Effects of conducting polymers on immobilized galactose oxidase

ELSEVIER

Synthetic Metals 87 (1997) 205-209

Effects of conducting polymers on immobilized galactose oxidase Kan Jinqing a, Xue Huaiguo a, Mu Shaolin a'*, Chen Hong b Department of Chemistry, Teachers College, Yangzhou University, Yangzhou 225002, People's Republic of China b State Key Laboratory of Physical Chemistry of the Solid Surface, Xiamen University,Xiamen, People's Republic of China Received 30 September 1996; revised 3t October 1996; accepted 4 December 1996

Abstract Based on the doping principle of conducting polymers, galactose oxidase immobilized in the polyaniline film shows a good bioelectrochemical response to galactose, but no bioelectrochemical response is observed for galactose oxidase immobilized in the polypyrrole film. The results from scanning tunneling microscopy (STM) images, Raman spectra and infrared reflectance spectra reveal that galactose oxidase is doped into the polyaniline film, but is not doped into the polypyrrole film. Keywords: Polyaniline; Polypyrrole; Galactose oxidase; Scanning tunnelling microscopy; Infrared and Raman spectroscopy

1. Introduction The immobilization of enzymes on electrodes is very important for electrochemical biosensors and flow reactors, which play a significant role in clinical, environmental and industrial applications. Conducting polymers, such as polypyrrole and polyaniline, have been used as new material for the immobilization of enzymes [ 1-8 ]. This is due to the fact that they have a high conductivity and stability in air and aqueous solution. The enzyme electrodes fabricated using conducting polymers have a good operational stability, long storage lifetime and fast response time [9-11]. Therefore, conducting polymers are promising support material for the immobilization of an enzyme, which has been reviewed extensively [ 12,13]. Most of the work on the immobilization of glucose oxidase (GOD) in conducting polymer films has concentrated on polypyrrole biosensors, which are formed during the electropolymerization of the monomer in the presence of GOD [ 16]. The enzyme is entrapped in a growing polymer film in a one-step process during electropolymerization. However, the activity of an enzyme may be affected by this immobilization method due to the presence of the monomer. Another method for the immobilization of an enzyme is based on the doping principle of conducting polymers and the isoelectric point of the enzyme [8-11 ]. The enzyme carrying a negative charge is doped into the polyaniline or polypyrrole film during the oxidation process of conducting *

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polymers. In this manner, a necessary condition is that the diameter of the hole between particles of conducting polymer should be a little larger than the diameter of an enzyme particle. In general, the diameter of an enzyme particle is in the range 100-1000 .~. There is galactose in human blood; its level in human blood relates to galactosemia and galactose intolerance. Therefore we have attempted to immobilize galactose oxidase in polyaniline films; this enzyme electrode has a good bioetectrochemical response to galactose [ 11 ]. However, we found that the response current of galactose oxidase immobilized on polypyrrole film is very small, and cannot even be detected under the same conditions of the polyaniline galactose oxidase electrode. In order to understand the effects of potyaniline and polypyrrole on the immobilized galactose oxidase, we employed some experiments to look for evidence of their difference. In this work, we report the scanning tunneling microscopy (STM), Raman spectra and infrared reflectance spectra (IRRS) of both kinds of enzyme electrodes.

2. Experimental The electrolysis cell for the preparation of polyaniline and polypyrrole consisted of two platinum electrodes (3 × 3 mm) and a saturated calomel electrode (SCE). The polyaniline film was obtained in a solution containing 0.2 M aniline and 1.0 M HC1 at a constant potential of 0.65 V. The polypyrrole film was synthesized electrochemically in a solution contain-

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J. Kan et aL / Synthetic Metals 87 (1997) 205-209

ing 0.1 M pyrrole and 0.1 M NaC1 at constant potentials of 0.65 and 0.75 V. A PAR model 173 potentiostat/galvanostat with a model 179 digital coulometer was used for the electrochemical polymerization of aniline or pyrrole. The galactose oxidase used for preparing the polyaniline galactose oxidase electrode or polypyrrole galactose oxidase electrode was obtained from Sigma Chemical Co. (EC 1.1.3.9). The solution used to prepare the enzyme electrodes contained 0.1 M N a H a P Q + NazHPO4 buffer + 0.4 ~M galactose oxidase, pH 7.50. The polyaniline and polypyrrole films were first reduced in 0.1 M phosphate buffer with pH 4 and at - 0 . 5 V for 10 min, and then the reduced polyaniline film or polypyrrole film was immediately immersed in the solution containing galactose oxidase and oxidized at 0.60 V for 20 min. The negatively charged galactose oxidase could be doped into films to form polyaniline galactose oxidase electrode or polypyrrole galactose oxidase electrode during the oxidation of polyaniline or polypyrrole. After that, the enzyme electrodes were washed thoroughly with the phosphate buffer, and kept in a refrigerator at 0 °C when not in use. The cell used to determine the response current of the enzyme electrode consisted of a galactose oxidase electrode, a platinum counter electrode, an SCE and 0.1 M phosphate buffer containing galactose. The hydrogen peroxide formed during the enzyme-catalyzed reaction was detected by the amperometric current method [ 14 ]. H202 ~ O2 + 2H + + 2e STM images of polyaniline and polypyrrole films were recorded on a Nanoscope III scanning tunnel microscope. IRRS were taken on a Nicolet 730 FT-IR spectrometer. Raman spectra were taken using a Confocal microprobe laser Raman system. The pretreatments of the polyaniline and polypyrrole films used to determine STM, IRRS and Raman spectra are the same as for the polyaniline and polypyrrole films used to fabricate the enzyme electrode, i.e., the films were first reduced at - 0.50 V and in 0.1 M phosphate buffer with pH 4 and then were immediately moved into 0.1 M phosphate buffer in the absence of galactose oxidase with pH 7.5 to oxidize for 20 min at 0.60 V. Therefore, these films were all at their oxidation states. In this paper, all potentials refer to the SCE. Aniline and pyrrole were purified by distillation before use. Other chemicals were analytical grade. Unless otherwise stated, the measurements were performed at room temperature.

3. Results and discussion 3.]. Effect of the substrate concentration on the response current

The relationship between the response currents of polyaniline galactose oxidase electrode and galactose concentration with pH 7.50 at 0.65 V is shown in Fig. 1. From Fig. t

~" 0,06

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0.03 0,00

I 4

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1

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12

[S] /mM Fig. 1. Relationshipbetweencurrent of polyanilinegalactoseoxidaseelectrode and galactoseconcentrationwith pH 7.50 at 0.65 V.

we can see that the response current of the enzyme electrode increases linearly with increasing galactose concentration from 0.2 to 10 mM, and the enzyme electrode has a good reproductivity, so the polyaniline galactose oxidase electrode can be used for the determination of galactose concentration. However, the response current of the polypyrrole galactose oxidase electrode was not detected in the above concentration range. This means that the amount of enzyme entering into the polypyrrole film is little. In order to prove this suggestion, the following experiments were performed. 3.2. STM

Fig. 2 shows the STM images of the polyaniline film (a), the polyaniline galactose oxidase electrode (b) and images of the polypyrrole films prepared at 0.65 V (c) and 0.75 V (d). From Fig. 2(a), we can see that the particle diameter of the polyaniline film is in the range 1000--2000 A, and many holes with a diameter of about 1200 ~. are located between particles. The result is similar to that obtained by MacDiarmid and co-workers [ 15]. The particle diameters of polypyrrole films obtained at 0.65 V (c) and 0.75 V (d) are about 20004300 and 4000-5000 A, respectively, but their particles are arranged tightly on the polypyrrole film surface with small holes of a diameter of about 50 ~. The diameter of an enzyme particle is in the range 1001000 A. The above result gives the idea that galactose oxidase could be doped into the polyaniline film during the oxidation process, and may not be doped into the polypyrrole film, but could be adsorbed on the polypyrrole film surface. From Fig. 2 (a) we can see that the boundaries between the particles and holes of the polyaniline film are very clear. However, their boundaries become ambiguous when the polyaniline film is covered with galactose oxidase (Fig. 2 ( b ) ) . The difference between Fig. 2(a) and (b) indicates that galactose oxidase is immobilized in the polyaniline film. This is similar to the result of the scanning electron micrograph where the polyaniline film was doped with uricase [ 10]. However, the STM image of the polypyrrole galactose oxidase electrode (omitted here) is the same as that of the polypyrrole film alone. Thus, little response current was observed for the polypyrrole galactose oxidase electrode. The adsorbed galactose oxidase o

o

J. Kan et at. / Synthetic Metals 87 (1997) 205-209

207

453,3 nN

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2 2 8 . 7 n~

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0,75

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Fig. 2. STM images: (a) polyaniline film; (b) polyaniline galactose oxidase electrode; (c) polypyrrole film obtained at 0.65 V; (d) polypyrrole film obtained at 0.75 V.

was very easily desorbed from polypyrrole film during the washing of the enzyme electrode. The evidence is that we were able to detect the presence of galactose oxidase in the buffer used to wash the enzyme electrode. It must be pointed out that xanthine oxidase was immobilized on the polypyrrole films; this enzyme electrode has a good bioelectrochemical response [ 16]. This means that the hole size in conducting polymers plays a key role in the immobilization of an enzyme using the doping method, but the characteristic of an enzyme is also important since xanthine oxidase can be immobilized on the polypyrrole film, but galactose oxidase was not immobilized on the polypyrrole film. 3.3. Raman spectra Raman spectra of polyanitine and polypyrrole electrodes with and without immobilized galactose oxidase are shown in Fig. 3. Comparing the Raman spectrum of polyaniline film (Fig. 3 ( a ) ) and that of the polyaniline galactose oxidase electrode (Fig. 3 (b)) we see that the Raman spectrum of the

polyanitine film with immobilized galactose oxidase shows a new peak at 1415 cm-~; the peak is a characteristic peak of the COa- symmetric stretching vibration in amino acid; this value is quite consistent with 1412 c m - 1 of amino acid obtained in [ 17]. This indicates that, when reduced polyaniline film is oxidized in the buffer (pH 7.50) containing galactose oxidase, the enzyme was doped into the polyaniline film. The band at 1379 cm -1 (Fig. 3 ( a ) ) shifts to 1382 cm -1 ( Fig. 3 (b) ), which indicates that there is interaction between the immobilized galactose oxidase and polyaniline. The Raman spectra of the polypyrrole films obtained at 0.65 and 0.75 V are shown in Fig. 3(c) and (e), respectively, and the Raman spectra of the galactose oxidase immobilized on the polypyrrole film obtained at 0.65 and 0.75 V are shown in Fig. 3(d) and (f), respectively. From Fig. 3 ( c ) - ( f ) we see that the Raman spectra of the films with and without immobilized galactose oxidase are not changed. The characteristic peak of amino acid does not appear. This indicates that little galactose oxidase remains on the polypyrrole films. This is why the response current of polypyrrole galactose oxidase electrode cannot be determined.

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J. Kan et al. /Synthetic Metals 87 (1997) 205-209

..J 800

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1100 1200 1300 1400 1500 1600 1700 Waven urnbers (cm-l)

800

900

|000 llOO 1200 1300 1400 1500 1600 t700 Wavenurnbers (era-l)

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800

1000 1200 1400 Wavenumbers (era.l)

1600

Fig. 3. Raman spectra: (a) potyanilinefilm; (b) polyaniline film with immobilizedgalactoseoxidase; (c) polypyrrolefilm obtained at 0.65 V; (d) galactose oxidase immobilized in the polypyrrolefilmobtained at 0.65 V; (e) polypyrrolefilmobtained at 0.75 V; (f) galactoseoxidaseimmobilizedin the potypyrrole film obtained at 0.75 V.

3.4, Infrared reflectance spectra (IRRS)

IRRS of polyaniline and polypyrrole electrodes with and without immobilized galactose oxidase are shown in Fig. 4. As described above, these films were all at their oxidation states. Comparing the infrared reflectance spectrum of polyaniline film (Fig. 4 (a)) and that of the polyaniline galactose oxidase electrode (Fig. 4 ( b ) ) we see that the spectrum of the polyaniline film with immobilized galactose oxidase

shows two new peaks at 2046 and 2120 c m - 1 (Fig. 4 ( b ) ) . It is clear that both new peaks are caused by the ammonia groups in amino acid. This reveals that when the reduced polyaniline film was immersed in the buffer (pH 7.50) containing galactose oxidase, the enzyme was doped into the polyaniline film during the oxidation process at 0.60 V. From Fig. 4 ( a ) - ( b ) we see that the peaks at 900 and 1270 cm -1 with immobilized galactose oxidase disappear, and the peaks at 1190 and 1360 e m - i shift to 1170 and 1342 c m - t, respec-

J. Kan et aI. / Synthetic Metals 87 (1997) 205-209

2000

1.500

1009

2000

Wavenumbers (cm-1)

209

1500

1000

Wavenumbers (cm-1)

Fig. 4. IRRS: (a) polyaniline film; (b) polyaniline film with immobilized galactose oxidase; (c) polypyrrole film obtained at 0.65 V; (d) galactose oxidase immobilized in the polypyrrole film obtained at 0.65 V. tively. This also provides evidence that there is interaction between the immobilized galactose oxidase and the polyaniline. Fig. 4 ( c ) - ( d ) shows the IRRS for the polypyrrole galactose oxidase electrode, which are the same as those of polypyrrole films obtained at 0.65 V. The results are consistent with those of the Raman spectra. This is more evidence that no galactose oxidase remains on the polypyrrole surface.

4. Conclusions Based on the doping principle of conducting polymers, galactose oxidase was d o p e d into the polyaniline film during the oxidation process. This is due to the fact that the hole size of polyaniline film is larger than the diameter of the galactose oxidase particle. However, galactose oxidase was not doped into polypyrrole film, mainly owing to the hole size of polypyrrole being too small. The question is why galactose oxidase cannot be strongly adsorbed on the polypyrrole surface. This m a y relate to the interaction between the enzyme and polypyrrole. Thus, further study o f this problem is required.

Acknowledgements The work is supported by the State Key Laboratory of Physical Chemistry o f the Solid Surface, Xiamen University.

The authors thank Dr Dian Zhongqun for the determination of Raman spectra, Dr Sun Shigang for the measurement o f IRRS, and Dr Mao Bingwei for making the STM experiments.

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