- Email: [email protected]
Radiation grafting of acrylic acid onto polytetrafluoroethylene films for glucose oxidase immobilization and its application in membrane biosensor
journal of MEMBRANE SCIENCE ELSEVIER
Journal of Membrane Science 127 (1997) 1-7
Radiation grafting of acrylic acid onto polytetrafluoroethylene films for glucose oxidase immobilization and its application in membrane biosensor S. Turmanova a, A. Trifonov b, O. Kalaijiev b, G. K o s t o v a'* aCenter for Electron Beam Technology, Professor Dr. A. Zlatarov University, Bourgas 8010, Bulgaria bResearch and Development Center for Special Polymers, Kl. Ochridski str. 4a, Sofia 1756, Bulgaria
Received 15 April 1996; received in revised form 16 September 1996; accepted 17 September 1996
Abstract Radiation induced grafting of acrylic acid (AA) onto 40 ~tm polytetrafluoroethylene (PTFE) films was carried out by the direct method of multiple (discrete) and single irradiation from 6°Co source at different doses up to 100 kGy and room temperature. Depending on the method, the grafting takes place either on the surface layer or within the polymer matrix. The graft copolymers synthesized (PTFE-g-PAA) were transformed into ionomers by treatment with KOH. Both forms were used as carriers for immobilization of enzymes. The copolymers in H- and K-forms were activated by the acylazide method and glucose oxidase (GOD) was immobilized on them. The most suitable proved to be the ionomers PTFE-g-COOK obtained by single irradiation, possessing activity of ca. 120 mU/cm2. Enzyme biosensor was designed based on Clark-type electrode and the active membranes prepared, where the membrane plays both the roles of enzyme and oxygen membrane. It can be used for determination of glucose in solutions. Keywords: Radiation grafting; Polytetrafluoroethylene grafted polyacrylic acid membrane; Immobilized glucose oxidase; Biosensor
1. Introduction In the recent 2-3 decades, much effort has been devoted to the development of various biosensors involving biologically sensitive component and transformers - devices with many fields of application [ 1]. One of the types of biosensors - the enzyme electrodes, where the sensitive component is an enzyme, most often immobilized and the transformer is usually *Corresponding author.
an ion-selective electrode - is an object of extensive studies [2,3]. Different types of enzyme electrode based on various materials such as polypyrrole [4-8], amino and carboxyl derivatives [9], carbon and epoxy resin composition [10], etc. as stable biosensors for analytical determination [11] were prepared and successfully used. The great variety of natural and synthetic polymers, as well as the versatile methods used for the immobilization provide a possibility to obtain biocatalysts
0376-7388/97/$17.00 © 1997 Publishedby Elsevier Science B.V. All rights reserved. PII S0376-7388(96)00277-3
2
S. Turmanova et al./Journal of Membrane Science 127 (1997) 1-7
in a form suitable for the biosensor. The requirements to the polymer carrier are determined both from the enzyme properties and the immobilization technique used [12]. The immobilization of enzymes onto grafted copolymers was reported in a number of papers [13-19]. Brett et al. studied the immobilization of enzymes catalase and cholesterol oxidase onto low density polyethylene grafted with polyacrylic acid (PAA) and PTFE-g-PAA membranes [18-23]. The authors discussed the possibility for covalent bonding of GOD onto nylon 66 and studied the effect of different factors (pH, ion force, etc.) on the activities of the free and bound (immobilized) enzyme [24]. The PTFE-g-PAA copolymers possess a number of properties incited their investigation as carriers. They can be prepared as membranes with high oxygen solubility, good hydrophilicity, biocompatibility and mechanical strength, possessing at the same time reactive groups for immobilization of enzymes. The present work aims to synthesize graft copolymers of PTFE-g-PAA and their ionomers, as well as to study their use for immobilization of glucose oxidase to prepare enzyme membrane which plays the additional role of diffusion (oxygen) membrane in the t r a n s f o r m e r - standard Clark-type oxygen electrode.
2. Experimental 2.1. Materials and reagents PTFE films (NPO "Plastchim", Russia) used were 40±2 ~tm thick. Acrylic acid (BASF, Germany) was vacuum distilled. Copolymers based on PTFE-g-PAA used were synthesized by the direct method of single and multiple irradiation (involving post-effect) by ,),-radiation from 6°Co source. Water solution of 40 mass% AA was used. Inhibitor NH4(FeSOa)2.6H20 with concentration 1.5 mass% versus the aqueous acid solution was added to reduce the homopolymerization of the monomer. The graft copolymerization was carried out in inert medium (N2) at 288 K. Dose rate was 5 kGy/h and the total dose was varied up to 100 kGy for the single and up to 25 kGy for the multiple irradiation. The postpolymerization periods for the multiple irradiation were 2 h after each exposition [25]. The grafting degree of PAA in the copolymers obtained by single (membranes type I, Table 1) and multiple irradiation was from 1.4 to 49.1 and from 7.5 to 50.5%, respectively. Part of the copolymers obtained by single and multiple irradiation were transformed into potassium salt (K-form) by treatment with 2.5% KOH at 373 K for 24 h [26] (membranes type II and III, Table 1, respectively).
Table 1 Immobilization of GOD onto PTFE-g-PAA membranes Group No.
Membrane type
Ionexchange group
Grafting degree, %
Active group conent, ~tmol/g
Enzyme activity, mU/crn2
I
PTFE-PAA (single irradiation)
-COOH
21 25 35
17 23 32
16 28 40
II
PTFE-g-PAA (single irradiation)
-COOK
3 8 17 24 35
2 5 115 118 80
12 18 112 120 98
III
PTFE-g-PAA (multiple irradiation)
-COOK
7 17 22 30
21 29 38 42
3 6 17 20
S. Turmanova et aL/Journal of Membrane Science 127 (1997) 1-7
3
The GOD used (E.C.1.1.3.4.) from Pen.chrysogenum, 200 U/mg, is a commercial product of Bioprogress, Bulgaria. All other reagents were of analytical grade.
2.2. Enzyme immobilization All the membranes shown in Table 1 were used for enzyme immobilization. They were activated by the acylazide method - a modification of the method of Guilbault [1]. Initial PTFE-g-PAA membrane (ca. 20 mg) was treated with 25 cm 3 2% HC1/CH3OH for 70 h at 298 K and then washed with water. The esterified membrane was immersed in 10 cm 3 30% solution of hydrazine in water for 2 h at 298 K, then washed with cooled to 277 K water, followed by treatment with 10 cm 3 0.5 M H N 0 2 for 20 min at 273 K and thorough washing. The membrane containing acylazide groups is then immersed in 2 c m 3 0.05 M phosphate buffer pH=7.5, containing 5 mg GOD, for 18 h at 277 K. The GOD bound membrane is then washed with 0.05 M phosphate buffer (pH----6.0) until no enzyme activity is detected in washing solution. The membranes were stored at 277 K.
2.2.1. Determination of the content of active groups in the membrane Part of the membrane (ca. 2 rag), activated by the acylazide method, was washed with 0.05 M phosphate buffer pH=7.5 and immersed in 2 cm 3 0.05 M phosphate buffer pH=7.5, containing 5 mg p-nitrophenol (PNP) for 18 h at 277 K. The membrane obtained was washed with dry acetone (<0.2% water) until absence of PNP in the acetone, followed by immersion in 20 cm 3 0.1 M water solution of aminopropanol for 30 min at 298 K. The content of aminolyzed PNP in the solution was determined spectrophotometrically at 410 nm.
2.2.2. Determination of GOD activity The activities of free and bound enzymes were determined spectrophotometrically, by using peroxidase and o-dianisine, according to [27].
,/
,/
Fig. 1. S c h e m a t i c d i a g r a m o f g l u c o s e electrode: 1 - E l e c t r o d e case; 2 - C a t h o d e (Pt); 3 - A n o d e ( A g / A g C I ) ; 4 - E n z y m e m e m b r a n e ( P T F E - g - P A A - G O D ) ; 5 - Electrolyte; 6 - M e m b r a n e fixture.
2.3. Glucose electrode The diffusion membrane of a standard electrode for determination of dissolved oxygen (Clark type, Zona Co., Bulgaria) is replaced by the enzyme membrane, so that the latter touches the cathode surface (Fig. 1). The enzyme electrode is mounted in 1.5 cm 3 thermostated reactor (3104-1 K) and polarized by potentiostat at - 7 5 0 m V . Samples of glucose solutions (5× 10 -2 cm 3) are pipetted into the reactor stirred medium of 0.05 M phosphate buffer. The change of the anode current is registered by nanoampermeter until a new equilibrium is established. 2.4. Apparatus Spectrophotometric studies were performed on Spectromom 195D, Hungary. The potentiostat and nanoampermeter were produced by Zona Co., Bulgaria.
3. Results and discussion Radiation graft copolymerization of AA onto PTFE films was carried out by using the single and multiple irradiation by "/-rays to prepare carboxyl containing copolymers of various grafting degrees. The - C O O H groups were fully neutralized with KOH to obtain the corresponding ionomers. The ion-exchange capacity A R of the membranes synthesized was determined by titration and was found to be from 2.0 to 2.9 meq/g.
4
S. Turmanova et al./Journal of Membrane Science 127 (1997) 1-7
0 6o
20 .
.
.
.
40 .
.
.
.
D.KGv 80 "100
60 .
.
.
.
.
.
.
.
.
30
.
"~ 20
40 10
Q
20 0 0
A
0 0
5
10
15
20 25 D,KGy
Fig. 2. Dependence of the grafting degree of AA onto PTFE on absorbed dose (D) by single (e) and multiple (O) irradiation,
Fig. 2 shows the dependence of the grafting degree of AA onto PTFE for both irradiation methods. It can be seen that, by single irradiation, the grafting degree reaches a limit value above which the increase of the dose has no effect. The constant yield of grafted polymer can be explained by two controversial effects taking place in the sample. The first one is connected with the increase of the yield resulting from the decrease of chain termination reaction rate due to the insolubility of PAA in the monomer and probably folding of the growing grafted chains with the reaction progress. The second effect decreases the yield and is determined by the reduced diffusion of monomer fi'om the solution to the PTFE film since the latter is coated by a thin layer of PAA. The grafting degree by multiple irradiation increases linearly from the beginning with irradiation dose. The reaction proceeds within the film and the hydrophilic monomer has enough time to diffuse through the amorphous layer of grafted PAA. The step-wise generation of radicals by the multiple irradiation additionally increases the copolymer yield. In this case, a continuous increase of the grafting degree with irradiation dose was observed. That is why, by multiple irradiation, the grafted copolymer yield reached are similar to those obtained by single irradiation but at significantly lower irradiation doses. The relative change of the grafted layer thickness (Ad) with the grafting degree is shown in Fig. 3. For PTFEg-PAA obtained by single irradiation, Ad is of the order of several percents (at high grafting degree)
.
A
.
.
-
10
i
20
r
30
.
.
.
40
50
Fig. 3. Dependenceof relative thickness of the grafted layer (Ad) on the graftingdegree (P) of PTFE-g-PAAmembranesobtainedby single (*) and multiple (O) irradiation. while by multiple irradiation the increase of Ad with P observed was much higher (up to 30%). The introduction of polar functional groups in hydrophobic polymer matrix stipulates the hydrophilization of the grafted membranes and, therefore, their swelling in water. The cation-exchange polar groups are hydrated by the water molecules to form associates and ion channels. The hydrophobic fluorocarbon phase and hydrated hydrophilic ion groups are incompatible. Therefore, they form separate phases to give internally hydrated ion clusters which determine the basic properties of the membranes. Fig. 4 shows the dependence of the water content on the grafting degree of PTFE membranes in H- and Kform. The water content was found to increase linearly 80
40
20
0
.
0
.
10
.
.
i
20
,
,
,
i
30
.
i
.
,
.
,
,
/,0
50 P,*/o
Fig. 4. Water uptake (W) versus grafting degree (P) for PTFE-gPAA membrane in H- (A, single; /k, multiple) and K-form (e, single; O, multiple) irradiation.
S. Turmanova et al./Journal of Membrane Science 127 (1997) 1-7
with P. The alkali treatment of the carboxyl-containing membranes improves its swelling degree due to the increased hydrophilicity of the membranes during the transformation o f - C O O H groups into their corresponding alkali salts. By single irradiation, the surface grafted carboxylic groups are more accessible by the water molecules, compared with the polar groups distributed within the film by the multiple irradiation. This should be taken into account for proper selection of membranes for enzyme immobilization. On the other hand, the intermediate non-grafted PTFE layer is responsible for the diffusion of oxygen through the membrane and suggests its use as a combined component in Clark-type electrode. For immobilization of GOD, the carboxylic groups in the grafted membranes should be activated in advance which was carried out by the acylazide method. The results are shown in Table 1. For the membranes of Group I, the active groups content was found to be from 17 to 32 tamol/g. The attempts to bind GOD onto these membranes gave membranes of comparatively lower enzyme activity (16-40 mU/cm 2) which delimits their use as biosensors.
The membranes of Group II showed clearly discernible maximum of the dependence of the active groups content on the grafting degree. This maximum is from 115 to 120~tmol/g corresponds to grafted polymer content from 17 to 24%. Similar dependence was observed for the enzyme activity which was measured to be from 112 to 120mU/cm 2 at the maximum. The membranes of this group are suitable for immobilization of GOD and can be used as biosensors. The membranes from Group III were also activated and the acylazide groups content was measured to be from 3 to 20 mU/cm 2. The analysis of these results showed that membranes of equal or similar grafting degree from different groups (I, II or III) acylazided under the same conditions possess different content of activated carboxylic groups. This is probably due to the different accessibility of the carboxylic groups in the membrane. The single irradiation gave surface grafted chains of PAA (Fig. 3). The transformation of the copolymers into their potassium salts leads to ionization of the carboxylic groups making them more accessible. By multiple irradiation, however, PAA is grafted within the film which leads to non-activation
5
of a part of the carboxylic groups even after their ionization. These suggestions were proved by the difference of the enzyme activity observed in membranes of different groups. Thus, for membranes of Group III, the large GOD molecules (160 000 Da) can not easily diffuse through the PAA layer due to steric hindrance to react through the c-NH2 groups of their lisine residues with the activated carboxylic groups, which results in their low enzyme activity. The enzyme is covalently bound to the activated carboxylic groups of the membranes studied. The enzyme bound membranes were washed several times with phosphate buffer to remove the adsorbed GOD until negative enzyme activity is measured in the washing solution. Therefore, the enzyme activity measured of the membranes is determined only by covalently bound GOD. Besides, the acylazide method is widely used for covalent binding of enzymes [1]. The analysis of the results obtained shows that membranes of Group II with grafting degrees from 17 to 24% are most suitable for immobilization of GOD and they were used further as enzyme sensor in the next experiments. The response of the electrode was registered after addition of glucose solutions of different concentrations (C). The cathode current measurements showed that it changes abruptly immediately after the glucose solution was pipetted, i.e. the oxygen in the membrane and near the cathode is rapidly consumed in the enzyme catalysed reaction, followed by a new equilibrium established in 15-20 s. After washing the reactor with fresh buffer, the enzyme electrode reaches its equilibrium for 20--30 s (Fig. 5). These results show that the response of the electrode to the pipetting of glucose is the difference between the cathode current before and after the pipetting (A/). As Fig. 6 shows, the dependence of A I on C is nonlinear but in the initial interval (0-0.7 mmol/1) it can be approximated by linear equation (AI=bC, where b is the slope of the line) with multiple correlation coefficient R=0.996. The estimated parameter value with their 95% confidence interval is b=32.00+0.78. The linear model reproduced experimental data in the concentration interval up to 0.7 mmol/1 with average relative error less than 5%. The mean relative experimental error AE is less than 1%.
6
S. Turmanova et al./Journal of Membrane Science 127 (1997) 1-7
120
4. C o n c l u s i o n
e-
100
T3
1
80 60 t, 0 20 0
i
i
i
i
i
10
20
30
40
50
60 LS
Fig. 5. Response of the enzyme electrode after the addition of 20 mM solution of glucose in the reactor: To - time of pipetting of glucose solution in the reactor; T1 - time to the new equilibrium reached by the electrode; T2 - start of the washing the electrode with fresh buffer; T3 - time to the new initial equilibrium reached by the electrode.
Carboxyl containing copolymers were synthesized by radiation graft copolymerization of AA onto PTFE films with different grafting degrees. Three types of membranes with different properties were prepared. Immobilization of the enzyme GOD onto functional carboxylic groups (activated by the acylazide method) of the membranes was carried out. The immobilization was found to proceed better when ionomers used are obtained by single irradiation and contain - C O O K groups, due to their high dissociation degree and accessibility of the activated groups. Enzyme sensor was designed and activated membranes synthesized were successfully used as a diffusion membrane of a Clark-type electrode together with immobilization of GOD in the biosensor. References
~35T a ~ 30
'
25
2O 15
10 5 0 0
0.1
0.2 0.3
0.4
0.5
0.6
0.7
0.8
o.g
1
C, mmoVI Fig. 6. Dependence of the electrode current 30 s after the addition of the glucose solution on the glucose concentration in the reactor (30-fold dilution of initial solution).
Glucose concentration of 0.7 mmol/1 is equivalent to 21 mmol/1 glucose in the solution pipetted (30-fold dilution), i.e. it is in the range of normal and pathological values of glucose in human urine and blood. When the electrode is stored in 0.05 M phosphate buffer, pH=6.0 and 277 K, the sensor sensibility remains unchanged for more than six months, provided a weekly pipetting of 10 mmol/1 glucose solutions.
[1] G.G. Guilbault, Immobilized enzymes and cells, in K. Mosbach (Ed.), Methods in Enzymology, Academic Press, New York, 1988. [2] A.P.F. Turner, I. Karube and G.S. Wilson, Biosensors. Fundamentals and Applications, Oxford University Press, Oxford, 1989. [3] J. Rodriguez-Flores and E. Lorenzo, Analytical voltammetry, in M.R. Smith and J.G. Vos (Eds.), Wilson and Wilson's Comprehensive Analytical Chemistry (Ed. G. Svehla), Elsevier, Amsterdam, 1992. [4] W. Schuhmann, R. Lammert, B. Uhe and H.L. Schmids, Polypyrrole: A new possibility for covalent binding of oxidoreductase to electrode surface as a base for stable biosensor, Sens. Actuators, B, Bl(l~5) (1990) 537. [5] T. Scalkhammer, E. Mann-Buxbaum, Fr. Pittner and G. Urban, Electrochemical glucose sensors of permselectivity non-conducting substituted pyrrole polymer, Sens. Actuators, B, B4(3-4) (1991) 273. [6] D.¥. Strike, N.E Rooij and M. Koudelkahep, Electrodeposition of glucose oxidase for the fabrication of miniature sensor, Sens. Actuators B, 13(1-3) (1993) 61. [7] C.G.Y. Koopal, B. De Ruiter and R.J.M. Nolte, Amperometric biosensor based on direct communication between glucose oxidase and a conducting polymer inside the pores of a filtration membrane, J. Chem. Soc., Chem. Commun., 23 (1991) 1691. [8] G. Fortier and D. Belanger, Characterization of the biochemical behavior of glucose oxidase entrapped in a polypyrrole film, Biotechnol. Bioeng., 37(9) (1991) 854. [9] M. Uo, M. Numata, M. Suzuki, E. Tamiya, I. Karube and A. Makishima, Preparation and properties of immobilized mercuric reductase in porous glass carriers, Nippon Seramkkusu Kyokai Gakjutsu Ronbushi, 100 (1992) 430.
S. Turmanova et al./Journal of Membrane Science 127 (1997) 1-7
[10] T. Wasa, K. Akimoto, T. Yao and S. Murao, Development of laccase membrane electrode by using carbon electrode i m p r e g n a t e d with e p o x y resin and its r e s p o n s e characteristics, Nippon Kagaku Kaishi, 9 (1984) 1398. [11] J.E. Frew, S.W. Bayliff, Ph.N.B. Gibbs and M.J. Green, Amperometric biosensor for the rapid determination of salycilate in whole blood, Anal. Chim. Acta, 224(1) (1989) 39. [12] O. Zaborsky, Immobilized Enzymes, CRC Press, Boca Ration, FL, 1973. [13] I.G. Beddows, M.H. Gil and J.T. Guthrie, The immobilization of enzymes, bovine serum albumin and phenylpropylamine to poly(acrylic acid)-polyethylene based copolymers, Biotechnol. Bioeng., 24(6) (1982) 1371. [14] El. Abdel-Hay, C.G. Beddows, M.H. Gil and J.T. Guthrie, The use of acrylic acid-co-nylon substrates for immobilizing enzymes, J. Polym. Sci., Polym. Chem. Ed., 21 (1983) 2463. [15] G.H. Hsiue, Z.S. Chou, N. Yu and K.P. Hsiung, Urease immobilized poly(vinyl-alcohol-g-butyl acrylate) membrane for urea sensor, J. Appl. Polym. Sci., 34(1) (1987) 319. [16] G.H. Hsiue and C.C. Wang, Glucose oxidase immobilized polyethylene-g-acrylic acid membranes for glucose oxidase sensor, Biotechnol. Bioeng., 36(8) (1990) 811. [17] K. Martensson, Preparation of an immobilized two-enzyme system/3-amylase-pullunase on an acrylic copolymer for the conversion of starch to maltose. 1. Preparation and stability of immobilized /3-amylase, Biotechnol. Bioeng., 16(5) (1974) 567. [18] T. Godjevargova and A. Dimov, Grafting of acrylonitrile copolymer membranes with hydrophilic monomers for immobilization of glucose oxidase, J. Appl. Polym. Sci., 57 (1995) 487. [19] T. Godjevargova, Radiation grafting of AMPSA and DMAEM onto membranes of acrylonitrile copolymer and
[20]
[21] [22]
[23]
[24]
[25]
[26]
[27]
7
polyamide for immobilization of glucose oxidase, J. Appl. Polym. Sci., 61 (1996) 343. A.M.O. Brett, M.H. Gil and A.P. Piedade, An electrochemical bienzyme membrane sensor for free cholesterol, Bioelectrochem. Bioenerg., 28 (1992) 105. A.M.O. Brett, M.H. Gil and A.P. Piedade, A cholesterol biosensor, Port. Electrochim. Acta, 9 (1991) 7. M. Alves da Silva, M.H. Gil, A.P. Piedade, J.S. Redinha, A.M.O. Brett and J.M.C. Costa, Immobilization of catalase on membranes of poly(ethylene-g-co-acrylic acid) and their application in hydrogen peroxide electrochemical sensors, J. Polym. Sci. Part A, Polym. Chem., 29 (1991) 269. J.S. Redinha, A.M.O. Brett, M. Alves da Silva and A.P. Piedade, A catalase membrane electrode for the determination of hydrogen peroxide, Port. Electrochim. Acta, 7 (1989) 17. M. Alves da Silva, M.H. Gil, J.S. Redinha, A.P. Piedade and J.L.C. Pereira, Immobilization of glucose oxidase on nylon membranes and its application in a flow-through glucose reactor, J. Polym. Sci. Part A, Polym. Chem., 29 (1991) 275. G.K. Kostov, S. Chr. Turmanova and A.N. Atanassov, Radiation induced grafting of acrylic acid onto low density polyethylene and polytetrafluoroethylene thin films, in J.M. Marshall, N. Kirov and A. Vavrek (Eds.), Electronic, Optoelectronic and Magnetic Thin Films, Wiley, New York, 1995, p. 672. E.A. Hegazy, I. Ishigaki, A. Rabie, A. Dessouki and J. Okamoto, Study on radiation grafting of acrylic acid onto fluorine containing polymers. II. Properties of membrane obtained by preirradiation grafting onto poly(tetrafluoroethylene), J. Appl. Polym. Sci., 26 (1981) 3871. A.S.G. Hugget and D.A. Nixon, Enzymatic determination of blood glucose, Biochem. J., 66 (1957) 12.
Journal of Membrane Science 127 (1997) 1-7
Radiation grafting of acrylic acid onto polytetrafluoroethylene films for glucose oxidase immobilization and its application in membrane biosensor S. Turmanova a, A. Trifonov b, O. Kalaijiev b, G. K o s t o v a'* aCenter for Electron Beam Technology, Professor Dr. A. Zlatarov University, Bourgas 8010, Bulgaria bResearch and Development Center for Special Polymers, Kl. Ochridski str. 4a, Sofia 1756, Bulgaria
Received 15 April 1996; received in revised form 16 September 1996; accepted 17 September 1996
Abstract Radiation induced grafting of acrylic acid (AA) onto 40 ~tm polytetrafluoroethylene (PTFE) films was carried out by the direct method of multiple (discrete) and single irradiation from 6°Co source at different doses up to 100 kGy and room temperature. Depending on the method, the grafting takes place either on the surface layer or within the polymer matrix. The graft copolymers synthesized (PTFE-g-PAA) were transformed into ionomers by treatment with KOH. Both forms were used as carriers for immobilization of enzymes. The copolymers in H- and K-forms were activated by the acylazide method and glucose oxidase (GOD) was immobilized on them. The most suitable proved to be the ionomers PTFE-g-COOK obtained by single irradiation, possessing activity of ca. 120 mU/cm2. Enzyme biosensor was designed based on Clark-type electrode and the active membranes prepared, where the membrane plays both the roles of enzyme and oxygen membrane. It can be used for determination of glucose in solutions. Keywords: Radiation grafting; Polytetrafluoroethylene grafted polyacrylic acid membrane; Immobilized glucose oxidase; Biosensor
1. Introduction In the recent 2-3 decades, much effort has been devoted to the development of various biosensors involving biologically sensitive component and transformers - devices with many fields of application [ 1]. One of the types of biosensors - the enzyme electrodes, where the sensitive component is an enzyme, most often immobilized and the transformer is usually *Corresponding author.
an ion-selective electrode - is an object of extensive studies [2,3]. Different types of enzyme electrode based on various materials such as polypyrrole [4-8], amino and carboxyl derivatives [9], carbon and epoxy resin composition [10], etc. as stable biosensors for analytical determination [11] were prepared and successfully used. The great variety of natural and synthetic polymers, as well as the versatile methods used for the immobilization provide a possibility to obtain biocatalysts
0376-7388/97/$17.00 © 1997 Publishedby Elsevier Science B.V. All rights reserved. PII S0376-7388(96)00277-3
2
S. Turmanova et al./Journal of Membrane Science 127 (1997) 1-7
in a form suitable for the biosensor. The requirements to the polymer carrier are determined both from the enzyme properties and the immobilization technique used [12]. The immobilization of enzymes onto grafted copolymers was reported in a number of papers [13-19]. Brett et al. studied the immobilization of enzymes catalase and cholesterol oxidase onto low density polyethylene grafted with polyacrylic acid (PAA) and PTFE-g-PAA membranes [18-23]. The authors discussed the possibility for covalent bonding of GOD onto nylon 66 and studied the effect of different factors (pH, ion force, etc.) on the activities of the free and bound (immobilized) enzyme [24]. The PTFE-g-PAA copolymers possess a number of properties incited their investigation as carriers. They can be prepared as membranes with high oxygen solubility, good hydrophilicity, biocompatibility and mechanical strength, possessing at the same time reactive groups for immobilization of enzymes. The present work aims to synthesize graft copolymers of PTFE-g-PAA and their ionomers, as well as to study their use for immobilization of glucose oxidase to prepare enzyme membrane which plays the additional role of diffusion (oxygen) membrane in the t r a n s f o r m e r - standard Clark-type oxygen electrode.
2. Experimental 2.1. Materials and reagents PTFE films (NPO "Plastchim", Russia) used were 40±2 ~tm thick. Acrylic acid (BASF, Germany) was vacuum distilled. Copolymers based on PTFE-g-PAA used were synthesized by the direct method of single and multiple irradiation (involving post-effect) by ,),-radiation from 6°Co source. Water solution of 40 mass% AA was used. Inhibitor NH4(FeSOa)2.6H20 with concentration 1.5 mass% versus the aqueous acid solution was added to reduce the homopolymerization of the monomer. The graft copolymerization was carried out in inert medium (N2) at 288 K. Dose rate was 5 kGy/h and the total dose was varied up to 100 kGy for the single and up to 25 kGy for the multiple irradiation. The postpolymerization periods for the multiple irradiation were 2 h after each exposition [25]. The grafting degree of PAA in the copolymers obtained by single (membranes type I, Table 1) and multiple irradiation was from 1.4 to 49.1 and from 7.5 to 50.5%, respectively. Part of the copolymers obtained by single and multiple irradiation were transformed into potassium salt (K-form) by treatment with 2.5% KOH at 373 K for 24 h [26] (membranes type II and III, Table 1, respectively).
Table 1 Immobilization of GOD onto PTFE-g-PAA membranes Group No.
Membrane type
Ionexchange group
Grafting degree, %
Active group conent, ~tmol/g
Enzyme activity, mU/crn2
I
PTFE-PAA (single irradiation)
-COOH
21 25 35
17 23 32
16 28 40
II
PTFE-g-PAA (single irradiation)
-COOK
3 8 17 24 35
2 5 115 118 80
12 18 112 120 98
III
PTFE-g-PAA (multiple irradiation)
-COOK
7 17 22 30
21 29 38 42
3 6 17 20
S. Turmanova et aL/Journal of Membrane Science 127 (1997) 1-7
3
The GOD used (E.C.1.1.3.4.) from Pen.chrysogenum, 200 U/mg, is a commercial product of Bioprogress, Bulgaria. All other reagents were of analytical grade.
2.2. Enzyme immobilization All the membranes shown in Table 1 were used for enzyme immobilization. They were activated by the acylazide method - a modification of the method of Guilbault [1]. Initial PTFE-g-PAA membrane (ca. 20 mg) was treated with 25 cm 3 2% HC1/CH3OH for 70 h at 298 K and then washed with water. The esterified membrane was immersed in 10 cm 3 30% solution of hydrazine in water for 2 h at 298 K, then washed with cooled to 277 K water, followed by treatment with 10 cm 3 0.5 M H N 0 2 for 20 min at 273 K and thorough washing. The membrane containing acylazide groups is then immersed in 2 c m 3 0.05 M phosphate buffer pH=7.5, containing 5 mg GOD, for 18 h at 277 K. The GOD bound membrane is then washed with 0.05 M phosphate buffer (pH----6.0) until no enzyme activity is detected in washing solution. The membranes were stored at 277 K.
2.2.1. Determination of the content of active groups in the membrane Part of the membrane (ca. 2 rag), activated by the acylazide method, was washed with 0.05 M phosphate buffer pH=7.5 and immersed in 2 cm 3 0.05 M phosphate buffer pH=7.5, containing 5 mg p-nitrophenol (PNP) for 18 h at 277 K. The membrane obtained was washed with dry acetone (<0.2% water) until absence of PNP in the acetone, followed by immersion in 20 cm 3 0.1 M water solution of aminopropanol for 30 min at 298 K. The content of aminolyzed PNP in the solution was determined spectrophotometrically at 410 nm.
2.2.2. Determination of GOD activity The activities of free and bound enzymes were determined spectrophotometrically, by using peroxidase and o-dianisine, according to [27].
,/
,/
Fig. 1. S c h e m a t i c d i a g r a m o f g l u c o s e electrode: 1 - E l e c t r o d e case; 2 - C a t h o d e (Pt); 3 - A n o d e ( A g / A g C I ) ; 4 - E n z y m e m e m b r a n e ( P T F E - g - P A A - G O D ) ; 5 - Electrolyte; 6 - M e m b r a n e fixture.
2.3. Glucose electrode The diffusion membrane of a standard electrode for determination of dissolved oxygen (Clark type, Zona Co., Bulgaria) is replaced by the enzyme membrane, so that the latter touches the cathode surface (Fig. 1). The enzyme electrode is mounted in 1.5 cm 3 thermostated reactor (3104-1 K) and polarized by potentiostat at - 7 5 0 m V . Samples of glucose solutions (5× 10 -2 cm 3) are pipetted into the reactor stirred medium of 0.05 M phosphate buffer. The change of the anode current is registered by nanoampermeter until a new equilibrium is established. 2.4. Apparatus Spectrophotometric studies were performed on Spectromom 195D, Hungary. The potentiostat and nanoampermeter were produced by Zona Co., Bulgaria.
3. Results and discussion Radiation graft copolymerization of AA onto PTFE films was carried out by using the single and multiple irradiation by "/-rays to prepare carboxyl containing copolymers of various grafting degrees. The - C O O H groups were fully neutralized with KOH to obtain the corresponding ionomers. The ion-exchange capacity A R of the membranes synthesized was determined by titration and was found to be from 2.0 to 2.9 meq/g.
4
S. Turmanova et al./Journal of Membrane Science 127 (1997) 1-7
0 6o
20 .
.
.
.
40 .
.
.
.
D.KGv 80 "100
60 .
.
.
.
.
.
.
.
.
30
.
"~ 20
40 10
Q
20 0 0
A
0 0
5
10
15
20 25 D,KGy
Fig. 2. Dependence of the grafting degree of AA onto PTFE on absorbed dose (D) by single (e) and multiple (O) irradiation,
Fig. 2 shows the dependence of the grafting degree of AA onto PTFE for both irradiation methods. It can be seen that, by single irradiation, the grafting degree reaches a limit value above which the increase of the dose has no effect. The constant yield of grafted polymer can be explained by two controversial effects taking place in the sample. The first one is connected with the increase of the yield resulting from the decrease of chain termination reaction rate due to the insolubility of PAA in the monomer and probably folding of the growing grafted chains with the reaction progress. The second effect decreases the yield and is determined by the reduced diffusion of monomer fi'om the solution to the PTFE film since the latter is coated by a thin layer of PAA. The grafting degree by multiple irradiation increases linearly from the beginning with irradiation dose. The reaction proceeds within the film and the hydrophilic monomer has enough time to diffuse through the amorphous layer of grafted PAA. The step-wise generation of radicals by the multiple irradiation additionally increases the copolymer yield. In this case, a continuous increase of the grafting degree with irradiation dose was observed. That is why, by multiple irradiation, the grafted copolymer yield reached are similar to those obtained by single irradiation but at significantly lower irradiation doses. The relative change of the grafted layer thickness (Ad) with the grafting degree is shown in Fig. 3. For PTFEg-PAA obtained by single irradiation, Ad is of the order of several percents (at high grafting degree)
.
A
.
.
-
10
i
20
r
30
.
.
.
40
50
Fig. 3. Dependenceof relative thickness of the grafted layer (Ad) on the graftingdegree (P) of PTFE-g-PAAmembranesobtainedby single (*) and multiple (O) irradiation. while by multiple irradiation the increase of Ad with P observed was much higher (up to 30%). The introduction of polar functional groups in hydrophobic polymer matrix stipulates the hydrophilization of the grafted membranes and, therefore, their swelling in water. The cation-exchange polar groups are hydrated by the water molecules to form associates and ion channels. The hydrophobic fluorocarbon phase and hydrated hydrophilic ion groups are incompatible. Therefore, they form separate phases to give internally hydrated ion clusters which determine the basic properties of the membranes. Fig. 4 shows the dependence of the water content on the grafting degree of PTFE membranes in H- and Kform. The water content was found to increase linearly 80
40
20
0
.
0
.
10
.
.
i
20
,
,
,
i
30
.
i
.
,
.
,
,
/,0
50 P,*/o
Fig. 4. Water uptake (W) versus grafting degree (P) for PTFE-gPAA membrane in H- (A, single; /k, multiple) and K-form (e, single; O, multiple) irradiation.
S. Turmanova et al./Journal of Membrane Science 127 (1997) 1-7
with P. The alkali treatment of the carboxyl-containing membranes improves its swelling degree due to the increased hydrophilicity of the membranes during the transformation o f - C O O H groups into their corresponding alkali salts. By single irradiation, the surface grafted carboxylic groups are more accessible by the water molecules, compared with the polar groups distributed within the film by the multiple irradiation. This should be taken into account for proper selection of membranes for enzyme immobilization. On the other hand, the intermediate non-grafted PTFE layer is responsible for the diffusion of oxygen through the membrane and suggests its use as a combined component in Clark-type electrode. For immobilization of GOD, the carboxylic groups in the grafted membranes should be activated in advance which was carried out by the acylazide method. The results are shown in Table 1. For the membranes of Group I, the active groups content was found to be from 17 to 32 tamol/g. The attempts to bind GOD onto these membranes gave membranes of comparatively lower enzyme activity (16-40 mU/cm 2) which delimits their use as biosensors.
The membranes of Group II showed clearly discernible maximum of the dependence of the active groups content on the grafting degree. This maximum is from 115 to 120~tmol/g corresponds to grafted polymer content from 17 to 24%. Similar dependence was observed for the enzyme activity which was measured to be from 112 to 120mU/cm 2 at the maximum. The membranes of this group are suitable for immobilization of GOD and can be used as biosensors. The membranes from Group III were also activated and the acylazide groups content was measured to be from 3 to 20 mU/cm 2. The analysis of these results showed that membranes of equal or similar grafting degree from different groups (I, II or III) acylazided under the same conditions possess different content of activated carboxylic groups. This is probably due to the different accessibility of the carboxylic groups in the membrane. The single irradiation gave surface grafted chains of PAA (Fig. 3). The transformation of the copolymers into their potassium salts leads to ionization of the carboxylic groups making them more accessible. By multiple irradiation, however, PAA is grafted within the film which leads to non-activation
5
of a part of the carboxylic groups even after their ionization. These suggestions were proved by the difference of the enzyme activity observed in membranes of different groups. Thus, for membranes of Group III, the large GOD molecules (160 000 Da) can not easily diffuse through the PAA layer due to steric hindrance to react through the c-NH2 groups of their lisine residues with the activated carboxylic groups, which results in their low enzyme activity. The enzyme is covalently bound to the activated carboxylic groups of the membranes studied. The enzyme bound membranes were washed several times with phosphate buffer to remove the adsorbed GOD until negative enzyme activity is measured in the washing solution. Therefore, the enzyme activity measured of the membranes is determined only by covalently bound GOD. Besides, the acylazide method is widely used for covalent binding of enzymes [1]. The analysis of the results obtained shows that membranes of Group II with grafting degrees from 17 to 24% are most suitable for immobilization of GOD and they were used further as enzyme sensor in the next experiments. The response of the electrode was registered after addition of glucose solutions of different concentrations (C). The cathode current measurements showed that it changes abruptly immediately after the glucose solution was pipetted, i.e. the oxygen in the membrane and near the cathode is rapidly consumed in the enzyme catalysed reaction, followed by a new equilibrium established in 15-20 s. After washing the reactor with fresh buffer, the enzyme electrode reaches its equilibrium for 20--30 s (Fig. 5). These results show that the response of the electrode to the pipetting of glucose is the difference between the cathode current before and after the pipetting (A/). As Fig. 6 shows, the dependence of A I on C is nonlinear but in the initial interval (0-0.7 mmol/1) it can be approximated by linear equation (AI=bC, where b is the slope of the line) with multiple correlation coefficient R=0.996. The estimated parameter value with their 95% confidence interval is b=32.00+0.78. The linear model reproduced experimental data in the concentration interval up to 0.7 mmol/1 with average relative error less than 5%. The mean relative experimental error AE is less than 1%.
6
S. Turmanova et al./Journal of Membrane Science 127 (1997) 1-7
120
4. C o n c l u s i o n
e-
100
T3
1
80 60 t, 0 20 0
i
i
i
i
i
10
20
30
40
50
60 LS
Fig. 5. Response of the enzyme electrode after the addition of 20 mM solution of glucose in the reactor: To - time of pipetting of glucose solution in the reactor; T1 - time to the new equilibrium reached by the electrode; T2 - start of the washing the electrode with fresh buffer; T3 - time to the new initial equilibrium reached by the electrode.
Carboxyl containing copolymers were synthesized by radiation graft copolymerization of AA onto PTFE films with different grafting degrees. Three types of membranes with different properties were prepared. Immobilization of the enzyme GOD onto functional carboxylic groups (activated by the acylazide method) of the membranes was carried out. The immobilization was found to proceed better when ionomers used are obtained by single irradiation and contain - C O O K groups, due to their high dissociation degree and accessibility of the activated groups. Enzyme sensor was designed and activated membranes synthesized were successfully used as a diffusion membrane of a Clark-type electrode together with immobilization of GOD in the biosensor. References
~35T a ~ 30
'
25
2O 15
10 5 0 0
0.1
0.2 0.3
0.4
0.5
0.6
0.7
0.8
o.g
1
C, mmoVI Fig. 6. Dependence of the electrode current 30 s after the addition of the glucose solution on the glucose concentration in the reactor (30-fold dilution of initial solution).
Glucose concentration of 0.7 mmol/1 is equivalent to 21 mmol/1 glucose in the solution pipetted (30-fold dilution), i.e. it is in the range of normal and pathological values of glucose in human urine and blood. When the electrode is stored in 0.05 M phosphate buffer, pH=6.0 and 277 K, the sensor sensibility remains unchanged for more than six months, provided a weekly pipetting of 10 mmol/1 glucose solutions.
[1] G.G. Guilbault, Immobilized enzymes and cells, in K. Mosbach (Ed.), Methods in Enzymology, Academic Press, New York, 1988. [2] A.P.F. Turner, I. Karube and G.S. Wilson, Biosensors. Fundamentals and Applications, Oxford University Press, Oxford, 1989. [3] J. Rodriguez-Flores and E. Lorenzo, Analytical voltammetry, in M.R. Smith and J.G. Vos (Eds.), Wilson and Wilson's Comprehensive Analytical Chemistry (Ed. G. Svehla), Elsevier, Amsterdam, 1992. [4] W. Schuhmann, R. Lammert, B. Uhe and H.L. Schmids, Polypyrrole: A new possibility for covalent binding of oxidoreductase to electrode surface as a base for stable biosensor, Sens. Actuators, B, Bl(l~5) (1990) 537. [5] T. Scalkhammer, E. Mann-Buxbaum, Fr. Pittner and G. Urban, Electrochemical glucose sensors of permselectivity non-conducting substituted pyrrole polymer, Sens. Actuators, B, B4(3-4) (1991) 273. [6] D.¥. Strike, N.E Rooij and M. Koudelkahep, Electrodeposition of glucose oxidase for the fabrication of miniature sensor, Sens. Actuators B, 13(1-3) (1993) 61. [7] C.G.Y. Koopal, B. De Ruiter and R.J.M. Nolte, Amperometric biosensor based on direct communication between glucose oxidase and a conducting polymer inside the pores of a filtration membrane, J. Chem. Soc., Chem. Commun., 23 (1991) 1691. [8] G. Fortier and D. Belanger, Characterization of the biochemical behavior of glucose oxidase entrapped in a polypyrrole film, Biotechnol. Bioeng., 37(9) (1991) 854. [9] M. Uo, M. Numata, M. Suzuki, E. Tamiya, I. Karube and A. Makishima, Preparation and properties of immobilized mercuric reductase in porous glass carriers, Nippon Seramkkusu Kyokai Gakjutsu Ronbushi, 100 (1992) 430.
S. Turmanova et al./Journal of Membrane Science 127 (1997) 1-7
[10] T. Wasa, K. Akimoto, T. Yao and S. Murao, Development of laccase membrane electrode by using carbon electrode i m p r e g n a t e d with e p o x y resin and its r e s p o n s e characteristics, Nippon Kagaku Kaishi, 9 (1984) 1398. [11] J.E. Frew, S.W. Bayliff, Ph.N.B. Gibbs and M.J. Green, Amperometric biosensor for the rapid determination of salycilate in whole blood, Anal. Chim. Acta, 224(1) (1989) 39. [12] O. Zaborsky, Immobilized Enzymes, CRC Press, Boca Ration, FL, 1973. [13] I.G. Beddows, M.H. Gil and J.T. Guthrie, The immobilization of enzymes, bovine serum albumin and phenylpropylamine to poly(acrylic acid)-polyethylene based copolymers, Biotechnol. Bioeng., 24(6) (1982) 1371. [14] El. Abdel-Hay, C.G. Beddows, M.H. Gil and J.T. Guthrie, The use of acrylic acid-co-nylon substrates for immobilizing enzymes, J. Polym. Sci., Polym. Chem. Ed., 21 (1983) 2463. [15] G.H. Hsiue, Z.S. Chou, N. Yu and K.P. Hsiung, Urease immobilized poly(vinyl-alcohol-g-butyl acrylate) membrane for urea sensor, J. Appl. Polym. Sci., 34(1) (1987) 319. [16] G.H. Hsiue and C.C. Wang, Glucose oxidase immobilized polyethylene-g-acrylic acid membranes for glucose oxidase sensor, Biotechnol. Bioeng., 36(8) (1990) 811. [17] K. Martensson, Preparation of an immobilized two-enzyme system/3-amylase-pullunase on an acrylic copolymer for the conversion of starch to maltose. 1. Preparation and stability of immobilized /3-amylase, Biotechnol. Bioeng., 16(5) (1974) 567. [18] T. Godjevargova and A. Dimov, Grafting of acrylonitrile copolymer membranes with hydrophilic monomers for immobilization of glucose oxidase, J. Appl. Polym. Sci., 57 (1995) 487. [19] T. Godjevargova, Radiation grafting of AMPSA and DMAEM onto membranes of acrylonitrile copolymer and
[20]
[21] [22]
[23]
[24]
[25]
[26]
[27]
7
polyamide for immobilization of glucose oxidase, J. Appl. Polym. Sci., 61 (1996) 343. A.M.O. Brett, M.H. Gil and A.P. Piedade, An electrochemical bienzyme membrane sensor for free cholesterol, Bioelectrochem. Bioenerg., 28 (1992) 105. A.M.O. Brett, M.H. Gil and A.P. Piedade, A cholesterol biosensor, Port. Electrochim. Acta, 9 (1991) 7. M. Alves da Silva, M.H. Gil, A.P. Piedade, J.S. Redinha, A.M.O. Brett and J.M.C. Costa, Immobilization of catalase on membranes of poly(ethylene-g-co-acrylic acid) and their application in hydrogen peroxide electrochemical sensors, J. Polym. Sci. Part A, Polym. Chem., 29 (1991) 269. J.S. Redinha, A.M.O. Brett, M. Alves da Silva and A.P. Piedade, A catalase membrane electrode for the determination of hydrogen peroxide, Port. Electrochim. Acta, 7 (1989) 17. M. Alves da Silva, M.H. Gil, J.S. Redinha, A.P. Piedade and J.L.C. Pereira, Immobilization of glucose oxidase on nylon membranes and its application in a flow-through glucose reactor, J. Polym. Sci. Part A, Polym. Chem., 29 (1991) 275. G.K. Kostov, S. Chr. Turmanova and A.N. Atanassov, Radiation induced grafting of acrylic acid onto low density polyethylene and polytetrafluoroethylene thin films, in J.M. Marshall, N. Kirov and A. Vavrek (Eds.), Electronic, Optoelectronic and Magnetic Thin Films, Wiley, New York, 1995, p. 672. E.A. Hegazy, I. Ishigaki, A. Rabie, A. Dessouki and J. Okamoto, Study on radiation grafting of acrylic acid onto fluorine containing polymers. II. Properties of membrane obtained by preirradiation grafting onto poly(tetrafluoroethylene), J. Appl. Polym. Sci., 26 (1981) 3871. A.S.G. Hugget and D.A. Nixon, Enzymatic determination of blood glucose, Biochem. J., 66 (1957) 12.