Disposable sub-microliter volume biosensor with enhanced sample inlet

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] 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 and requires only about 0.2 microliter of fluid sample. Still even more particularly, this invention relates to disposable electrochemical sensors which are used for performing electrochemical assays for the accurate determination of analytes in physiological fluids.

[0003] 2. Description of the Prior Art

[0004] Biosensors have been used in the determination of concentrations of various analytes in fluids for more than three decades. 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's blood sugar, or glucose levels, have become everyday necessities for many of the nation'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, this can result in vision loss, kidney failure and vascular diseases. 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.

[0005] 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.

[0006] One type of blood glucose biosensors 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.

[0007] However, many of the prior art devices require a test sample volume of greater than 2 microliters. This volume of test sample can only be obtained from a patient, for example, using a needle and syringe, or by lancing a portion of the skin such as the fingertip and “milking” the area to obtain a useful sample volume. These procedures are inconvenient for the patient, and often painful, particularly when frequent samples are required. Less painful methods for obtaining a sample are known such as lancing the arm or thigh, which have a lower nerve ending density. However, lancing the body in the arm or thigh typically produces submicroliter sample volumes of blood because these areas are not heavily supplied with near-surface capillary blood vessels. Because the present invention requires as little as 0.2 microliters of blood, it allows not only sampling from the finger tip with much less pain, but also a possibility to obtain adequate blood samples from alternate sites.

[0008] Additional shortcomings of the prior art devices are that they have a more limited linear range, usually up to about 600 mg/dL. Further, they require a relatively longer waiting time before a reading can be achieved. Another shortcoming of the biosensor 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.

[0009] Because of the importance of obtaining accurate glucose readings, it would be highly desirable to develop a reliable and user-friendly biosensor strips that do not have all of the shortcomings mentioned above.

[0010] Therefore, what is needed is an electrochemical sensor which requires less sample volume than previously required by the prior art. What is further needed is an electrochemical sensor which has a wide linear measurement range; that is, a sensor useable over a wider glucose concentration. What is still further needed is an electrochemical sensor which has a relatively short wait time for development of a steady-state response. 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

[0011] It is an object of the present invention to provide an improved electrochemical sensor which combines an enzyme and a mediator. It is a further object of the present invention to provide an electrochemical sensor which requires less sample volume than previously required by the prior art. It is still another object of the present invention to provide an electrochemical sensor which can measure a small volume of sample without the use of a mesh layer in the sample path. It is yet another object of the present invention to provide an electrochemical sensor which has a wide linear measurement range and a relatively short wait time for development of a steady-state response. It is another object of the present invention to provide an electrochemical sensor that has a modified inlet port to facilitate sample introduction.

[0012] The present invention achieves these and other objectives by providing an electrochemical sensor which requires a sample size of only about 0.2 microliters and does not use a mesh layer in the sample path as a means of achieving a reduced size of the sample. Further the present invention uses a reagent composition which allows readings, which correlate very closely to the analyte concentration in the fluid sample, to be taken 5 seconds after the fluid sample enters the sample channel.

[0013] 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. The sample fluid channel is sized to optimize the quick flow of a sample such as whole blood into the channel. Within the fluid channel lies at least one working electrode and a reference electrode, preferably a working electrode, a reference electrode and a pseudo-working electrode. The arrangement of the working electrode and the reference electrode is not important for purposes of the results obtained from the electrochemical sensor. The working electrode, the reference electrode and the pseudo-working electrode are each in electrical contact with separate conductive conduits, respectively. 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.

[0014] The laminated body has a base insulating layer made from a plastic material. The base insulating layer has a conductive layer on one side. The conductive layer 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 and substantially covers all of the base insulating layer. The vapor-deposited conductive layer is separated into conductive conduits by etching/scribing the conductive layer. 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/layer.

[0015] It should be pointed out that the gold film or tin oxide/gold film itself cannot function as a reference electrode. To make the reference electrode work, there must be a redox reaction (e.g., Fe(CN)₆³⁻+e⁻→Fe(CN)₆⁴⁻) 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.

[0016] The unique feature of the present invention is its ability to measure sample sizes as small as 0.10 microliters, or smaller, without using opposing working and reference electrodes and a sorbent/mesh layer therebetween to reduce the required sample volume for measurement. This is is achieved by reducing the width and length of the U-shaped cutout and by using a thinner middle layer.

[0017] The laminated body also has a middle insulating layer on top of the base layer. The 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 electrode portion of the conductive conduits on the base layer with the open end corresponding to the open end of the laminated body described earlier.

[0018] The middle layer must be of sufficient thickness for loading a sufficient amount of chemical reagent for use as an electrochemical sensor while maintaining a flow-channel dimension having optimum blood flow characteristics. The U-shaped cutout contains chemical reagent. The chemical reagent has a redox mediator with at least one of a stabilizer, a binder, a surfactant, a buffer, and 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. It also makes the reference electrode function.

[0019] 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 exposing a portion of the chemical reagent of the middle insulating 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 of the 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.

[0020] 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.

[0021] The electrode portions, located within the sample fluid channel, contain reagent material for the working electrode (W), the reference electrode (R) and the pseudo-working electrode (W₀). A reagent mix is disposed into the fluid channel thus covering the electrode portions of the base insulating layer and the conductive conduits. A sufficient amount of reagent mix is deposited within the U-shaped cutout of the middle insulating layer to substantially cover all of the conductive surface delineated by the U-shaped cutout. The amount of the reagent mix used is such that the reagent matrix created upon drying is sufficient for use as an electrochemical sensor yet provides enough empty space above the reagent matrix to allow rapid blood flow through the fluid channel. The reagent matrix has a redox mediator with at least one of a stabilizer, a binder, a surfactant, a buffer, and an enzyme capable of catalyzing a reaction involving a substrate for the enzyme.

[0022] The possible electrode arrangements within the sample fluid channel may be W-R-W₀, W-W₀-R, R-W-W₀, R-W₀-W, W₀-W-R or W₀-R-W 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-R-W₀; that is, as the sample fluid entered the open end of the laminated body, the fluid would cover W first, then R, then W₀.

[0023] The pseudo-working electrode, W₀, is positioned so that the sample fluid reaches it last. The resulting current at W₀thus triggers the reading meter to start the measurement and analyte concentration determination process. Such an arrangement obviates reliability and accuracy problems due to an insufficient sample fluid size. It should be pointed out that W₀can also be used as a counter electrode. The resulting three-electrode system (i.e. working electrode, reference electrode and counter electrode) would be used in the case of a sample fluid having a large IR drop. It should also be pointed out that impedance between any two electrodes could be used to estimate the hematocrit of a blood sample and therefore to correct the hematocrit interference.

[0024] 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

[0025]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.

[0026]FIG. 2 is an exploded, perspective view of the present invention showing the various layers of the laminated body.

[0027]FIG. 3 is a cross-sectional view of the present invention of FIG. 1

[0028]FIGS. 4A, 4B and <b>4</b>C are top views of a segment of a strip of each layer of the present invention showing the patterns for making multiple sensors of the present invention.

[0029]FIG. 4D 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

[0030] The preferred embodiment of the present invention is illustrated in FIGS. <b>1</b>-<b>4</b>. FIG. 1 shows a sensor <b>10</b> of the present invention. Sensor <b>10</b> has a laminated body <b>100</b>, a fluid sampling end <b>110</b>, an electrical contact end <b>120</b>, and a vent opening <b>42</b>. Fluid sampling end <b>110</b> includes a sample fluid channel <b>112</b> between a sampling end aperture <b>114</b> and vent opening <b>42</b>. Sampling end <b>110</b> also includes an inlet notch <b>44</b>. Electrical contact end <b>120</b> has three discreet conductive contacts <b>122</b>,<b>123</b> and <b>124</b>.

[0031] Referring now to FIG. 2, laminated body <b>100</b> is composed of a base insulating layer <b>20</b>, a middle layer <b>30</b>, and a top layer <b>40</b>. 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 <b>20</b> has a conductive layer <b>21</b> on which is delineated a first conductive conduit <b>22</b>, a second conductive conduit <b>23</b> and a third conductive conduit <b>24</b>. Conductive conduits <b>22</b>, <b>23</b> and <b>24</b> may be formed by scribing or scoring the conductive layer <b>21</b> as illustrated in FIG. 2 and shown as scribe line <b>27</b> and <b>28</b> or by silk-screening the conductive conduits <b>22</b>, <b>23</b> and <b>24</b> onto base layer <b>20</b>. Scribing or scoring of conductive layer <b>21</b> may be done by mechanically scribing the conductive layer <b>21</b> sufficiently to create the three independent conductive conduits <b>22</b>, <b>23</b> and <b>24</b>. The preferred scribing or scoring method of the present invention is done by using a carbon dioxide (CO₂) laser, a YAG laser or an eximer laser. An additional scoring line <b>29</b> (enlarged and not to scale; for illustrative purposes only) may be made, but is not necessary to the functionality of sensor <b>10</b>, along the outer edge of base layer <b>20</b> in order to avoid potential static problems which could give rise to a noisy signal. Conductive layer <b>21</b> may be made of any electrically conductive material, preferably gold or tin oxide/gold. A useable material for base layer <b>20</b> 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.

[0032] Middle layer <b>30</b> has a U-shaped channel cutout <b>32</b> located at middle layer sensor end <b>31</b>. The U-shaped cutout can be made by using a laser or diecut. The length of channel cutout <b>32</b> is such that when middle layer <b>30</b> is layered on top of base layer <b>20</b>, electrode areas W, R and W₀are within the space defined by channel cutout <b>32</b>. The thickness of middle layer <b>30</b> 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 <b>112</b>, which is filled by capillary action of the sample fluid. Channel cutout <b>32</b> holds the reagent matrix <b>50</b>, more clearly shown in FIG. 3, forming the working electrode, the reference electrode and the pseudo-working electrode. Typically, the reagent matrix <b>50</b> must be loaded with a redox mediator to make the reference electrode function. If R is not loaded with a redox reagent or mediator, working electrode W and W₀will not work. Electrode areas W, W₀and R are loaded preferably with the same chemical reagent. The reagents preferably contain an oxidized form of a redox mediator, a stabilizer, a binder, a surfactant, a buffer, and an enzyme. Typically, the redox mediator may be at least one of ferrocene, potassium ferricyanide, other ferrocene derivatives, or other organic and inorganic redox mediators. 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. The enzyme is 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. The enzyme could be glucose oxidase, lactate oxidase, cholesterol oxidase and creatinine amidohydrolase.

[0033] Top layer <b>40</b>, which is placed over and coextensive with middle layer <b>30</b>, has a vent opening <b>42</b> spaced from fluid sample end <b>110</b> of sensor <b>10</b> to insure that sample fluid in fluid channel <b>112</b> will completely cover electrode areas W, R and W₀. Vent opening <b>42</b> is placed in top layer <b>40</b> so that it will align somewhat with the bottom of channel cutout <b>32</b> of middle layer <b>30</b>, the bottom meaning the channel cutout <b>32</b> located furthest from sensor end <b>31</b>. Preferably, vent opening <b>42</b> will expose a portion of and partially overlay the bottom of the U-shaped cutout <b>32</b> of middle layer <b>30</b>.

[0034] Top layer <b>40</b> also includes an inlet notch <b>44</b> at fluid sample end <b>110</b> of sensor <b>10</b>. Inlet notch <b>44</b> is included to facilitate sample loading in fluid channel <b>112</b> where sampling end aperture <b>114</b> could be inadvertently blocked thus preventing the sample fluid from entering fluid channel <b>112</b> if sample notch <b>44</b> were absent. Sample notch <b>44</b> may have any shape and is not limited to the semi-circular shape shown. The vent opening and the inlet notch can be made by using a laser or by diecut.

[0035]FIG. 3 shows an enlarged cross-sectional view of the various layers of the present invention. The layers are not to scale in order that the relationship of each component of the present invention may be better understood by those skilled in the art, especially scribe lines <b>27</b> and <b>28</b>.

Preparation of Electrode Reagent Matrix

[0036] The electrode reagent matrix comprises the oxidized form of a redox mediator, a stabilizer, a binder, a surfactant, a buffer, and an enzyme. The oxidized form of the redox mediator, potassium ferricyanide, was found to be stable in the matrix. Suitable potassium ferricyanide is available from Sigma Chemical, St. Louis, Mo. (Cat. No P3667). 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 preferred stabilizer is polyethylene glycol (Cat. No. P4338, Sigma Chemicals, St. Louis, Mo.). The binder should be capable of binding all other chemicals in the reagent matrix in electrode areas W, R and W₀to the conductive surface/layer <b>21</b> of base layer <b>20</b>. 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. Citric acid and sodium citrate used in making the citrate buffer may be obtained from Sigma Chemical. The surfactant is necessary to facilitate dispensing of the electrode reaction matrix into channel cutout <b>32</b> as well as for quickly dissolving the dry chemical reagents involved in forming the reagent matrix. 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 available from Fluka Chemical, Milwaukee, Wis. (Cat. No. 94443). The reagent matrix is obtained by preparing a reagent mix as follows:

[0037] 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.

[0038] Step 2: Prepare a 1% methocel 60 HG solution by stirring 1 gram of methocel in 100 ml of citrate buffer from Step 1 for 12 hours.

[0039] Step 3: Add 0.3 ml of 10% Triton X-100 into the methocel solution.

[0040] Step 4: Add 2.5 grams of polyethylene glycol into the solution from Step 3.

[0041] Step 5: While stirring, add 6.5 grams potassium ferricyanide to the solution of Step 4.

[0042] 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

[0043] A piece of a gold or tin oxide/gold polyester film available from Courtaulds FOR Performance Films is cut to shape, as illustrated in FIG. 2, forming base layer <b>20</b> of sensor <b>10</b>. A CO₂laser is used to score the gold or tin oxide/gold polyester film (25W laser available from Synrad, Inc., San Diego, Calif.). As illustrated in FIG. 2, the film is scored by the laser creating scoring line <b>27</b> and <b>28</b> such that two electrodes at sample fluid end <b>110</b> and three contact points <b>122</b>, <b>123</b> and <b>124</b> were formed at electrical contact end <b>120</b>. The scoring line is very thin but sufficient to create two separate electrical conductors. An additional scoring line <b>29</b> made be made, but is not necessary, along the outer edge of base layer <b>20</b> to avoid potential static problems which could cause a noisy signal from the finished sensor <b>10</b>.

[0044] A piece of double-sided tape (Arcare® 7840) available from Adhesive Research, Glen Rock, Pa., is cut to size and shape forming middle layer <b>30</b> with U-shaped channel <b>32</b> so that it will cover a majority of the conductive layer <b>21</b> of base layer <b>20</b> except for exposing a small electrical contact area at electrical contact end <b>120</b> illustrated in FIG. 1. The U-shaped channel <b>32</b> is cut by using the CO₂laser. Middle layer <b>30</b> is then layered onto base layer <b>20</b>. As mentioned earlier, this middle layer <b>30</b> serves as a spacer and defines the size of the fluid sample channel <b>112</b>. It also defines the electrode area <b>26</b> which holds the electrode reagent matrix <b>50</b>. Its width and length is optimized to provide for a relatively quick moving fluid sample. The size of U-shaped channel 32 is about 0.039 in. (1.0 mm) wide by about 0.134 in. (3.4 mm) long. However, the channel width and length can be reduced so that sample volume can be as little as 0.1 microliter.

[0045] 1.0 microliters of reagent mix is dispensed into channel <b>32</b> to form electrodes W, R and W₀. The reagent mix is a mixture of a redox mediator, a stabilizer, a binder, a surfactant, a buffer, and an enzyme. The preferred composition for the reagent mix 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 reagent mix, the device was dried in an oven at 55° C. for about 2 minutes.

[0046] After drying, a piece of a transparency film (Cat. No. PP2200 or PP2500 available from 3M) is fashioned into top layer <b>40</b>. A rectangular vent hole <b>42</b> and a semi-circular notch <b>44</b> are made using the CO₂laser previously mentioned. The preferred size of vent hole <b>42</b> is about 0.039 in. (1.0 mm) by about 0.051 in. (1.30 mm). Vent hole <b>42</b> is located approximately 0.087 in. (2.2 mm) from fluid end <b>110</b> of sensor <b>10</b>. Semi-circular notch <b>44</b> has a radius of approximately 0.030 in. (0.75 mm) and is recessed from fluid end <b>110</b> of sensor <b>10</b>. Top layer <b>40</b> is aligned and layered onto middle layer <b>30</b> to complete the assembly of sensor <b>10</b>, as illustrated in FIG. 1.

[0047] 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 of each layer material as shown in FIGS. <b>4</b>A-<b>4</b>C. This is accomplished by starting with a relative large piece of base layer <b>20</b> having conducting layer <b>21</b> thereon. A plurality of scored lines <b>27</b> and <b>28</b> are made into conductive layer <b>21</b> such that a repetitive pattern, as illustrated in FIG. 4A, is created using the preferred scribing method described previously whereby each pattern will eventually define the three conductive paths <b>22</b>, <b>23</b> and <b>24</b> for each sensor. Similarly, a large piece of middle layer <b>30</b> having a plurality of elongated cutouts <b>32</b> in a repetitive pattern and illustrated in FIG. 4B is layered onto base layer <b>20</b>. The large piece of middle layer <b>30</b> is sized to fit over base layer <b>20</b> in such that the plurality of elongated cutouts <b>32</b> are aligned over the areas where the scribe lines <b>27</b> and <b>28</b> intersect exposing three distinct electrode areas W, R and W₀, and exposing the plurality of conductive contacts <b>122</b>, <b>123</b> and <b>124</b> located at the opposite edge of the strip. The size of each cutout and the amount of reagent mix disposed in each channel <b>32</b> are similar to that disclosed above. After dispensing the reagent mix into the respective cutouts, the reagent mix is dried such that each elongated cutout <b>32</b> of middle layer <b>30</b> contains a thin layer of the reagent matrix. A top layer <b>40</b> comparably-sized to and coextensive with middle layer <b>30</b> having a plurality of vent openings <b>42</b> and notch forming openings <b>44</b>′ in a repetitive pattern, as shown in FIG. 4C, is layered onto middle layer <b>30</b>. FIG. 4D is a top view of the combined layers. The laminated strip created by the three layers <b>20</b>, <b>30</b> and <b>40</b> has a plurality of sensors <b>10</b> that can be cut from the laminated strip. The laminated strip is cut longitudinally along line A-A′ at fluid sampling end <b>210</b> to form a plurality of sampling apertures <b>114</b> and longitudinally along line B-B′ at electrical contact end <b>220</b> to form a plurality of conductive contacts <b>122</b>,<b>123</b> and <b>124</b>. The laminated strip is cut at predetermined intervals along lines C-C′ forming a plurality of individual sensors <b>10</b>. Shaping of the fluid sampling end <b>120</b> of each sensor <b>10</b>, 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.

[0048] A more inclusive description of the wide linear range, the precision and the minimum sample volume features of the present invention along with additional test parameters and examples is provided in U.S. Pat. No. 6,258,229, which is incorporated herein by reference in its entirety.

[0049] 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.