Assessment and optimisation of
`dip-coating procedure for the
`preparation of electroenzymic
`glucose transducers
`
`P.A. Rea*
`
`P. Rolfe
`
`P. J. Goddard
`
`Bioengineering Unit, Department of Paediatrics, John Radcliffe Hospital. University of Oxford,
`Oxford OX3 9DU, England
`
`Abstract-The details of construction and the performance characteristics of a dip (cid:173)
`coated electroenzyn:ic glucose transducer comprising an H 2 0 2 electrode coated with a
`layer of glucose ox1dase encapsulated in cellulose acetate and overlaid with a layer of
`polyurethane are presented. The steady-state current increases when the glucose
`oxidase and cellulose acetate concentrations of the dip-coating solutions are increased,
`but high cellulose acetate concentrations yield thick and mechanically unstable
`membranes. A compromise between current yield and mechanical stability can,
`however, be achieved by employing glucose oxidase and cellulose acetate concen (cid:173)
`, respectively. A polyurethane concentration
`trations of 200 mg m1- 1 and 2 · 5 g 100 m1- 1
`of 6 g 1 OOml- 1 is optimal both in terms of the current yield and the linearity of response.
`The relationship between steady-state current and D -glucose concentration is ' ap(cid:173)
`proximately linear over the concentration range 0·5 to 11 ·5 mM, and if correction is
`made for deviations from linearity at higher glucose concentrations, concentrations in
`excess of 20 mM can readily be quantified. The steady-state current is pH and
`temperature dependent, but the dependencies are relatively small in the physiological
`range. The mean rate of decrease of the glucose current during long-term operation of
`the optimised transducer is 0·83 per cent of the initial current per hour at 37°C. The
`hydrated electrodes perform satisfactorily after storage for more than two weeks at room
`temperature. The transducers have a mean response time [t90%]of 50s or less.
`
`Keywords-Dip-coated electroenzymic glucose transducer, In vitro performance,
`Optimisation
`
`Med. & Biol. Eng . & Comput., 1985, 23, 108- 115
`
`1 Introduction
`APPROXIMATELY 0·5 per cent of the population of the
`developed nations suffer from type I insulin-dependent dia(cid:173)
`betes mellitus (IDDM) (CARISTY et al., 1979). Rapid death
`from the disease is uncommon because of the availability of
`monocomponent insulins, but IDDM remains a major
`clinical problem because of the complications that attend the
`condition: blindness, gangrene, renal damage, neuropathy,
`impotence and micovascular disease. These complications
`have a reported incidence of 30-50 per cent among long(cid:173)
`standing sufferers (DECKERT et al., 1978). Animal experi(cid:173)
`ments and epidemiological studies indicate that the compli(cid:173)
`cations oflDDM are associated with abnormal excursions of
`blood glucose and metabolite concentrations as a result of
`the relative infrequency with which diabetics administer
`insulin to themselves during the conventional management
`of the disease (TCHOBROTSKY, 1978; ENGERMAN et al., 1977).
`Thus, in an attempt to regulate the rate of insulin infusion in
`a more controlled and continuous manner, and thereby
`abrogate the long-term complications of IDDM, open-loop
`
`*Present address and address for correspondence: Cell Physiology
`Laboratory, Department of Biology, McGill University, 1205 Avenue
`Docteur Penfield, Montreal, PO H3A 181, Canada
`
`First received 11th November 1983 and in final form 21st May 1984
`© IFMBE: 1985
`
`continuous subcutaneous insulin infusion pumps (CSII)
`have been developed (ALBISSER et al., 1974a; b).
`Precise control of blood glucose concentrations necessi(cid:173)
`tates a closed-loop device which delivers insulin to the
`systemic blood supply in proportion to the difference
`between the desired and measured physiological glucose
`concentration (SoELDNER, 1982). There is therefore a need for
`in vivo glucose transducers for clinical applications.
`A large number of glucose transducers based on the
`'enzyme electrode principle', in which a glucose oxidase
`membrane and an electrochemical detector are combined,
`have been described (SoELDNER, 1982). The detection meth(cid:173)
`ods include potentiometric (MALMSTADT and PARDUE, 1965)
`and amperometric (GUILBAULT, 1980) measurements of the
`H 20 2 and H + ions generated in the glucose oxidase(cid:173)
`mediated oxidation of D-giucose, direct (UPDIKE and HICKS,
`1967) or differential (BESSMAN and ScHULTZ, 1973) measure(cid:173)
`ments of the 0 2 consumed and amperometric measurements
`of the redox state of the flavin moiety of glucose oxidase via
`an intermediary redox couple (ScttLAPFER et al., 1974). The
`enzyme immobilisation methods examined include entrap(cid:173)
`ment of the enzyme solution with a perm-selective mem(cid:173)
`brane (GUILBAULT and LUBRANO, 1973), gel (HICKS and
`UPDIKE, 1966) or microporous matrix (Sttu and WILSON,
`1979), coreticulation (BAUMAN et al., 1965), adsorption
`(SILVER, 1976) or covalent coupling
`to a membrane
`
`108
`
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`
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`
`Dexcom Inc. v. WaveForm Technologies, Inc.
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`
`

`

`(THEVENOT et al., 1982). However, our experience of these
`techniques is that only a few can readily be adapted to the
`mass fabrication of small transducers: two conditions that
`must be met before in vivo glucose monitoring can become
`widespread. Of the procedures we have investigated, the
`encapsulation of glucose oxidase in a cellulose acetate matrix
`has proved to be one of the most suitable for transducer
`miniaturisation and bulk production. Independently de(cid:173)
`veloped by SmcHIRI et al. (1982; 1983), this method involves
`the dip-coating of an H 20 2 electrode with a glucose
`oxidase/cellulose acetate mixture and polyurethane.
`SmcHIRI et al. (1982), however, provide only brief details of
`their method of transducer fabrication and little indication of
`the influence of the fabrication method on the response
`characteristics of the transducer. Thus in this report we
`describe the details of construction of such a dip-coated
`glucose transducer and summarise the performance charac(cid:173)
`teristics of the optimised system.
`Three features of the dip-coat are examined:
`
`(a) the influence of the composition of the dip-coat on the
`glucose response of the transducer in terms of its current
`yield and linearity of response
`(b) the pH and temperature dependencies of the steady(cid:173)
`state current
`(c) the stability of the transducer during Jong-term storage
`and operation.
`
`2 Materials and methods
`2.1 Electrodes
`The glucose transducer is a conventional glucose oxidase(cid:173)
`coupled electrode in which D-glucose is measured as the
`steady-state rate of formation of H 20 2 upon the glucose
`oxidase-mediated
`oxidation
`of
`D-glucose
`to
`D-gluconolactone.
`
`b-D-glucose+0 2 -----> D-gluconolactone.
`
`. (1)
`
`The rate of formation of H 20 2 is measured as the steady(cid:173)
`state current resultant on its oxidation at a platinum anode.
`
`. (2)
`
`platinum anode
`
`,..... ...... ~-../epoxy resin
`--------- Ag/AgCI cathode
`
`)
`
`polyurethane
`dip- coat
`
`glucose
`oxidase I cellulose
`acetate dip-coat
`
`O·l25mm
`
`5 mm
`
`The experimental electrodes consisted of a 0· 125 mm dia(cid:173)
`meter platinum anode (purity 99·9 per cent, from Good(cid:173)
`fellow Metals Ltd., Cambridge, England) and a 5 mm
`diameter Ag/ AgCI reference electrode of hemispherical
`geometry set into PVC (Fig. 1). The working and reference
`electrodes were insulated with epoxy resin. PVC was chosen
`as the material for the body of the electrode because it bonds
`tightly with polyurethane.
`
`2. 2 Electrode pretreatment
`The uncoated platinum working electrode was pretreated
`before each experiment by applyigg a potential of - 200 m V
`(w.r.t. Ag/AgCl) until the cathodic current had decayed to a
`minimum. The electrode was then polarised at + 50 m V
`(w.r.t. Ag/AgCI) and the current allowed to decay to about
`0 nA. This pretreatment procedure, recommended by GUIL(cid:173)
`BAULT and LUBRANO (1973), was found to yield reproducible
`H 20 2 currents from the onset of measurement.
`The dip-coated electrodes were pretreated before each
`experiment by applying a potential of +600mV (w.r.t.
`Ag/AgCl); only when the current had decayed to a minimum
`were measurements made. The electrodes were rinsed with
`buffer at the end of each experiment to remove unreacted
`glucose and reaction products. Unless stated to the contrary,
`the freshly coated transducers were hydrated in buffer for
`12 h before any measurements were made.
`
`2. 3 Dip-coating
`The glucose transducers were prepared in a manner
`similar to that described by SmcHIRI et al. (1982). The
`electrode tip was lightly polished with 0·25 µm diamond
`paste (Engis Ltd., Maidstone, England) and dipped into a
`suspension of glucose oxidase (18300U/g; type II from A.
`niger, EC 1.1.3.4 from Sigma Chemical Co. Ltd., Poole,
`England) prepared in a solution of cellulose acetate (acetic
`acid content 53·5-54·5 per cent from BDH, Poole, England).
`The lyophilisate of glucose oxidase was milled to a fine
`powder with a mortar and pestle before its suspension in the
`cellulose acetate solution to improve the evenness and
`reproducibility of film formation. The cellulose acetate
`solutions were made up in 100 per cent acetone or a 1 : 1
`mixture of acetone and ethanol. Immediately after their
`withdrawal from the dip-coating solution, the electrodes
`were inverted and allowed to dry for 45 min at room
`temperature. The polyurethane coat was applied. by dipping
`the glucose oxidase/cellulose acetate-coated electrode into a
`solution of polyurethane (Estane 5701 Fl) dissolved in a
`l: 18 (v/v) mixture of dimethylformamide and tetrahydro(cid:173)
`furan. The electrodes were finally left to dry in air at room
`temperature for 12-18 h before hydration.
`For most of the studies described below, the glucose
`oxidase/cellulose acetate dip-coating solution consisted of
`200 mg ml - 1 glucose oxidase and 2·5 g 100 ml - l cellulose
`acetate suspended in a 1 : 1 mixture of acetone and ethanol.
`The polyurethane dip-coating solution consisted of
`6 g lOOm1- 1 polyurethane dissolved in a 1 : 18 (v/v) mixture
`of dimethylformamide and tetrahydrofuran. These com(cid:173)
`positions were found to be optimal.
`
`2 .4 Note on stirring dependence
`The glucose concentration dependence of the steady-state
`current was measured in unstirred media. In the absence of
`diffusion limitation, the concentration dependence of the
`steady-state current should approximate
`the
`intrinsic
`kinetics of the immobilised enzyme such that
`
`Fig. 1 Schematic diagram of experimental dip-coated glucose
`transducer
`
`iss = (imax[D-glucose])/(Km + [D-glucose])
`
`Medical & Biological Engineering & Computing
`
`March 1985
`
`. (3)
`
`109
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`
`

`

`not lower, in the unstirred condition. Indeed, the results of
`rotating disk-electrode experiments where
`the hydro(cid:173)
`dynamics of the bulk medium can be precisely defined
`demonstrate that H 20 2 effiux from the surface of the
`transducer is a more important determinant of the steady(cid:173)
`state current at high rotation speeds*. Thus, to evaluate the
`influences of diffusion limitation and catalytic limitation it is
`necessary to measure the rates of both glucose and H 20 2
`mass transport to and from the transducer surface over a
`wide range of stirring speeds; a procedure which would be
`impracticable for a large number of routine tests. Con(cid:173)
`sequently, for reasons ofreproducibility and practicality, the
`measurements of the glucose current were made in unstirred
`media.
`
`2. 5 Determination of D-glucose concentration dependence
`All the measurements were made in Miller and Golder's
`constant ionic strength buffer. The buffer consisted of
`80 mM NaCl, 5·68 mM Na2 HP0 4 and 3·2 mM NaH 2 P04 .
`Unless stated otherwise, the final pH of the buffer was 7·44
`units and its ionic strength was 0·11; this approximates the
`pH and ionic strength of blood plasma (DAWSON et al., 1959).
`Aliquots of a stock solution of 2 M D-glucose were added
`to the buffer and the medium stirred to ensure homogeneity.
`The steady-state current was then measured 3-5 min after
`the cessation of stirring. The measurements were made in a
`thermojacketed reaction cell and the D-glucose solutions
`were prepared at least 18 h before their use to ensure an
`equilibrium mixture of the a- and b-anomers. All the
`D-glucose concentrations are expressed as total D-glucose
`(a- plus b-anomers). An electrode potential of +600mV
`(w.r.t. Ag(AgCI) was employed throughout.
`
`5
`
`4
`
`""" c
`
`11 mM
`D - glucose
`
`5 mM
`D - glucose
`
`where iss = steady-state current at glucose concentration
`[D-glucose], imax = maximum steady-state current and
`Km = glucose concentration at which iss = imaxf2.
`Multiplying both sides of expr. 3 by issimax and rearranging
`
`(EADIE, 1942; HoFSTEE, 1952)
`
`. (4)
`
`A plot of iss against iss/[D-glucose] should therefore yield a
`straight line of slope - Km and intercept
`imax· Since
`imax = k+ 2 e 0 , where e0 =enzyme activity, and Km is a
`constant, (k _ 1+k+ 2 )/k + 1 , determination of these two para(cid:173)
`meters from the Eadie-Hofstee transformation ( expr. 4)
`should enable the definition of the performance charac(cid:173)
`teristics of the transducer in terms of its maximum glucose
`current imax and the glucose concentration range over which
`linearity applies Km. However, direct measurements of the
`glucose current show that rein. 4 does not hold for dip-coated
`transducers (Fig. 2). Neither in the stirred nor the unstirred
`condition does a plot of iss against iss/[D-glucose] yield a
`straight-line relationship (Fig. 2b ). Although the deviations
`from linearity are greatest in the unstirred condition, inspec(cid:173)
`tion of the arithmetic plots (Fig. 2a) shows that diffusion
`limitation with respect to the mass transport of glucose to the
`transducer surface is not limiting on the measured steady(cid:173)
`state current. The measured steady-state currents are higher,
`
`a Arithmetic Plot
`
`Unstirred
`,,,,,,---0--()--0
`.... ~
`,...
`,...'1
`
`Stirred
`
`3·0
`
`2. 5
`
`2·0
`
`1·5
`
`"""
`c
`
`~
`
`~
`
`l · 0
`
`0·5
`
`/ A
`
`/
`o/
`7
`
`/
`
`/
`
`/
`9/
`I
`I
`I
`
`I t
`
`I
`I
`I
`I
`I
`I
`I
`p
`I
`I
`I
`q
`I
`I •
`I
`I
`l•
`
`b
`
`<!
`c
`in
`Ill
`
`Eadie-Hofstee Plot
`<><>a.
`"-._Unstirred
`"'
`"
`'
`
`II.
`\
`Stirred\
`\
`\
`o\
`~
`
`•
`
`O·O
`
`0 •
`O·l
`0·4
`0·3
`0·2
`i ss ID- glucose, nA mM-1
`
`0
`
`4
`
`6
`
`14
`12
`10
`D- glucose, mM
`
`16
`
`18
`
`20
`
`22
`
`0
`
`50
`
`200
`100
`150
`Glucose oxidase,mg ml-I
`
`Fig. 2
`
`Influence of stirring the bulk medium on the relationship
`between steady-state current iss and D-glucose concent(cid:173)
`ration. The dip-coat was prepared from dip-coating solutions
`containing 100 mg ml- 1 glucose oxidase suspended in
`2·5 g 100 m1- 1 cellulose acetate dissolved in a 1 : 1 mixture of
`acetone and ethanol. A polyurethane concentration of
`2 g 100 m1- 1 was employed. The measurements were made at
`20°C in Miller & Golder's buffer (pH 7·44; o-11). The buffer
`was stirred with a magnetic stirrer set at maximum velocity.
`(a) arithmetic plot (iss against [D-glucose], (b) Eadie(cid:173)
`Hofstee plot (iss against issf[D-glucose])
`
`Fig. 3 Effect of the glucose oxidase concentration of the glucose
`oxidase/cellulose acetate dip-coating solution on the steady(cid:173)
`state current at D-glucose concentrations of l, 5 and 11 mM.
`The dip-coats were prepared from glucose oxidase sus(cid:173)
`pended in 2·5g100 ml- 1 cellulose acetate dissolved in a 1 : l
`mixture of acetone and ethanol. Polyurethane was employed
`at a concentration of 2g lOOm1- 1
`. Conditions were 37°C,
`pH7·44, 0·1 I. Mean ±SE (n = 5) are shown
`
`* Unpublished observations by Rea
`
`110
`
`Medical & Biological Engineering & Computing
`
`March 1985
`
`Dexcom Inc. v. WaveForm Technologies, Inc.
`IPR2017-01051
`Exhibit 1037
`
`

`

`The pH and temperature dependencies of the glucose
`current were assessed in a similar manner.
`
`2.6 Stability to long-term storage and long-term operation
`These were determined as described in the legends of the
`corresponding figures.
`
`3 Results
`3. l Optimisation of composition of dip-coating solutions
`The effects of the glucose oxidase, cellulose acetate and
`polyurethane concentrations of the dip-coating solutions on
`the current yield of the final transducer are summarised in
`Figs. 3, 4 and 5. The influences of each of the three dip-coat
`components were measured at D-glucose concentrations of
`1, 5 and 11 mM to determine the concentration dependence
`as well as the overall current yield of the transducer.
`An increase in the glucose oxidase concentration of the
`2·5 g 100 ml - 1 cellulose acetate dip-coating solution from 50
`to 200mgm1- 1 causes a 7-10-fold increase in the steady(cid:173)
`state current at D-glucose concentrations of 5 and 11 mM
`(Fig. 3). The effects of glucose oxidase concentrations in
`excess of 100 mg ml - 1 are less marked at lower D-glucose
`concentrations; this is assumed to result from diffusion
`limitation with respect to the glucose current at low bulk
`D-glucose concentrations.
`
`11 mM
`D- glucose
`
`D- glucose
`
`5
`
`I
`I
`I
`I
`
`L
`
`/ D- qlucose
`
`/
`
`/
`
`/
`
`/
`
`/
`
`lmM
`D- glucose
`
`0
`
`Fig. 4
`
`4
`Cellulose acetate, g 100 ml-1
`
`Effect of cellulose acetate concentration on steady-state
`current at D-glucose concentrations of 1, 5 and 11 mM.
`Glucose oxidase and polyurethane concentrations of
`200 mg ml- 1 and 2 g 100 ml- 1, respective[ y, were employed
`throughout. Conditions were otherwise as for Fig. 3. Dissol(cid:173)
`ution of cellulose acetate in 1 : 1 mixture of acetone and
`ethanol(---); dissolution of cellulose acetate in 100 per
`cent acetone (---)
`
`The steady-state current increases with cellulose acetate
`concentration, and this is more marked at the higher glucose
`concentrations and when the cellulose acetate dip-coating
`solutions are prepared in 100 per cent acetone rather than a
`1 : 1 mixture of acetone and ethanol (Fig. 4). The more
`positively eccentric relationship between the steady-state
`current and cellulose acetate concentration when the latter is
`dissolved in 100 per cent acetone rather than a 1 : 1 mixture
`of acetone and ethanol was considered to make reproduc(cid:173)
`ibility between transducers more difficult to achieve. Con(cid:173)
`sequently, a 1 : l mixture of acetone and ethanol was
`employed as the solvent.
`
`<(
`c
`.;, 3
`
`~
`
`10
`
`12
`
`14
`
`16
`
`18
`
`Polyurethane, g/lOOml
`
`Fig. 5 Effect of polyurethane concentration of dip-coating solution
`on steady-state current at D-glucose concentrations of 1, 5
`and 11 mM. Glucose oxidase and cellulose acetate concen(cid:173)
`trations of200mgml- 1 and 2·5g lOOml- 1 were employed
`throughout. Conditions were otherwise as for Fig. 3
`
`An important factor when constructing transducers is
`their mechanical stability. Thus, although high concen(cid:173)
`trations of glucose oxidase and cellulose acetate give higher
`current yields, they also yield thicker films which have a
`spongy appearance upon hydration. As these were con(cid:173)
`sidered to be less stable mechanically than thinner films and
`unnecessarily increase the response time, a compromise
`between current yield and mechanical stability was struck by
`employing a glucose oxidase concentration of 200 mg ml - 1
`and a cellulose acetate concentration of 2·5 g 100 ml - i.
`The concentration of polyurethane employed for the
`second dip-coating solution is crucial (Fig. 5). Concen(cid:173)
`trations of polyurethane in excess of 6 g 100 ml - 1 result in a
`severe attenuation of current yield, whereas concentrations
`of less than 6 g 100 ml - l result in large deviations from
`linearity at the higher D-glucose concentrations. A poly(cid:173)
`urethane concentration of 6 g 100 ml - l was
`therefore
`employed.
`
`3. 2 Glucose concentration dependence
`The relationship between steady-state current and
`D-glucose concentration for a glucose transducer prepared
`from dip-coating solutions containing 200 mg ml - i glucose
`oxidase, 2·5 g 100 m1- 1 cellulose acetate and 6 g 100 m1- 1
`polyurethane is shown in Fig. 6. D-glucose concentrations of
`0·5 mM to in excess of20 mM can readily be determined with
`this transducer. If it is assumed that the relationship between
`steady-state current and D-glucose concentration is ap(cid:173)
`proximately linear over the range 0-11 ·5 mM, a slope of
`0·335±0·083nAmM- 1 (r = 0·995) is obtained. Since a
`blood glucose concentration of 8-11 mM is very suggestive
`of diabetes and a concentration of greater than 11 mM is
`
`Medical & Biological Engineering & Computing
`
`March 1985
`
`111
`
`Dexcom Inc. v. WaveForm Technologies, Inc.
`IPR2017-01051
`Exhibit 1037
`
`

`

`almost diagnostic (SINCLAIR, 1979), the glucose concen-
`tration range over which approximate linearity applies
`appears to be sufficiently wide to include both normal and
`pathological glucose concentrations.
`
`5
`
`4
`
`3
`
`2
`
`<(
`c:
`
`~
`~
`
`6
`
`5
`
`4
`
`3
`
`<(
`c:
`;:,,
`
`~
`
`II· 5 mM
`·o - glucose
`
`5 · 5 mM
`D - glucose
`
`2·5 mM
`D - glucose
`
`I
`
`I
`
`! -f I· 0 mM
`
`D - glucose
`
`7. 0
`
`7. 5
`
`5·5
`
`6·0
`
`6·5
`pH
`Fig. 7 pH dependence of steady-state current at D-glucose concen(cid:173)
`trations of 1, 2·5, 5·5 and 11·5 mM. Miller & Golder's buffer
`adjusted to the required pH with NaH 2 P0 4 or Na 2 HP0 4
`was employed throughout. Conditions were otherwise as for
`Fig.6. Mean ±SE (n = 5) are shown
`
`0 I 2 3 4 5 6 7 8 9 10 11 I2 13 I4 15 I6 I7 I8 19 20 21 22
`D-glucose, mM
`
`Fig. 6 Relationship between steady-state current and D-glucose
`concentration for transducer prepared from 200 mg ml- 1
`glucose oxidase, 2·5g100ml- 1 cellulose acetate and
`6g lOOm1- 1 polyurethane. - - - line drawn through data
`points; -
`- -
`line computed by linear regression (least(cid:173)
`squares method) for D-glucose concentrations of 0·5
`11 ·5 mM.
`The
`computed
`regression
`is
`to
`y = (0·335 ±0·083)x +0·338 nA where y = i,, (nA) and
`x = D-glucose concentration (mM). r = 0·995
`
`3. 3 pH and temperature dependence
`The steady-state current of the transducer is maximal in
`the pH range 6·5-7·0 units and decreases when the pH is
`increased above or decreased below this range (Fig. 7). The
`mean ratios of the steady-state currents obtained at pH 6-97
`w.r.t. 7·33 and pH 6-97 w.r.t: 5·87 are 1·1 and 1 ·0, respectively.
`The temperature dependence of the steady-state current is
`dependent on the D-glucose concentration (Fig. 8). The
`approximate mean temperature coefficients are 0·017, 0·017,
`0·009, 0·075, and 0·098 nA 0 c- 1 over the temperature range
`32-44°C for D-glucose concentrations of 1 ·0, 2·5, 5·5, 11-5
`and 19·5 mM, respectively.
`
`3 .4 Response time
`The time required for the current to attain 90 per cent of its
`final value upon transfer of the transducer from buffer
`lacking glucose to buffer containing 5 mM D-glucose is
`48·8 ± 7·38 s (n = 5). A typical response curve is shown in
`Fig.9.
`
`3. 5 Stability of glucose current
`The dip-coated transducers are relatively stable to storage
`in buffer at 21°C. The glucose current decreases from 100 to
`70 per cent of the initial value during the first 3 days of
`storage in buffer but from there on decreases at only
`approximately l per cent per day (Fig. 10). As the percentage
`loss of the initial current is similar for all three of the
`
`19·5 mM
`D - glucose
`
`11· 5 mM
`D - glucose
`
`l55mM
`D - glucose
`
`~2·5mM
`11 OmM
`
`- D - glucose
`
`D - glucose
`
`4
`
`<(
`c:
`
`~
`
`~
`
`t
`t
`r !
`!
`
`f-
`
`30
`
`32
`
`34
`
`40
`38
`36
`Temperature, °C
`Fig. 8 Temperature dependence of steady-state current at
`D-glucose concentrations ofl·O, 2·5, 5·5, 11·5and19·5 mM.
`Conditions were otherwise as for Fig. 6. Mean ±SE (n = 5)
`are shown
`
`42
`
`44
`
`46
`
`112
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`Medical & Biological Engineering & Computing
`
`March 1985
`
`Dexcom Inc. v. WaveForm Technologies, Inc.
`IPR2017-01051
`Exhibit 1037
`
`

`

`D-glucose concentrations tested ( 1, 5 and 11 mM), single(cid:173)
`point calibrations may be sufficient to correct for the
`atterluation of current yield during storage. Continuous
`
`<l'.
`c:
`
`<l'.
`c::
`"."'
`
`0
`
`40
`
`80
`
`120
`
`160
`Ti me, s
`
`200
`
`240
`
`280
`
`320
`
`Fig.9
`
`Time dependence of the response of the transducer. The
`transducer was tran~ferred from buffer lacking glucose to
`buffer containing 5 mM D-glucose at the time indicated.
`Conditions were otherwise as
`for Fig. 6.
`t9o% =
`48·8 ± 7·38 s (n = 5)
`
`operation of the transducer at 37°C in buffer containing
`5 mM D-glucose results in a 55 per cent decrease in the
`measure current after 50 h (Fig. 11). 17 per cent of the initial
`current is lost during the first 5h of operation, but from this
`time onwards the drift is approximately linear with a mean
`value of0·83 per cent of the initial current per h (r = 0·891).
`
`4 Conclusions and discussion
`The development of a dip-coating procedure employing
`polyurethane for the preparation of electroenzymic trans(cid:173)
`ducers was considered to be of high priority for several
`reasons.
`
`(a) The application of dip-coats rather than preformed
`membranes generally yields a
`smoother surface
`geometry. Since surface irregularities encourage throm(cid:173)
`bus formation and foreign-body responses (WooDWARD,
`1982), dip-coated surfaces might diminish the incidence
`of thrombus formation during intravascular use and
`inflammation during subcutaneous use.
`(b) Dip-coating procedures combine high mechanical
`stability with comparative simplicity of membrane ap-
`
`c::
`"' L
`
`L
`:J
`u
`
`-ro
`·-c:
`
`Ii'-
`
`r·, ::o·,
`
`I
`
`Ill
`
`100
`
`90
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`r------------- ~ ·---
`
`=.
`
`·---·----·---
`
`·--·-- ..
`
`.. I
`
`0
`
`2
`
`3
`
`4
`
`5
`6
`7
`Time. days
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`Fig. IO Stability of hydrated dip-coated trans(cid:173)
`ducers to long-term storage at 21°C.
`The transducers were stored in Miller &
`Golder's buffer (pH 7-44; 0·1 I) and at
`the times indicated were tested to de(cid:173)
`termine their response to D-glucose at
`concentrations of l, 5 and 11 mM. Each
`bar is the mean ±SE (n = 5) of the
`initial current. The smooth line(---)
`is drawn through the mean percentage of
`the initial current calculated from the
`sum of the percentage initial currents for
`each of the three D-glucose concent(cid:173)
`rations. Conditions were otherwise as for
`Fig.6
`
`~ 1 mM D-glucose
`D 5 mM D-glucose
`ml 11 mM D-glucose
`
`100
`90
`
`-:= .. 80
`
`=>
`u
`
`m
`
`70
`60
`50
`40
`c: ·-
`30
`~ 20
`10
`
`(c)
`
`(d)
`
`plication and thereby facilitate the fabrication of small
`electrodes.
`Of the polymers that are available commercially, the
`polyurethanes appear to be among the most suitable for
`in vivo use. They are mechanically stable, elicit a
`comparatively small tissue reaction and are elastomeric
`and consequently do not leach plasticisers.
`The polyurethanes are very resistant to g-irradiation
`(Irradiation Products Ltd., Swindon, England); a
`common procedure for the sterilisation of transducers
`for in vivo applications. (Providing that the dip-coated
`transducers are dry when irradiated, there is only a 20-
`40 per cent loss of enzyme activity after sterilisationt. As
`the response characteristics of sterilised transducers are
`similar to unsterilised ones, irradiation may therefore be
`an appropriate method of sterilisation.)
`
`The results of the in vitro tests of the dip-coated glucose
`trransducers described above are therefore encouraging.
`When the dip-coating solutions are optimised with respect to
`
`t Unpublished observations by Rea
`
`10
`
`15
`
`20
`
`25
`Time. h
`
`30
`
`35
`
`40
`
`45
`
`50
`
`Fig.11 Stability of glucose current during continuous operation of
`the transducers in the presence of 5 mM D-glucose at 37°C.
`The transducers were hydrated for approximately 1 h in
`Miller & Golder's buffer and then transferred to buffer
`containing 5 mM D-glucose. Conditions were otherwise as
`for Fig. 6. Data taken from continuous chart recorder
`recordings, mean ±SE (-.-.); line of best
`fit,
`y = -0·83x+(85-48±6·98) where y =percentage initial
`current and x =time (h), computedfrom 4·5h onwards by
`least-squares method(--) (r = 0·891; n = 5)
`
`Medical & Biological Engineering & Computing
`
`March 1985
`
`113
`
`Dexcom Inc. v. WaveForm Technologies, Inc.
`IPR2017-01051
`Exhibit 1037
`
`

`

`glucose oxidase, cellulose acetate and polyurethane concen(cid:173)
`tration an electroenzymic glucose transducer with satisfac(cid:173)
`tory performance characteristics can readily be fabricated.
`The range over which the steady-state current is propor(cid:173)
`tional to D-glucose concentration encompasses both normal
`and abnormal blood glucose concentrations, thus enabling
`diagnostic applications, and the pH and temperature de(cid:173)
`pendencies of the glucose current are sufficiently small in the
`physiological range for pH and temperature compensation
`to be unnecessary. The range of linearity may have to be
`extended, by, for example, increasing the polyurethane
`content of the second dip-coat, for the continuous monitor(cid:173)
`ing of a known diabetic where blood glucose concentrations
`are frequently in excess of 12 mM (SINCLAIR, 1979). The
`response time of the transducer is sufficiently short to ensure
`continuous and accurate measurements of changes in blood
`glucose concentration. Perhaps the major shortcoming of
`the transducer, and one that is common to most electro(cid:173)
`enzymic transducers (SoELDNER, 1982), is its stability during
`prolonged use. When the transducer is operated continu(cid:173)
`ously in the presence of glucose at body temperature there is
`a steady and persistent decrease in current. As more than half
`of the initial current is lost after 50 h of operation, the
`transducer is clearly not suitable for long-term implantation.
`However, for short-term experimental or diagnostic pur(cid:173)
`poses the transducer is satisfactory on the condition that drift
`can be compensated by periodic calibrations. We are
`currently developing polyurethane bilumen catheter glucose
`transducers for intravascular use, and these are being tested
`in vivo. The flow and p0 2 dependence of the transducer will
`be evaluated in this measurement situation. Since H 20 2
`efflux from the surface of the transducer is an important
`determinant of the steady-state current (Section 2.4), the
`influence of tissue and blood catalase activities, which will
`contribute to the outwardly directed diffusion potential for
`H 20 2 and therefore change the response characteristics of
`the transducer, will also be determined.
`
`Acknowledgments-The authors wish to thank the MRC for
`financial support during the course of this investigation and the two
`anonymous reviewers for useful comments.
`
`References
`ALBISSER, A. M., LEIBEL, B. S., EWART, T. G., DAVIDOVAC, Z.,
`BOTZ, C. K. and ZINGG, W. (1974a) An artificial pancreas.
`Diabetes, 23, 389-396.
`ALBISSER, A. M., LEIBEL, B. S., EWART, T. G., DAVIDOVAC, Z.,
`BOTZ, c. K., ZINGG, W., SCHIPPER, H. and GANDER, R. (1974b)
`Clinical control of diabetes by the artificial pancreas. Ibid., 23,
`397-404.
`BAUMAN, E. K., GOODSON, L. H., GUILBAULT, G. G. and KRAMER,
`D. N. ( 1965) Preparation of immobilized cholinesterase for use in
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`HESSMAN, S. P. and SCHULTZ, R. D. (1973) Prototype glucose
`oxygen sensor for the artificial pancreas. Trans. Am. Soc. Artif
`Int. Organs, 19, 361.
`CARISTY, M. GREEN, A. CHRISTAU, B., KROMANN, H. and NERUP,
`K. (1979) Epidemiological studies of insulin dependent diabetes
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`DAWSON, R. M. C., ELLIOTT, D. C., ELLIOTT, w. H. and JONES,
`K. M. (1959) Data for Biochemical Research. Oxford University
`Press, London.
`DECKERT, T., POULSON, J.E. and LARSEN, M. (1978) Prognosis of
`diabetics with .diabetes onset before the age of thirty-one. I:
`Survival, causes of death and complications. Diabetologia, 14,
`363-370.
`
`EADIE, G. S. (1942) The inhibition of cholinesterase by physostig(cid:173)
`mine and prostigmine. J. Biol. Chem., 146, 85-93.
`ENGERMAN, R., BLOODWORTH, J.M. B., and NELSON, S. (1977)
`Relationship of microvascular disease in diabetics to metabolic
`control. Diabetes, 26, 760- 769.
`GUILBAULT, G. G. (1980) Use of enzyme electrodes in biomedical
`investigations. In Medical and biological applications of electro(cid:173)
`chemical devices. KORYTA, J. (Ed.), John Wiley & Sons Ltd.,
`London.
`GUILBAULT, G. G. and LUBRANO, G. J. (1973) An enzyme electrode
`for the aperometric determination of glucose. Anal. Chim. Acta,
`64, 439-455.
`HICKS, G. P. and UPDIKE, S. J. (1966) The preparation and
`characterisation of lyophilised poly