United States Patent [19J
`Wilson et al.
`
`111111111111111111111111111111111111111111111111111111111111111111111111111
`US005165407A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,165,407
`Nov. 24, 1992
`
`[54]
`[75]
`
`IMPLANTABLE GLUCOSE SENSOR
`Inventors: George S. Wilson; Dilbir S. Bindra;
`Brian S. Hill, all of Lawrence, Kans.;
`Daniel R. Thevenot, Paris Cedex,
`France; Robert Sternberg, Thiais,
`France; Gerard Reach, Paris Cedex,
`France; Yanan Zhang, Lawrence,
`Kans.
`[73] Assignee: The University of Kansas, Lawrence,
`Kans.
`[21] Appl. No.: 682,560
`[22] Filed:
`Apr. 9,1991
`
`Related U.S. Application Data
`[63] Continuation-in-part of Ser. No. 511,049, Apr. 19,
`1990, abandoned.
`Int. CJ.s ................................................ A61B 5/00
`[51]
`[52] U.S. Cl ..................................... 128/635; 204/403;
`204/415
`[58] Field of Search ................. 128/635; 204/403, 415
`[56]
`References Cited
`U.S. PATENT DOCUMENTS
`3,249,103 5/1966 Woodhouse ........................ 128/635
`3;726,777 4/1973 Macur ............................. 128/635 X
`4,545,382 10/1985 Higgins et a1 ....................... 128/635
`4,671,288 6/1987 Gough ................................ 128/635
`
`FOREIGN PATENT DOCUMENTS
`0320109 6/1989 European Pat. Off ............. 128/635
`0169668 10/1982 Japan .................................. 128/635
`60-33644 2/1986 Japan .................. , ................ 128/635
`
`0261341 11/1987 Japan ................................... 128/635
`0274254 11/1987 Japan ................................... 128/635
`1296913 3/1987 U.S.S.R ............................... 204/403
`
`OTHER PUBLICATIONS
`Updike et al., "The Enzyme Electrode", Nature, vol.
`214, Jun. 1967, pp. 986-988.
`Salkind et al., "Improving ... Stability", Med. Inst., val.
`15, No.2, Mar.-Apr. 1981; pp. 126-127.
`Primary Examiner-Lee S. Cohen
`Attorney, Agent, or Firm-Hovey, Williams, Timmons &
`Collins
`ABSTRACT
`[57]
`Implantable enzymatic sensors (25, 43, 44) for biochemi(cid:173)
`cals such as glucose are provided having an ideal size
`and geometry for optional long term implantation and
`linear responses over the concentration ranges of inter(cid:173)
`est. The sensors (25, 43, 44) include an elongated body
`(10, 26, 46) supporting an indicating electrode section
`having an appropriate enzyme immobilized thereon to
`present an enzymatic indicating surface (21, 33, 54). A
`permeable synthetic polymer membrane (24, 42, 56) is
`applied over the sensor body (10, 26, 46) to protect the
`enzyme and regulate diffusion of analyte therethrough,
`to ensure linearity of sensor response. The sensors (25,
`43) are of flexible design and can be implanted using a
`catheter. Alternately, the sensor (44) includes an inter(cid:173)
`nal indicating electrode body (46) housed within an
`apertured, hollow needle (48). A holder (66) afftxed to
`the needle (48) allows for easy manipulation and im(cid:173)
`plantation of the sensor (44).
`
`9 Claims, 3 Drawing Sheets
`
`12
`
`AGAMATRIX, INC.‎
`Exhibit 2002-1 (IPR2016-01679)‎
`Dexcom, Inc. v. AgaMatrix, Inc.
`
`

`
`U.S. Patent
`
`Nov. 24, 1992
`
`Sheet 1 of 3
`
`5,165,407
`
`18
`
`12
`
`28
`
`Fig. 1.
`
`43 J
`
`42
`
`Fig.2.
`
`AGAMATRIX, INC.‎
`Exhibit 2002-2 (IPR2016-01679)‎
`Dexcom, Inc. v. AgaMatrix, Inc.
`
`

`
`U.S. Patent
`
`Nov. 24, 1992
`
`Sheet 2 of 3
`
`5,165,407
`
`~ 50
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`~ g 10
`z
`l.1J
`({)
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`o~--~----~--~----~--~--~
`30
`0
`10
`2.0
`(m M)
`GLUCOSE CONCENTRATION
`FfG. 3.
`FfG. 4.
`
`~
`
`l\JE
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`i 4-.0
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`4:
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`. 10
`20· .
`30
`40
`STORAGE PERIOD
`(DA.YS)
`
`AGAMATRIX, INC.‎
`Exhibit 2002-3 (IPR2016-01679)‎
`Dexcom, Inc. v. AgaMatrix, Inc.
`
`

`
`U.S. Patent
`
`Nov. 24, 1992
`
`Sheet 3 of 3
`
`5,165,407
`
`.
`lQ
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`<J)
`lf)
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`CX)
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`
`AGAMATRIX, INC.‎
`Exhibit 2002-4 (IPR2016-01679)‎
`Dexcom, Inc. v. AgaMatrix, Inc.
`
`

`
`1
`
`5,165,407
`
`IMPLANTABLE GLUCOSE SENSOR
`
`This is a continuation-in-part of application Ser. No.
`07/511,049, filed Apr. 19, 1990, now abandoned.
`
`2
`SUMMARY OF THE INVENTION
`The present invention overcomes the problems out(cid:173)
`lined above, and provides a greatly improved enzymatic
`5 sensor specifically designed for long-term implantation
`m a patient. The sensor is adapted for positioning in an
`BACKGROUND OF THE INVENTION
`environment characterized by the presence of biologi-
`cal molecules which are substrates for or products pro-
`1. Field of the Invention
`The present invention is broadly concerned with a
`duced by enzymes, in order to determine the presence
`subcutaneously implantable enzymatic sensor charac- 10 of such biological molecules. While the principles of the
`terized by small size, optimum geometry and linearity of
`invention may be used in the fabrication of glucose
`sensor response over the concentration range of inter-
`sensors, the invention is not so limited. Indeed, the sen-
`est. More particularly, it is preferably concerned with
`sors in accordance with the invention may be produced
`an implantable glucose sensor of this type designed to
`using a wide variety of immobilized enzymes, for the
`provide, in conjunction with a suitable signal processing 15 detection of an equally large number of analytes. Exem-
`platy enzymes and their corresponding substrates are
`unit, a current which is proportional to subcutaneous
`given in U.S. Pat. No. 4,721,677 to Clark, and this pa-
`glucose concentration. In preferred forms, glucose sen-
`sors of the invention are based on the enzyme-catalyzed
`tent is incorporated by reference herein.
`oxidation of glucose to gluconic acid and hydrogen
`In any event, the enzymatic sensors in accordance
`peroxide, the latter being monitored amperometrically 20 with the invention preferably are in the form of an
`by the sensors.
`elongated body supporting at least an indicating elec-
`2. Description of the Prior Art
`trode, with the indicating electr~e p~esentin~ a section
`adap~ed. fo~ exposure to the. biological environment.
`There have been a great many attempts in the past to
`develop viable implantable sensors for continuous in 25 The. mdica~I?g electrode section has an enzyme. o~er~-
`bly. Immobilized thereon to pre.sent an enzy~atic mdi-
`vivo measurements of biochemicals. For example, con-
`catmg surface. A number of vanants are possible for the
`siderable effort has been made to devise reliable im-
`reference electrode. For example, use may be made of
`plan table sensors for monitoring glucose concentrations
`an externally applied electrocardiogram skin electrode
`in blood. Such determinations are useful in a variety of
`(an 8 mm disk covered with silver chloride and avail(cid:173)
`applications, e.g., in the treatment of diabetics. One 30
`able as Model E-243 from the Phymep Company, 21
`difficulty in providing a reliable implantable glucose
`Rue Campofo~io, Paris, France), or a reference elec(cid:173)
`sensor is that glucose levels in the bloodstream of a
`trode which is implanted with the indicating electrode.
`patient vary on a time basis ana are normally dependent
`In one specific embodiment employing an implanted
`upon the physical activity of the individual, his food,
`reference electrode, the indicating surface of the indi(cid:173)
`beverage and sugar intake, his metabolic rate, and other 35
`cating electrode and the reference electrode are later(cid:173)
`individualized factors. Furthermore, the geometry of
`ally spaced apart along the length of the body and each
`the sensor must be such as. to adapt to implantation in a
`substantially circumscribes the latter and is substantially
`living patient.
`exposed to the biological environment when the sensor
`Glucose sensors have been proposed in the past
`is placed therein. Use of such circumferentially extend(cid:173)
`which rely upon the well-established enzyme-catalyzed 40
`ing enzymatic indicating surfaces and reference elec(cid:173)
`oxidation of glucose wherein glucose and oxygen func(cid:173)
`trodes sections is believed to be an important aspect of
`tion as substrates for the enzyme glucose oxidase in the
`this embodiment. Alternately, the reference electrode
`production of gluconic acid and hydrogen peroxide, the
`section may comprise a conductive salt bridge circum(cid:173)
`latter being monitored amperometrically. See, for ex(cid:173)
`scribing the body and lying in a plane transverse to the
`ample, U.S. Pat. Nos. 3,539,455 to Clark and 4,671,288 45
`longitudinal axis of the body; in this case, a reference
`to Gough.
`electrode is placed in electrical contact with the salt
`Although the idea of an implantable enzymatic glu(cid:173)
`bridge, through use of a buffered electrolyte. In another
`cose sensor is not per se new, considerable difficulty has
`embodiment, the reference electrode is simply placed
`been encountered in producing reliable, cost-efficient 50
`adjacent the indicating electrode as a part of the overall
`devices of this character. For example, many proposed
`sensor.
`sensor geometries are simply not realistically implant(cid:173)
`In preferred practice, the sensor body advanta(cid:173)
`able, at least for the periods of time required for ade(cid:173)
`geously comprises an electrically conductive noble
`quate clinical glucose monitoring. Thus, the devices
`metal (e.g. platinum or platinum-iridium) electrode cov(cid:173)
`proposed in the '288 Gough Patent, because of a re- 55
`ered with electrically insulative material, with a portion
`quirement of multiple electrodes carried ·within a tubu-
`of this material removed from the electrode to defme an
`lar needle, inevitably are of such diameter as to be un(cid:173)
`enzyme-receiving zone. Thus, a short length of Teflon
`comfortable to the user and not practical for extended
`(polytetrafluoroethylene) coated platinum-iridium wire
`implantation. Furthermore, many prior sensors do not
`may be provided, with a short section of the insulation
`exhibit a stable and linear response, particularly over 60
`removed intermediate the ends of the wire, so that re(cid:173)
`extended times of implantation, and do not give accu(cid:173)
`spective segments of the insulating material are on op(cid:173)
`rate and reliable results. Finally, fabrication of prior
`posite sides of and define a recessed enzyme-receiving
`glucose sensors has presented formidable difficulties, to
`circumferential zone. Alternately, the endmost portion
`the extent that only about one in five sensors produced
`of the Teflon may be removed, leaving a protruding
`by conventional techniques are deemed usable. This 65
`exposed stretch of wire which defines the enzyme(cid:173)
`obviously represents a considerable inefficiency, to the
`receiving zone. An enzyme is operably immobilized on
`point that no truly successful implantable glucose sen(cid:173)
`the exposed section of the platinum-iridium wire, by
`known means such as adsorption of the enzyme on a
`sor has heretofore been produced on a large scale.
`
`AGAMATRIX, INC.‎
`Exhibit 2002-5 (IPR2016-01679)‎
`Dexcom, Inc. v. AgaMatrix, Inc.
`
`

`
`5,165,407
`
`25
`
`4
`to form the desired polymer solution droplet, followed
`by passing the electrodes through the loop to achieve
`uniform coating along the length of the sensor body.
`The enzymatic sensors of the invention have an ideal
`geometry for implantation. Generally speaking, the
`flexible units not housed within a needle are equivalent
`in size and shape to a 26-gauge needle (i.e., about 0.45
`mm. outside diameter). Moreover, "their geometry per(cid:173)
`mits the reproducible deposition of films and materials
`and allows careful control of the amount and orienta(cid:173)
`tion of the enzyme onto the indicating electrode. Fi(cid:173)
`nally, the preferred sensors are effectively "capped"
`with insulation (Teflon) which prevents the sensors
`from penetrating further into the tissue than is required.
`Thus, the insertion of the sensor causes minimal trauma
`to the tissue and to the sensor itself. The sensor can flex
`laterally, and this again minimizes tissue damage caused
`by movement of the patient.
`In the case of implantable glucose sensors, response
`times of less than two minutes and linearities over glu(cid:173)
`cose· concentrations of 0-25 mM can be achieved. At
`the same time, through use of the fabrication techniques
`of the invention, the rejection rate upon initial manufac(cid:173)
`ture is drastically reduced.
`In the case of sensors received within a needle hous(cid:173)
`ing, such can be readily manipulated by the patient for
`implantation purposes. These sensors typically have a
`slightly larger diameter than the flexible sensors de(cid:173)
`scribed previously, but are not so large as to cause sig(cid:173)
`nificant discomfort. This relatively small size is assured
`because of the sensor construction, making use or· a
`small Teflon-coated Pt-Ir wire and immobolized en-
`zyme.
`
`3
`cellulose acetate or Nafion layer (1-3 microns thick(cid:173)
`ness), followed by cross linking with glutaraldehyde.
`Another important aspect of the present invention
`resides in the preferred use of a synthetic polymer mem(cid:173)
`brane disposed over the enzymatic indicating surface to 5
`serve as a permeable protective layer. In particular, a
`layer of polyurethane is advantageously applied as a
`thin coating over at least the indicating surface (and
`preferably the entire indicating electrode) in order to
`protect the enzymatic reaction surface from the biologi- 10
`cal environment. Moreover, this layer provides a diffu(cid:173)
`sional barrier for glucose which slows down the flow of
`glucose and creates a linear sensor response over the
`concentration ranges of interest. In particular, in order
`to achieve the desired linear response, use is made of an 15
`active enzyme layer and a relatively thin protective
`membrane. It is important that the membrane regulate
`the passage of molecules therethrough to an extent that
`the enzymatic reaction between the indicating surface
`and these molecules is determined by the rate of diffu- 20
`sian through the membrane, and not the enzymatic
`reaction kinetics. In practice using the methods of sen(cid:173)
`sor construction herein described, an optimal balance
`between the competing goals of linear response and
`sensitivity and response times may be achieved.
`The use of an additional, negatively charged inner
`membrane layer immediately adjacent the Pt-Ir wire
`also retards the diffusion of negatively charged species
`(e.g. ascorbate and urate) in the biological environment
`which are interfering species. Of course, this inner 30
`membrane does not significantly exclude hydrogen
`peroxide, and electrically neutral species.
`Although the thickness of the outermost polyure(cid:173)
`thane membrane has not been specifically ascertained, it
`is estimated that the membrane has a thickness of from 35
`about 5 to 10 microns in the preferred glucose sensors
`hereof.
`The sensors described above are, by virtue of their
`construction, relatively flexible and therefore comfort-
`able in use. However, this same characteristic flexibility 40
`makes it necessary to employ a catheter to implant the
`sensors. In an alternative embodiment, sensors may be
`provided which can be readily implanted without the
`need of a catheter, even by the patient himself. In such
`embodiments, use is made of an elongated, tubular, 45
`metallic housing, typically a conventional hypodermic
`needle; the sensor apparatus is inserted within the nee-
`die, and includes an indicating electrode having a sec-
`tion thereof provided with immobilized enzyme. In
`order to expose the enzyme to the biological environ- 50
`ment, the needle sidewall is apertured in registry with
`the enzyme. A holder is also provided adjacent the
`rearward end of the needle body in order to facilitate
`DESCRIPTION OF THE PREFERRED
`manipulation and insertion of the sensor. This holder
`EMBODIMENTS
`advantageously is in the form of a transversely extend- 55
`The following examples illustrate the construction of
`ing flag-like plastic body secured to the needle housing.
`glucose sensors depicted in FIGS. 1 and 2, and are
`The invention also comprehends a novel method of
`described with particular reference to these drawings. It
`applying the polyurethane membrane described previ-
`will be understood, however, that the examples are
`ously. That is to say, a real difficulty in the production
`of enzymatic sensors stems from the difficulty of apply- 60 illustrative only, and nothing therein should be taken as
`ing various materials uniformly to a very small, implant-
`the limitation upon the overall scope of the invention.
`able device. This difficulty has been overcome in the
`EXAMPLE 1-FIG. 1
`context of the present invention, by applying to the
`sensor surface a well-defined volume of a polymer dis(cid:173)
`One end of a 10 em section 10 of Medwire Corpora(cid:173)
`solved in an organic solvent such that the film is uni- 65
`tion Teflon-Coated platinum-iridium wire is provided.
`The section 10 includes a central platinum-iridium wire
`formly distributed across the surface, In practice, this
`12 (0.18 mm o.d.) and a coating ofinsulative Teflon 14
`method is carried out by providing a wire loop, and
`(0.035 mm thickness) therearound. The central wire 12
`holding the coating liquid in the loop by surface tension
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 is an enlarged, sectional view illustrating a
`glucose sensor in accordance with the invention;
`FIG. 2 is an enlarged, sectional view of another glu-
`case sensor in accordance with the invention;
`FIG. 3 is a graph showing the linear sensor response
`of the FIG. 1 glucose sensor over a glucose concentra-
`tion range of 0-25 mM;
`FIG. 4 is a graph illustrating the storage stability of
`the FIG. 1 glucose sensor;
`FIG. 5 is a sectional view depicting another sensor
`embodiment wherein the indicating electrode is housed
`within an implantable needle;
`FIG. 6 is a perspective view of the sensor illustrated
`in FIG. S; and
`FIG. 7 is a perspective view of an embodiment simi-
`·lar to that of FIG. 6, but depicting the use of an implant-
`able reference electrode.
`
`AGAMATRIX, INC.‎
`Exhibit 2002-6 (IPR2016-01679)‎
`Dexcom, Inc. v. AgaMatrix, Inc.
`
`

`
`15
`
`s
`forms the indicating electrode from the sensor. A cavity
`16 (1-3 mm in length) is formed in the wire 10 as shown
`in FIG. 1. This is achieved by first putting a circular cut
`on the Teflon coating with a paper cutter and then
`pulling the Teflon out to create a cavity of about 1 5
`millimeter in length, exposing a corresponding section
`of the wire 12. The excess Teflon extending beyond the
`left end of the wire 12 is then trimmed off with the
`cutter.
`The reference electrode 18 is formed on the Teflon 10
`surface, about 1.5 millimeters to the right of the exposed
`platinum iridium surface as viewed in FIG. 1. A thin
`silver wire (0.1 mm o.d., 15 em length) is tightly
`wrapped around the TEFLON TM surface covering to
`form a coil 20 of about 5 millimeters in length. A wire
`wrapping tool may be utilized for this purpose. The
`trailing portion of the wire to the right of coil 20 is
`covered with a section 22 of heat shrinkable Teflon
`tubing (5 em long, 1.5 mm o.d., Zeuss Industrial Prod- 20
`ucts Inc.), leaving small lengths of the silver wire and
`platinum iridium wires uncovered to serve as electrical
`leads. A heat gun operating at 600° C. is employed for
`shrinking the Teflon tubing. A layer of silver chloride is
`formed on the coil 20 by passing current (0.4 mA/cm2) 25
`for 60 minutes through the wrapped silver wire while it
`is dipped in a stirred O.lN HCL solution. The exposed
`portions of reference electrode 18 are then rinsed with
`de-ionized water for 6 hours. The reference electrodes
`prepared in this manner show a potential of -64±3 30
`mV (n=lO) vs. Ag/AgC1(3M NaCI) in 0.15M NaCl at
`37° c.
`In order to immobilize glucose oxidase (GOx,
`E.C.l.l.3.4) on the exposed portion of wire 12, an inner,
`negatively charged membrane is first applied to the 35
`exposed wire section. Tl}ereafter, a circumferentially
`extending enzymatic indicating layer 21 is formed
`within cavity 16. Two different apflroaches have been
`employed to achieve these ends.
`
`A. Attachment of GOx to Bovine Serum Albumin
`Coupled Cellulose Acetate
`The exposed platinum iridium surface within cavity
`16 is degreased by washing with acetone. It is then
`rinsed with de-ionized water and dried in cold air 45
`stream before polymer deposition.
`The left hand part of the sensor (portion to the left of
`the reference electrode coil20) is dipped into 5% cellu(cid:173)
`lose acetate (39.8% acetyl content) in 50% acetone and
`50% ethanol for 10 seconds and is withdrawn slowly. It
`is then exposed to the vapor above the cellulose acetate
`solution for 5 seconds and is dipped again into the cellu(cid:173)
`lose acetate solution for 10 seconds. The sensor is then
`removed and dried in air at room temperature (23° C.) 55
`for one minute and placed in deionized water for 6
`hours to permit displacement by water of entrapped
`solvent in the membrane pores. The cellulose acetate
`membrane prepared in this fashion shows good long(cid:173)
`term stability and also discriminates well against ascor- 60
`bate and urate. Bovine serum albumin (BSA) is then
`covalently coupled to cellulose acetate. and a subse(cid:173)
`quent reaction of the membrane with GOx, which has
`previously been activated with an excess of p-benzoqui(cid:173)
`none, is carried out. The detailed procedure for this 65
`reaction is described in the literature, Sternberg et. al.,
`Anal. Chern. 1988, 60, 2781, which is incorporated
`herein by reference.
`
`5,165,407
`
`6
`B. Physical Adsorption of Enzyme on Cellulose
`Acetate or Nation Followed by Crosslinking with
`Glutaraldehyde
`1. The sensor is coated with cellulose acetate in ex(cid:173)
`actly the same manner as described above to create
`membrane. The GOx (270 U/mg) is physically ad(cid:173)
`sorbed by dropping 5 fLl of GOx solution ( 40 mg/Ml in
`O.lM phosphate buffered saline) on the indicating ele(cid:173)
`ment within cavity 16, and is allowed to dry for 10
`minutes at room temperature. To immobilize the en-
`zyme and form circumferential surface 21, the sensor is
`exposed to glutaraldehyde Vjlpor generated from 25%
`glutaraldehyde solution placed at the bottom of an en(cid:173)
`closed glass chamber for 12 hours at room temperature.
`The sensor is then rinsed in de-ionized water and dried
`in air for 2 hours. The crosslinking with glutaraldehyde
`protects the enzyme from heat degradation, proteolytic
`enzymes and hydrolysis, E. M. Salona, C. Saronio, and
`S. Garattini (eds), "Insolubilized Enzymes." Raven,
`New York, 1974, incorporated by reference herein.
`2. Nation (Perfluorosulfonic acid polymer, obtained
`from E. I. DuPont de Nemours and Co., may also be
`used as an alternate for cellulose acetate for the inner
`membrane. After cleaning the sensing portion of the
`sensor as above, it is electrocoated with Nation using
`the method described by Adams et al, Neurosci. Meth.
`Vol. 22, 1987, pp 167-172, incorporated by reference
`herein. One drop of Nation (5% solution, Aldrich) is
`placed in a 2 mm loop formed at one end of a copper
`wire. A DC potential of + 3 V is applied to the working
`electrode with respect to the loop for 10 seconds. The
`sensor is pulled out of the loop before turning off the
`potential and is dried in air for 2 hours, and the GOx
`enzyme is applied as described above.
`Alternate polymers may be used in lieu of or in com(cid:173)
`bination with cellulose acetate or Nation for coating of
`the exposed Pt-Ir wire surface. For example, polyani(cid:173)
`line and polyphenol derivatives can be electrochemi-
`40 cally deposited onto the exposed indicating electrode
`surface. Oxidative electropolymerization of aniline and
`phenol monomer yields stable and adhesive coating
`over the exposed wire. These materials moreover have
`good size selectivity which can be utilized to further
`improve the sensor selectivity against electrochemical
`interferences in biological environments. The combina-
`tion of a size selective coating with a charge selective
`fllm (e.g. cellulose acetate) may reduce the in vivo back(cid:173)
`ground current and the risk of electrochemical interfer(cid:173)
`ence. Electropolymerization of aniline and phenol is
`well known, see for example Ohsaka et al. Anal Chern.
`1987, 59, 1758-61, and Malitesta et al. Anal. Chern. 1990,
`62, 2735-40, both of.which are incorporated by refer(cid:173)
`ence herein.
`Finally, Eastman-Kodak AQ 29-D polymer (poly( es(cid:173)
`ter-sulfonic acid)) has both charge and size selective
`features, and may be applied to the exposed indicating
`electrode wire in lieu of Nation. A coating of this type
`applied to the indicating electrode with a cellulose ace(cid:173)
`tate layer thereover should improve overall selectivity.
`Combined coatings made from mixtures of cellulose
`acetate and the AQ 29-D polymer should also provide
`advantages in terms of sensor selectivity.
`In order to complete the preparation of the sensor,
`the whole assembly, including the reference electrode,
`is dip coated with 4% polyurethane (Thermedics, SG
`85A) dissolved in 98% tetrahydrofuran (THF) and 2%
`dimethylformamide (DMF) to form an outer membrane
`
`50
`
`AGAMATRIX, INC.‎
`Exhibit 2002-7 (IPR2016-01679)‎
`Dexcom, Inc. v. AgaMatrix, Inc.
`
`

`
`5,165,407
`
`7
`24. The polyurethane solution (10 uL) is held in a wire
`loop (2 mm i.d.) by surface tension and the sensor is
`passed through the loop. This leaves a uniform polymer
`film on the completed sensor 25 to the appropriate ex(cid:173)
`tent depicted in FIG. 1. This method provides a good S
`control over the amount of polymer which is applied to
`the sensor. The sensor is dried in air for 6 hours at room
`temperature and then left in O.lM phosphate buffered
`saline, pH=7.4 for 72 hours for the various outer mem(cid:173)
`branes to condition fully. It is possible to recoat the 10
`sensor with polyurethane if the desired linear range of
`glucose sensitivity is not obtained after the first coating.
`
`EXAMPLE 2-FIG. 2
`One end of a 10 em section 26 of Teflon-coated plati- 15
`num-iridium wire is provided having a 0.18 mm o.d., a
`central Pt-Ir wire 28 and a teflon sheath 30 (0.035 mm
`thickness). The left hand end of the wire is stripped to
`form a cavity 32 as described in Example 1. The right
`hand end of section 26 is then inserted into a 5 centime- 20
`ters long polyethylene tube 34 (0.67 mm o.d., 0.30 mm
`i.d.). The left hand extremity of the polyethylene tube is
`sealed by putting a drop of 4% cellulose acetate solution
`(in acetone) into the opening. The acetone is allowed to
`dry while holding the Teflon-coated wire in the middle 25
`of the polyethylene tube. This permits the formation of
`a circumferential salt bridge deposit 36 which effec(cid:173)
`tively acts as the terminal part of the reference elec(cid:173)
`trode, lies in a plane transverse to the longitudinal axis
`of the wire 28 and establishes electrical contact between 30
`the reference and sensing electrodes. The empty annu-
`lar space between the Teflon-c9ated wire and the poly(cid:173)
`ethylene tube is then filled under vacuum with O.lM
`phosphate buffer, pH=7.4 containing 9 g/L NaCI. A
`chloridized silver wire 38 (0.05 mm o.d., 5 em long 35
`prepared as described in Example 1), is introduced into
`the polyethylene tube from. the right hand end thereof
`and this opening is also sealed as described above to
`present a sealing deposit 40. The reference electrode
`shows a potential of -60±10 mV (n=6) vs, Ag/AgCl 40
`(saturated KCL) at 37" C. The enzyme immobilization
`and polyurethane deposition steps are then carried out
`using the procedures described in Example 1 to give the
`inner, negatively charged membrane 32a, the circum(cid:173)
`ferential indicating enzyme layer 33, and outer permea- 45
`ble membrane 42 illustrated in FIG. 2. The complete
`sensor 43 is then ready for calibration and use with
`electrical connections afforded by the axially extending
`ends of the wires 28, 38.
`The sensors described in the above example are cali- 50
`brated by dipping into a thermostated cell (at 37" C.)
`containing 10 ml of stirred O.lM phosphate buffered
`saline, pH=7.4, and a potential of +600mV (for hydro(cid:173)
`gen peroxide detection) is applied between the working
`and the reference/indicating electrodes. The back- 55
`ground current is allowed to stabilized for 20 minutes.
`The calibration of the sensor is carried out by adding
`increasing amounts of glucose to the stirred buffer. The
`current is measured at the plateau (steady state re(cid:173)
`sponse) and is related to the concentration of the ana- 60
`lyte. Following the calibration procedure, the sensors
`are stored in O.lM phosphate buffered saline, pH=7.4 at
`room temperature.
`A typical response curve to the glucose addition is
`shown in FIG. 3, for a sensor made in accordance with 65
`FIG. 1. As illustrated, the response characteristics of
`the sensor over the concentration range of interest
`(0-25 mM) are essentially linear, and are especially so
`
`8
`over the range of 0-15 mM. The sensor output is also
`essentially independent of the stirring rate. The in vitro
`characteristics of the sensor are summarized in the fol(cid:173)
`lowing Table. A typical storage stability curve for the
`sensor is shown in FIG. 4. During the first few days of
`sensor preparation,
`the polyurethane membrane
`changes its permeability for glucose as a result of hydro(cid:173)
`lytic and swelling processes, leading to the increased
`passage of glucose and an increased current. After this
`initial period, however, the stability is excellent.
`The sensors of the invention are in use electrically
`coupled with suitable signal processing equipment, and
`implanted into a desired subcutaneous site. Glucose and
`oxygen diffusing through the outer synthetic polymer
`membrane are enzymatically catalyzed by the GOx at
`the indicating surface, resulting in production of glu(cid:173)
`conic acid ·and hydrogen peroxide. The latter is mea(cid:173)
`sured amperometrically, which is a measurement of
`glucose concentration.
`
`TABLE
`In Vitro Characteristics of FIG. I Glucose Sensor
`Value
`Parameter
`0.7 ± 0.2
`Residual current (nA/mm2)0
`1.8 ± 0.8
`Sensitivity (nA/mM · mm2)
`IS± 3
`Linear Range (upper limit) (mM)
`3.5 ±I
`Response time (min.), T 90%
`Results shown above are expressed as mean ± SD for six sensors.
`~esidua1 currents are measured after J hour of polarization.
`
`FIGS. 5 and 6 illustrate another sensor 44 in accor(cid:173)
`dance with the invention. In this case, the sensor body
`46 is received within a stainless steel hollow tubular
`needle 48.
`The sensor body 46 includes an innermost, Teflon(cid:173)
`coated, platinum-iridium wire 50 (90% Pt/10% Ir) hav(cid:173)
`ing a total O.D. of about 0.2 mm and a cavity 52 formed
`therein as described in Example 1. The cavity 52 is
`approximately 1.0 mm in length and is located about 3.0
`mm from the tip of the wire 50. A glucose oxidase layer
`54 is immobilized within the cavity 52, and comprises a
`cellulose acetate polymer layer attached to the surface
`of the Pt-Ir wire, with glucose oxidase crosslinked
`through glutaraldehyde onto the cellulose acetate. This
`procedure is in accordance with Example l.B.l. above.
`The entirety of the indicating electrode is then covered
`by a membrane 56 of polyurethane, again using the
`method set forth in Example 1.
`The sensor body 46 is thereupon inserted into a 25-
`gauge disposable stainless steel hypodermic needle, the
`latter having an aperture 58 adjacent the forward,
`sharpened insertion end 60 thereof. The sensor body 46
`is installed in such manner that the glucose oxidase layer
`54 comes into registfY with the sidewall opening 58,
`thereby exposing the layer 54 to the biological environ(cid:173)
`ment. A silicone rubber plug 62 is installed in the for(cid:173)
`ward end of the needle 48 as shown.
`As illustrated in FIG. 5, the wire 50 extends rear(cid:173)
`wardly out of the end of needle 48, and is adapted to be
`connected with appropriate instrumentation for mea(cid:173)
`suring glucose concentrations. In order to seal the rear(cid:173)
`ward end of the sensor 44, a bead 64 of epoxy is applied
`around the wire 50 and the butt end of the needle 48 and
`sensor body 46.
`The overall sensor 44 is completed by provision of a
`holder 66 extending transversely of the needle 48. The
`holder 66 is preferably in the form of a plastic sheet
`wrapped around the rearward end of the needle 48 as
`shown, and secured by means of epoxy 'Or polycyanoa-