US20100081152A1 - Small Volume In Vitro Analyte Sensor and Methods of Making - Google Patents
Small Volume In Vitro Analyte Sensor and Methods of Making Download PDFInfo
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- US20100081152A1 US20100081152A1 US12/569,428 US56942809A US2010081152A1 US 20100081152 A1 US20100081152 A1 US 20100081152A1 US 56942809 A US56942809 A US 56942809A US 2010081152 A1 US2010081152 A1 US 2010081152A1
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Definitions
- This invention relates to analytical sensors for the detection of bioanalytes in a small volume sample.
- Analytical sensors are useful in chemistry and medicine to determine the presence and concentration of a biological analyte. Such sensors are needed, for example, to monitor glucose in diabetic patients and lactate during critical care events.
- Sensors capable of electrochemically measuring an analyte in a sample are known in the art. Some sensors known in the art use at least two electrodes and may contain a redox mediator to aid in the electrochemical reaction. However, the use of an electrochemical sensor for measuring analyte in a small volume introduces error into the measurements. One type of inaccuracy arises from the use of a diffusible redox mediator. Because the electrodes are so close together in a small volume sensor, diffusible redox mediator may shuttle between the working and counter electrode and add to the signal measured for analyte. Another source of inaccuracy in a small volume sensor is the difficulty in determining the volume of the small sample or in determining whether the sample chamber is filled. It would therefore be desirable to develop a small volume electrochemical sensor capable of decreasing the errors that arise from the size of the sensor and the sample.
- the sensors of the present invention provide a method for the detection and quantification of an analyte in submicroliter samples.
- the invention includes a method and sensor for analysis of an analyte in a small volume of sample by, for example, coulometry, amperometry and/or potentiometry.
- a sensor of the invention utilizes a non-leachable or diffusible redox mediator.
- the sensor also includes a sample chamber to hold the sample in electrolytic contact with the working electrode.
- the sensor also contains a non-leachable or diffusible second electron transfer agent.
- the working electrode faces a counter electrode, forming a measurement zone within the sample chamber, between the two electrodes, that is sized to contain no more than about 1 ⁇ L of sample, preferably no more than about 0.5 ⁇ L, more preferably no more than about 0.25 ⁇ L, and most preferably no more than about 0.1 ⁇ l of sample.
- a sorbent material is optionally positioned in the sample chamber and measurement zone to reduce the volume of sample needed to fill the sample chamber and measurement zone.
- a biosensor which combines coulometric electrochemical sensing with a non-leachable or diffusible redox mediator to accurately and efficiently measure a bioanalyte in a submicroliter volume of sample.
- the preferred sensor includes an electrode, a non-leachable or diffusible redox mediator on the electrode, a sample chamber for holding the sample in electrical contact with the electrode and, preferably, sorbent material disposed within the sample chamber to reduce the volume of the chamber.
- the sample chamber together with any sorbent material, is sized to provide for analysis of a sample volume that is typically no more than about 1 ⁇ L, preferably no more than about 0.5 ⁇ L, more preferably no more than about 0.25 ⁇ L, and most preferably no more than about 0.1 ⁇ L.
- the sensor also contains a non-leachable or diffusible second electron transfer agent.
- One embodiment of the invention includes a method for determining the concentration of an analyte in a sample by, first, contacting the sample with an electrochemical sensor and then determining the concentration of the analyte.
- the electrochemical sensor includes a facing electrode pair with a working electrode and a counter electrode and a sample chamber, including a measurement zone, positioned between the two electrodes.
- the measurement zone is sized to contain no more than about 1 ⁇ L of sample.
- the invention also includes an electrochemical sensor with two or more facing electrode pairs. Each electrode pair has a working electrode, a counter electrode, and a measurement zone between the two electrodes, the measurement zone being sized to hold no more than about 1 ⁇ L of sample.
- the sensor also includes a non-leachable redox mediator on the working electrode of at least one of the electrode pairs or a diffusible redox mediator on a surface in the sample chamber or in the sample.
- One aspect of the invention is a method of determining the concentration of an analyte in a sample by contacting the sample with an electrochemical sensor and determining the concentration of the analyte by coulometry.
- the electrochemical sensor includes an electrode pair with a working electrode and a counter electrode.
- the sensor also includes a sample chamber for holding a sample in electrolytic contact with the working electrode. Within the sample chamber is sorbent material to reduce the volume sample needed to fill the sample chamber so that the sample chamber is sized to contain no more than about 1 ⁇ L of sample.
- the sample chamber also contains a non-leachable or diffusible redox mediator and optionally contains a non-leachable or diffusible second electron transfer agent.
- Sensors can be configured for side-filling or tip-filling.
- the sensor may be part of an integrated sample acquisition and analyte measurement device.
- the integrated sample acquisition and analyte measurement device may include the sensor and a skin piercing member, so that the device can be used to pierce the skin of a user to cause flow of a fluid sample, such as blood, that can then be collected by the sensor.
- the fluid sample can be collected without moving the integrated sample acquisition and analyte measurement device.
- One method of forming a sensor includes forming at least one working electrode on a first substrate and forming at least one counter or counter/reference electrode on a second substrate.
- a spacer layer is disposed on either the first or second substrates.
- the spacer layer defines a channel into which a sample can be drawn and held when the sensor is completed.
- a redox mediator and/or second electron transfer agent are disposed on the first or second substrate in a region that will be exposed within the channel when the sensor is completed.
- the first and second substrates are then brought together and spaced apart by the spacer layer with the channel providing access to the at least one working electrode and the at least one counter or counter/reference electrode.
- the first and second substrates are portions of a single sheet or continuous web of material.
- FIG. e 1 is a schematic view of a first embodiment of an electrochemical sensor in accordance with the principles of the present invention having a working electrode and a counter electrode facing each other;
- FIG. 3 is a schematic view of a third embodiment of an electrochemical sensor in accordance with the principles of the present invention having a working electrode and a counter electrode facing each other and having an extended sample chamber;
- FIG. 4 is a not-to-scale side-sectional drawing of a portion of the sensor of FIG. 1 or 3 showing the relative positions of the redox mediator, the sample chamber, and the electrodes;
- FIG. 5 is a top view of a fourth embodiment of an electrochemical sensor in accordance with the principles of the present invention, this sensor includes multiple working electrodes;
- FIG. 6 is a perspective view of an embodiment of an analyte measurement device, in accordance with the principles of the present invention, having a sample acquisition means and the sensor of FIG. 4 ;
- FIG. 7 is a graph of the charge required to electrooxidize a known quantity of glucose in an electrolyte buffered solution (filled circles) or serum solution (open circles) using the sensor of FIG. 1 with glucose oxidase as the second electron transfer agent;
- FIG. 8 is a graph of the average glucose concentrations for the data of FIG. 7 (buffered solutions only) with calibration curves calculated to fit the averages; a linear calibration curve was calculated for the 10-20 mM concentrations and a second order polynomial calibration curve was calculated for the 0-10 mM concentrations;
- FIG. 9 is a Clarke-type clinical grid analyzing the clinical relevance of the glucose measurements of FIG. 7 ;
- FIG. 10 is a graph of the charge required to electrooxidize a known quantity of glucose in an electrolyte buffered solution using the sensor of FIG. 1 with glucose dehydrogenase as the second electron transfer agent;
- FIGS. 11A , 11 B, and 11 C are top views of three configurations for overlapping working and counter electrodes according to the present invention.
- FIGS. 12A and 12B are cross-sectional views of one embodiment of an electrode pair formed using a recess of a base material, according to the invention.
- FIGS. 13A and 13B are cross-sectional views of yet another embodiment of an electrode pair of the present invention formed in a recess of a base material;
- FIG. 16 is a graph of charge delivered by a sensor having a diffusible redox mediator for several glucose concentrations
- FIG. 17 is a graph of charge delivered by sensors with different amounts of diffusible redox mediator over time.
- FIG. 18B illustrates a top view of a spacer for placement on the first film of FIG. 18A ;
- FIG. 19A illustrates a top view of a first film with a working electrode for use in a sixth embodiment of a sensor according to the invention
- FIG. 19C illustrates a bottom view of a second film (inverted with respect to FIGS. 19A and 19B ) with counter electrodes placement over the spacer of FIG. 19B and first film of FIG. 19A ;
- FIG. 20C illustrates a bottom view of a second film (inverted with respect to FIGS. 20A and 20B ) with counter electrodes placement over the spacer of FIG. 20B and first film of FIG. 20A ;
- FIG. 21B illustrates a top view of a spacer for placement on the first film of FIG. 21A ;
- FIG. 22A illustrates a top view of a first film with a working electrode for use in a ninth embodiment of a sensor according to the invention
- FIG. 22B illustrates a top view of a spacer for placement on the first film of FIG. 22A ;
- FIG. 23A illustrates a top view of a first film with a working electrode for use in a tenth embodiment of a sensor according to the invention
- FIG. 23C illustrates a bottom view of a second film (inverted with respect to FIGS. 23A and 23B ) with counter electrodes placement over the spacer of FIG. 23B and first film of FIG. 23A ;
- FIG. 24B illustrates a top view of a spacer for placement on the first film of FIG. 24A ;
- FIG. 25 illustrates a top view of a twelfth embodiment of an electrochemical sensor, according to the invention.
- FIG. 26 illustrates a perspective view of one embodiment of an integrated analyte acquisition and sensor device
- FIG. 28 illustrates a graph comparing measurements of analyte concentration in blood samples collected from a subject's arm made by a sensor of the invention with those determined by a standard blood test;
- FIG. 29 illustrates a graph comparing measurements of analyte concentration in blood samples collected from a subject's finger made by a sensor of the invention with those determined by a standard blood test;
- a “counter electrode” refers to one or more electrodes paired with the working electrode, through which passes an electrochemical current equal in magnitude and opposite in sign to the current passed through the working electrode.
- the term “counter electrode” is meant to include counter electrodes which also function as reference electrodes (i.e. a counter/reference electrode) unless the description provides that a “counter electrode” excludes a reference or counter/reference electrode.
- an “effective diffusion coefficient” is the diffusion coefficient characterizing transport of a substance, for example, an analyte, an enzyme, or a redox mediator, in the volume between the electrodes of the electrochemical cell.
- the cell volume may be occupied by more than one medium (e.g., the sample fluid and a polymer film). Diffusion of a substance through each medium may occur at a different rate.
- the effective diffusion coefficient corresponds to a diffusion rate through this multiple-media volume and is typically different than the diffusion coefficient for the substance in a cell filled solely with sample fluid.
- An “electrochemical sensor” is a device configured to detect the presence of and/or measure the concentration of an analyte via electrochemical oxidation and reduction reactions. These reactions are transduced to an electrical signal that can be correlated to an amount or concentration of analyte.
- a compound is “immobilized” on a surface when it is entrapped on or chemically bound to the surface.
- An “indicator electrode” includes one or more electrodes that detect partial or complete filling of a sample chamber and/or measurement zone.
- a “layer” includes one or more layers.
- the “measurement zone” is defined herein as a region of the sample chamber sized to contain only that portion of the sample that is to be interrogated during an analyte assay.
- the “potential of the counter/reference electrode” is the half cell potential of the reference electrode or counter/reference electrode of the cell when the solution in the cell is 0.1 M NaCl solution at pH7.
- Patentiometry and “chronopotentiometry” refer to taking a potentiometric measurement at one or more points in time.
- a “reference electrode” includes a reference electrode that also functions as a counter electrode (i.e., a counter/reference electrode) unless the description provides that a “reference electrode” excludes a counter/reference electrode.
- a “second electron transfer agent” is a molecule that carries electrons between a redox mediator and the analyte.
- a “surface in the sample chamber” includes a surface of a working electrode, counter electrode, counter/reference electrode, reference electrode, indicator electrode, a spacer, or any other surface bounding the sample chamber.
- the small volume, in vitro analyte sensors of the present invention are designed to measure the concentration of an analyte in a portion of a sample having a volume no more than about 1 ⁇ L, preferably no more than about 0.5 ⁇ L, more preferably no more than about 0.25 ⁇ L, and most preferably no more than about 0.1 ⁇ L.
- the analyte of interest is typically provided in a solution or biological fluid, such as blood or serum.
- a small volume, in vitro electrochemical sensor 20 of the invention generally includes a working electrode 22 , a counter (or counter/reference) electrode 24 , and a sample chamber 26 (see FIG. 4 ).
- the working electrode 22 may be formed from a molded carbon fiber composite or it may consist of an inert non-conducting base material, such as polyester, upon which a suitable conducting layer is deposited.
- the conducting layer typically has relatively low electrical resistance and is typically electrochemically inert over the potential range of the sensor during operation.
- Suitable conducting layers include gold, carbon, platinum, ruthenium dioxide, palladium, and conductive epoxies, such as, for example, ECCOCOAT CT5079-3 Carbon-Filled Conductive Epoxy Coating (available from W.R. Grace Company, Woburn, Mass.), as well as other non-corroding materials known to those skilled in the art.
- the electrode e.g., the conducting layer
- the electrode is deposited on the surface of the inert material by methods such as vapor deposition or printing.
- a tab 23 may be provided on the end of the working electrode 22 for easy connection of the electrode to external electronics (not shown) such as a voltage source or current measuring equipment.
- external electronics such as a voltage source or current measuring equipment.
- Other known methods or structures such as contact pads may be used to connect the working electrode 22 to the external electronics.
- a dielectric 40 may be deposited on the electrode over, under, or surrounding the region with the redox mediator, as shown in FIG. 4 .
- Suitable dielectric materials include waxes and non-conducting organic polymers such as polyethylene.
- Dielectric 40 may also cover a portion of the redox mediator on the electrode. The covered portion of the redox mediator will not contact the sample, and, therefore, will not be a part of the electrode's working surface.
- the diffusible components may be independently or jointly disposed on any surface of the sample chamber and/or placed in the sample (i.e., each diffusible component need not be disposed on the same surface of the sample chamber or placed in the sample).
- the redox mediator whether it is diffusible or non-leachable, mediates a current between the working electrode 22 and the analyte and enables the electrochemical analysis of molecules which may not be suited for direct electrochemical reaction on an electrode.
- the mediator functions as an electron transfer agent between the electrode and the analyte.
- the redox mediator and second electron transfer agent are diffusible and disposed on the same surface of the sample chamber, such as, for example, on the working electrode. In this same vein, both can be disposed on, for example, the counter electrode, counter/reference electrode, reference electrode, or indicator electrode. In other instances, the redox mediator and second electron transfer agent are both diffusible and independently placed on a surface of the sample chamber and/or in the sample. For example, the redox mediator may be placed on the working electrode while the second electron transfer agent is placed on any surface, except for the working electrode, or is placed in the sample.
- the reverse situation in which the second electron transfer agent is disposed on the working electrode and the redox mediator is disposed on any surface, except for the working electrode, or is placed in the sample is also a suitable embodiment.
- the redox mediator may be disposed on the counter electrode and the second electron transfer agent is placed on any surface except for the counter electrode or is placed in the sample. The reverse situation is also suitable.
- the diffusible redox mediator and/or second electron transfer agent may diffuse rapidly into the sample or diffusion may occur over a period of time. Similarly, the diffusible redox mediator and/or second electron transfer agent may first dissolve from the surface on which it was placed as a solid and then the diffusible redox mediator and/or second electron transfer agent may diffuse into the sample, either rapidly or over a period of time. If the redox mediator and/or second electron transfer agent diffuse over a period of time, a user may be directed to wait a period of time before measuring the analyte concentration to allow for diffusion of the redox mediator and/or second electron transfer agent.
- a diffusible redox mediator may shuttle back and forth from the working electrode to the counter electrode even in the absence of analyte. This typically creates a background signal. For coulometric measurements, this background signal is referred to herein as “Q Back .”
- the background signal corresponds to the charge passed in an electrochemical assay in the absence of the analyte.
- the background signal typically has both a transient component and a steady-state component. At least a portion of the transient component may result, for example, from the establishment of a concentration gradient of the mediator in a particular oxidation state. At least a portion of the steady-state component may result, for example, from the redox mediator shuttling between the working electrode and counter or counter/reference electrode.
- the steady-state component of the background signal may contain noise associated with (a) variability in, for example, the temperature of the sample, the sample viscosity, or any other parameter on which the background signal depends during the duration of the assay, or (b) imperfections in the electrochemical cell, such as, for example, non-uniform separation between the working electrode and the counter or counter/reference electrode, variations in electrode geometry, or protrusions from the working electrode, the counter electrode, and/or the counter/reference electrode.
- Additional analytes include, for example, acetyl choline, amylase, bilirubin, cholesterol, chorionic gonadotropin, creatine kinase (e.g., CK-MB), creatine, DNA, fructosamine, glucose, glutamine, growth hormones, hormones, ketones, lactate, peroxide, prostate-specific antigen, prothrombin, RNA, thyroid stimulating hormone, and troponin.
- concentration of drugs such as, for example, antibiotics (e.g., gentamicin, vancomycin, and the like), digitoxin, digoxin, drugs of abuse, theophylline, and warfarin, may also be determined.
- Another method includes controlling the applied potential such that for an electrooxidative assay the redox mediator is not readily reduced at the counter or counter/reference electrode or for an electroreductive assay the redox mediator is not readily oxidized at the counter or counter/reference electrode.
- This can be accomplished, for example, in an electrooxidative assay by using a sensor having a diffusible redox mediator with a potential, relative to a reference or counter/reference electrode, that is negative with respect to the potential of the counter electrode (relative to a reference electrode) or the counter/reference electrode.
- a population may include people lacking a particular condition or alternatively, having a particular condition, such as, for example, diabetes.
- Reference intervals pertaining to average or expected values such as, for example, those provided in Tietz Textbook of Clinical Chemistry , supra, may be used as guidelines, but a physical examination or blood chemistry determination may also determine an average or expected value.
- the physiological average molar amount of analyte may depend on the health or age of the person from whom the sample is obtained. This determination is within the knowledge of a skilled physician.
- C m is the minimum concentration of mediator required
- d is the distance between a working electrode and a counter or counter/reference electrode in a facing arrangement
- C A is the average analyte concentration in the sample
- D m is the effective diffusion coefficient of the mediator in the sample
- t is the desired measurement time.
- the analyte being measured is 5 mM glucose
- the redox mediator effective diffusion coefficient is 10 ⁇ 6 cm 2 /see and the desirable response time is no more than about 1 minute, then the minimum redox mediator concentration is 2.08 mM. Under these conditions the background signal will be less than the signal from the electrooxidation of the analyte.
- redox mediator any organic or organometallic redox species can be used as a redox mediator
- one type of suitable redox mediator is a transition metal compound or complex.
- suitable transition metal compounds or complexes include osmium, ruthenium, iron, and cobalt compounds or complexes.
- the transition metal is coordinatively bound to one or more ligands.
- the ligands are typically mono-, di-, tri-, or tetradentate.
- the most preferred ligands are heterocyclic nitrogen compounds, such as, for example, pyridine and/or imidazole derivatives.
- Multidentate ligands may include multiple pyridine and/or imidazole rings.
- metallocene derivatives such as, for example, ferrocene, can be used.
- amino as used herein includes alkylamino, such as methylamino, diethylamino, N,N-methylethylamino and the like; alkoxyalkylamino, such as N-(ethoxyethyl)amino, N,N-di(methoxyethyl)amino, N,N-(methoxyethyl)(ethoxyethyl)amino, and the like; and nitrogen-containing rings, such as piperidino, piperazino, morpholino, and the like.
- R 8 is hydrogen, alkyl, alkoxy, or alkenyl.
- alkyl alkoxy
- alkenyl alkenyl
- almido alkenyl
- amino amino
- keto sulfur-containing group
- the measurement zone (i.e., the region containing the volume of sample to be interrogated) is generally sized to contain a volume of sample of no more than about 1 ⁇ L, preferably no more than about 0.5 ⁇ L, more preferably no more than about 0.25 ⁇ L, and most preferably no more than about 0.1 ⁇ L.
- One particularly useful configuration of this embodiment positions working electrode 22 and counter electrode 24 facing each other, as shown in FIG. 3 .
- the measurement zone corresponding to the region containing the portion of the sample which will be interrogated, is the portion of sample chamber 26 bounded by the working surface of the working electrode and disposed between the two facing electrodes.
- the depth of the recess 104 and the volume of the conductive layer 100 , sensing layer 32 , and the portion, if any, of the second electrode 107 in the recess 104 determines the volume of the measurement zone.
- the predictability of the volume of the measurement zone relies on the extent to which the formation of the recess 104 is uniform.
- FIGS. 18A to 18C illustrate one alternative sensor design for formation of thin film sensors.
- the sensor includes a first substrate 500 upon which a working electrode 502 is formed.
- the working electrode 502 includes a contact region 503 for connection with external electronics.
- a spacer 504 FIG. 18B ), such as, for example, a layer of adhesive or a double-sided tape defines a channel 506 to produce a sample chamber for the sensor.
- Two counter (or counter/reference) electrodes 510 , 512 are formed on a second substrate 508 , as shown in FIG. 18C (inverted with respect to FIGS. 18A and 18B to show the electrode side up).
- This multiple counter electrode arrangement may provide a fill indicator function, as described below.
- the skin piercing member 704 may include an actuator (not shown) that includes a mechanism that allows for cocking and releasing the skin piercing member 704 or the skin piercing member may be actuated externally.
- an actuator not shown
- a sensor reader (not shown) or other device may be coupled to the sample acquisition and sensor device, the sensor reader or other device including a mechanism that cocks and/or releases the skin piercing member 704 .
- a sensor may be configured to have the desired ratio by choosing D M , C M , and d accordingly. For example, the concentration of the redox mediator may be reduced (i.e., C M may be reduced).
- At least 90% of the analyte is typically electrolyzed, preferably, at least 95% of the analyte is electrolyzed and, more preferably, at least 99% of the analyte is electrolyzed.
- the analyte be electrolyzed quickly.
- the speed of the electrochemical reaction depends on several factors, including the potential that is applied between the electrodes and the kinetics of reactions (1) and (2). (Other significant factors include the size of the measurement zone and the presence of sorbent in the measurement zone.) In general, the larger the potential, the larger the current through the cell (up to a transport limited maximum) and therefore, the faster the reaction will typically occur. However, if the potential is too large, other electrochemical reactions may introduce significant error in the measurement.
- the magnitude of the background current, I back is given by the following expression:
- a thermal heating element such as, for example, a wire or an ink that is capable of converting electrical energy into heat energy
- This wire or ink can be applied, for example, on the opposite side of a base material, such as a polymer film, from one or more of the working, counter, reference, or counter/reference electrodes, or applied around the periphery of the working, counter, reference, or counter/reference electrodes.
- the sample may be heated up to 5 to 20° C. above an initial temperature. In other instances, the temperature of the sample may not be known but a constant amount of power or current may be applied to the wire or ink.
- Table 1 The results of the analyses are shown in Table 1 and in FIG. 7 .
- Q avg is the average charge used to electrolyze the glucose in 3-6 identical test samples ( FIG. 7 graphs the charge for each of the test samples) and the 90% rise time corresponds to the amount of time required for 90% of the glucose to be electrolyzed.
- the data show a sensor precision of 10-20%, indicating adequate sensitivity of the sensor for low glucose concentrations, as well as in the physiologically relevant range (30 ⁇ g/dL-600 ⁇ g/dL).
- the redox mediator was formed by complexation of poly(1-vinyl imidazole) with Os(4,4′-dimethoxy-2,2′-bipyridine) 2 Cl 2 in a ratio of 15 imidazole groups per Os cation followed by cross linking the osmium polymer with glucose oxidase using polyethylene glycol diglycidyl ether.
- the sensors Upon application of the potential to the electrodes, a charge of 2.5 ⁇ C and 3.8 ⁇ C was passed in the two sensors, respectively. Subsequent reduction and reoxidation of the redox mediators yielded oxidation charges of 27.9 ⁇ C and 28.0 ⁇ C, respectively. Therefore, the sensors originally contained 91% and 86% of the Os cations in the desirable oxidized Os(III) state.
- FIG. 29 shows the data for 23 small volume sensors tested on blood obtained from the finger.
- R 2 was 0.986.
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US12/723,169 Expired - Fee Related US8425743B2 (en) | 1998-10-08 | 2010-03-12 | Small volume in vitro analyte sensor and methods of making |
US14/281,640 Expired - Fee Related US9234863B2 (en) | 1998-10-08 | 2014-05-19 | Small volume in vitro analyte sensor |
US14/281,642 Expired - Fee Related US9291592B2 (en) | 1998-10-08 | 2014-05-19 | Small volume in vitro analyte sensor |
US14/281,405 Expired - Fee Related US9341591B2 (en) | 1998-10-08 | 2014-05-19 | Small volume in vitro analyte sensor |
US14/281,647 Expired - Fee Related US9316609B2 (en) | 1998-10-08 | 2014-05-19 | Small volume in vitro analyte sensor |
US15/155,681 Expired - Fee Related US9891185B2 (en) | 1998-10-08 | 2016-05-16 | Small volume in vitro analyte sensor |
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