- Email: [email protected]
Cyro-hydrogel for the construction of a tyrosinase-based biosensor
ANALmcA CHIMICA ACTA Analytica Chimica Acta 319 (1996) 71-77
Cyro-hydrogel for the construction of a tyrosinase-based biosensor Qing Deng, Yizhu Guo, Shaojun Dong Laboratory
of Electroanalytical
Chemistry, Changchun Institute
*
ofApplied Chemistry, Chinese Academy of Sciences, Changchun 130022, China
Received 3 June 1994; revised 20 October 1994; accepted 7 November
1994
Abstract A new immobilization method for construction of a tyrosinase based biosensor is described. A simple physical freezing technique was adopted for preparation. The immobilized enzyme yields specific activities that are more than 22% of the soluble enzyme. The enzyme electrode can be stored in dry state for more than three months without any loss of activity. The biosensor was applied to the determination of several phenols and o-diphenols. The lowest detect limit is 0.02 pmol/l and the linear range was 1.0 X lo-‘-1.0 X 10m4 mol/l for catechol. The kinetic parameters have also been calculated. Keywords:
Biosensors;
Cyro-hydrogel;
Tyrosinase
1. Introduction Recently the amperometric determination of phenolic compounds and catecholamine neurotransmitters has received much attention in biosensor Tyrosinase (polyphenoloxidase, E.C. research. 1.14.18.1) has a broad specificity for o-diphenol compounds and can catalyze the oxidation of catechol to 1,2-benzoquinone. Two main stratagems have been used in the amperometric detection of these compounds. In the first one an oxygen electrode was used to detect the decrease of dissolved oxygen. There are some reports based on the Clark oxygen electrode and especially on some tissue electrodes [l-4], but the detection limit is only 1O-5 mol/l. Recently Uchiyama et al [51 amplified the response
* Corresponding
author.
0003-2670/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0003-2670(95)00498-X
by combining the enzymatic reaction and the chemical reduction, but the response time was too long and the stability of the sensor was not satisfactory. The other method is to directly detect the quinonoid compound on a carbon electrode. This requires good immobilization methods because tyrosinase is not as robust as glucose oxidase and the stability of the electrode is not easy to control. Wang et al. [6,7] and Bonakdar et al. [8] used carbon paste mixing with tyrosinase or tissues to obtain good responses. Cosnier and Innocent [9] reported a method based on the electropolymerization of a pyrrole amphiphilic monomer-tyrosinase mixture previously adsorbed on an electrode. The detect limit is 2 nmol/l. However, their immobilized method is not so simple. Recently we developed a novel immobilized freezing method [lo]. A certain quantity of polyvinyl alcohol and polyhydroxyl cellulose were mixed to form a gel. When tyrosinase was mixed with the gel
72
Q. Deng et al. /Analytica
and stored in refrigerator below - 4°C a cryo-hydrogel enzyme layer was formed on the electrode. The apparent activity of immobilized enzyme was 22% of the value of soluble enzyme. Here some preliminary results of the characteristics of the immobilization method are reported. The enzyme electrode was used to detect catechol, pcresol, phenol and dopamine. The stability, kinetic response and effects of pH and temperature are described.
Chimica Acta 319 (1996) 71-77
glassy carbon electrode. The resulting electrode was put into a refrigerator and kept at - 5°C for 12 hours and then thawed at room temperature (15 f 2°C) for 1 h. This process was repeated three times to obtain adequate mechanical robustness [ 111. The resulting enzyme electrode (electrode 1, 420 U of enzyme) was stored at 4°C in dry state or in phosphate buffer (0.1 M, pH 6.5). The bare film electrode, and two other enzyme electrodes (electrode 2, 250 U, and electrode 3, 800 U) were prepared in the same way though no enzyme or different amounts of enzyme were used.
2. Experimental 2.4. Determination
of apparent activity
of immobi-
2.1. Reagents
lized enzyme
Tyrosinase (EC. 1.14.18.1 from mushroom, 4200 U mg-‘) was obtained from Sigma. Polyvinyl alcohol was purchased from GuangZhou Chemical Reagents. Polyhydroxyl cellulose was synthesized in our laboratory. The gel was prepared according to Ref. [lo]. All other reagents were analytical grade. All solutions were prepared with doubly distilled water. The supporting electrolyte were 0.1 M phosphate buffer.
The apparent activity of immobilized enzyme in the cryo-hydrogel modified electrode was determined using spectrophotometry. The enzyme electrode was inserted into a cuvette containing 2 ml 0.1 M pH 6.5 phosphate buffer and 1O-4 M catechol. The solution was agitated. After 3 min the enzyme electrode was taken from the solution and the absorbance was measured at 380 nm. The absorbance change per minute was calculated. A tyrosinase calibration curve was obtained by repeating the above measurement using a soluble enzyme. The apparent activity of the amount of enzyme presented on the enzyme electrode can be determined from the calibration curve.
2.2. Apparatus All amperometric measurements were performed with a PARC 370 electrochemical system combined with a Gould 6000 x-y recorder. A conventional three-electrode system was used. The working electrode was a glassy carbon disk (diameter 4 mm) covered with an enzyme layer. An Ag/AgCl (saturated KCl) electrode was used as a reference electrode and platinum wire as auxiliary electrode. The 15ml electrochemical cell was thermostated at 25 f O.l”C. The enzyme electrode was poised at -0.2 V in air-saturated phosphate buffer (0.1 M, pH 6.9) under stirring. After the sensor had reached a stable background current, successive aliquots of substrate solution were added to establish the linear range. 2.3. Enzyme electrode preparation 0.5 mg tyrosinase was dissolved into 0.5 g (50 ~1) of gel. 10 ~1 of the mixture were spread on a
3. Results and discussion 3.1. Characteristic
of the immobilization
method
The solution formed a cross-linked (via hydrogen bonding and van der Waals forces) amorphous, netted polymer gel material through the process of freeze-thaw cycling. We call this material cryo-hydrogel. There are three types of water in the cryo-hywater’ ’ which has drogel [ 12-141. “Unfreezing strong interaction with the polymer, it will not freeze until - 40°C; “bond water” which exits near the hydrophobic group of the polymer (the amount of this kind of water was related to the component of the polymer); and “free water” which has the same character as natural water. The amount of free water
73
Q. Deng et al./Analytica Chimica Acta 319 (1996) 71-77
changes with humidity, but the other types of water were quite stable. If the cryo-hydrogel was put in a dry atmosphere, it shrinks ca. 40%, but when contacted with a small amount of water, the material will recover to its original state. When the gel was cryo-desiccated dried with enzyme, it tends to stabilize the activity of the enzyme. This is thought to be due to the hydroxyl group in the polymer holding or substituting for the “bound” water which is necessary for the retention of the tertiary structure of the enzyme and the subsequent activity of the molecule [15]. That is to say, the enzyme was retained in the network of the polymer with its perfect tertiary structure. In order to obtain adequate mechanical robustness, the freeze-thaw process should be repeated several times. However, the repeated freeze thawing process will denature some of the enzyme. Adding protein will make the gel loosen. Therefore we decided to perform the freeze-thaw process three times. The immobilized enzyme yields specific apparent activity that is more than 22% of the soluble enzyme from the spectrophotometric results. In addition, the enzyme will not denature when the polymer shrivelled. When supplied with some water, the enzyme layer was rehydrated and the activity of the enzyme can be fully stimulated. 3.2. The responds of the enzyme electrode Tyrosinase catalyses the oxidation of phenol and o-diphenol to quinones. The electrode response is based on the amperometric detection of the biocatalytically generated quinone products. The catalytic and electrode reaction are shown below: OH
tyrotiase 2
+ 2Hz0
(1)
-0.4
-0.2
0.0
E (V vs.
0.2
0.4
0.6
Ag/AgCl)
Fig. 1. Cyclic votammograms of bare film electrode (- - -) and 800 U enzyme electrode (-) in pH 6.5 buffer containing 5 X 10m3 M p-cresol
for 5 min.
Fig. 1 shows the cyclic voltammetric (CV) curves of the bare film and enzyme electrode (electrode 3) in pH 6.5 buffer solution containing 5 X 1O-3 M p-cresol for 5 min. p-Cresol was electroinactive in the potential range examined and there is no response at the bare film electrode. At the enzyme electrode according to reactions 1 and 2 the reactions start quickly, and the response can be easily detected. The hydrodynamic voltammograms of the sensor are shown in Fig. 2. The reduction of oxygen at the electrode will inference with the response if the applied potential was more negative than - 0.35 V. We chose -0.2 V vs. Ag/AgCl as the optimum value. Specific substrates were injected to the air saturated stirring electrolyte and the current-time curves were recorded (shown in Fig. 3). The enzyme electrode shows efficient activity to both catechol and phenol. The response time measured from Fig. 3 was within 50 s. 3.3. pH effect
OH
0
+2S + 2H+
OH
(2)
Fig. 4 illustrates the effect of electrolyte pH value on the response of the sensor. Curve a represents the immobilized enzyme and curve b the soluble enzyme. The response of the immobilized enzyme varies a little between pH 6 and 8. At pH 9, a
74
Q. Deng et al. /Analytica Chitnica Acta 319 (1996) 71-77
250
250
ZDO
200
- 1.5
hlJO
,150
$
d V
-
-
3
- 1.0 _
-100 100
- 0.5
50 50 i I , I I 0 -0.4 -0.3 -0.2 -0.1 0.0
0.1
I
I
0.2
0.3
:.oo
7.00
of the sensor. pH 6.5 buffer
response of 82% was obtained, while the response of the soluble enzyme was only 10%. At pH 12 the noise was still quite small, therefore the low response of the sensor was due to denaturation of the enzyme. The polymer was quite stable. From Fig. 4 we can see the effect of pH on both the immobilized enzyme and the soluble enzyme as a bell-shaped response curve. At pH range 4.5 to 9, the soluble enzyme was active. This was in accordance with the literature [16]. The bell shape of the response curve of the immobilized enzyme was wider and it moves more to the basic direction than the curve of the soluble enzyme. The reason for this is that the microenvironment of the enzyme had been changed
Fig. 3. Steady state current vs. time responses of the 420 U enzyme electrode with increasing (a) catechol and (b) phenol in 3 pmol/l steps. Potential: -0.2 V vs. Ag/AgCl; electrolyte: 25°C air-saturated 0.1 M phosphate buffer (pH 6.9) under stirring.
9.00
11.00
PH
0.4
E (V vs. Ag/AgCl) Fig. 2. Hydrodynamic voltammogram containing lo-’ M catechol.
0.0 5.00
Fig. 4. Dependence of current response for catechol (1 pmol/l) on (a) the free enzyme (54 U/ml) and (b) the immobilized enzyme electrode.
by the immobilization. The hydrogen bond between
the polymer and enzyme changes the charge distribution of the enzyme surface. The binding ability of the enzyme to hydrogen ion was decreased. In addition the gel was composed using a buffer solution, and this also gives the enzyme a more stable pH microenvironment. In the following experiment we chose pH 6.9 as the optimum value. 3.4. Temperature effect The effect of temperature on the response also shows a bell-shaped curve. In air-saturated solution the response increased with a temperature increase from 10 to 45°C. The response time is gradually decreased, because the activity of the enzyme increases at higher temperatures. However, the signal decreased at values > 50°C. At 60°C the response was 60% of the highest value. At this time the sensor was put in a 30°C solution again. The response was the same as the original though the response time required was 150 s. The enzyme was denatured irreversibly beyond 65°C. At all temperature ranges tested the polymer film stayed quite stable. Therefore, the noise is quite small, according to the Arhenius equation. The apparent activation energy for the rate-determining step was calculated to be 35.7 kJ/mol. In order to maintain the stability of the sensor, we chose 25°C as the optimum temperature.
Q. Deng et al./Analytica
75
Chimica Acta 319 (1996) 71-77
oa
0
4.0
/
-b
1.0 IL I
0.0
I
I
I
50.0
100.0
150.0
C
enzyme loading as in Fig. 3. (a)
Different amounts of the enzyme were used to detect catechol under optimum conditions. Fig. 5 shows the effect of enzyme loading on the response of the biosensor. The sensitivity (slope of linear portion) enlarged with the increase of enzyme. Under the optimum conditions we obtained calibration curves of catechol, p-cresol, phenol and dopamine using electrode 1. In order to give a more obvious description of all concentration ranges, we constructed a log Z-log C curve (shown in Fig. 6). The parameters of the curve, the sensitivity of the electrode and the lowest detected substrate concentrations are listed in Table 1. The lowest detected limit was obtained according with a signal-to-noise
Slope Intercept Corr. coeff. Low limit ( PM) Linear range (M) Sensitivity (mA/M)
curves (log I-log
Fig. 6. Calibration curve (log I-log C) of catechol (a), p-cresol (b), phenol (c) and dopamine (d). Experimental conditions as in Fig. 3.
3.5. Enzyme loading effect
of calibration
-3.5 -3.0
Log C 04)
(W)
Fig. 5. Calibration curves (I-C) of different electrodes to catechol. Experimental conditions 250 U, (b) 420 U, (c) 800 U.
Table 1 Parameters
-7.0 -6.5 -6.0 -5.5 -5.0 -4.5 4.0
200.0
ratio > 2. The response of 0.02 hmol/l catechol was 5 nA. The sensitivity of different substrates can be calculated from the slope of the linear section of the I-C curve (not shown). 3.6. Kinetic analysis The response on a substrate and sensitivity are the most important parameters for appreciating a biosensor. The use of the Eadie-Hofstee form of the Michaelis-Menten equation is quite efficient in the kinetic analysis of a biosensor. For amperometric biosensors the reaction rates are substituted with steady-state currents.
C), the lowest detected substrate concentration,
linear range and sensitivity
of the sensor
Catechol
p-Cresol
Phenol
Dopamine
0.981 f 0.012 8.049 It 0.033 0.9993 0.020 1.0 x lo-‘-l.0 86.9
0.935 * 0.013 7.357 + 0.040 0.9983 0.020 5.0 x 10-‘-5.0x 39.6
1.077 + 0.051 7.686 f 0.070 0.9903 0.10 1.0 x 10-6-1.0 23.1
1.090 f 0.016 7.265 f 0.033 0.9988 0.10 2.0 x lo- 6-5.0 x lo- 4 8.1
x 10-4
10-5
x 1o-4
76
Q. Deng et al./Analytica
Chimica Acta 319 (1996) 71-77
enzyme loading is high, the enzyme is loose and the diffusion of substrate to the film was the main part; when enzyme loading is small, the diffusion of substrate and product in the more compact film became obvious. From Fig. 5 we can see that the response of electrode 2 was quite small but the linear range was nearly the same as for electrode 1. This shows the effect of diffusion. c(
3.7. Stability and the reproducibility of the electrode
1.00 I
I
o~“Po.o
I
I
20.0
I/C Fig. 7. Eadie-Hofstee Fig. 6.
I
40.0
30.0
I 50.0
bA/mM)
plot of electrode
1. Experimental
data from
Here Z, is the steady-state current, C, the concentration of substrate, Ksp the apparent MichaelisMenten constant, and ZSmaxthe intercept on the current axis [17]. In the case of kinetic control, the enzymatic reaction is the rate controlling process; K$‘P can be obtained from the slope of the Z, - IS/C, plot according to Eq. 3. Fig. 7 shows the Eadie-Hofstee plot of electrode 1 for catechol. The plot was a line with a slope of -0.07 mmol/l. Apparently it was kinetically controlled. The Eadie-Hofstee plots of phenol, p-cresol and dopamine can be obtained from the kinetic data in Fig. 6. The KEP values of catechol, p-cresol, phenol and dopamine were obtained as 0.07, 0.08, 0.12, and 0.61 mmol/l, respectively. The KZP value of catechol was the smallest, i.e., catechol was the most appropriate substrate of tyrosinase. Diffusion control is complex in our system. The immobilization method reported here is a thick-film technique. The diffusion can be divided into at least two parts: (i) the diffusion of substrate to the enzyme film and (ii> diffusion of substrate and product to the electrode surface in the film. Here the rate of stirring is constant, but the structures of the high and low enzyme film were different. The gel will loosen when freezing it together with protein [ll]. When
The immobilization method described in this article maintained the tertiary structure of the enzyme. The storage time of the sensor is therefore satisfactory. The activity of the electrode maintained 98% after not being used but only stored in dry state in 4°C for 3 months. This is quite good for the preservation and transfer of biosensors. The sensor could be used repeatedly for at least two weeks without deterioration of the response when used 20 times a day and stored dry in the refrigerator when not in use. The response lost 40%, however, if stored in pH 6.5 buffer for 1 month. The reason is that the aperture of the amorphous netted texture of the gel was bigger than the enzyme molecule. The material needs to be improved further. The enzyme electrode shows good reproducibility: for seven replicate determinations of 3 pmol/l catechol in pH 6.9 buffer, the response was 190,190, 185, 185, 195, 185 and 190 nA, respectively. The relative deviation was less than 3.8%. However, the relative standard deviation of eight equivalently prepared electrodes was not quite well (20%).
4. Conclusions
The construction of a simple and cheap biosensor based on freezing enzyme and polyhydroxyl gel has been demonstrated. This method opens up a new way for the preparation and preservation during the fabrication of biosensors. This technique will work for glucose oxidase, alcohol dehydrogenase and probably a whole range of other enzymes as well. Moreover, the material can retain a certain percentage of its water in some organic solvent. The application of this material in organic phase enzyme electrodes seems to be attractive.
Q. Deng et al./Analytica Chimica Acta 319 (1996) 71-77
Acknowledgements Support by the National Natural Science Foundation of China is gratefully acknowledged.
References [l] U. Wollenberger and (1983) 239. [2] J.S. Sidwell and G.A. 419. [3] F. Mazzei, M. Lanzi, Sensors Actuators, B7 [4] S. Uchiyama 361.
F.W.
Scheller,
Rechnitz,
Biotechnol.
Biotechnol.
G. Lorenti, F. Procelli (1992) 427.
Lett.,
5
L&t., 7 (1985) and C. Botre,
and S. Suzuki, Anal. Chim. Acta, 261 (1992)
[5] S. Uchiyama, Y. Hasebe, H. Shimizu and H. Ishihara, Anal. Chim. Acta, 276 (1993) 341.
71
[6] J. Wang and K. Varughese, Anal. Chem., 62 (1990) 318. [7] J. Wang and MS. Lin. Anal. Chem., 60 (1988) 1545. [8] M. Bonakdar, J. Vilchez and H. Mottola, J. Electroanal. Chem., 266 (1989) 47. [9] S. Cosnier and C. Innocent, Bioelectrochem. Bioenerg., 31 (1993) 147. [lo] S. Dong and Y. Guo, J. Electroanal. Chem., 375 (1994) 405. 1111 Minamibe, Kobunshi Kako, 32 (1983) 523. [12] Y.D. Feng, J. Sichuan University (Natural Science Edition), 26 (1989) 470. [13] M.A. Frommer and D. Lancet, J. Appl. Polymer Sci., 16 (1972) 1295. [14] G. Huang, Y. Feng and J. He, Acta Polym. Sinica, 3 (1992) 6. [15] T.D. Gibson and J.R. Wordward, in P.R. Edelman and J. Wang (Eds.), Biosensors and Chemical Sensors, American Chemical Society, Washington, DC, 1992, Chap. 5. [16] D. Kertesz and R. Zito, Biochim. Biophys. Acta, 96 (1965) 447. [17] R.A. Kamin and G.S. Wilson, Anal. Chem., 52 (1980) 1198.
Cyro-hydrogel for the construction of a tyrosinase-based biosensor Qing Deng, Yizhu Guo, Shaojun Dong Laboratory
of Electroanalytical
Chemistry, Changchun Institute
*
ofApplied Chemistry, Chinese Academy of Sciences, Changchun 130022, China
Received 3 June 1994; revised 20 October 1994; accepted 7 November
1994
Abstract A new immobilization method for construction of a tyrosinase based biosensor is described. A simple physical freezing technique was adopted for preparation. The immobilized enzyme yields specific activities that are more than 22% of the soluble enzyme. The enzyme electrode can be stored in dry state for more than three months without any loss of activity. The biosensor was applied to the determination of several phenols and o-diphenols. The lowest detect limit is 0.02 pmol/l and the linear range was 1.0 X lo-‘-1.0 X 10m4 mol/l for catechol. The kinetic parameters have also been calculated. Keywords:
Biosensors;
Cyro-hydrogel;
Tyrosinase
1. Introduction Recently the amperometric determination of phenolic compounds and catecholamine neurotransmitters has received much attention in biosensor Tyrosinase (polyphenoloxidase, E.C. research. 1.14.18.1) has a broad specificity for o-diphenol compounds and can catalyze the oxidation of catechol to 1,2-benzoquinone. Two main stratagems have been used in the amperometric detection of these compounds. In the first one an oxygen electrode was used to detect the decrease of dissolved oxygen. There are some reports based on the Clark oxygen electrode and especially on some tissue electrodes [l-4], but the detection limit is only 1O-5 mol/l. Recently Uchiyama et al [51 amplified the response
* Corresponding
author.
0003-2670/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0003-2670(95)00498-X
by combining the enzymatic reaction and the chemical reduction, but the response time was too long and the stability of the sensor was not satisfactory. The other method is to directly detect the quinonoid compound on a carbon electrode. This requires good immobilization methods because tyrosinase is not as robust as glucose oxidase and the stability of the electrode is not easy to control. Wang et al. [6,7] and Bonakdar et al. [8] used carbon paste mixing with tyrosinase or tissues to obtain good responses. Cosnier and Innocent [9] reported a method based on the electropolymerization of a pyrrole amphiphilic monomer-tyrosinase mixture previously adsorbed on an electrode. The detect limit is 2 nmol/l. However, their immobilized method is not so simple. Recently we developed a novel immobilized freezing method [lo]. A certain quantity of polyvinyl alcohol and polyhydroxyl cellulose were mixed to form a gel. When tyrosinase was mixed with the gel
72
Q. Deng et al. /Analytica
and stored in refrigerator below - 4°C a cryo-hydrogel enzyme layer was formed on the electrode. The apparent activity of immobilized enzyme was 22% of the value of soluble enzyme. Here some preliminary results of the characteristics of the immobilization method are reported. The enzyme electrode was used to detect catechol, pcresol, phenol and dopamine. The stability, kinetic response and effects of pH and temperature are described.
Chimica Acta 319 (1996) 71-77
glassy carbon electrode. The resulting electrode was put into a refrigerator and kept at - 5°C for 12 hours and then thawed at room temperature (15 f 2°C) for 1 h. This process was repeated three times to obtain adequate mechanical robustness [ 111. The resulting enzyme electrode (electrode 1, 420 U of enzyme) was stored at 4°C in dry state or in phosphate buffer (0.1 M, pH 6.5). The bare film electrode, and two other enzyme electrodes (electrode 2, 250 U, and electrode 3, 800 U) were prepared in the same way though no enzyme or different amounts of enzyme were used.
2. Experimental 2.4. Determination
of apparent activity
of immobi-
2.1. Reagents
lized enzyme
Tyrosinase (EC. 1.14.18.1 from mushroom, 4200 U mg-‘) was obtained from Sigma. Polyvinyl alcohol was purchased from GuangZhou Chemical Reagents. Polyhydroxyl cellulose was synthesized in our laboratory. The gel was prepared according to Ref. [lo]. All other reagents were analytical grade. All solutions were prepared with doubly distilled water. The supporting electrolyte were 0.1 M phosphate buffer.
The apparent activity of immobilized enzyme in the cryo-hydrogel modified electrode was determined using spectrophotometry. The enzyme electrode was inserted into a cuvette containing 2 ml 0.1 M pH 6.5 phosphate buffer and 1O-4 M catechol. The solution was agitated. After 3 min the enzyme electrode was taken from the solution and the absorbance was measured at 380 nm. The absorbance change per minute was calculated. A tyrosinase calibration curve was obtained by repeating the above measurement using a soluble enzyme. The apparent activity of the amount of enzyme presented on the enzyme electrode can be determined from the calibration curve.
2.2. Apparatus All amperometric measurements were performed with a PARC 370 electrochemical system combined with a Gould 6000 x-y recorder. A conventional three-electrode system was used. The working electrode was a glassy carbon disk (diameter 4 mm) covered with an enzyme layer. An Ag/AgCl (saturated KCl) electrode was used as a reference electrode and platinum wire as auxiliary electrode. The 15ml electrochemical cell was thermostated at 25 f O.l”C. The enzyme electrode was poised at -0.2 V in air-saturated phosphate buffer (0.1 M, pH 6.9) under stirring. After the sensor had reached a stable background current, successive aliquots of substrate solution were added to establish the linear range. 2.3. Enzyme electrode preparation 0.5 mg tyrosinase was dissolved into 0.5 g (50 ~1) of gel. 10 ~1 of the mixture were spread on a
3. Results and discussion 3.1. Characteristic
of the immobilization
method
The solution formed a cross-linked (via hydrogen bonding and van der Waals forces) amorphous, netted polymer gel material through the process of freeze-thaw cycling. We call this material cryo-hydrogel. There are three types of water in the cryo-hywater’ ’ which has drogel [ 12-141. “Unfreezing strong interaction with the polymer, it will not freeze until - 40°C; “bond water” which exits near the hydrophobic group of the polymer (the amount of this kind of water was related to the component of the polymer); and “free water” which has the same character as natural water. The amount of free water
73
Q. Deng et al./Analytica Chimica Acta 319 (1996) 71-77
changes with humidity, but the other types of water were quite stable. If the cryo-hydrogel was put in a dry atmosphere, it shrinks ca. 40%, but when contacted with a small amount of water, the material will recover to its original state. When the gel was cryo-desiccated dried with enzyme, it tends to stabilize the activity of the enzyme. This is thought to be due to the hydroxyl group in the polymer holding or substituting for the “bound” water which is necessary for the retention of the tertiary structure of the enzyme and the subsequent activity of the molecule [15]. That is to say, the enzyme was retained in the network of the polymer with its perfect tertiary structure. In order to obtain adequate mechanical robustness, the freeze-thaw process should be repeated several times. However, the repeated freeze thawing process will denature some of the enzyme. Adding protein will make the gel loosen. Therefore we decided to perform the freeze-thaw process three times. The immobilized enzyme yields specific apparent activity that is more than 22% of the soluble enzyme from the spectrophotometric results. In addition, the enzyme will not denature when the polymer shrivelled. When supplied with some water, the enzyme layer was rehydrated and the activity of the enzyme can be fully stimulated. 3.2. The responds of the enzyme electrode Tyrosinase catalyses the oxidation of phenol and o-diphenol to quinones. The electrode response is based on the amperometric detection of the biocatalytically generated quinone products. The catalytic and electrode reaction are shown below: OH
tyrotiase 2
+ 2Hz0
(1)
-0.4
-0.2
0.0
E (V vs.
0.2
0.4
0.6
Ag/AgCl)
Fig. 1. Cyclic votammograms of bare film electrode (- - -) and 800 U enzyme electrode (-) in pH 6.5 buffer containing 5 X 10m3 M p-cresol
for 5 min.
Fig. 1 shows the cyclic voltammetric (CV) curves of the bare film and enzyme electrode (electrode 3) in pH 6.5 buffer solution containing 5 X 1O-3 M p-cresol for 5 min. p-Cresol was electroinactive in the potential range examined and there is no response at the bare film electrode. At the enzyme electrode according to reactions 1 and 2 the reactions start quickly, and the response can be easily detected. The hydrodynamic voltammograms of the sensor are shown in Fig. 2. The reduction of oxygen at the electrode will inference with the response if the applied potential was more negative than - 0.35 V. We chose -0.2 V vs. Ag/AgCl as the optimum value. Specific substrates were injected to the air saturated stirring electrolyte and the current-time curves were recorded (shown in Fig. 3). The enzyme electrode shows efficient activity to both catechol and phenol. The response time measured from Fig. 3 was within 50 s. 3.3. pH effect
OH
0
+2S + 2H+
OH
(2)
Fig. 4 illustrates the effect of electrolyte pH value on the response of the sensor. Curve a represents the immobilized enzyme and curve b the soluble enzyme. The response of the immobilized enzyme varies a little between pH 6 and 8. At pH 9, a
74
Q. Deng et al. /Analytica Chitnica Acta 319 (1996) 71-77
250
250
ZDO
200
- 1.5
hlJO
,150
$
d V
-
-
3
- 1.0 _
-100 100
- 0.5
50 50 i I , I I 0 -0.4 -0.3 -0.2 -0.1 0.0
0.1
I
I
0.2
0.3
:.oo
7.00
of the sensor. pH 6.5 buffer
response of 82% was obtained, while the response of the soluble enzyme was only 10%. At pH 12 the noise was still quite small, therefore the low response of the sensor was due to denaturation of the enzyme. The polymer was quite stable. From Fig. 4 we can see the effect of pH on both the immobilized enzyme and the soluble enzyme as a bell-shaped response curve. At pH range 4.5 to 9, the soluble enzyme was active. This was in accordance with the literature [16]. The bell shape of the response curve of the immobilized enzyme was wider and it moves more to the basic direction than the curve of the soluble enzyme. The reason for this is that the microenvironment of the enzyme had been changed
Fig. 3. Steady state current vs. time responses of the 420 U enzyme electrode with increasing (a) catechol and (b) phenol in 3 pmol/l steps. Potential: -0.2 V vs. Ag/AgCl; electrolyte: 25°C air-saturated 0.1 M phosphate buffer (pH 6.9) under stirring.
9.00
11.00
PH
0.4
E (V vs. Ag/AgCl) Fig. 2. Hydrodynamic voltammogram containing lo-’ M catechol.
0.0 5.00
Fig. 4. Dependence of current response for catechol (1 pmol/l) on (a) the free enzyme (54 U/ml) and (b) the immobilized enzyme electrode.
by the immobilization. The hydrogen bond between
the polymer and enzyme changes the charge distribution of the enzyme surface. The binding ability of the enzyme to hydrogen ion was decreased. In addition the gel was composed using a buffer solution, and this also gives the enzyme a more stable pH microenvironment. In the following experiment we chose pH 6.9 as the optimum value. 3.4. Temperature effect The effect of temperature on the response also shows a bell-shaped curve. In air-saturated solution the response increased with a temperature increase from 10 to 45°C. The response time is gradually decreased, because the activity of the enzyme increases at higher temperatures. However, the signal decreased at values > 50°C. At 60°C the response was 60% of the highest value. At this time the sensor was put in a 30°C solution again. The response was the same as the original though the response time required was 150 s. The enzyme was denatured irreversibly beyond 65°C. At all temperature ranges tested the polymer film stayed quite stable. Therefore, the noise is quite small, according to the Arhenius equation. The apparent activation energy for the rate-determining step was calculated to be 35.7 kJ/mol. In order to maintain the stability of the sensor, we chose 25°C as the optimum temperature.
Q. Deng et al./Analytica
75
Chimica Acta 319 (1996) 71-77
oa
0
4.0
/
-b
1.0 IL I
0.0
I
I
I
50.0
100.0
150.0
C
enzyme loading as in Fig. 3. (a)
Different amounts of the enzyme were used to detect catechol under optimum conditions. Fig. 5 shows the effect of enzyme loading on the response of the biosensor. The sensitivity (slope of linear portion) enlarged with the increase of enzyme. Under the optimum conditions we obtained calibration curves of catechol, p-cresol, phenol and dopamine using electrode 1. In order to give a more obvious description of all concentration ranges, we constructed a log Z-log C curve (shown in Fig. 6). The parameters of the curve, the sensitivity of the electrode and the lowest detected substrate concentrations are listed in Table 1. The lowest detected limit was obtained according with a signal-to-noise
Slope Intercept Corr. coeff. Low limit ( PM) Linear range (M) Sensitivity (mA/M)
curves (log I-log
Fig. 6. Calibration curve (log I-log C) of catechol (a), p-cresol (b), phenol (c) and dopamine (d). Experimental conditions as in Fig. 3.
3.5. Enzyme loading effect
of calibration
-3.5 -3.0
Log C 04)
(W)
Fig. 5. Calibration curves (I-C) of different electrodes to catechol. Experimental conditions 250 U, (b) 420 U, (c) 800 U.
Table 1 Parameters
-7.0 -6.5 -6.0 -5.5 -5.0 -4.5 4.0
200.0
ratio > 2. The response of 0.02 hmol/l catechol was 5 nA. The sensitivity of different substrates can be calculated from the slope of the linear section of the I-C curve (not shown). 3.6. Kinetic analysis The response on a substrate and sensitivity are the most important parameters for appreciating a biosensor. The use of the Eadie-Hofstee form of the Michaelis-Menten equation is quite efficient in the kinetic analysis of a biosensor. For amperometric biosensors the reaction rates are substituted with steady-state currents.
C), the lowest detected substrate concentration,
linear range and sensitivity
of the sensor
Catechol
p-Cresol
Phenol
Dopamine
0.981 f 0.012 8.049 It 0.033 0.9993 0.020 1.0 x lo-‘-l.0 86.9
0.935 * 0.013 7.357 + 0.040 0.9983 0.020 5.0 x 10-‘-5.0x 39.6
1.077 + 0.051 7.686 f 0.070 0.9903 0.10 1.0 x 10-6-1.0 23.1
1.090 f 0.016 7.265 f 0.033 0.9988 0.10 2.0 x lo- 6-5.0 x lo- 4 8.1
x 10-4
10-5
x 1o-4
76
Q. Deng et al./Analytica
Chimica Acta 319 (1996) 71-77
enzyme loading is high, the enzyme is loose and the diffusion of substrate to the film was the main part; when enzyme loading is small, the diffusion of substrate and product in the more compact film became obvious. From Fig. 5 we can see that the response of electrode 2 was quite small but the linear range was nearly the same as for electrode 1. This shows the effect of diffusion. c(
3.7. Stability and the reproducibility of the electrode
1.00 I
I
o~“Po.o
I
I
20.0
I/C Fig. 7. Eadie-Hofstee Fig. 6.
I
40.0
30.0
I 50.0
bA/mM)
plot of electrode
1. Experimental
data from
Here Z, is the steady-state current, C, the concentration of substrate, Ksp the apparent MichaelisMenten constant, and ZSmaxthe intercept on the current axis [17]. In the case of kinetic control, the enzymatic reaction is the rate controlling process; K$‘P can be obtained from the slope of the Z, - IS/C, plot according to Eq. 3. Fig. 7 shows the Eadie-Hofstee plot of electrode 1 for catechol. The plot was a line with a slope of -0.07 mmol/l. Apparently it was kinetically controlled. The Eadie-Hofstee plots of phenol, p-cresol and dopamine can be obtained from the kinetic data in Fig. 6. The KEP values of catechol, p-cresol, phenol and dopamine were obtained as 0.07, 0.08, 0.12, and 0.61 mmol/l, respectively. The KZP value of catechol was the smallest, i.e., catechol was the most appropriate substrate of tyrosinase. Diffusion control is complex in our system. The immobilization method reported here is a thick-film technique. The diffusion can be divided into at least two parts: (i) the diffusion of substrate to the enzyme film and (ii> diffusion of substrate and product to the electrode surface in the film. Here the rate of stirring is constant, but the structures of the high and low enzyme film were different. The gel will loosen when freezing it together with protein [ll]. When
The immobilization method described in this article maintained the tertiary structure of the enzyme. The storage time of the sensor is therefore satisfactory. The activity of the electrode maintained 98% after not being used but only stored in dry state in 4°C for 3 months. This is quite good for the preservation and transfer of biosensors. The sensor could be used repeatedly for at least two weeks without deterioration of the response when used 20 times a day and stored dry in the refrigerator when not in use. The response lost 40%, however, if stored in pH 6.5 buffer for 1 month. The reason is that the aperture of the amorphous netted texture of the gel was bigger than the enzyme molecule. The material needs to be improved further. The enzyme electrode shows good reproducibility: for seven replicate determinations of 3 pmol/l catechol in pH 6.9 buffer, the response was 190,190, 185, 185, 195, 185 and 190 nA, respectively. The relative deviation was less than 3.8%. However, the relative standard deviation of eight equivalently prepared electrodes was not quite well (20%).
4. Conclusions
The construction of a simple and cheap biosensor based on freezing enzyme and polyhydroxyl gel has been demonstrated. This method opens up a new way for the preparation and preservation during the fabrication of biosensors. This technique will work for glucose oxidase, alcohol dehydrogenase and probably a whole range of other enzymes as well. Moreover, the material can retain a certain percentage of its water in some organic solvent. The application of this material in organic phase enzyme electrodes seems to be attractive.
Q. Deng et al./Analytica Chimica Acta 319 (1996) 71-77
Acknowledgements Support by the National Natural Science Foundation of China is gratefully acknowledged.
References [l] U. Wollenberger and (1983) 239. [2] J.S. Sidwell and G.A. 419. [3] F. Mazzei, M. Lanzi, Sensors Actuators, B7 [4] S. Uchiyama 361.
F.W.
Scheller,
Rechnitz,
Biotechnol.
Biotechnol.
G. Lorenti, F. Procelli (1992) 427.
Lett.,
5
L&t., 7 (1985) and C. Botre,
and S. Suzuki, Anal. Chim. Acta, 261 (1992)
[5] S. Uchiyama, Y. Hasebe, H. Shimizu and H. Ishihara, Anal. Chim. Acta, 276 (1993) 341.
71
[6] J. Wang and K. Varughese, Anal. Chem., 62 (1990) 318. [7] J. Wang and MS. Lin. Anal. Chem., 60 (1988) 1545. [8] M. Bonakdar, J. Vilchez and H. Mottola, J. Electroanal. Chem., 266 (1989) 47. [9] S. Cosnier and C. Innocent, Bioelectrochem. Bioenerg., 31 (1993) 147. [lo] S. Dong and Y. Guo, J. Electroanal. Chem., 375 (1994) 405. 1111 Minamibe, Kobunshi Kako, 32 (1983) 523. [12] Y.D. Feng, J. Sichuan University (Natural Science Edition), 26 (1989) 470. [13] M.A. Frommer and D. Lancet, J. Appl. Polymer Sci., 16 (1972) 1295. [14] G. Huang, Y. Feng and J. He, Acta Polym. Sinica, 3 (1992) 6. [15] T.D. Gibson and J.R. Wordward, in P.R. Edelman and J. Wang (Eds.), Biosensors and Chemical Sensors, American Chemical Society, Washington, DC, 1992, Chap. 5. [16] D. Kertesz and R. Zito, Biochim. Biophys. Acta, 96 (1965) 447. [17] R.A. Kamin and G.S. Wilson, Anal. Chem., 52 (1980) 1198.