`
`Anal. Chem. 19”. 60. 2781—2786
`
`2781
`
`transition time in the submillisecond region can be obtained
`by this technique. The transition time obtained with a 40-uA
`preset current is 885 us. Since the geometry of the gold
`microwire electrode is uncertain, the calculation of the the-
`oretical value for the transition time is not attempted. The
`efi'ective scan rate for this chmnopotentiogram is about 1100
`We. Cyclic voltammograma at similar scan rates were only
`obtained with an ultramicroelectrcde after signal averaging
`(25). It also shows that the top and bottom portions of the
`tramient are partially removed due to the decay of the current
`transient to the preset level in less than one cycle time (125
`us). The cycle time and thus the background overcorrecticn
`corddbeminimizedhycollecting dataatshorlertimolntervala
`with smaller potential pulses. We also note that the shape
`of the transients is sharper then described by eq 2. Similar
`behavior was predicted in potentlometric stripping analysis
`(26) and 000 with an ultramicroelectrods (27}.
`In conclusion. the very low background achieved by sam-
`pling the current on the pulse and by the use of a dynamic
`background correction is essential for the characterization of
`chronopotentiogrems in the millisecond level. The analytical
`utility of SPPC is clearly demonstrated by the fast snodic
`stripping analysis of ppb-level Odin) and Phill) in aqueous
`solution.
`It appears that an accurate measurement of a
`submlllisecond transition time may require a faster data ac-
`quisition frequency.
`
`LITERATURE CITED
`J.
`J. fiscltnanal. Chem. 1900. 1.879.
`.
`(1) Lingano.
`(2) Rm. W H. Anal. Gram. 1.01. 83. 485—487.
`(a) Anson. F. c Anal. Chem. 1 01. 83. 1123-1124.
`(4) ley. FL 2!. McIntyre. J. D. E. J. Am. Glam. Soc. 1085. 87.
`sacs—cu .'
`
`(5) emu. W. D.; Hogs. F. E.: Mueller. 1’. FL: Jonas. H. c. Anal. dram.
`1.05. 57. 1415-1410.
`(6) Stu-rout. P. E.; Hiram. J. L.: Vaudeuii. 3.: O'Brien. 6. E. J. Electro-
`cham. Soc. 1075. 122. 1195—1200.
`1311431 .
`(7) Sorrow.
`Es Vainwil. a. J. Elem. Soc. 1015. 122.
`(B) Shrrock. P. E.: Ellison. R. H. J. Ebolrodram. Soc. 101'. 128.
`629—031.
`(ll) Kale. Y.: Yamada. Ar. Yoshido. H.: Unowm K.; Van-ha. N. M.
`Chem. Soc. Jpn. till. 5!. 176-180.
`(10) admirer. or. A: Last. T. A. Anal. Gram. int. 53. 2095—2100.
`11) ransom. A: Quanta. (3. Anal. Groin. loll. so. EDS-50?.
`12) fiance. o. W; Rowena. T. Anal. Mom. 108?. 59. l31—134.
`13} Sawyer. D. “L: Roberts. J. L. Jr. W! Warning: for
`mu: Wiley: New Your. 19'“: p 7?.
`(14} Aubersai. E. 5.: Old-ism. K. B. 9m February 19”. inf-218.
`(15} Olrnstsad, M. L.; Nifllolaon. R. 8. J. Flt”. M. 19H.
`1350-1550.
`
`.72.
`
`17,
`.
`(16) do v“. T. J. Electroanal. Glam. InMaclaIEieclrocham. ‘Ilsl.
`(17) Rodgers. R. 8.: Melisa. L. J. Eloctmanal. Glam. lnl‘erlaclalEIsm-
`chsm. 10“. 19. 1—11.
`
`(20)
`
`Hoard-rial. Chem. [Meal Eben-odious. 1072. 34.
`(18)
`(19) Porous. S. P.: annlioid. A. J. Eisetrcanal. am. Inmwlficlm-
`cilsm. 1967. 18. 124-131.
`l-hiiiang. H.: Yam. 0.; Emu. L. Anal. OM". Act 10.7. 202.
`111-122.
`1 9.
`:téflg-iang. H.: Yagner. D.: Rsrvrssn. L. Anal. cm. Aces 1sa1. 202.
`(21)
`(22) Baranaski. A. 8. Anal. Chem. "IT. 50. 862—666.
`(23) Kcu-Iavea. 8. P.: O'Dea. J. J.; Chem. P.; Ostsryomg. J. Anal.
`Chem. 19". 59. 386—339.
`(24) Mid. it: arm-ole. K.: Tokens. K.: “much. .i.: Octal-young. J. J. Elec-
`mm.amn.rmwchcaem.1sss, rar. 231.
`(25)
`tiowoii. J. 0.; Wignman. B. M. Ami. Gum. 1|“. as. 524429.
`(26) balsam. it: Casinos. .i. F. Anal. mam. was. 5?. senses.
`(27) Gan. 2.: SM. J. 0.: Adam. R. N. J. Esciroanal. Glam. 1m-
`claiHsclmdrsm. 1932. 1.95. 1.
`
`RECEIVED for review April 11. 1988. Accepted September 23,
`1988.
`
`Covalent Enzyme Coupling on Cellulose Acetate Membranes
`for Glucose Sensor Development
`
`Robert Sternherg.l Dilblr S. Bindra,‘ George S. Wilson." and Daniel R. 'l‘hévenot*-l
`
`Laboratoire de Biaélectrochimie et d’Analyse du Milieu. U.F.R. de Sciences et Technologie, Université Paris—Val de
`Marne, Avenue du General de Gaulie, 94010 Créteii Cedex, France, and Department of Chemistry, University of
`Kansas. Lawrence. Kansas 66045
`
`Maillodslorhlnoblzirigoiucossoxldass(60x)oncsiiuioss
`acslala(cA)msmhranosarocomparsd. momma-rod
`hvoivssoovaisnioorplngolbcvhsssnmalbuninflsnio
`0A membrane and a subseqqu reaction of the mombrans
`with 60x. which has previously been activated with an ox-
`ossa oi' p-bonzoqulnons. This coupling procedure is fairly
`rsproducibis and allows the praparaflon of thin membranes
`(5-20 pm) showhg him sis-lacs activities (1-3 U/a'n') which
`arsslabisovsrapsriodoH-amonlhs. Elselrochanlcaiand
`rsaolabslhg experiments show that snzyms Inactivation as
`a result of immobilization Is negligible. A good correlation
`between sulaco activity of membranes and their 60:: load
`is observed.
`
`INTRODUCTION
`The performance of an enzyme electrode is ultimater de-
`pendent on the ability of its enzymatic membrane to sustain
`
`‘Author to whom coma
`lUniversité Paris—Val
`3 University of Kansas.
`
`ndence should be directed.
`e Mama.
`
`and protect the enzyme. Among the available methods of
`enzyme immobilization. four methods are currently used:
`adsorption followed by reticulation with bifunctionsl magenta
`such as glutaraldehyde (I), covalent coupling between enzyme
`and activated support (2—5) or activated enzyme and support
`(6-8), and reversible immunological coupling (9). Those in-
`volving covalent coupling to solid supports are of great interest
`since they generally yield the best activity stabllities (10).
`Nevertheless two difficulties may be encountered:
`low levels
`of activatable or activated surface groups on the support and
`denaturation of enzyme if covalent coupling is accomplished
`through functional groups of the enzyme which are essential
`to its catalytic activity. Highly active and stable membranes
`may be prepared by say] azide activation of reconstituted
`collagen films (2, 11). However such membranes have been
`found to be too thick and too fragile, especially at 37 °C. to
`be recommended for in vivo applications of enzyme electrodes.
`As cellulose acetate (CA) membranes of different thickness
`and permeability may easily be prepared by film casting or
`coating and because they exhibit significant permselectivity
`toward anions (12), we have studied their ability to support
`enzyme and be used for an in vivo implantable glucose sensor.
`
`0003-2700/85/0360-2181801.60/0 © 1988 American Chemical Society
`
`
`
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`2782 I ANALYTICAL CHEMISTRY. VOL. 80, NO. 24. DECEMBER 15. 1988
`
`
`
`(a)
`Figure 1. Schematic dlagram oi rotating membrane electrode:
`misting disk electrode shaft and electrical contact. (b) Kel-F body. (a)
`threaded cehr. (d) membrane sipped cap. (e) putimm electrode, and
`(f) membrane.
`
`The literature is abundant with reports of glucose assets
`and many of these are used routinely for in vitro clinical
`determinations. Despite a history of more than 25 years (13),
`no completely reliable implantable sensor has yet been de-
`veloped (14). For such applications a miniaturized sensor is
`needed and the fabrication of such devices through multilayer
`film deposition is a major problem. It is quite evident that
`miniaturized sensors will yield high sensitivity and stability
`only if the enzyme layer and the associated polymer layers
`can be deposited in a reproducible fashion. Commercially
`available membranes are not suitable for this purpose. The
`enzyme can be immobilized and protective polymer films
`deposited by dip coating (15). Because of the low success rate
`(«0%) associated with such a technique. there exists a need
`for optimization of procedures for both enzyme and protective
`layer preparation. Starting with an intact membrane provides
`a defined system for the optimization process.
`This paper proposes and evaluates a method of glucose
`oxidase (G01) coupling to CA membranes. These membranes
`have initially been activated and coupled to bovine serum
`albumin (BSA) in order to increase the number of functional
`groups to which GOx may be coupled and to impart a protein
`environment to the enzyme. 60: is coupled to CA—BSA
`membranes by using p-benzoquinons (PBQ) as bifunctional
`reagent (9, 16, 17). Use of radiolabeled GOx, in order to keep
`a track of enzymatic activity and investigate the reasons for
`the loss of sensor response with time, is also discussed. This
`work allows for the correct choice of coupling procedure and
`support before the whole glucose sensor is assembled.
`
`EXPERIMENTAL SECTION
`
`instrumentation. G0: activity of membranes was determined
`by continuous monitoring of enzymaticslly generated hydrogen
`peroxide on a combined platinum disk electrode (anode diameter
`2 mm) (YSI Model 2150) covered with a general purpose cellulose
`acetate membrane. This electrode was connected to a YSI Model
`25 Oxidsse Meter (Yellow Springs Instruments, Yellow Springs,
`01'!) and to s potantiometric recorder (Curken). Determinations
`of solution and membrane radioactivity were performed on a LKB
`Waller: 80000 automated gamma counter. 60: membranes were
`mounted on a rotated platinum disk electrode (anode diameter
`3 min) using a specifically designed membrane holder tip (Figure
`1). The rotation speed of the membrane and anode was controlled
`by a Soles-Tscussel (Lyon, France) EDI motor and Controvit
`power unit. A Ag/AgCl reference and a platinum wire auxiliary
`electrode were connected. together with the platinum disk anode,
`to a BAS LC4A amperometric detector (Bioanalytical Systems.
`West Lafayette. IN) and potentiometric recorder (Curken). All
`experiments were performed in thermostated cells at 37 ’0.
`
`Chemicals. Reagents. Pure p-benzoquinone (PBQ) was ob-
`telan from Merck and was recrystallised from petroleum ether.
`Sodium borobydrids from Sigma [98%) was used and Ceriumil'v')
`sulfate—Z-(sulfuric acid) was obtained from Ali‘s Division (Ven-
`tron). Acetone and ethanol (both AR 100%) were obtained from
`Mallinokrodt. Cellulose acetate (CA) with 39.8% aoetyl content,
`was purchased from Aldrich Jansen Chimioa. Glucose. hydrogen
`peroxide. and phosphate salts were reagent grade and all solutions
`were prepared in doubly distilled water.
`Proteins. Glucose oxidase (GOx) isolated from Aepergillus
`niger, (grade VII. 136000 U/E) and bovine serum albumin (BSA)
`fraction V were obtained from Sigma. Rodiochemicals. Naml
`(23 mCi/mL in 0.01 N NaOH) was obtained from ICN Radio-
`chcmicals (Irvine, CA).
`Glucose Oxidase Membrane Preparation. 0. Cellulose
`Acetate Membrane Preparation. With stirring. 1.8 g of CA was
`dissolved in 15.8 g of acetone. and 2.8 mL of distilled water was
`added. After homogenization, this solution was cast on a glass
`plate and evaporated for 60 s at 22 °C to form a thin membrane
`(0.015 mm in the wet state and 0.010 mm in the dry state). The
`CA membrane was removed from the glass plate by immersing
`the plate in distilled water. The membrane was out into smaller
`pieces and stored in distilled water.
`b. Cellulose Acetate Membrane Activation. Procedure A.
`Four CA membranes (2.5 cm x 2.5 cm) were suspended for 3 h
`in 100 mL of 1 mM Ce(IV) solution in 0.1 M nitric acid prepared
`just prior to use. Then the membranes were washed in distilled
`water for 5 min and immersed in 10 mL of 10 mg/mL BSA in
`0.1 M borats buffer, pH 9.0. The volume of BSA solution was
`reduced to about 1 mL and three additions of 2 mg of sodium
`borohydride were made every 20 min at room temperature. After
`a second washing of 5 min in distilled water, the membranes were
`stored in 0.1 M phosphate buffered saline solution (PBS). pH 7.4,
`at room temperature until the GO! coupling reaction was per-
`formed.
`Procedure B. This method is the same as procedure A except
`that a higher Ce(IV) concentration (100 mM) and a shorter ox-
`idation time (20 min) were employed.
`Procedure C.Inthiscase100mLof100mMsodiumperiodate
`(20 min) was employed as oxidizing agent. The membranes were
`washed and BSA added as in procedure A. The Scbifl’base formed
`was reduced for 2 h by adding 4 mg of sodium cysnoborohydride
`to the 1 mL of BSA solution containing the membrane.
`c. Glucose Oxidaae Activation. Freshly prepared PBQ solution
`0.1 mL (15 mg in 1 mL ethanol) was introduced in an alumi-
`num-foil-oovered tube containing 0.5 mL of 60: solution (20 mg
`in 0.1 M PBS pH 7.4). After 30 min of incubation at 37 °C. the
`mixture was filtered through a G-25 Sepbadex column (1 x 10
`cm) coupled with a peristaltic pump (20 mL/h) and eduilibrated
`with 0.15 M sodium chloride. The that fraction, a pink-brown
`band of 2—3 mL, was collected and used as the enzyme coupling
`solution.
`d. Coupling Reaction. The CA-BSA membranes were sus-
`pended in 2—3 mL of activated GO: solution. whose pH was
`adjusted to 8.0—9.0 with 0.25 mL of 1.0 M sodium carbonate
`solution. After 38 h of incubation at room temperature. the
`membranes were removed. washed in stirred 0.15 M potassium
`chloride solution for 24 h and finally stored in 0.1 M PBS, pH
`7.4, containing 1.5 mM sodium azide. The aside was added to
`all storage and experimental buffers to limit microbial contam-
`ination and catalase activity.
`Radiolabeled Glucose Oxidase Preparation. The iodine
`monochloride method described by Helmkamp, Contreras, and
`Bale (18} was chosen to incorporate 1"51 into 60:. A 0.5 mM lCl
`reagent was prepared by adding 4 uL of pure 1C1 to 100 mL of
`2 M NaCl. In a l-mL conical vial was placed 80 pl. of 0.5 mM
`101 and the desired amount of Naml solution. Typically, 1.0-2.5
`uCi of "‘1 was used to iodinate 1.0 mg of protein, depending upon
`the specific activity needed. The desired amount of glucose
`oxidase in a volume of 0.20 mL was added to the vessel with the
`room lights turned off. The contents of the vessel were mixed
`by tapping and allowed to reset for 2 min In the dark. The
`reaction mixture was applied to the top of a G-25 Sephada:
`column (25 X 1 cm) that had been aquilibratad with PBS. pH
`7.4. Elution was continued with PBS at a flow rate of 0.5 mL/min
`and l-mL fractions were collected. The effluent was monitored
`
`
`
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`ANALYTICAL CHEMISTRY. VOL. 60, NO. 24, DEGEWER 15, 1988 e 2183
`
`with a gamma counter and the first fraction, containing labeled
`protein, was collected. The pooled protein was concentrated to
`about 1.5 mL by using pressure dialysis. The ratio of protein-
`bound iodide to total iodide was determined by the triahloroacetic
`acid (TCA) precipitation method (19). The value, expressed as
`efficiency of labeling, was usually 95-98%. The same data were
`used to calculate the specific activity of the iodineted glucose
`oxidase in microcuries of 1"‘I attached per milligram of glucose
`oxidase. The spectrophotometric determination of enzymatic
`activity of rsdiolabeled glucose oxidase solution showed retention
`of “5% of the original activity.
`Glucose Oxidase Membrane Characterization. a. Enzy-
`matic Surface Activity Determination. The hydrogen peroxide
`anodic sensor, covered with a nonenzymatic CA membrane, was
`immersed into a thermostated cell (37 ’0) containing 10 mL of
`PBS. When the background current was stabilized, is. after 10
`minoi
`on,0.1mLof1Mgiucoeestandsrdsoiutionwas
`added to the buffer solution. The incremental production of
`hydrogen peroxide was measured by placing the (EA-BSA-
`PBQ—GO: membrane in the stirred buffer solution and then
`removing it. This process was repeated several times. The re-
`sulting increase in current. after suitable calibration with hydrogen
`peroxide standards, could be used to estimate the membrane
`surface enzymatic activity.
`b. Sensor Response. The rotating membrane electrode was
`dipped into a thermostated cell at 37 °C containing 25 mL of PBS.
`When the background current was stabilized, Le. after about 20
`min of polarisation, several 0.026~OJ25 mL additions of 1 M
`glucose standard solution were performed until a 15—20 mM final
`concentration was obtained. Then one or two 26-uL aliquots of
`0.1 M hydrogen peroxide were added to the buffer solution.
`Response was calculated by comparing steady-state current either
`to the background current (i5) prior to any glucose addition or
`to the steady-state unrent corresponding to the previous addition.
`Thus either I—Igvs Cor MIMI“ Goring Courses were plotted.
`c. Immobilized Enzyme Determination. The mass of immo-
`bilized ‘30: was estimated from the 1 activity of the membranes
`prepared by using rsdioiabeled GO: and the specific activity of
`radiolabeied enzyme solution. With the initial enzyme activity
`orb-ophiiised powder (Ufg) and a 15-20% decrease ofthis activity
`due to the radiolsbeling procedure taken into account, a further
`estimation of membrane activity expressed in U/cm' was ac-
`oomplished.
`
`RESULTS AND DISCUSSION
`
`The purpose ofthis work was to find a suitable method that
`allows the efficient and reproducible coupling of GOx to a
`support suitable for an in vivo glucose sensor. We were
`particularly concerned with the methods and support mate-
`rials that could easily be adaptable to the preparation of
`needle-type microsensors. Thus all commercially available
`solid membranes were avoided and we restricted ourselves to
`cast CA films as support material.
`Cellulose Acetate Membrane Activation. Bovine serum
`albumin (BSA) (1. 3, 6, 7), gelatin (20), or collagen (2, 8) are
`frequently used in enzyme immobilization techniques (21).
`They bring to the enzyme a proteic environment which seems
`favorable for its stability (11). As CA membranes usually
`possess low levels of accessible hydroxyl groups for covalent
`enzyme coupling, we have decided to increase the number of
`reacting groups by fuel: covalently coupling BSA to CA (Figure
`2). BSA contains more than 57 active amino groups per
`molecule and may be covalently grafted onto the free aldehyde
`groups obtained by CA hydroxyl group oxidation (Figure 3).
`The imine functions are subsequently reduced with boro-
`hydride. We have compared three different procedures for
`CA membrane activation and CA—BSA membranes from each
`procedure which were reacted with identical activated G0:
`solutions (vide supra). Comparisons of the properties of GOx
`membranes prepared by these three procedures (Table D show
`that the best activities are obtained with procedure C, is.
`periodate oxidation followed by cyanoborohydride reduction.
`The use of cyanoborohydride, which is a milder reducing agent
`
`OH —.—.—p
`
`cHi-NHO
`
`NH:
`
`NH.
`
`NH,
`
`NH!
`NH.
`
`H-PBO-NH/
`
`3‘
`
`CHfliH-
`
`NH:
`9 NH,
`
`NH:
`
`'
`
`H-PBQ-N
`
`'p
`
`N'H
`33°
`
`N‘H
`
`Figure 2. General scheme of 60x coupling on CA membranes: en-
`hancement of CA surface groups by covalent BSA coupling.
`
`Table I. Activation Procedures of CA Membranes and
`Properties of CA—BSA-PBQ-GOx Membranes Obtained‘
`
`activation
`
`CA membrane oxidation
`reactant
`concentration
`duration (min‘)
`reaction with BSA
`concentration (g/L)
`reduction
`reactant
`concentration (g/L)
`duration (min)
`radiolsbeled GOx attached
`(pg-cm")
`
`G0x surface activity
`(U’cnfl)
`
`CA—BSA—PBQ—GOx membrane
`preparation
`procedure procedure
`b
`c
`
`procedure
`a
`
`Ce(lV)
`1
`180
`
`10
`
`BI'L'
`8 X 2
`3 x 20
`
`Ce(IV)
`100
`20
`
`10
`
`BH.‘
`3 X 2
`3 x 20
`
`I0"
`100
`20
`
`10
`
`BH,CN'
`4
`120
`
`3.8 i 1.5
`(n=3)
`0.50 m 0.10
`(n I 3)
`
`8.0 i 2.8
`("'5)
`1.0 i 0.40
`(n = 5)
`
`13 :i: 1.8
`(rt-‘5)
`2.8 i 0.40
`(n = 5)
`
`“Amounts of GO: and activities are expressed as mean :2
`stande deviation for a set of membranes prepared identically.
`All GO: masses and activities are referred to membrane surface,
`is. 0.8 cm .
`
`than borohydride (procedure A and B), probably prevents
`breaking of disulfide bonds of the BSA protein.
`Glucose Oxidase Activation and Coupling. Following
`a procedure described by Avrameas et a1. (16'), we used p-
`benzoquinone (PBQ) for activating GOx solutions. 60: was
`treated with an excess of PBQ, reagent known to react with
`proteins (17), and the excess PBQ was separated by size ex-
`clusion chromatography (Figure 3). Most of the above-men-
`tioned coupling reactions take place at low temperature, low
`ionic strength, and within the physiological pH range. The
`reason for treating GO: and not the amino groups of the
`CA-BSA support with PBQ is that it allows GOx-PBQ to react
`
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`2704 I ANALYTICAL CI'EMISTRY, VOL. 60, NO. 24. DECEWER 15. 1988
`untrue activation
`
`34)” Ce(lV)eri0i
`exllstles
`
`34M)
`
`Ill-II,
`
`3'CH=N'BSA 4' H30
`
`i-cam-esa
`
`BH.‘ er BH,CN'
`————->
`rel-atlas
`
`i-CHrNH-BSA-NH,
`
`enzyme activation
`
`GOx-NH,
`
`stapling relation
`
`p-teusqslssls 60" ‘NH
`
`later-allele
`
`tern
`
`H
`
`H
`
`O
`l
`l
`
`use" of 'IQ 60x -NH
`
`activated : :
`
`feral
`
`10'
`
`a CH NH BSA NH
`'
`1'
`‘
`'
`
`activated to:
`: "“——"
`
`i-CHfNH-BSA-HH
`
`Figure 3. Chemical reactions involved In CA-BSA-PBQ-GOX membrane preparation.
`
`separation of use” of p" (septum I25)
`
`OH
`
`OH
`
`NH—GOx
`
`S
`
`‘9
`
`
`
`canlambs-useActivity(U/cznz b
`
`n
`
`D
`
`I
`
`I
`
`Ll
`
`with CA—BSA as well as with other GO: molecules allowing
`multilayer enzymatic fixation. In order to check the possible
`effect of PBQ on the enzyme activity. G0x activities of all
`solutions were measured during different steps of this acti-
`vation procedure. Taking into account variation of solution
`volumes, spectrophotometric and electrochemical determi-
`nation of G0: activities in 0.1 M glucose solutions showed
`that GOx-PBQ solutions yield 92-97 % initial activity. This
`result indicates that no significant reduction of GQx activity
`is occurring during this activation procedure. As reaction
`between G01 and PBQ is performed in an excess of PBQ, the
`G0x-PBQ product exhibits quinone properties and may
`continue to react with proteins such as CA—BSA to yield
`CA—BSA-PBQ—GOx membranes. Control experiments with
`nonactivated CA membranes showed no significant attach-
`ment of the enzyme.
`Characteristics of Glucose Oxidase Membranes Used
`for Glucose Benson. The characterization of the enzymatic
`membranes is important for the evaluation of various enzyme
`attachment procedures. We used three different approaches
`for such a characterization:
`(a) membrane surface activity,
`determination using continuous electrochemical monitoring
`of hydrogen peroxide in a stirred 0.1 M glucose solution; (b)
`mounting the membrane on a rotating disk electrode tip
`(Figure 1), for well-defined hydrodynamics, and determination
`of sensor calibration curve to glucose: (c) incorporation of a
`radiolabel in GOx and determination of the mass of G0: in
`solution or on the membrane.
`
`The first method is simple and rapid but not very accurate
`since hydrodynamic conditions are not well defined when
`membranes are freely stirred in the measuring cell. The
`second method is more accurate but takes into account
`membrane permeability to substrates and reaction products;
`furthermore a good contact between these thin membranes
`andtlieplafinumdiskisnottriviaL'Finallyfihethirdmethod
`is definitely the most complicated because it requires n‘I—GOx
`preparation and characterization, but it is also the most ac-
`curate and sensitive. Figure 4 shows the correlation between
`enzymatic activity, as monitored by surface activity, and the
`mass of immobilized enzyme. The mass of enzyme on the
`membrane of different membranes Was controlled such that
`the amount of enzyme immobilized could be varied. All these
`data obtained just after coupling show an excellent correlation
`between surface activity and the mass of GQx immobilized.
`Furthermore when enzyme activities of these membranes,
`
`Immobiliser! radiohbelsd cox ( ur/cmz )
`F we 4. Correlation oi GOx surface activities with immobilized
`‘
`l—GOx amount In dlflereni CA—BSA—PBQ—GOx membranes.
`
`estimated from the amount of 60x immobilized, and the
`specific activity of the initial lyophilized GOx powder. were
`compared to experimental activity values, an excellent cor-
`relation was obtained. In fact the measured/ calculated ac-
`tivity ratio was equal to 0.84 :i: 0.29 for six membranes and
`22 experiments indicating that the radiolabeling procedure
`allows a good evaluation not only of total G0: mass but also
`of its enzymatic activity.
`Several experiments involving the use of radiolabeled GO:
`were carried out in order to understand the reasons for the
`loss of enzymatic activity with time, a phenomenon observed
`for most membranes prepared. Figure 5 compares the
`time-dependent loss of enzymatic activity with the loss of
`enzyme mass from the membrane surface. For the first 7 days.
`the removal of enzyme from the membrane is directly reflected
`in the loss of biological activity. After this time, however, the
`biological activity decays much more rapidly. It was surmised
`that this latter phenomenon was due to time dependent de-
`naturation of the immobiliud enzyme resulting from multiple
`covalent attachment. It is possible for the activated groups
`on the membrane surface to react with protein amine functions
`even after the coupling reaction is supposedly terminated.
`Such further BSA—GOx or GOx—GOx coupling may modify
`the enzyme configuration and produce some denatm'ation. To
`prevent this from occurring, 1 M lysine was reacted with
`membranes for 6 h immediately after coupling. The purpose
`of this procedure is to deactivate remaining groups on the
`
`
`
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`
`ANALYTICAL CHEMISTRY. VOL. 60. NO. 24. DECEMBER 15. 1988 0 2705
`
`Table II. Pertinent Parameters for G0: Membranes
`
`parameters
`
`collagen-GO:
`
`Col-GO: + CA
`
`CA + G01
`
`CA—BSA—PBQ—GOI
`
`reconstituted
`collagen
`acyl azi'de
`activation
`
`collagen +
`cel acetate
`acyl azide
`activation
`
`cellulose
`acetate
`entra mant
`wi
`CA
`
`cellulose
`acetate
`covalent coup-
`ling with PBQ
`
`membrane
`material
`attachment
`procedure
`membrane thickness (um)
`
`swollen
`glucose responses:
`sensitivity‘ (mA‘M'l-cm")
`linear range (mM)
`0.001
`0.1
`0.001
`lower limit
`2-3
`5-30
`2—3
`upper limit
`10—60
`10-60
`20—50
`response time transient (s)
`0.5-3
`2—4
`2-4
`steady state (min)
`10-160
`4-8
`120-2000
`stability” (days)
`'All sensor sensitivities are referred to anode area, La. 7.06 mm'. ‘Stabilities are evaluewd for a 50% decrease of sensitivity.
`
`100
`200-400
`
`8-10
`
`100 + 15
`300 + 15
`
`0.01-0.1
`
`0.1
`40
`50-90
`3-6
`
`5-25
`5-25
`
`0.01-0.1
`
`5-25
`5-25
`
`4-10
`
`l h
`
`
`
` hRelativeGOXactivityandamount
`
`3
`
`andannual!
`Relative60Xearth-it:
`
`
`5.
`
`Is
`
`b I
`
`n
`
`e
`
`e
`
`is
`fine ( days )
`
`is
`
`I
`
`as
`
`Flows 0. Relative evolution of mince (30): activity (0) and 1"I-GOX
`amount (X) In a CA-BSAa—PBO-GOX membrane treated wlth lysine
`after coupling.
`2.!
`
`\
`
`0
`
`II
`Tim ( dur- )
`
`II
`
`I.
`
`Flgure a. Relative evolnion of eui‘ace GOx actMty (c) and 11'14an
`(X) In a CA—BSA~PBO-GOX membrane not treated with lyslne after
`coupling.
`
`membrane, thus preventing further coupling. The effect of
`this treatment is shown in Figure 6 where it will be noted that
`the enzymatic activity tracks the mass of immobilized protein.
`Protein is still lost from the membrane at an approximately
`comparable rate. However. in the absence of rsdiochemical
`measurements, membranes showad significant improvement
`on these stability values, probably because of decreased
`handling. A relative activity value as high as 80-85% was
`frequently observed after passage of 2—3 weeks (Figure 7). As
`the membranes were very fragile and unsupported, handling
`them on a routine basis must have caused their partial dis-
`integration. These results demonstrate that enzyme inacti-
`vation is negligible after coupling and that any slight surface
`activity decrease. when observed. is probably related to im-
`mobilized enzyme loss from the membrane.
`Microbial degradation as a possible explanation for the
`observed behavior was ruled out on the basis that azide was
`present as an inhibitor in all storage buffers. The fact that
`no significant release (<1%) of iodide was observed. as con-
`firmed by the repetitive TCA assays (16') on stored iodinated
`enzyme solution also supports this belief.
`Table II presents pertinent analytical parameters for G0:
`membranes when mounted on a platinum anode. The sensor
`sensitivities, as referred to anode area. of the CA—BSA-
`PBQ—GO: membranes are very close to those for highly active
`acyl aside activated collagen membranes (2). As CA mem-
`branes are significantly thinner than reconstituted collagen,
`especially when the latter are swollen, both transient and
`steady-state response times for them are correspondineg much
`shorter. The stability at 37 °C and anion permselectivity of
`CA membranes are also preferable to those of collagen for
`
`(U/cmZ)
`
`SurfaceacIivlly
`
`0
`
`2
`
`‘
`
`I
`O
`Time (days)
`Figure 7. Evolution of surface 60x activity In a CAeBSA—PBQ—GOX
`membranetreatedwlmlysheafiercoupmaandnotrouhelyhendled
`tor radoehemlcal measwemenis.
`
`‘0
`
`‘2
`
`H
`
`development. of in vivo glucose sensors.
`Figure 8 presents calibration curves of sensors prepared with
`such membranes. The linear ranges usually reach 2—3 mM
`glucose for such active collagen-G0! or CA—BSA—PBQ—GO:
`membranes indicating that, for higher glucose concentrations,
`the enzymatic reaction is the rate-limiting step. High linear
`ranges have been obtained either with less active CA mem-
`branes using a GO: entrapment procedure or with collagen—
`GOx membranes covered with nonenzymatic CA membranes
`allowing external diffusion restriction (see Table 11). Indeed
`linear ranges as high as 15 mM are needed for potentially
`implantable glucose sensors (14).
`
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`27“ 0 ANALYTICAL Cl-EMISTRY. VOL. 80. NO. 24. DECEMBER 15. 1988
`
`"#fi
`
`
`
`8SSSan-orrupmo(u/cmz)
`
`G
`a
`I
`re
`II
`It
`Glucose concentration ( In! )
`
`I
`
`Is
`
`In
`
`Flute I. Glucose cellar-Hon caves of sensors prepared mm (0)
`colleen-Gm. (X) CA wllh entrapped G0x. and (A) CA-BSA-PBG—
`GOx membranes.
`
`This study underlines the importance of careful control of
`coupling conditions in order to achieve reproducible immo-
`bilization of enzyme with high activity and stability. The
`coupling methods described here are successfully adapted to
`needle-type microsensors (22). An extended linear range.
`necessary for subcutaneous implantation, is achieved by
`adding an external membrane (polyurethane) with concom-
`itant sacrifice of sensitivity and to some extent response time.
`A complete evaluation of these microsensors is currently in
`progress.
`
`ACKNOWLGMENT
`We thank Uditha de Alwia for numerous discussions and
`suggestions concerning the immobilization chemistry.
`LITERATURE CITED
`(1) Arnold. M. A.; Rechnflz. G. A. Anal. Charm 1082. 54. 2315-2311.
`(2) Mount. D. H.: 00ml. P. H.: Siemberp. H.: Qumran. D. 0. Anal.
`Glam. 10". 61. 90-100.
`(s) Who-rd. L. 3.. Jr.: Cashier. J. F.; Yeo. S. J.; Wolfson. 8. K.. 1.:
`Draeh. A. L.: Uu. G. 0. Appl. Elam. W. 10“. 9. 95—104.
`
`(4) Moody. G. J.: Sandi“. G. 8.: 11m). J. 0. Fl. WI (Lanna)
`13“. 1‘11. 605-609.
`. G. 8.: “sum-.3. J. D. R. W“ (W)
`[5) Moody. G. J.:
`1m. 1‘". 1285-1235.
`[5) Km. J. J.: (as-Moe. V. w. Laws-mick. V. 11.: mm. a. v.
`Bioeenm ms. 2. 35—44.
`(1') Milan. W. H.: Kandy. F. H.: Guarantee. 8. .I.: Vsdparna. P. M. Anal.
`mm. Acts 1|". 1‘58. 60-60.
`161 Nlasclrll. 1.1.: Motown-I. M. A.: P11101011. R.W. m.
`1'88. 15. 143-157.
`(9) De AMI, w. U.: Hill. B. 8.: lulu-lam. B. 1.: Wis-on. G. 5. Mai.
`Gnarn. 1M7. 59. 2585-2601.
`(10) Thivenol. D. R. Diabetes M1002. 5(3). 184-139.
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`(13) Sdielsr. P. w.; Pislfler. D.: Schubert. F4 Rem H.: mm. D.
`lumen: thand floor:7w.A.P.F..Ka-
`rubs. 1.. Wilson. G. 9.. Eds: Oxford Universny Press: New York.
`198?;
`315—346.
`(14) Valve.
`:fieech. EL: mm. D. R. in Room: Fwd-mule
`Wm; Turner, A. P. F.. Karma. 1.. ‘Msm. G. 8.. £115.;
`Lmhrerslly Press: New York. 1981': pp 390—408.
`(16) Shun H.: Kawamorl. H.: Yermsakl. Y. In Home: m.
`his “Apple-flora: Tm. A. P. F.. Knobs. 1.. Wish. G. 8.. Erin:
`Outed unscrew Press: New York. 198?: pp 400—424.
`{1a) Ternynck. 7.: AIR-mess. 3. Ann. rm. (Peril) 1!". 1270.
`197—208.
`(11) Webb. J. L. In Ordnance In Enrymeand Met-bola mom "I: Aca-
`demlc: New York. London. 1985; pp 421-594.
`(15) Momma. R. w.: Camus. M. A.: Bale. w. F. m. J. Appl. Radial.
`feet. 1067.
`(19) Du-Bahn. G. P. Mai. Harem. 10811. 1‘08. 411-418.
`(20) We. J. 1...: Yang. J. 8.: Kuaairabe. H.: ‘I'hnrms. D. W.
`Ebony. 1m. 25. 255145“.
`(21) Met. 8. A. In More. WNWM: Timer.
`A. P. F.. Rambo. 1.. Wilson. G. 3.. Erie: Orlerd University Press: New
`York. 1937: pp 36-99.
`(22) Sternberg. H.: Barren. M.-B.: Ganplotu. L.; Wench D. H.: Bind-a, D.
`5.: Wlleon. G. 8.: Velio. 6.: Froguel. P.: Reach. G Blosmears. In
`press.
`
`RECEIVED for review December 9. 1987. Accepted September
`26, 1988. This work has been supported by “Caisse Nationale
`de’Assurance Maladie des .Travailleurs Salaries” (Grant 0.
`N.A.M.T.S.-I.N.S.E.R.M. No. 85.3.54.8.E). French Ministry
`of Foreign Affairs. National Institutes of Health (U.S.) (Grant
`DK 30718) and “Aide aux Jeunes Diabétiques”. Their fi-
`nancial help is gratefully acknowledged.
`
`
`
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