DISPOSABLE SENSOR WITH ENHANCED
SAMPLE PORT 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 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.
[0003] 2. Description of the Prior Art
[0004] 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'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, 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.
[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 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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
[0013] 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.
[0014] 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.
[0015] 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., ... 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] 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.
[0017] 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.
[0018] 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.
[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 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.
[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 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.
[0022] 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.
[0023] In the three-cutout embodiment, two cutouts contain material for the working electrodes (Wl and W2) and one for the reference electrode (R). W2 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 W2 is minimized. The possible electrode arrangements within the sample fluid channel may be W1-W2-R, W1-R-W2, R-W1-W2, W2-W1R, W2-R-W1, or R-W2-W1 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 W1-W2-R; that is, as the sample fluid entered the open end of the laminated body, the fluid would cover Wl first, then W2, 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 (W2) to non-enzymeloaded working electrode (Wl).
[0024] 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 Wl (first working electrode) from the current response from W2 (second working electrode) to calculate the analyte concentration in the sample fluid. This is achieved by maintaining the surface area of Wl substantially equal to the surface area of W2. Also important is the composition of the reagents disposed on Wl and W2. 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.
[0025] The hematocrit interference is reduced by using a two-step process. First, the resistance (r-value) between any two electrodes is measured. The r-value is then used to estimate the hematocrit level in the sample fluid. The following equation represents this relationship:
r=k1/(l-H) Eq. (1)
[0026] where
[0027] r is resistance value measured in Ohms or Kilo-Ohms
[0028] H is hematocrit level
[0029] k1 is a constant
[0030] 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):
... Eq. (2)
[0031] where
[0032] Ccorr is the corrected analyte concentration [0033] Cmea is the measured analyte concentration [0034] k2-k5 are constants
[0035] H is the calculated hematocrit level from Eq. (1)
[0036] Constants Kj-kj are derived from empirical data.
[0037] 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
[0038] 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.
[0039] FIG. 2 is an exploded, perspective view of the present invention showing the various layers of the laminated body.
[0040] FIGS. 3A, 3B, 3C, and 3D are top views of a strip of each layer of the present invention showing the patterns for making multiple sensors of the present invention.
[0041] 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
[0042] 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.
[0043] 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
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