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Cryoimmobilized enzyme for biosensor construction
ELSEVIER
Sensors and ActuatorsB 31 (1996) 103-196
Cryoimmobilized enzyme for biosensor construction Ying Wang, Yizhu Guo, Guoyi Zhu *, Shaojun Dong Laboratoryof ElectroanalyticalChemistry.ChangchunInstituteofAppliedChemistry.ChineseAcademyof Sciences. Changchun130022.China Received 17 February 1995;in revisedform I August 1995;accepted ~,I August 1995
Abstract
A new immobilization material and an immobilization method for a glucose sensor with HEFc (hydmxyethylferrocene) as mediator is described. In the course of three months, the enzyme electrode shows almost no deterioration in its response characteristics. The response time is less than 30 s. The electrode has a wide linear range up to 10 mmol I- t with good repeatability. The kinetic parameters have also been calculated.
Keywords:Biosensors;Cryohydrogel,Glucoseoxidase; Hydroxyethylferrocene
1. Introduction One of the most important problems in the development of commercial biosensors is the stabilization of the enzymes. We use cryohydrogel [ 1 ] to construct enzyme electrodes, because such electrodes can be conveniently stored in the dry state and can be simply rehydrated before use. The results were obtained imply that this novel cryoimmobilization is a useful method for keeping the catalytic activity of the enzyme. We prepared a kind of polyhydroxyl cellulose (PHC), a mixture of polyvinyl alcohol (PVA) and carboxymethyl hydroxyethyl cellulose (CMHEC) (with C M H E C / PVA = 1 / 5 - 1 / 3 w / w ) , the aqueous solution of which can be frozen to produce a hydrogel; we call it cryohydrogel [ 1]. It has a semi-interpenetrating network (SIPN) and a relatively high mechanical strength and it can retain its water molecules even in organic solvents [2]. When glucose oxidase (GOD) and hydroxyethylferrocene (HEFc) are mixed with the PHC aqueous solution and stored in a refrigerator below - 4 °C, a cryohydrogel enzyme layer formed. The electrode constructed in this way has greatly improved stability.
mutarotate for 24 h before use. The PHC was prepared in our laboratory. All reagents were analytical grade. All solutions were prepared with doubly distilled water. Supporting electrolytes were 0.05 mol 1- t phosphate buffer (PB).
2.2. Apparatus 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 eloctrode was a eryoimmobilized enzyme electrode. An Ag/AgCI (saturated KC1) electrode was used as a reference electrode and a Pt plate as a counter electrode. Unless otherwise indicated, the electrolyte solutions contained 0.05 mol I - ' PB (pH 6.98) and 0.05 mol 1- t KCI. The Pt electrode was polished with 0.3 and 0.5 tzm aAI203 powder, respectively, washed with distilled water then ultrasonicated in deionized water and acetone successively, and .finally dried at room temperature. GOD (5 mg) and HEFc( 1.23 mg) were dissolved in 200/zl 10% PHC solution. 10 tzl of this solution was spread over the surface of the platinmn electrode. The electrode was then frozen at - 4 °C for 24 h and can be stored at 4 °C either in the dry state or in PB.
2. Experimental
2.1. Reagents
3. Results and discussion
GOD and HEFc were purchased from Sigma Chemical Co. A stock solution of D-glucose was prepared and allowed to
3.1. Properties of the enzyme-mediator electrode
* Correspondingauthor. 0925-4005/96/$15.00 © 1996ElsevierScienceS.A. All rights reserved SSD10925-4005(95)0181 I-7
The properties and behaviour of this kind of electrode depend on the concentration of the PHC and the refrigeration
194
Y. Wang et al. /Sensors and Actuators B 31 (1996) 193-196
conditions. There are three kinds of water in the cryohydrogel [3], i.e., 'non-freezing water', which has strong interaction with the polymer, 'bond water', which exits near the hydrophilic group of the polymer and 'free water', which has exactly the same character as natural water. In quite a dry environment, the cryohydrogel will shrink about 40% [4 ]; when it is in contact with water it can recover to its original state. When the gel is cryodesiccated with enzyme, it tends to stabilize the activity of the enzyme. This was thought to be due to the hydroxyl group in the polymer holding oi substituting the 'bound water', which is necessary for the retention of the tertiary structure of the enzyme and the subsequent activity of the molecule [5]. That is to say, the enzyme was retained in the network of the polymer with its perfect tertiary structure. A hydrodynamic voltammogram of the enzyme electrode showed that a stable and sensitive response can be obtained in the vicinity of 0.5 V; this is somewhat higher than the formal potential of HEFc. The positive shift in the potential may be due to the limited mobility of HEFt molecules in the cryohydrogel, which increases the resistance of the redox mediator. Fig. ! shows the current-time curve of glucose response obtained at the enzyme electrode in phosphate buffer. A stable base current was obtained after an equilibration time of about 10 min. The response time is less than 30 s. Fig. 2 gives the calibration curve of the current at the GOD-HEFc-cryohydrogel enzyme electrode with various concentrations of glucose. A linear range up to 10 mmol I- ~ is obtained and the curve passes through zero.
4rain
S i)
,
.S
1_ Time Fig. I. Amperometricresponseof the enzymeelectrode.Expenmeatal~;ondition: in 0.05 tool I- ~ PB and 0.05 mol I- J KCI under stilting; applied potential,0.5 V.
0
2
4
6
8
I0
12
14
I0
Concentration (retool I-1) Fig. 2. Calibrationplot of the enzymeelectrode.Conditionsare the same as in Fig. 1.
3.2. pH and temperature effect The response of the enzyme electrode varies little between pH 6.0 and 7.5, and pH 6.98 was chosen as the optimum value in the experiment. The effect of temperature on the enzyme electrode response was studied in the range 10--60 °C. The amperometric response increases with the temperature between I 0 and 45 0(2. At 55 *C the response is 60% of the highest value; this may be due to the denaturation of the enzyme at high temperature. At all temperatures tested the polymer film remains stable, and the noise is quite small. In order to maintain the stability of the sensor, we chose 25 °C as the operating temperature. The favourable temperature for GOD in solution is about 20 *(2; however, more than 40 0(2 can be obtained for the cryohydrogei-immobilized GOD (see Fig. 3). The results imply that enhanced thermal stability could be obtained for an enzyme electrode prepared in this way.
3.3. Kinetic analysis The response to substrate and the sensitivity are the most important parameters for evaluating a biosensor. The EadieHofstee form of the Michaelis-Menten equation is quite effi-
8
b
0
10
~0
3~0 T('C
A
5'0
do
)
Fig. 3. Temperature effect on the amperometric response. ( a ) G O D and HEFc are in solution; (b) G O D and HEFc are immobilized in the cryohy-
drogel. Conditionsare the same as in Fig. I. cient in kinetic analysis of a biosensor. In an amperometric biosensor the reaction rate is replaced by the steady-state current;
E Wang et al./Sensors and Actuators B 31 (1996) 193-196
195
(a) oo
,
,min
(b) 4
2
20o
~00
4'oo
;0o
6OO
Sensitivity(FC, pA/mmol I-I) Fig. 4. The Eadie-Hofstee plot of experimental data from Fig. 1.
Here Is is the steady-state current, C, is the concentration of substrate, ~ is the apparent Michaelis-Menten constant and/~,~ is the intercept on the current axis [6]. Fig. 4 shows the Eidie-Hofstee plot of the enzyme electrode. K~pp is 27.5 mmol I - ~. It is somewhat higher than that of the soluble enzyme or that of immobilized enzyme in conducting polymers (e.g., polypyrrole). In this case, the diffusion rate of redox mediator is somewhat slow due to the resistance of hydrogel, which may be the critical step of the enzymatic reaction.
3.4. Stability of the enzyme electrode The enzyme electrode has a prolonged lifetime, for this cryoimmobilization method can maintain the tertiary structure of the enzyme. After being stored in the dry state in a refrigerator at 4 °C for three months, the enzyme electrode shows almost no deterioration in its response characteristics. This GOD-HEFc-cryobydrogel electrode shows high stability. The sensitivity of the enzyme electrode still kept 75% of its initial value after intermittent use for two months.
3.5. FIA application Fig. 5(a) shows the flow-injection biosensing response of the cryoimmobilized enzyme electrode. The enzyme electrode was operated continuously in the flow system with 20 injections of 8 mmol I- t standard glucose solution, the relative standard deviation being less than 1.5%. The sensor activity dropped to 16.5% of its initial value after 300 analyses performed for 5 h, which implied that the enzyme and mediator would not readily leach out of the cryohydrogel and the response is stable and repeatable. Fig. 5(b) displays typ-
Fig, 5. How-injectionbiosensingof glucose at the enzymeelectrode. (a) Detectionpeaks for repeated injectionsof 8 mrnol I-l glucose. (b) Flowinjectionpeaks for increasinglevelsof glucose: I, 4.0; 2. 8.0; 3, 12.0;4, 16.0;5, 20.0;6, 22.0mmolI- ~.Carrier/electrolyte,0.05 reel I- ~phosph~e buffer (pH 6.98); flowrate.0.5 ml min- ~;operatingpotential. +0.5 V. ical flow-injection detection peaks for different glucose concentrations at the enzyme electrode. The peak half-width ( 8 20 s) allows injection rates of 60-80 samples per hour. From the current peaks, the relationship between the glucose concentration and the current can he obtained: S(mmol l - t ) ffi0.72i(p.A) - 0.32 with a linear related coefficient of 0.9898.
4. Conclusions Above all, this cryoimmobilization method gives a new way to prepare and preserve sensors. Compared with physical adsorption and entrapping immobilization, the cryohydroge! immobilization has a distinct improvement on the enzyme electrode stability. Because the enzyme and mediator are physically and chemically entrapped in the three-dimensional interpenetrating network, they do not readily leach out of the hydrogel film that adheres tightly to the base electrode surface. Furthermore, in the physical or chemical cross-linking methods, radical-producing chemicals, such as initiator, pbotosensitizer and cross-linker, and/or an activating process, such as heat, UV light or radiation, are needed, and the immobilized enzyme is readily deactivated to some extent. However, in this cryoimmobilized process, neither the abovementioned chemicals nor activating process is necessary and the cryohydrogel is formed under favourable conditions. Thus, the immobilized enzyme suffers no activation or denaturation. This is a unique advantage of cryoimmobilization over other immobilization methods already reported. Therefore, the enzyme thus immobilized has and can retain a high and stable catalytic activity.
196
E Wang et al. / Sensors and Actuators B 31 (1996) 193-196
Acknowledgement The financial support of the National Natural Science Foundation of China is gratefully appreciated. Thanks are also due to Ms. Anhua Liu for her advice.
References [ 1] S. Dong and Y. Guo, A novel enzyme electrode for the water-free organic phases,& Electroanal. Chem., 375 (1994) 405. [ 2] S. Dongand Y. Guo, Organicphaseenzymeelectrodeoperatedin waterfree solvents,Anal. Chem.. 66 (1994) 3895. [3] Y.D. Feng. Study on state of water in cryo-hydrogel. J. Sichuan University Natural Sci. Edition, 26 ( 1989) 470 (in Chinese). [4l S. Dong and Y. Guo, An organic phase enzyme electrodebased on an apparent direct electron transfer between graphite electrode and immobilized horseradishperoxidase,J. Chem. Soc., Chem. Commun., (1995) 483. [5] T.D. Gibson and J.R. Wordward,in P.G. Edelmanand J. Wang (eds.), Biosensors and Chemical Sensors, American Chemical Society, Waslungton DC, 1992,Ch. 5. [6] R.A. Kamin and G.S. Wilson, Rotatingring-diskenzyme electrode for biocatalysis kinetic studies and characterizationof the immobilized enzyme layer. Anal. Chem.. 52 (1980) ! 198.
Biographi~ Ying Wang is working for her Ph.D. in analytical chemistry at CIAC; her dissertation is 'Biosensor and application of QCM in electrochemistry'. Yizhu G u o is working for his Ph.D. degree in analytical chemistry at CIAC and will graduate by the end of 1995. He received his M.Sc. (1992) in material science at C I A C . His dissertation is on 'Bioelectrochemistry and (organic phase) biosensor'. G u o y i Zhu is professor of electroanalytical chemistry. He received his M.Sc. ( 1981 ) at Changchun Institute of Applied Chemistry (CIAC), Chinese Academy of Sciences. His research interests include the basic theory of electroanalytical chemistry. Shaojun D o n g is professor of analytical chemistry mid director of the Laboratory of ElectroanalyticalChemistry. Her research interests include chemically modified elecu'odes,bioelectrochemistryand spectroelectrochemistry.
Sensors and ActuatorsB 31 (1996) 103-196
Cryoimmobilized enzyme for biosensor construction Ying Wang, Yizhu Guo, Guoyi Zhu *, Shaojun Dong Laboratoryof ElectroanalyticalChemistry.ChangchunInstituteofAppliedChemistry.ChineseAcademyof Sciences. Changchun130022.China Received 17 February 1995;in revisedform I August 1995;accepted ~,I August 1995
Abstract
A new immobilization material and an immobilization method for a glucose sensor with HEFc (hydmxyethylferrocene) as mediator is described. In the course of three months, the enzyme electrode shows almost no deterioration in its response characteristics. The response time is less than 30 s. The electrode has a wide linear range up to 10 mmol I- t with good repeatability. The kinetic parameters have also been calculated.
Keywords:Biosensors;Cryohydrogel,Glucoseoxidase; Hydroxyethylferrocene
1. Introduction One of the most important problems in the development of commercial biosensors is the stabilization of the enzymes. We use cryohydrogel [ 1 ] to construct enzyme electrodes, because such electrodes can be conveniently stored in the dry state and can be simply rehydrated before use. The results were obtained imply that this novel cryoimmobilization is a useful method for keeping the catalytic activity of the enzyme. We prepared a kind of polyhydroxyl cellulose (PHC), a mixture of polyvinyl alcohol (PVA) and carboxymethyl hydroxyethyl cellulose (CMHEC) (with C M H E C / PVA = 1 / 5 - 1 / 3 w / w ) , the aqueous solution of which can be frozen to produce a hydrogel; we call it cryohydrogel [ 1]. It has a semi-interpenetrating network (SIPN) and a relatively high mechanical strength and it can retain its water molecules even in organic solvents [2]. When glucose oxidase (GOD) and hydroxyethylferrocene (HEFc) are mixed with the PHC aqueous solution and stored in a refrigerator below - 4 °C, a cryohydrogel enzyme layer formed. The electrode constructed in this way has greatly improved stability.
mutarotate for 24 h before use. The PHC was prepared in our laboratory. All reagents were analytical grade. All solutions were prepared with doubly distilled water. Supporting electrolytes were 0.05 mol 1- t phosphate buffer (PB).
2.2. Apparatus 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 eloctrode was a eryoimmobilized enzyme electrode. An Ag/AgCI (saturated KC1) electrode was used as a reference electrode and a Pt plate as a counter electrode. Unless otherwise indicated, the electrolyte solutions contained 0.05 mol I - ' PB (pH 6.98) and 0.05 mol 1- t KCI. The Pt electrode was polished with 0.3 and 0.5 tzm aAI203 powder, respectively, washed with distilled water then ultrasonicated in deionized water and acetone successively, and .finally dried at room temperature. GOD (5 mg) and HEFc( 1.23 mg) were dissolved in 200/zl 10% PHC solution. 10 tzl of this solution was spread over the surface of the platinmn electrode. The electrode was then frozen at - 4 °C for 24 h and can be stored at 4 °C either in the dry state or in PB.
2. Experimental
2.1. Reagents
3. Results and discussion
GOD and HEFc were purchased from Sigma Chemical Co. A stock solution of D-glucose was prepared and allowed to
3.1. Properties of the enzyme-mediator electrode
* Correspondingauthor. 0925-4005/96/$15.00 © 1996ElsevierScienceS.A. All rights reserved SSD10925-4005(95)0181 I-7
The properties and behaviour of this kind of electrode depend on the concentration of the PHC and the refrigeration
194
Y. Wang et al. /Sensors and Actuators B 31 (1996) 193-196
conditions. There are three kinds of water in the cryohydrogel [3], i.e., 'non-freezing water', which has strong interaction with the polymer, 'bond water', which exits near the hydrophilic group of the polymer and 'free water', which has exactly the same character as natural water. In quite a dry environment, the cryohydrogel will shrink about 40% [4 ]; when it is in contact with water it can recover to its original state. When the gel is cryodesiccated with enzyme, it tends to stabilize the activity of the enzyme. This was thought to be due to the hydroxyl group in the polymer holding oi substituting the 'bound water', which is necessary for the retention of the tertiary structure of the enzyme and the subsequent activity of the molecule [5]. That is to say, the enzyme was retained in the network of the polymer with its perfect tertiary structure. A hydrodynamic voltammogram of the enzyme electrode showed that a stable and sensitive response can be obtained in the vicinity of 0.5 V; this is somewhat higher than the formal potential of HEFc. The positive shift in the potential may be due to the limited mobility of HEFt molecules in the cryohydrogel, which increases the resistance of the redox mediator. Fig. ! shows the current-time curve of glucose response obtained at the enzyme electrode in phosphate buffer. A stable base current was obtained after an equilibration time of about 10 min. The response time is less than 30 s. Fig. 2 gives the calibration curve of the current at the GOD-HEFc-cryohydrogel enzyme electrode with various concentrations of glucose. A linear range up to 10 mmol I- ~ is obtained and the curve passes through zero.
4rain
S i)
,
.S
1_ Time Fig. I. Amperometricresponseof the enzymeelectrode.Expenmeatal~;ondition: in 0.05 tool I- ~ PB and 0.05 mol I- J KCI under stilting; applied potential,0.5 V.
0
2
4
6
8
I0
12
14
I0
Concentration (retool I-1) Fig. 2. Calibrationplot of the enzymeelectrode.Conditionsare the same as in Fig. 1.
3.2. pH and temperature effect The response of the enzyme electrode varies little between pH 6.0 and 7.5, and pH 6.98 was chosen as the optimum value in the experiment. The effect of temperature on the enzyme electrode response was studied in the range 10--60 °C. The amperometric response increases with the temperature between I 0 and 45 0(2. At 55 *C the response is 60% of the highest value; this may be due to the denaturation of the enzyme at high temperature. At all temperatures tested the polymer film remains stable, and the noise is quite small. In order to maintain the stability of the sensor, we chose 25 °C as the operating temperature. The favourable temperature for GOD in solution is about 20 *(2; however, more than 40 0(2 can be obtained for the cryohydrogei-immobilized GOD (see Fig. 3). The results imply that enhanced thermal stability could be obtained for an enzyme electrode prepared in this way.
3.3. Kinetic analysis The response to substrate and the sensitivity are the most important parameters for evaluating a biosensor. The EadieHofstee form of the Michaelis-Menten equation is quite effi-
8
b
0
10
~0
3~0 T('C
A
5'0
do
)
Fig. 3. Temperature effect on the amperometric response. ( a ) G O D and HEFc are in solution; (b) G O D and HEFc are immobilized in the cryohy-
drogel. Conditionsare the same as in Fig. I. cient in kinetic analysis of a biosensor. In an amperometric biosensor the reaction rate is replaced by the steady-state current;
E Wang et al./Sensors and Actuators B 31 (1996) 193-196
195
(a) oo
,
,min
(b) 4
2
20o
~00
4'oo
;0o
6OO
Sensitivity(FC, pA/mmol I-I) Fig. 4. The Eadie-Hofstee plot of experimental data from Fig. 1.
Here Is is the steady-state current, C, is the concentration of substrate, ~ is the apparent Michaelis-Menten constant and/~,~ is the intercept on the current axis [6]. Fig. 4 shows the Eidie-Hofstee plot of the enzyme electrode. K~pp is 27.5 mmol I - ~. It is somewhat higher than that of the soluble enzyme or that of immobilized enzyme in conducting polymers (e.g., polypyrrole). In this case, the diffusion rate of redox mediator is somewhat slow due to the resistance of hydrogel, which may be the critical step of the enzymatic reaction.
3.4. Stability of the enzyme electrode The enzyme electrode has a prolonged lifetime, for this cryoimmobilization method can maintain the tertiary structure of the enzyme. After being stored in the dry state in a refrigerator at 4 °C for three months, the enzyme electrode shows almost no deterioration in its response characteristics. This GOD-HEFc-cryobydrogel electrode shows high stability. The sensitivity of the enzyme electrode still kept 75% of its initial value after intermittent use for two months.
3.5. FIA application Fig. 5(a) shows the flow-injection biosensing response of the cryoimmobilized enzyme electrode. The enzyme electrode was operated continuously in the flow system with 20 injections of 8 mmol I- t standard glucose solution, the relative standard deviation being less than 1.5%. The sensor activity dropped to 16.5% of its initial value after 300 analyses performed for 5 h, which implied that the enzyme and mediator would not readily leach out of the cryohydrogel and the response is stable and repeatable. Fig. 5(b) displays typ-
Fig, 5. How-injectionbiosensingof glucose at the enzymeelectrode. (a) Detectionpeaks for repeated injectionsof 8 mrnol I-l glucose. (b) Flowinjectionpeaks for increasinglevelsof glucose: I, 4.0; 2. 8.0; 3, 12.0;4, 16.0;5, 20.0;6, 22.0mmolI- ~.Carrier/electrolyte,0.05 reel I- ~phosph~e buffer (pH 6.98); flowrate.0.5 ml min- ~;operatingpotential. +0.5 V. ical flow-injection detection peaks for different glucose concentrations at the enzyme electrode. The peak half-width ( 8 20 s) allows injection rates of 60-80 samples per hour. From the current peaks, the relationship between the glucose concentration and the current can he obtained: S(mmol l - t ) ffi0.72i(p.A) - 0.32 with a linear related coefficient of 0.9898.
4. Conclusions Above all, this cryoimmobilization method gives a new way to prepare and preserve sensors. Compared with physical adsorption and entrapping immobilization, the cryohydroge! immobilization has a distinct improvement on the enzyme electrode stability. Because the enzyme and mediator are physically and chemically entrapped in the three-dimensional interpenetrating network, they do not readily leach out of the hydrogel film that adheres tightly to the base electrode surface. Furthermore, in the physical or chemical cross-linking methods, radical-producing chemicals, such as initiator, pbotosensitizer and cross-linker, and/or an activating process, such as heat, UV light or radiation, are needed, and the immobilized enzyme is readily deactivated to some extent. However, in this cryoimmobilized process, neither the abovementioned chemicals nor activating process is necessary and the cryohydrogel is formed under favourable conditions. Thus, the immobilized enzyme suffers no activation or denaturation. This is a unique advantage of cryoimmobilization over other immobilization methods already reported. Therefore, the enzyme thus immobilized has and can retain a high and stable catalytic activity.
196
E Wang et al. / Sensors and Actuators B 31 (1996) 193-196
Acknowledgement The financial support of the National Natural Science Foundation of China is gratefully appreciated. Thanks are also due to Ms. Anhua Liu for her advice.
References [ 1] S. Dong and Y. Guo, A novel enzyme electrode for the water-free organic phases,& Electroanal. Chem., 375 (1994) 405. [ 2] S. Dongand Y. Guo, Organicphaseenzymeelectrodeoperatedin waterfree solvents,Anal. Chem.. 66 (1994) 3895. [3] Y.D. Feng. Study on state of water in cryo-hydrogel. J. Sichuan University Natural Sci. Edition, 26 ( 1989) 470 (in Chinese). [4l S. Dong and Y. Guo, An organic phase enzyme electrodebased on an apparent direct electron transfer between graphite electrode and immobilized horseradishperoxidase,J. Chem. Soc., Chem. Commun., (1995) 483. [5] T.D. Gibson and J.R. Wordward,in P.G. Edelmanand J. Wang (eds.), Biosensors and Chemical Sensors, American Chemical Society, Waslungton DC, 1992,Ch. 5. [6] R.A. Kamin and G.S. Wilson, Rotatingring-diskenzyme electrode for biocatalysis kinetic studies and characterizationof the immobilized enzyme layer. Anal. Chem.. 52 (1980) ! 198.
Biographi~ Ying Wang is working for her Ph.D. in analytical chemistry at CIAC; her dissertation is 'Biosensor and application of QCM in electrochemistry'. Yizhu G u o is working for his Ph.D. degree in analytical chemistry at CIAC and will graduate by the end of 1995. He received his M.Sc. (1992) in material science at C I A C . His dissertation is on 'Bioelectrochemistry and (organic phase) biosensor'. G u o y i Zhu is professor of electroanalytical chemistry. He received his M.Sc. ( 1981 ) at Changchun Institute of Applied Chemistry (CIAC), Chinese Academy of Sciences. His research interests include the basic theory of electroanalytical chemistry. Shaojun D o n g is professor of analytical chemistry mid director of the Laboratory of ElectroanalyticalChemistry. Her research interests include chemically modified elecu'odes,bioelectrochemistryand spectroelectrochemistry.