.

Q_back= (AFD_MC_M/ d) (tn_M) However, A is the area of the working electrode, F is the Faraday constant (about 96, 500 coulombs / equivalent), and D._MIs the effective diffusivity of the mediator in the sample, C_MIs the concentration of the redox mediator in the measurement range, d is the distance between the opposing electrodes, t is the total measurement time, n_MRepresents the number of electrons gained or lost by the redox mediator .

Further, the charge of the analyte is expressed by the following equation when the electrooxidation of the analyte is completed by about 90% within the measurement time. Q_G= Ad (0.90) C_Gn_GF where A is the area of the working electrode, d is the distance between the opposing electrodes, C_GIs the glucose concentration, n is the number of electrons required for electrolysis of the analyte (eg, 2 electrons per glucose molecule), and F is the Faraday constant. For glucose, C_GIs 5 mM (or 5 x 10)^-6Mol / cm³), T is 60 seconds, n_GIs 2, n_M1. The ratio of the charge from the redox mediator to the charge due to electrolytic oxidation of the analyte is expressed by the following formula.

Q_back/ Q_G= (D_MC_M/ d²) (Tn_M/ (0.9n_GC_G)) = (D_MC_M/ d²)×(6.7×10⁶) For example, Q_back/ Q_GIf the ratio of is 5, (D_MC_M) / d²Is 7.5 × 10^-7Mol / (cm³Seconds). Also, for example, Q_back/ Q_GIf the ratio of is 1, (D_MC_M) / d²Is 1.5 x 10^-7Mol / (cm³Seconds). As yet another example, when the above ratio is 0.1, (D_MC_M) / d²Is 1.5 x 10^-8Mol / (cm³Seconds). Therefore, according to the desired ratio, D_M, C_MWith proper selection of, and d, the sensor can be configured to have the desired ratio.

This ratio can be increased, for example, by reducing the redox mediator concentration (ie, C)._MMay be reduced) can be affected. Alternatively or additionally, the spread of redox mediators may be reduced. Other sensor configurations are also suitable for controlling the ratio of background signals to signals generated by the analyte. This will be described below.

Background charge Q_backCan be calculated by various methods. For example, by using a limited amount of diffusible redox mediator; by forming a film on the counter electrode to limit the diffusion of the redox mediator to the counter electrode; or by the potential difference between the working electrode and the counter electrode. By making Q relatively small_backCan be made smaller. Q_{ba ck}Redox mede is a suitable sensor configuration and method for reducing The reaction rate of the yater is significantly faster at the counter electrode than at the working electrode; immobilization of the redox mediator on the working electrode; the reaction at the counter electrode or counter / reference electrode causes the redox mediator to be on the counter electrode or counter / reference electrode. Immobilization in; or delaying the spread of the redox mediator.

In coulometric measurements, at least 20% of the analyte is electrolyzed. Preferably, at least 50%, more preferably at least 80%, even more preferably at least 90% of the analyte is electrolyzed. In one embodiment of the invention, the analyte is completely or nearly completely electrolyzed. The charge can be calculated from the measured current value obtained during the electrochemical reaction, and the concentration of the analyte can be determined using equation (3a) or equation (3b). Completion of the electrochemical reaction is generally indicated by the current reaching a steady-state value. This indicates that all or almost all of the analyte was electrolyzed. In this type of measurement, generally at least 90% of the analyte is electrolyzed, preferably at least 95% of the analyte is electrolyzed, and more preferably at least 99% of the analyte is electrolyzed.

With respect to coulometry, it is generally desirable for the analyte to be rapidly electrolyzed. The rate of the electrochemical reaction depends on several factors, including the potential applied between the electrodes and the kinetics of reactions (1) and (2). Another important factor is the size of the measurement range. In general, the higher the potential, the greater the current passing through the cell, and thus, in general, the reaction occurs more quickly. However, if the potential is too high, it will be measured by other electrochemical reactions. There is a significant error in the fixed value. In general, based on the predicted concentration of the analyte in the sample, not only a particular redox mediator and any second electron transfer agent, but also between the electrodes so that the analyte is almost completely electrolyzed in less than 5 minutes. The potential is also selected. The analyte is preferably electrolyzed within about 2 minutes, more preferably within about 1 minute.

Coulometry has the disadvantage that the volume of the sample to be measured must be known, but coulometry is, for example, non-temperature dependent on the measured value, non-enzymatically dependent on the enzyme activity of the measured value, measurement. It is a preferable method for analyzing a small amount of sample because it has advantages such as no dependence of the value on the redox mediator activity and no error in the measured value due to the consumption of the analyte in the sample. As described above, coulometry is a method of measuring the amount of charge that has passed or is believed to pass during complete or near-complete electrolysis of an analyte. One of the methods by coulometry involves electrolyzing the analyte at the working electrode and measuring the current generated between the working electrode and the counter electrode more than once during the electrolysis. When the current reaches a steady state, the electrolysis is complete. The charge used to electrolyze the sample is calculated by integrating the measured current over time and calculating any background signal. Since the charge is directly related to the amount of analysis material in the sample, there is no temperature dependence of the measured value. In addition, the activity of the enzyme does not affect the measurements, only the time required to obtain the measurements (ie, according to the less active enzyme, it takes a long time to completely electrolyze the sample. (It takes time), so the measurement of the analysis product concentration will not be inaccurate due to the decay of the enzyme over time. Finally, the consumption of the analyte in the sample due to electrolysis is not the cause of the error, but rather the purpose of this method (however, the electrolysis curve is based on known electrochemical principles). When extrapolated from a partial electrolysis curve, the analyte does not need to be completely electrolyzed).

In some examples, it is desirable to use an assay other than coulometry, such as an amperometry or potentiometry assay. "Amperometry" or "chronopotency metric" is a measurement of potency metric at a point of time of 1 or more. These measuring methods are useful when the volume of the measured sample is unknown. The volume of a sample within the measurement range of a small volume sensor (ie, 1 microliter or less) is difficult to accurately reproduce if the fabrication tolerances for one or more dimensions of the measurement range vary significantly. Amperometry or potentiometry measurements, etc., are described, for example, in US Patent Application No. 09 / 295,962 (filed April 21, 1999) for information on measurements that use non-Culometry.

(Sample heating) The sample can be heated to increase the rate of diffusion, oxidation, or reduction of the analyte. This heating can be achieved by a variety of methods, including placing the sensor in a heating environment or applying a heating element to the sensor.

Another method includes, for example, providing the sensor with a thermal heating element such as a wire or ink element capable of converting electrical energy into thermal energy. The wire or ink can be applied to a substrate such as a polymer film, eg, on the opposite side of one or more working electrodes, counter electrodes, reference electrodes, or counter / reference electrodes, or working electrodes, counter electrodes, reference electrodes, or It can be applied around the facing / reference electrode. In some examples, the temperature of the sample can be heated to rise in the range of 5-20 ° C above the initial temperature. In another example, the temperature of the sample does not have to be known, but a certain amount of power or current should be applied to the wire or ink.

(General example) The present invention is further characterized by the following general examples. These examples have been described so far It is not intended to limit the scope of the invention as fully described by the description in. Changes within the concept of the present invention will be apparent to those skilled in the art.

One method for making a sensor with a working electrode, multiple counter / reference electrodes and a side-filled sample chamber, shown in FIGS. 5A-5C, is shown below. According to the method described here, a plurality of sensors can be made and later separated.

A first non-conductive substrate, preferably polyester, is formed. For example, a conductive carbon ink such as Graphite # 4491, which can be purchased from Ercon, Wareham, Massachusetts (MA), is applied to the pattern shown in Figure 5A on the first polymer substrate. Screen-printed on one side of 1 and dried to provide working electrode 422, trace 453 and contact pad 423. After cutting the substrate web to final size, each working electrode 422 has a trace 453 that extends to the width of the sensor and extends to the far end of the sensor. As an example, the sensor has a width of about 6.1 mm (0.240 inches). A typical width is about 2 mm to 20 mm, preferably about 4 mm to 10 mm, but wider or narrower sensors can be used if desired.

The sensing layer described in US Patent Application No. 09 / 295,962 containing a diffusible mediator and a diffusible glucose dehydrogenase enzyme is applied to the working electrode in the sample chamber region. The sensing layer can be formed on the electrodes before or after forming the sample chamber. On the other half of the polyester substrate, conductive silver / silver chloride ink, such as the trade name "Silver / Silver Chloride # R414", is applied to the pattern shown in Figure 5B. Screen print on the second half of the polymeric substrate and allow to dry. As shown in FIG. 5B, the Ag / AgCl ink provides a first indicator or reference electrode 441, a counter electrode 424, and a second indicator electrode 442. The counter electrode may extend over the entire surface of the sensor, or may extend over a part thereof. As an example, the width of the counter electrode is about 1 mm to 20 mm, preferably about 2 to 10 mm. In one configuration of the sensor, the counter electrode 424 has a width of approximately 3.71 mm (0.146 inches). The indicator electrode has a width of, for example, about 0.1 mm to 5 mm, preferably about 0.25 mm to about 1 mm.

In one configuration of the sensor, a working electrode such as the working electrode 422 shown in FIG. 5A extends to the width of the sensor. It is about 6.1 mm (0.240 inches). Opposite electrodes, such as counter electrode 424, have a width of approximately 3.71 mm (0.146 inches). Indicator electrodes such as the indicator electrodes 441,442 in FIG. 5B have a width of approximately 0.51 mm (0.020 inches). There is a space of 0.30 mm (0.012 inch) between these indicator and counter electrodes. Each of the indicator electrodes may be formed behind the final side of the sensor strip, eg, about 0.38 mm (0.015 inch), respectively. As a result, the process of slitting the substrate into individual sensors prevents the electrodes 441,442 from being damaged, for example, by being rubbed, striped, chipped, or the like.

The present invention has been described using a variety of specific, preferred embodiments and methods. However, it will be apparent to those skilled in the art that many variations and variations may be made as long as they are within the spirit and scope of the present invention.

<figref num="1">The schematic diagram of the 1st Embodiment of the electrochemical sensor by this invention.</figref><figref num="2">An exploded view of the sensor shown in Figure 1, showing each layer individually.</figref><figref num="3">The schematic diagram of the 2nd Embodiment of the electrochemical sensor based on the principle of this invention.</figref><figref num="4">Top view of the third embodiment of the electrochemical sensor based on the principle of the present invention.</figref><figref num="5A">Top view of a first substrate provided with a working electrode used in the sensor of the fourth embodiment according to the present invention.</figref><figref num="5B">Bottom view of a second substrate with counter electrodes placed on opposite sides of the substrate of FIG. 5A (inverted view of FIG. 5A).</figref><figref num="5C">Top view of the spacer placed between the first substrate of FIG. 5A and the second substrate of FIG. 5B.</figref><figref num="6A">Top view of a first film with a working electrode used in the sensor of the fifth embodiment of the present invention.</figref><figref num="6B">Top view of the spacer placed on the first film of FIG. 6A.</figref><figref num="6C">Bottom view of a second film with counter electrodes placed on top of the spacer of FIG. 6B and the first film of FIG. 6A (inverted view of FIGS. 6A and 6B).</figref><figref num="7A">Top view of a first film with a working electrode used in the sensor of the sixth embodiment of the present invention.</figref><figref num="7B">Top view of the spacer placed on the first film of FIG. 7A.</figref><figref num="7C">Bottom view of a second film with counter electrodes placed on top of the spacer of FIG. 7B and the first film of FIG. 7A (inverted view of FIGS. 7A and 7B).</figref><figref num="8A">Top view of a first film having a working electrode used in the sensor of the seventh embodiment of the present invention.</figref><figref num="8B">Top view of the spacer placed on the first film of FIG. 8A.</figref><figref num="8C">Bottom view of a second film with counter electrodes placed on top of the spacer of FIG. 8B and the first film of FIG. 8A (inverted view of FIGS. 8A and 8B).</figref><figref num="9A">Top view of a first film with a working electrode used in the sensor of the eighth embodiment of the present invention.</figref><figref num="9B">Top view of the spacer placed on the first film of FIG. 9A.</figref><figref num="9C">Bottom view of a second film with counter electrodes placed on top of the spacer of FIG. 9B and the first film of FIG. 9A (inverted view of FIGS. 9A and 9B).</figref><figref num="10A">Top view of a first film with a working electrode used in the sensor of the ninth embodiment of the present invention.</figref><figref num="10B">Top view of the spacer placed on the first film of FIG. 10A.</figref><figref num="10C">Bottom view of a second film with a spacer of FIG. 10B and a counter electrode placed on top of the first film of FIG. 10A (inverted view of FIGS. 10A and 10B).</figref><figref num="11A">Top view of a first film having a working electrode used in the sensor of the tenth embodiment of the present invention.</figref><figref num="11B">Top view of the spacer placed on the first film of FIG. 11A.</figref><figref num="11C">11C is a bottom view of a second film having a spacer of FIG. 11B and a counter electrode placed on top of the first film of FIG. 11A (inverted view of FIGS. 11A and 11B).</figref><figref num="12A">Top view of a first film having a working electrode used in the sensor of the eleventh embodiment according to the present invention.</figref><figref num="12B">Top view of the spacer placed on the first film of FIG. 12A.</figref><figref num="12C">Bottom view of a second film with a spacer of FIG. 12B and a counter electrode placed on top of the first film of FIG. 12A (inverted view of FIGS. 12A and 12B).</figref><figref num="13A">Top view of an embodiment of a sheet of sensor components according to the present invention.</figref><figref num="13B">Top view of another embodiment of a sheet of sensor components according to the present invention.</figref><figref num="14A">Sectional drawing of a continuous process for providing a sample chamber in a spacer layer.</figref><figref num="14B">Sectional drawing of a continuous process for providing a sample chamber in a spacer layer.</figref><figref num="14C">Sectional drawing of a continuous process for providing a sample chamber in a spacer layer.</figref><figref num="14D">Sectional drawing of a continuous process for providing a sample chamber in a spacer layer.</figref><figref num="14E">Sectional drawing of a continuous process for providing a sample chamber in a spacer layer.</figref><figref num="14F">Sectional drawing of a continuous process for providing a sample chamber in a spacer layer.</figref><figref num="14G">Bottom view of Figure 14B.</figref><figref num="15A">A graph showing the relationship between current and time used to calculate the concentration of an analyte.</figref><figref num="15B">A graph showing the relationship between the logarithm of current and time used to calculate the concentration of an analyte.</figref><figref num="16A">Top perspective view of a sensor positioned for insertion into the electrical connector device of the present invention.</figref><figref num="16B">Exploded view of the electrical connector device in Figure 16A.</figref><figref num="17A">Top perspective view of the sensor fully positioned within the electrical connector device of Figure 16A.</figref><figref num="17B">Exploded view of the electrical connector device in Figure 17A.</figref><figref num="18A">Bottom perspective view of the electrical connector device of FIGS. 16A and 16B.</figref><figref num="18B">Bottom perspective view of the electrical connector device of FIGS. 17A and 17B.</figref><figref num="19A">The figure which shows the example of the suitable conductive path between contact pads.</figref><figref num="19B">The figure which shows the example of the suitable conductive path between contact pads.</figref><figref num="19C">The figure which shows the example of the suitable conductive path between contact pads.</figref><figref num="19D">The figure which shows the example of the suitable conductive path between contact pads.</figref><figref num="19E">The figure which shows the example of the suitable conductive path between contact pads.</figref><figref num="19F">The figure which shows the example of the suitable conductive path between contact pads.</figref><figref num="19G">The figure which shows the example of the suitable conductive path between contact pads.</figref><figref num="19H">The figure which shows the example of the suitable conductive path between contact pads.</figref><figref num="19I">The figure which shows the example of the suitable conductive path between contact pads.</figref><figref num="19J">The figure which shows the example of the suitable conductive path between contact pads.</figref><figref num="19K">The figure which shows the example of the suitable conductive path between contact pads.</figref><figref num="19L">The figure which shows the example of the suitable conductive path between contact pads.</figref><figref num="20">The cross-sectional view from the inside of the connector to the sensor of the present invention positioned in the connector.</figref><figref num="21">Perspective view of the ring for use with the lancet device.</figref>