Amperometric enzyme electrode with fast response to glucose using a layer of lipid-modified glucose oxidase and Nafion anionic polymer

Andy&a

Chmca Acra, 274 (1993) 201-207 Elsevier Science Pubhshers B V , Amsterdam

201

Amperometric enzyme electrode with fast response to glucose using a layer of lipid-modified glucose oxidase and Nafion anionic polymer Fumlo Mlzutam, Solchl Yabulu and Tatsuo Katsura Natwnal Inmtute of Bwscmce

and Human-Technology,

1-I Hlgasht, Tsukuba, lbarakr 305 (Japan)

(Received 21st August 1992, revised manuscnpt recewed 12th October 1992)

AbStIBCt

An amperometnc enzyme electrode for glucose was prepared by usmg a hpld-modlfied glucose ondase and a pexfluonnated monomer(Nafion) lkt, a glassy carbon base electrode was dipped mto a benzene solution of the modlfied enzyme and dned, then the electrode was dipped mto a Nafion solution and dned The water-Insoluble, modlfied enzyme was thus mnnolxhzed on the electrode surface vnth a strongly adhenng, thm polymer coatmg The anodlc current (at 0 9 V vs Ag/AgCl) of this electrode Increased Imme&ately after the addltlon of glucose and the response time was shorter than 2 s A hnear response to glucose was observed up to 3 mM with a detectlon hnut of 0 2 PM The enzyme electrode was apphed to the determmatlon of glucose m fnut ounces, the Nafion coatmg was effectlve m suppressmg the electrochermcal Interference by t-ascorbate m the samples The electrode could be used for at least 6 weeks Keywords Amperometry, Enzymatic methods, Enzyme electrodes, Glucose

AnalytIcal devices combmmg the specticlty of enzymatic reactions with high sensltlvlty of electrochenncal transduction have attracted mcreasmg interest m the last decade [1,2] Particular efforts m this field have been directed to the reahzatlon of an amperometnc sensor for the analytically significant substrate glucose by usmg glucose oxldase (GOD) [3] In conventional amperometrlc glucose-sensing electrodes, three membranes are attached on the surface of base electrode the first 1s used for protecting against foulmg of electrode, the second for munoblllzmg GOD and the third for ehmmatmg electroactrve mterferents Vanous approaches to developmg simple, rapid and reproducrble procedures for Correspondence to F &tam, National Institute of BIOscience and Human-Technology, l-l mgaslu, Tsukuba, lbaralo 305 (Japan)

the construction of glucose-sensmg electrodes have been presented For nnmoblllzmg GOD easily and effectively, mcorporatlon of GOD by murmg the enzyme with carbon paste matrices [4-81, electrodeposition of GOD durmg the anodlc growth of polymers [9-161 and that during the cathodic growth of platmum black [17,18] have been exammed Most recently, Wang et al [19] developed a unique method for unmoblllzmg GOD on an electrode surface with the use of an aqueous solution contammg the enzyme and a poly(ester-sulphomc acid) (AQ-55D, Eastman) the solution 1s placed on an electrode and then dried wtth a heat gun to form a GOD-polymer-coated electrode They also reported the use of a perfluormated monomer (Nafion, DuPont) as the matrix for enzyme unmoblllzatlon [201 Amomc polymers such as Nafion and AQ polymers have been re-

0003~2670/93/$06 00 0 1993 - Elsevler Science Pubbshers B V All nghts reserved

202

ported to serve as protective and selective coatmgs on enzyme electrodes [5,21,22] the polymer coatmg 1s effective for protecting the electrode surface from fouling agents present m blologlcal and food samples [5], and also for excludmg anlomc mterferents (e g , L-ascorbate) m these samples [21-271 The use of such a functional polymer layer as the matnx for enzyme nnmoblhzatron [19,20] 1s an interesting approach to reahzmg enzyme electrodes with a snnple membrane structure A simple and thm enzyme membrane-based system would give a fast response and high sensltlvlty m electrochermcal measurements [ 15,281 However, an electrode using a GOD-anionic polymer system [19] showed poor stability owmg to leaching of GOD out of the polymer matrix It seems natural that water-soluble, negatively charged [29] GOD molecules will leach out of the layer of an anionic lmear polymer In order to circumvent this stab&y problem, the use of a lipid-modlfied GOD [30], instead of native GOD, 1s considered to be suitable the water-msoluble, modified enzyme would be far more stably ammoblhzed m the polymer matrix In this paper, the preparation and use of an enzyme electrode based on such a lipid-modified GOD and an anionic polymer coating is described The lipid-modified GOD was placed on an electrode surface by dip-coating from a benzene solution of the modified enzyme, then the polymer coatmg was applied by dip-coatmg from a Nafion solution The resultmg electrode exhlblted high performance characteristics such as rapid response (100% response, less than 2 s), high sensltlvlty (detection limit lower than 0 2 pm) and high stab&y (usable for at least 6 weeks)

EXPERIMENTAL Mater&s

The enzymes used were GOD (EC 1 13 4, from Aspergdlus sp , Grade II, Toyobo, Osaka) and peroxldase (EC 1 11 17, from horseradish, Grade I-C, Toyobo) An amphlphlle, iV-(cu-tnmethylammomoacetylMldodecyl-L-glutamate chloride [(C,,),gluN+Cl-I, was obtained from

F Muutanr et al /Anal Chm Acta 274 (1993) 201-207

Sogo Pharmaceutical (Tokyo) and Nafion [5% solution, 1100 equlv wt I, from Aldnch (Milwaukee, WI) F-luts (Boehrmger Mannhelm, Indlanapohs, IN) were used for the spectrophotometric measurement of glucose and L-ascorbate The ktt for glucose uses the enzyme pair hexohnase-glucose-6-phosphate dehydrogenase Other reagents were of analytical-reagent grade DelonIzed, doubly dlstllled water was used throughout L~phnod$ed enzyme GOD modified with (C,&,gluN+ was prepared according to the procedure of Okahata et al [30] A buffer solution (5 ml, 0 02 M potassmm acetate buffer plus 0 1 M KCl, pH 6) containing 25 mg of GOD was mixed with an aqueous dlsperslon (50 ml> of 100 mg of (C,,),gluN+ClThe precipitate formed after mcubatton of the mixture at 4°C for 24 h was lyophrllzed A light yellow powder (ca 100 mg) was obtained The GOD content m the modified enzyme was determined by measurmg the absorption of flavm ademne dmucleotlde (FAD) m a benzene solution of the lyophlhzed product at 450 nm The enzyme actlvltles of the unmodified and modified GOD were measured spectrophotometncally by using a peroxldase-phenol-4-ammoantlpyrme chromogenic system [311 The solution (or dlspersed medium m the case of the modified enzyme) used for measuring the GOD activity was a 0 1 M potassmm phosphate buffer (pH 7 0,25”C) Enzyme electrode system A glassy carbon disc electrode (GCE) (Bloanalytrcal Systems, West Lafayette, IN) 3 mm m diameter was polished with a 0 05-pm alumma slurry, rinsed with water and then somcated m water for 2 mm The electrode was activated by anodlzatlon at 15 V vs Ag/AgCl for 30 mm m a 0 05 M potassium phosphate buffer solution (pH 7 5) [32], rinsed with water and dried After the pretreatment, the GCE was dipped into a benzene solution of the modified GOD [2% (w/v)1 and the solvent was allowed to evaporate at room temperature for 5 mm Finally, Naflon membrane coatings were made by dip-coating the enzymeattached GCE m 0 5% (w/v) Nafion solution, which was prepared by diluting the 5% solution

F iUuutanr et al /And

Chm Acta 274 (1993) 201-207

as recerved with a mtiure of 2-propanol [50% (v/v)] and water 1211, and the electrode was allowed to dry mth the surface facing down [51 at room temperature for at Ieast 1 h The Naflon coatmg and drymg process was repeated three times unless stated otherwise The thickness of the resultmg modlfied GOD-Naflon layer obtamed m this way was determmed as ca 0 5 ,um by using a stylus-type apparatus (Talystep, Taylor-Hobson, Lelcester) Other enzyme electrodes, an electrode with a native GOD-Naflon layer and one with a modlfled GOD-acetylcellulose layer, were prepared as follows The electrode based on native GOD was made by casting 5 ~1 of GOD solution [0 5% (w/v)] on the GCE surface, allowing the enzyme solution to dry for 1 h and by coating three times with the 0 5% Naflon solution The electrode with an acetylcellulose coating was prepared by the use of an acetone solution of 0 5% (w/v) acetylcellulose instead of 0 5% Naflon solution A potentlostat (I-W-502, Hokuto Denko, Tokyo) was used m a three-electrode conflguratlon for amperometrlc measurements the enzyme electrode, an Ag/AgCl reference electrode (Bloanalytical Systems) and a platmum auxdrary electrode were unmersed m 10 ml of a test solution of 0 1 M potassium phosphate buffer (pH 7) m a cyhndrlcal cell The solution was saturated with air and stirred by using a magnetic bar The temperature of the solution was kept at 25°C

RESULTS AND DISCUSSION

Proper&s of modtfted enzyme The GOD content m the lyophlhzed powder of

the modified enzyme was calculated to be 33% In neutral media, GOD 1s highly amomc (expressed as GODWe) [28], hence it 1s assumed that II molecules of lipid attached on the surface of GOD form a complex expressed as n[(C,,), gluN+l GOD”- (n - 80X1- From the GOD content m the complex and the molecular weight of native GOD (15 X 10’1, n 1s calculated to be 490 The cross-sectional area of (C,&luN+ can be calculated to be 0 45 nm’, as that of an analogue, N-(a-tr~methylammomoacetyl)d~hexade-

203

-I Time

Fig 1 Current-time curves for (A) the GCE coated with a modtiled GOD-Naf~on layer and (B) that with a native GOD-Nafion-layer, obtained on mcreasmg the glucose concentratlon m 0 2 mM steps

cyl-L-glutamate [(C&gluN+], has been evaluated as 0 45 mn2 from the surface pressure-area isotherm of (C,,)&N+ on a water subphase [301 GOD 1s a globular protein with a diameter of 8 nm [331, hence the number of (C12),gluN+ that can be directly attached to the GOD surface is calculated to be 450 This value 1s compatible with the n value obtained, (C,,),gluN+ molecules are considered to form a monolayer on the GOD surface The GOD actlvlty of the modtiled enzyme was determined to be 38 U mg-’ solid, which corresponded to 115 U mg- ’ GOD The GOD activity of the unmodified enzyme was determmed to be 110 U mg-’ These results show that the enzyme actlvlty does not decrease during the modlficatlon process and that the enzyme substrates, glucose and oxygen, permeate easily mto the hpld layer to reach the active site of the enzyme Glucose response of enzyme electrode

Figure 1 shows the current-tune curves for the GCE coated Hrlth the modrfled GOD-Naflon layer and that with the native GOD-Naflon layer The potential of each electrode was set at 0 9 V vs Ag/AgCl The current on each electrode mcreased unmehately after the addition of glucose and reached another steady state wlthm 2 s As the Nafion layer is thm, the glucose added 1s expected to diffuse quickly through the layer so

204

as to be oxldrzed by the enzymatic reaction Hydrogen peronde produced through the GOD reaction m the vlcmlty of the GCE surface 1s nnmedlately oxldlzed to give an anodlc current These rapid processes of substrate diffusion and electrochemical oxtdatlon result m a fast response on the enzyme electrodes As shown m Fig 1, the glucose response of the electrode based on the modified GOD was far larger than that on the electrode using native GOD When native GOD was used, the enzyme readily leaked out of the Naflon matrrx and only a small amount of GOD remained munoblhzed m the matnx After the GCE coated with the native GOD-Nafion layer had been unmersed m the test solution for 30 mm, the solution exhibited an enzyme actlvlty of 0 27 U, which corresponded to the amount of native GOD used for preparing the enzyme electrode (2 5 pug), and the GOD activity on the GCE surface was lower than 1 mU cmm2 In contrast, such a significant leakage of the enzyme from the GCE surface was not observed with the modified GOD-Nafion system About 20% of the modified GOD placed on the electrode surface leaked out of the enzyme-Nafion layer wlthm 30 mm after soakmg the electrode m the test solution, but the leakage of the enzyme was almost negligible after the 30-mm soakmg The GOD actlvlty retamed m the modlfled enzyme-Naflon layer was as high as 200 mU cm-’ The much higher enzyme activity on the GCE coated with the modified GOD-Naflon layer 1s responsible for the larger glucose response than that wth the electrode using native GOD Figure 2 shows the relationship between the steady-state current response for 1 mM glucose and the potential of the GCE coated with a modified GOD-Naflon layer The currentpotential profile obtained with the same electrode m the glucose-free test solution 1s also shown m Fig 2 The current response for glucose increased with increasing electrode potential applied and reached a plateau above 10 V vs Ag/AgCl On the other hand, the background (residual) current increased appreciably above 10 V vs Ag/AgCl Subsequent experiments were therefore done at 0 9 V vs Ag/AgCl The shape

F Mzutam et al /Anal

Chtm Acta 274 (1993) 201-207

06

12’

06

08

MentlallVvs

10 Ag/AqCI

Fig 2 Effect of potential on (01 the current response for 1 mM glucose of the GCE coated with a modified GGD-NafIon-layer and (0) the background current of the same electrode

of the glucose response-potential profile shown m Fig 2 was almost identical with that of the current-potential curve for the oxldatlon of hydrogen peroxide on the same electrode, as expected For the construction of the present glucase-sensing system, glassy carbon was selected as the base electrode material because the hydrophobic surface of the material was considered to be suitable for munoblhzmg the hydrophobic lipid-coated enzyme On the other hand, carbon electrodes are known to show poor activity for the oxldatlon of hydrogen peroxide 134,351 A high electrode potential 1s hence required for operating the present enzyme electrode as cornpared with conventional glucose sensors based on platinum electrodes, the operating potential of which 1s usually m the range 0 6-O 7 V vs Ag/AgCl The relative standard devratron for ten successive measurements of 0 2 mM glucose on the modified GOD-Nafion-coated GCE was 13% Figure 3 shows a cahbratlon graph obtained wrth the modlfied GOD-Nafion-coated GCE The enzyme electrode gave a linear current response up to 3 mM glucose, and a significant increase m the response with an increase m the glucose concentratlon was observed m the range 3-10 mM The detection limit was as low as 0 2 PM (slgnal-tonoise ratlo = 5)

F Muutaru et al /AML!

205

Chm. Acta 274 (1993) 201-207 TABLE 1

Comparison of results obtamed for glucose m fruit ~mces by different methods a Glucose concentration (mM)

Samnle

Orange juice 1 Orange juice 2 Orange Jmce 3 Apple ~ulce Mixed fruit JUNX 1 Mured fruit juice 2 ‘0

2

1,

6

6

10

12

Gbneconcentrat~n/mM

Fig 3 Cahbratlon graph for glucose on the GCE coated with a modtied GOD-Nafion layer The inset shows an enlargement of the mltlal part of the graph

Detemmatwn of glucose m beverages Figure 4 shows the enzyme electrode responses for the successive addltlon of 0 2 mM glucose and 0 1 mM L-ascorbate, which IS a typlcal electroactrve mterferent present m blologlcal and food samples The enzyme electrodes used were (a) the electrode coated three tunes with 0 5% Nafion solution, (b) that coated once with 0 5% Naflon solution and (c) that coated three tnnes with 0 5% acetylcellulose solution As shown, the use of a Nafion layer was very effective m reducing the ratio of L-ascorbate response to glucose response as compared wth the use of

Time

Fig 4 Current-tune curves for the enzyme electrodes (a) electrode coated three times wth 0 5% Nafion solution, (b) electrode coated once Hrlth 0 5% Nafion solution, (c) electrode coated three tnnes wrth 0 5% acetylcellulose solution tI and t2 are the time pomts when 0 2 mM glucose and 0 1 mM L-ascorbate were added, respectively

proposed method (x)

F-lot method(y)

155 148 142 144 175 160

155 145 138 142 171 160

x/ y

100 102 103 101 102 100

a L-Asaxbate concentrations m the samples were determined to be m the range 0 3-2 1 mM by using the F-lot

acetylcellulose, and repetition of the Naflon coatmg brought about a decrease m the ratlo, as expected The ratlo of response for glucose to that for the same concentration of L-ascorbate on electrode (a) was 14 The concentrations of glucose and L-ascorbate m normal sera are ca 5 and ca 0 05 mM, respectlvely, and those in fruit JUNX are reported to be 100-200 [36,37] and lower than 3 mM [38], respectlvely, the L-ascorbate concentration IS less than 2% of the glucose concentration m these samples For measuring glucose m such samples with electrode (a), the level of electrochenncal mterference by ascorbate IS expected to be wlthm a few percent of the signal output

O0

10

Days Fu 5 Long-term stabtity of the modified GOD-Nafioncoated GCE The electrode response for 1 mM glucose was measured 30 trmes a day The average value of the 30 measurements IS plotted agamst the number of days after the preparation of the electrode

206

Table 1 gives the results for the determmatlon of glucose m fruit Juices from the current mcrease on electrode (a) Each sample (50 ~1) was added to the test solution vvlthout any pretreatment The results were compared with those given by the F&t method The agreement was excellent, the regression equation between the results obtamed by the present electrode method (x) and those by the F-lut method (y) was y = 1003x - 2 573 and the correlation coefficient was 0 989 for the SIXsamples given m Table 1 These results show that the present enzyme electrode IS useful for the simple and rapid determmatlon of glucose concentration The use of a pair of electrodes, the present enzyme electrode and a deactivated enzyme-based electrode, for differential measurement of electrode current would be a suitable approach to the more accurate determmatlon of glucose Stab&y of mod#ied GOD-Nafin-attached GCE The long-term stability of the GCE coated with the modified GOD-Naflon layer was exammed by determining 1 mM glucose 30 times a day each day for 6 weeks The average value of the electrode responses for the 30 measurements did not decrease for 3 weeks, as shown m Fig 5 The electrode responses gradually decreased after 3 weeks, but they are still measurable until the end of 6 weeks The detection limit was lower than 1 PM even on the 42nd day The modified GODNaflon-coated GCE thus showed a high stability Concluson The combmatlon of a water-msoluble, lipidmodified GOD with a thm coating of Naflon amomc polymer provided a glucose-sensing electrode with high performance characterrstlcs such as rapid response, high sensltlvlty and high stablllty These characterlstlcs are particularly promlsmg for the application of the electrodes m flow injection systems Work m this direction 1s m progress The simple procedure for the preparation of enzyme electrodes 1s another attractive feature of the present enzyme electrodes, the dip-coating procedure may be of particular mterest because it 1s highly suitable for preparing

F

Muutant

et al

/AMY Chtm Acta 274 (1993) 201-207

micro-enzyme electrodes Such a micro-enzyme sensor has already been prepared with the use of an ultramicro GCE (diameter 10 pm) as the base transducer

REFERENCES 1 F W Scheller, F Schubert, R Renneberg, H-G Muller, M Janchen and H Welse, Bmsensors, 1 (1985) 135 2 F Mlzutam and M Asa~, m D L Wise (Ed 1, Blomstrumentatlon, Butterworths, Boston, 1990, p 317 3 R Wdson and A P F Turner, Blosensors Bmelectron , 7 (1992) 165 4 W Matsuszewslu and M Trojanowicz, Analyst, 113 (1988) 735 5 L Gorton, H I Karan, P D Hale, T Inagakt, Y Okamoto and T A Skotheun, Anal Chum Acta, 228 (1990) 23 6 J Wang, L-H Wu, Z Lu, R LI and J Sanchez, Anal Chum Acta, 228 (1990) 251 I A Amme, J-M Kaufmann and G J Patnache, Talanta, 38 (1991) 107 8 F Mlzutam, S Yabulu, A Okuda and T Katsura, Bull Chem Sot Jpn ,64 (1991) 2849 9 M Umana and J Wailer, Anal Chem ,58 (1986) 2979 10 NC Foulds and CR Lowe, J Chem Sot, Faraday Trans 1, 82 (1986) 1259 11 P N Bartlett and R G Whltaker, J Electroanal Chem , 224 (1987) 37 12 C Iwakura, Y KaJlya and H Yoneyama, J Chem Sot, Chem Commun , (1988) 1019 13 N C Foulds and C R Lowe, Anal Chem ,60 (1988) 2473 J Chem Sot, 14 S Yabulu, H Shmohara and M hawa, Chem Commun , (1989) 945 15 C Mahtesta, F , Palmlsano, L Torso and P G Zambonm, Anal Chem , 62 (1990) 2735 16 Y KaJlya, H Sugar, C Iwakura and H Yoneyama, Anal Chem ,63 (1991) 49 17 K Ikanyama, S Yamauchl, T Yuluhashl and H Ushloda, Anal L&t, 20 (1987) 1791 18 K. Ikanyama, S Yamauchl, T Y&hash] and H Ushloda, J Electrochem Sot , 136 (1989) 702 19 J Wang, D Leech, M Ozsoz, S Martmez and M R Smyth, Anal Chnn Acta, 245 (1991) 139 20 J Wang, E Dempsey, M Ozsoz, and M R Smyth, Analyst, 116 (1991) 997 21 D J HarrIson, R FB Turner and HP Bakes, Anal Chem , 60 (1988) 2002 22 S Dong, B Wang and B Lm, Blosensors Bloelectron , 7 (1991) 215 23 GA Gerhardt, kF Oke, F Nagy, B Moghaddam and R N Adams, Brain Res , 290 (1984) 390 24 F Nagy, GA Gerhardt, A F Oke, ME Rice, RN Adams, R B Moore, M N Szentnmay and C R Martm, J Electroanal Chem , 188 (1985) 85 25 J Wang and P Tuzhl, Anal Chem ,58 (1986) 3257

F Mmtanr et al /Anal

Chm Acta 274 (1993) 201-207

26 A Aolu, T Matsue and I U&da, Denlu Kagaku, 56 (1988) 1120 27 T Matsue, A Aolu, T Abe and I U&da, Chem Lett , (1989) 133 28 J Voet and E C Andersen, Bmchem Blophys,233 (1984) 88 29 P W Carr and L D Bowers, Immobdlzed Enzymes m AnalytIcal and Clmlcal Chenustry, Wdey, New York, 1980, p 197 30 Y Okahata, T Tsuruta, K IJUO and K. Arlga, Thm Solid Films, 180 (1989) 65 31 B B Baummger, J Clm Path01 , 27 (1974) 1015

207 32 J Wang and P Tuzhl, Anal Chem ,58 (1986) 1787 33 S Nakamura, S Hayashl and K. Koga, Blochun Wophys Acta, 445 (1976) 294 34 L Gorton, Anal Chun Acta, 178 (1985) 247 35 L Gorton and T Svensson, J Mol Catal, 38 (1986) 49 36 K. Matsumoto, H Karmkado, H Matsubara and Y OsaJlma, Anal Chem ,60 (1988) 147 37 F Mlzutam and M Asal, Anal Chum Acta, 236 (1990) 245 38 K. Matsumoto, K Yamada, and Y Osqlma, Anal Chem , 53 (1981) 1974