Abstract:
A disposable biosensor for testing a fluid sample including a laminated strip with a first and second end, a reference electrode embedded in the laminated strip proximate to the first end, at least one working electrode embedded in the laminated strip proximate to the first end and the reference electrode, an open path for receiving a fluid sample beginning from the first end and connecting to a vent spaced from the first end, the open path being sufficiently long to expose the reference electrode and the working electrode to the fluid sample, and conductive contacts located at the second end of the laminated strip. The laminated strip has a base layer with a conductive coating, a reagent holding layer, a channel forming layer and a cover having an inlet notch at the first end. The working electrode contains a reagent having an enzyme.

Description:
BACKGROUND OF THE INVENTION 
   1. Field of the Invention 
   The present invention relates generally to electrochemical sensors that can be used for the quantification of a specific component or analyte in a liquid sample. Particularly, this invention relates to a new and improved electrochemical sensor and to a new and improved method of fabricating electrochemical sensors. More particularly, this invention relates to a disposable electrochemical sensor that is inexpensive to manufacture. Even more particularly, this invention relates to a disposable electrochemical sensor that gives accurate readings in the presence of interferents and varying red blood cells (hematocrit). Still even more particularly, this invention relates to disposable electrochemical sensors that are used for performing electrochemical assays for the accurate determination of analytes in physiological fluids. 
   2. Description of the Prior Art 
   Biosensors have been known for more than three decades. They are used to determine concentrations of various analytes in fluids. Of particular interest is the measurement of blood glucose. It is well known that the concentration of blood glucose is extremely important for maintaining homeostasis. Products that measure fluctuations in a person&#39;s blood sugar, or glucose levels have become everyday necessities for many of the nation&#39;s millions of diabetics. Because this disorder can cause dangerous anomalies in blood chemistry and is believed to be a contributor to vision loss and kidney failure, most diabetics need to test themselves periodically and adjust their glucose level accordingly, usually with insulin injections. If the concentration of blood glucose is below the normal range, patients can suffer from unconsciousness and lowered blood pressure, which may even result in death. If the fasting blood glucose concentration is higher than the normal range, it can result in vision loss, kidney failure and vascular disease. Thus, the measurement of blood glucose levels has become a daily necessity for diabetic individuals who control their level of blood glucose by insulin therapy. 
   Patients who are insulin dependent are instructed by doctors to check their blood-sugar levels as often as four times a day. To accommodate a normal life style to the need of frequent monitoring of glucose levels, home blood glucose testing was made available with the development of reagent strips for whole blood testing. 
   One type of blood glucose biosensor is an enzyme electrode combined with a mediator compound, which shuttles electrons between the enzyme and the electrode resulting in a measurable current signal when glucose is present. The most commonly used mediators are potassium ferricyanide, ferrocene and its derivatives, as well as other metal-complexes. Many sensors based on this second type of electrode have been disclosed. 
   However, the prior art devices suffer from various shortcomings. One of these shortcomings is interference with biosensor readings caused by other substances in the sample fluid, which can oxidize at the same potential. Prevalent among these is ascorbic acid, uric acid and acetaminophen. As these and other interfering substances oxidize, the current resulting from their oxidation is added to and indistinguishable from the current resulting from the oxidation of the blood analyte being measured. An error therefore results in the quantification of the blood analyte. 
   Another shortcoming is the interference caused by red blood cells (the hematocrit effect). This interference tends to cause an artificially high response rate for low hematocrit levels and, conversely, an artificially low response rate for high hematocrit levels. 
   Additional shortcomings of the prior art devices are that they have a more limited linear range and require a relatively large quantity of sample volume. Further, they require a relatively longer waiting time for development of a steady-state response before a reading can be achieved. Another shortcoming of biosensors having an end or side inlet for direct introduction of the blood sample to the sample chamber from the source of the blood droplet is the inadvertent blockage or partial blockage of the inlet by the blood source. Users tend to push the biosensor hard against the blood sampling point such as at the finger or the arm. Because the entrance to the capillary channel of the biosensor is small, such action typically blocks or partially blocks the inlet. The result is that (1) the blood does not enter the capillary channel at all, or (2) the blood partially enters the channel but does not fill it up sufficiently, or (3) the blood fills up the capillary channel very slowly. Under scenario (1), the meter may not be triggered and thus not reading is made. Under scenarios (2) and (3), the meter may not be triggered or it may be triggered but gives inaccurate test results due to insufficient sample or the slowness of the capillary filling action. 
   Each of these shortcomings may, either individually or when combined with one or more of the other shortcomings, contribute to erroneous measurement readings during analysis. 
   Because of the importance of obtaining accurate glucose readings, it would be highly desirable to develop a reliable and user-friendly electrochemical sensor, which does not have one or more of the drawbacks mentioned above. 
   Therefore, what is needed is an electrochemical sensor that incorporates an interference-correcting electrode to minimize the interference caused by oxidizable substances present in the sample fluid. What is further needed is an electrochemical sensor whose response is substantially independent of the hematocrit of the sample fluid. What is still further needed is an electrochemical sensor that requires less sample volume than previously required by the prior art. Yet, what is still further needed is an electrochemical sensor that has a wide linear measurement range; that is, a sensor having a reduced or negligible interference effect and useable over a wider glucose concentration. What is also needed is an electrochemical sensor with a modified inlet port to facilitate introduction of the sample into the sample chamber of the electrochemical sensor. 
   SUMMARY OF THE INVENTION 
   It is an object of the present invention to provide an improved electrochemical sensor that combines an enzyme and a mediator. It is a further object of the present invention to provide an electrochemical sensor that incorporates an interference-correcting electrode to minimize the interference caused by oxidizable substances present in the sample fluid. It is a further object of the present invention to provide an electrochemical sensor whose response is substantially independent of the hematocrit levels of the sample fluid. It is still another object of the present invention to provide an electrochemical sensor that has a wide linear measurement range. It is yet another object of the present invention to provide an electrochemical sensor that has a modified inlet port to facilitate sample introduction. 
   The present invention achieves these and other objectives by providing an electrochemical sensor that has a modified sample inlet port for facilitating sample introduction and that requires a smaller sample size and compensates for interference from oxidizable species in the sample and from varying hematocrit levels. The present invention has a laminated, elongated body having a sample fluid channel connected between an opening on one end of the laminated body and a vent hole spaced from the opening. Within the fluid channel lies at least one working electrode and a reference electrode. The working electrode and the reference electrode are each in electrical contact with separate conductive conduits. The separate conductive conduits terminate and are exposed for making an electrical connection to a reading device on the end opposite the open channel end of the laminated body. 
   The laminated body has a base insulating layer made from a plastic material. Several conductive conduits are delineated on the base insulating layer. The conductive conduits may be deposited on the insulating layer by screen printing, by vapor deposition, or by any method that provides for a conductive layer, which adheres to the base insulating layer. The conductive conduits may be individually disposed on the insulating layer, or a conductive layer may be disposed on the insulating layer followed by etching/scribing the required number of conductive conduits. The etching process may be accomplished chemically, by mechanically scribing lines in the conductive layer, by using a laser to scribe the conductive layer into separate conductive conduits, or by any means that will cause a break between and among the separate conductive conduits required by the present invention. The preferred conductive coatings are gold film or a tin oxide/gold film composition. It should be pointed out that although the same electrically conducting substance (gold film or tin oxide/gold film) after scoring is used as conducting material for both working electrodes and the reference electrode, this material itself cannot function as a reference electrode. To make the reference electrode work, there must be a redox reaction (e.g., Fe(CN) 6   3− +e − →Fe(CN) 6   4− ) at the electrically conducting material when a potential is applied. Therefore, a redox couple or mediator must be present at the conducting material used for the reference electrode. 
   On top of the base insulating layer and the conductive conduits, the laminated body has a first middle insulating layer or a reagent holding layer containing cutouts for at least one working electrode and a reference electrode. If a second working electrode is included, it and the reference electrode may share the same cutout. Where three cutouts are used, each cutout corresponds to and exposes a small portion of a single conductive conduit. The cutouts for the working electrodes can be the same or different size. The cutout for the reference electrode may be the same or different size as the cutouts for the working electrodes. The placement of all of the cutouts is such that they will all co-exist within the sample fluid channel described above. This reagent holding layer is also made of an insulating dielectric material, preferably plastic, and may be made by die cutting the material mechanically or with a laser and then fastening the material to the base layer. An adhesive, such as a pressure-sensitive adhesive, may be used to secure the reagent holding layer to the base layer. Adhesion may also be accomplished by ultrasonically bonding the reagent holding layer to the base layer. The reagent holding layer may also be made by screen printing the first middle insulating layer over the base layer. 
   The thickness of the reagent holding layer must be of sufficient thickness for loading a sufficient amount of electrode material for use as an electrochemical sensor. Each cutout contains electrode material. The electrode material has a redox mediator with at least one of a stabilizer, a binder, a surfactant, and a buffer. At least one of the cutouts also contains an enzyme capable of catalyzing a reaction involving a substrate for the enzyme. The redox mediator is capable of transferring electrons between the enzyme-catalyzed reaction and the working electrode. 
   The laminated body also has a second middle insulating layer, or channel forming layer, on top of the reagent holding layer. The second middle layer is also made of a plastic insulating material and creates the sample fluid channel of the laminated body. It contains a U-shaped cutout on one end which overlays the cutouts in the reagent holding layer with the open end corresponding to the open end of the laminated body described earlier. 
   The laminated body of the present invention has a top layer with a vent opening and an inlet notch. The vent opening is located such that at least a portion of the vent opening overlays the bottom of the U-shaped cutout of the channel forming layer. The vent allows air within the sample fluid channel to escape as the sample fluid enters the open end of the laminated body. The inlet notch facilitates sample introduction through the inlet by creating a top inlet aperture, which is in communication with the end inlet of the sensor. In the event that the sample inlet port is inadvertently blocked by the source of the blood sample such as a finger, the inlet notch remains open for receiving the sample fluid. 
   The sample fluid generally fills the sample fluid channel by capillary action. In small volume situations, the extent of capillary action is dependent on the hydrophobic/hydrophilic nature of the surfaces in contact with the fluid undergoing capillary action. This is also known as the wetability of the material. Capillary forces are enhanced by either using a hydrophilic insulating material to form the top layer, or by coating at least a portion of one side of a hydrophobic insulating material with a hydrophilic substance in the area of the top layer that faces the sample fluid channel between the open end of the laminated body and the vent opening of the top layer. It should be understood that an entire side of the top layer may be coated with the hydrophilic substance and then bonded to the second middle layer. 
   The number of cutouts in the reagent holding layer can be one, two and three or more. To use only one cutout, the single cutout must expose portions of at least two conductive conduits. Such an arrangement allows for testing a smaller sample volume compared to a two or a three cutout embodiment. However, this embodiment lacks the interference correction features of the other embodiments. 
   An embodiment having two cutouts is an alternative to the single cutout version. It has one cutout serving as the working electrode and the other one serving as a reference electrode. Another embodiment of the two cutout version combines the features of making the single cutout with that of the two cutout version. One of the cutouts containing electrode material is scored into two parts, one part serving as a first working electrode and the second part serving as the reference electrode. The second cutout serves as a second working electrode. Such a design is an alternative embodiment of the preferred embodiment of the present invention. This version of the two-cutout embodiment has the interference and hematocrit correction features but also allows for measuring an even smaller sample volume than that of the three-cutout embodiment. 
   In the three-cutout embodiment, two cutouts contain material for the working electrodes (W 1  and W 2 ) and one for the reference electrode (R). W 2  further contains the enzyme capable of catalyzing a substrate of the enzyme. The three electrodes are positioned and sized in such a way that the resistance of the fluid sample can be precisely measured and the possible carry-over from W 2  is minimized. The possible electrode arrangements within the sample fluid channel may be W 1 -W 2 -R, W 1 -R-W 2 , R-W 1 -W 2 , W 2 -W 1 -R, W 2 -R-W 1 , or R-W 2 -W 1  with the arrangement listed as the arrangement of electrodes would appear from the open end of the laminated body to the vent opening. The preferred position was found to be W 1 -W 2 -R; that is, as the sample fluid entered the open end of the laminated body, the fluid would cover W 1  first, then W 2 , then R. The preferred position allows for the precise measurement of blood sample resistance. This is necessary for good correlation between the resistance and hematocrit level in the blood sample. The preferred position also obviates reliability and accuracy problems due to an insufficient sample fluid size. The meter will not be triggered until the sample reaches the R. Such an arrangement also obviates possible carryover problems from enzyme-loaded working electrode (W 2 ) to non-enzyme-loaded working electrode (W 1 ). 
   As mentioned earlier, oxidizable interferents such as ascorbic acid, uric acid and acetaminophen, to name a few, cause inaccurate readings in the output of an electrochemical biosensor. The present invention negates this effect by subtracting the current response at W 1  (first working electrode) from the current response from W 2  (second working electrode) to calculate the analyte concentration in the sample fluid. This is achieved by maintaining the surface area of W 1  substantially equal to the surface area of W 2 . Also important is the composition of the reagents disposed on W 1  and W 2 . The reagents are designed to have a minimal effect on the response of the interferences which also contributes to the accuracy of the analyte measurement. 
   The hematocrit interference is reduced by using a two-step process. First, the resistance (r-value) between any two electrodes is measured. The revalue is then used to estimate the hematocrit level in the sample fluid. The following equation represents this relationship:
 
 r=k   1 /(1 −H )  Eq. (1) 
         where
           r is resistance value measured in Ohms or Kilo-Ohms   H is hematocrit level   k 1  is a constant   
               

   Second, the hematocrit level value is then used to mathematically correct the enzyme concentration reading obtained from above. The following equation represents the calculation performed using the calculated hematocrit level from Eq. (1):
 
 C   corr   =C   mea /( k   2   +k   3   C   mea +( k   4   +k   5   C   mea )(1 −H ))  Eq. (2) 
         where
           C corr  is the corrected analyte concentration   C mea  is the measured analyte concentration   k 2 -k 5  are constants   H is the calculated hematocrit level from Eq. (1)
 
Constants K 1 -K 5  are derived from empirical data.
   
               

   All of the advantages of the present invention will be made clearer upon review of the detailed description, drawings and appended claims. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
       FIG. 1  is a perspective view of the present invention showing the open end, the vent and the electrical contact points of the laminated body. 
       FIG. 2  is an exploded, perspective view of the present invention showing the various layers of the laminated body. 
       FIGS. 3A ,  3 B,  3 C, and  3 D are top views of a strip of each layer of the present invention showing the patterns for making multiple sensors of the present invention. 
       FIG. 3E  is a top view of a segment of the laminated strip of the present invention showing the patterns for making multiple sensors of the present invention. 
   

   DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
   The preferred embodiment of the present invention is illustrated in  FIGS. 1-3 .  FIG. 1  shows a sensor  10  of the present invention. Sensor  10  has a laminated body  100 , a fluid sampling end  110 , an electrical contact end  120 , and a vent opening  52 . Fluid sampling end  110  includes a sample fluid channel  112  between a sampling end aperture  114  and vent opening  52 . Sampling end  110  also includes an inlet notch  54 . Electrical contact end  120  has at least three discreet conductive contacts  122 ,  124  and  126 . 
   Referring now to  FIG. 2 , laminated body  100  is composed of a base insulating layer  20 , a first middle layer or reagent holding layer  30 , a second middle layer or channel forming layer  40 , and a top layer  50 . All layers are made of a dielectric material, preferably plastic. Examples of a preferred dielectric material are polyvinyl chloride, polycarbonate, polysulfone, nylon, polyurethane, cellulose nitrate, cellulose propionate, cellulose acetate, cellulose acetate butyrate, polyester, acrylic and polystyrene. Base insulating layer  20  has a conductive layer  21  on which is delineated a first conductive conduit  22 , a second conductive conduit  24  and a third conductive conduit  26 . Conductive conduits  22 ,  24  and  26  may be formed by scribing or scoring the conductive layer  21  as illustrated in  FIG. 2  or by silk-screening the conductive conduits  22 ,  24  and  26  onto base layer  20 . Scribing or scoring of conductive layer  21  may be done by mechanically scribing the conductive layer  21  sufficiently to create the three independent conductive conduits  22 ,  24  and  26 . The preferred scribing or scoring method of the present invention is done by using a carbon dioxide (CO 2 ) laser, a YAG laser or an eximer laser. An additional scoring line  28  (enlarged and not to scale; for illustrative purposes only) may be made, but is not necessary to the functionality of sensor  10 , along the outer edge of base layer  20  in order to avoid potential static problems which could give rise to a noisy signal. Conductive layer  21  may be made of any electrically conductive material, preferably gold or tin oxide/gold. A useable material for base layer  20  is a tin oxide/gold polyester film (Cat. No. FM-1) or a gold polyester film (Cat. No. FM-2) sold by Courtaulds Performance Films, Canoga Park, Calif. 
   First middle layer  30  has a first electrode cutout  32  which exposes a portion of first conductive conduit  22 , a second electrode cutout  34  which exposes a portion of second conductive conduit  24  and a third electrode cutout  36  which exposes a portion of third conductive conduit  26 . First layer  30  is made of a plastic material, preferably a medical grade one-sided tape available from Adhesive Research, Inc., of Glen Rock, Pa. Acceptable thickness of the tape for use in the present invention are in the range of about 0.002 in. (0.051 mm) to about 0.005 in. (0.127 mm). One such tape, Arcare® 7815, is preferred because of its ease of handling and shows good performance in terms of its ability to hold a sufficient quantity of chemical reagents and to promote a favorable blood flood speed (capillary action) through sample fluid channel  112  of sensor  10 . It should be understood that the use of a tape is not required. A plastic insulating layer may be coated with a pressure sensitive adhesive, or may be ultrasonically-bonded to base layer  20 , or may be silk-screened onto base layer  20  to achieve the same results as using the polyester tape mentioned. 
   The three cutouts  32 ,  34  and  36  define electrode areas W 1 , W 2  and R, respectively, and hold chemical reagents forming two working electrodes and one reference electrode. Typically, electrode area R must be loaded with a redox reagent or mediator to make the reference electrode function. If R is not loaded with a redox reagent or mediator, working electrodes W 1  and W 2  will not work property. The reagents preferably contain an oxidized form of a redox mediator, a stabilizer, a binder, a surfactant, and a buffer. Typically, the redox mediator may be at least one of ferrocene, potassium ferricyanide and other ferrocene derivatives. The preferred stabilizer is polyethylene glycol, the preferred binder is methyl cellulose, the preferred surfactant is t-octylphenoxypolyethoxyethanol, and the preferred buffer is a citrate buffer. Electrode area W 2  is preferably loaded with the same chemical reagents loaded into electrode areas W 1  and R but with the addition of an enzyme capable of catalyzing a reaction involving a substrate for the enzyme or a substrate catalytically reactive with an enzyme and a mediator capable of transferring electrons transferred between the enzyme-catalyzed reaction and the working electrode to create a current representative of the activity of the enzyme or substrate and representative of the compound. It should be pointed out that R can also be loaded with the same chemisty as W 2 . The enzyme could be glucose oxidase, lactate oxidase, cholesterol oxidase and creatinine amidohydrolase. 
   The cutouts and electrode areas of first layer  30  are positioned relative to each other and to the flow of the sample fluid in sample fluid channel  112  such that the resistance of the sample fluid may be precisely measured and the possible carryover from electrode area W 2  to electrode area W 1  could be minimized. Using fluid sample end  110  of sensor  10  as a reference point, the arrangements of the electrode areas could be W 1 -W 2 -R, W 1 -R-W 2  or R-W 1 -W 2 . The preferred position was found to be W 1 -W 2 -R. 
   Second middle layer  40  has a U-shaped channel cutout  42  located at second layer sensor end  41 . The length of channel cutout  42  is such that when second middle layer  40  is layered on top of first middle layer  30 , electrode areas W 1 , W 2  and R are within the space defined by channel cutout  42 . The thickness of second middle layer  40  was found to be critical for the volume of the capillary channel and for the speed of the sample fluid flow into sample fluid channel  112 , which is filled by capillary action of the sample fluid. 
   Top layer  50 , which is placed over second middle layer  40 , has a vent opening  52  spaced from fluid sample end  110  of sensor  10  to insure that sample fluid in fluid channel  112  will completely cover electrode areas W 1 , W 2  and R. Vent opening  52  is placed in top layer  50  so that at least a portion of vent opening  52  exposes a portion of bottom of channel cutout  42  of second middle layer  40 . Preferably, vent opening  52  will expose a portion of and partially overlay a portion of the U-shaped cutout  42  of second middle layer  40  that is furthest from fluid sampling end  110  of sensor  10 . 
   Top layer  50  also includes an inlet notch  54  at fluid sample end  110  of sensor  10 . Inlet notch  54  is included to facilitate sample loading in fluid channel  112  where sampling end aperture  114  could be inadvertently blocked if sample notch  54  were absent. Sample notch  54  may have any shape and is not limited to the semi-circular shape shown. 
   Preparation of Reagents 1 &amp; 2 
   Reagents 1 and 2 comprise the oxidized form of a redox mediator, a stabilizer, a binder, a surfactant, and a buffer. Reagent 2, in addition, contains an enzyme. The oxidized form of the redox mediator, potassium ferricyanide, was found to be stable in the matrices. The quantity used in the formulation must be sufficient to attain a workable linear range. The enzyme must also have sufficient activity, purity and stability. A commercially available glucose oxidase may be obtained from Biozyme, San Diego, Calif. as Cat. No. G03A, about 270 U/mg. The stabilizer must be sufficiently water-soluble and be capable of stabilizing both the mediator and the enzyme. The binder should also be capable of binding all other chemicals in the reagents in electrode areas W 1 , W 2  and R to the conductive surface/layer  21  of base layer  20 . The preferred stabilizer is polyethylene glycol (Cat. No. P4338, Sigma Chemicals, St. Louis, Mo.). The preferred binder is Methocel 60 HG (Cat. No. 64655, Fluka Chemical, Milwaukee, Wis.). The buffer solution must have sufficient buffer capacity and pH value to optimize the enzyme reaction. A 0.05M citrate buffer is preferred. The surfactant is necessary to facilitate dispensing of Reagents 1 and 2 into cutouts  32 ,  34  and  36  of middle layer  30  as well as for quickly dissolving the dry chemical reagents. The amount and type of surfactant is selected to assure the previously mentioned functions and to avoid a denaturing effect on the enzyme. The preferred surfactant is Triton X-100. The reagents are prepared as follows: 
   Reagent 1 
   
       
       Step 1: Prepare 50 mM citrate buffer (pH 5.7) by dissolving 0.1512 grams citric acid and 1.2580 grams sodium citrate in 100 ml of deionized water. 
       Step 2: Prepare a 1% methocel 60HG solution by stirring 1 gram of methocel in 100 ml of citrate buffer from Step 1 for 12 hours. 
       Step 3: Add 0.3 ml of 10% Triton X-100 into the methocel solution. 
       Step 4: Add 2.5 grams of polyethylene glycol into the solution from Step 3. 
       Step 5: While stirring, add 1 gram of potassium ferricyanide to the solution from Step 4. 
     
  
   Reagent 2 
   
       
       Step 1-Step 4: same steps as Reagent 1. 
       Step 5: While stirring, add 6.5 grams potassium ferricyanide to the solution of Step 4. 
       Step 6: Add 1.0 gram of glucose oxidase to the solution of Step 5 and stir for 10 minutes or until all solid materials are completely dissolved. 
     
  
   Electrode Construction 
   A piece of a gold or tin oxide/gold polyester film available from Courtaulds Performance Films is cut to shape, as illustrated in  FIG. 2 , forming base layer  20  of sensor  10 . A CO 2  laser is used to score the gold or tin oxide/gold polyester film. As illustrated in  FIG. 2 , the film is scored by the laser such that three electrodes at sample fluid end  110  and three contact points  122 ,  124  and  126  are formed at electrical contact end  120 . The scoring line is very thin but sufficient to create three separate electrical conductors. A scoring line  28  can be made, but is not necessary, along the outer edge of base layer  20  to avoid potential static problems which could cause a noisy signal from the finished sensor  10 . 
   A piece of one-sided adhesive tape is then cut to size and shape forming first middle layer  30  so that it will cover a majority of the conductive layer  21  of base layer  20  except for exposing a small electrical contact area illustrated in FIG.  1 . Three rectangular, square or circular cutouts  32 ,  34  and  36  of substantially equal size are punched by CO 2  laser (25W laser available from Synrad, Inc., San Diego, Calif.). Cutouts  32 ,  34  and  36  define the electrode areas W 1 , W 2  and R, which hold chemical reagents. The size of the cutouts is preferred to be made as small as possible in order to make the fluid sample channel  112  of sensor  10  as short as possible while still being capable of holding sufficient chemical reagent for the electrodes to function properly. The preferred hole size for the present invention has a typical dimension of about 0.033 in. (0.84 mm) by about 0.043 in. (1.09 mm). As illustrated in  FIG. 2 , cutouts  32 ,  34  and  36  are aligned with each other and having a spacing of about 0.028 in. (0.71 mm) between them. The rectangular cutouts are for illustrative purposes only. It should be understood that the shape of the cutouts is not critical provided that the size of the cutouts is big enough to hold sufficient chemical reagents for the electrodes to function properly but small enough to allow for a reasonably small sample channel. As noted earlier, changing the shape of the cutouts or the surface area of the cutouts may require changing the constant values k 1 -k 5  for Eq. 1 and Eq. 2. As stated previously, the preferred arrangement of the electrodes formed in cutouts  32 ,  34  and  36  is W 1  (working electrode  1 ), W 2  (working electrode  2 ) and R (reference electrode). 
   0.4 microliters of Reagent 1 is dispensed into each electrode area W 1  and R. Reagent 1 is a mixture of a redox mediator, a stabilizer, a binder, a surfactant, and a buffer. The preferred mixture for Reagent 1 is made by mixing the following components in the described percentages: about 1 wt % potassium ferricyanide, about 2.5 wt % polyethylene glycol, about 1 wt % methocel 60 HG, about 0.03 wt % Triton X-100 and about 0.05M citrate buffer (pH 5.7). 0.4 microliters of Reagent 2 is dispensed into electrode area W 2 . 
   Reagent 2 is a mixture similar to that of Reagent 1 but with the addition of an enzyme capable of catalyzing a reaction involving a substrate of the enzyme. The preferred enzyme is glucose oxidase. The preferred mixture for Reagent 2 is made by mixing the following percentages of the following ingredients: about 6.5 wt % potassium ferricyanide, about 2.5 wt % polyethylene glycol, about 1 wt % methocel 60 HG, about 0.03 wt % Triton X-100, about 0.05M citrate buffer (pH 5.7), and about 1 wt % glucose oxidase. After the addition of the reagents, the device was dried for about 2 minutes at 55° C. in an oven. After drying, a piece of double-sided tape available from Adhesive Research was fashioned into second middle layer  40  with U-shaped channel  42 . Second middle layer  40  is then layered onto first middle layer  30 . As mentioned earlier, this second middle layer  40  serves as a spacer and defines the size of the fluid sample channel  112 . Its width and length is optimized to provide for a relatively quick moving fluid sample. The preferred size of U-shaped channel  42  is about 0.063 in. (1.60 mm) wide by about 0.248 in. (6.30 mm) long. 
   A piece of a transparency film (Cat. No. PP2200 or PP2500 available from 3M) is fashioned into top layer  50 . A rectangular vent hole  52  and a semi-circular flu notch  54  are made using the CO 2  laser previously mentioned. The preferred size of vent hole  52  is about 0.075 in. (1.91 mm) by about 0.059 in. (1.50 mm). Vent hole  52  is located approximately 0.130 in. (3.3 mm) from fluid end  110  of sensor  10 . Semi-circular notch  54  has a radius of approximately 0.030 in. (0.75 mm) and is recessed from fluid end  110  of sensor  10 . Top layer  50  is aligned and layered onto second middle layer  40  to complete the assembly of sensor  10 , as illustrated in FIG.  1 . 
   Although the description of electrode construction above describes construction for a single sensor, the design and materials used are ideal for making multiple sensors from one piece, or a continuous strip, of each layer material as shown in  FIG. 3A-3E . This would be accomplished by starting with a relative large piece of base layer  20  having conducting layer  21  thereon. A plurality of scored lines are made into conductive layer  21  such that a repetitive pattern, as illustrated in  FIG. 3A , is created using the preferred scribing method described previously whereby each pattern will eventually define the three conductive paths  22 ,  24  and  26  for each sensor. Similarly, a large piece of first middle layer  30 , which is illustrated in  FIG. 3B  and which also has a plurality of cutouts  32 ,  34 , and  36  in a repetitive pattern, is sized to fit over base layer  20  in such a way that a plurality of sensors  10  will be had when completed. The size of each cutout and the electrode material disposed in the plurality of electrode areas W 1 , R and W 2  are similar to that disclosed above. After disposing Reagents 1 &amp; 2 in their respective cutouts and dried, a large piece of second middle layer  40  having a plurality of elongated cutouts  42  and illustrated in  FIG. 3C  is layered onto first middle layer  30  such that each elongated cutout  42  of second middle layer  40  contains corresponding cutouts  32 ,  34  and  36  of first middle layer  30 . A comparably-sized top layer  50  having a plurality of vent openings  52  and notch forming openings  54 ′ in a repetitive pattern, as shown in  FIG. 3D , is layered onto second middle layer  40 .  FIG. 3E  is a top view of the combined layers. The laminated strip created by the four layers  20 ,  30 ,  40  and  50  has a plurality of sensors  10  that can be cut from the laminated strip. The laminated strip is cut longitudinally along line A-A′ at fluid sampling end  210  to form a plurality of sampling apertures  114  with sample notches  54  and longitudinally along line B-B′ at electrical contact end  220  to form a plurality of conductive contacts  122 ,  124  and  126 . The laminated strip is also cut at predetermined intervals along line C-C′ forming a plurality of individual sensors  10 . Shaping of the fluid sampling end  120  of each sensor  10 , as illustrated in  FIG. 1 , may be performed if desired. It should be understood by those skilled in the art that the order in which the laminated strip can be cut is not important. For instance, the laminated strip may be cut at the predetermined intervals (C-C′) and then the cuts along A-A′ and B-B′ can be made to complete the process. 
   A more inclusive description of the compensation characteristics of the present invention along with additional test parameters and examples is provided in U.S. Pat. No. 6,287,451, which is incorporated herein by reference in its entirety. 
   Although the preferred embodiments of the present invention have been described herein, the above description is merely illustrative. Further modification of the invention herein disclosed will occur to those skilled in the respective arts and all such modifications are deemed to be within the scope of the invention as defined by the appended claims.