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A novel glucose sensor system with Au nanoparticles based on microdialysis and coenzymes for continuous glucose monitoring
Sensors and Actuators A 108 (2003) 258–262
A novel glucose sensor system with Au nanoparticles based on microdialysis and coenzymes for continuous glucose monitoring Min Pan∗ , Xishan Guo, Qiang Cai, Guang Li, Yuquan Chen Biosensor National Special Laboratory, Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, PR China Received 12 September 2002; received in revised form 12 August 2003; accepted 25 August 2003
Abstract A novel glucose sensor based on microdialysis technique has been developed for continuous glucose monitoring. It includes: (1) The microdialysis sampling and interval perfusion system, it can prevent electrode from being fouled and it also provides enough time for glucose molecule traversing the microdialysis membrane to establish a dynamic balance, and the sensor can be recovered well, too. (2) A “sandwich” structure glucose sensor based on an aqueous glucose oxidase (GOD) and catalase solution, it is easy to replace the inactive enzyme solution. Au interdigital array (IDA) microelectrodes and microgrooves were fabricated on the silicon wafer for electrochemistry determinations. (3) Continuous glucose monitoring system. The results of in vitro experiments show this glucose sensor has short response time, high sensitivity and good linearity. To improve the stability of liquid enzyme, aqueous colloidal gold nanoparticles was mixed with coenzymes solution. The result of experiments shows the repeatability of sensor was improved, and the sensitivity of sensor was enhanced. To the best of our knowledge, this is the first demonstration that aqueous colloidal gold nanoparticles enhance the activity of aqueous enzymes. © 2003 Elsevier B.V. All rights reserved. Keywords: Microdialysis; Microelectrode; Glucose sensor; Electrochemistry; Enzyme; Gold nanoparticles
1. Introduction Recently many studies have been done for in vivo continuous glucose monitoring system. A feasible method to monitor the blood glucose concentration is the implantation of a biosensor in the subcutaneous (s.c.) tissue. However, the function of glucose electrode such as a hydrogen peroxide detecting electroenzymatic electrode is markedly impaired in human s.c. tissue and plasma. Even if the surface of the electrode were coated with biocompatible membrane, the insulating protein shell surrounding the sensor would also affect sensor sensitivity soon after it was implanted in s.c. tissue, and the glucose sensor would be clogged [1]. Moreover, the implanted glucose sensor is usually fabricated by immobilized glucose oxidase (GOD) membrane technique, and this kind of sensor leads to decreasing enzyme’s activity and sensitivity. It also takes a long response time for this kind of sensor to reach steady-state and the enzyme is not easy to replace when losing activity. To cope with these problems, a new glucose sensor system developed here includes: (1) The microdialysis sampling ∗ Corresponding author. Fax: +86-571-87951676. E-mail address: [email protected] (M. Pan).
0924-4247/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.sna.2003.08.009
system, it can prevent electrode from being insulated by protein in the s.c. tissue for longtime monitoring, this technique was reported somewhere [2–5]. (2) The “sandwich” structure microfabricated sensor system (Fig. 3a), it integrates three layers: upper silicon chip, biocompatible microdialysis membrane and bottom silicon chip with Au interdigital array (IDA) electrodes. In order to avoid the middle dialysis membrane is crushed, the upper silicon chip and the bottom silicon chip are cross-bracing placed together. Two flow chambers with microgrooves are separately fabricated on the upper and bottom silicon wafers. It is easy to change the inactive enzyme solution just by injecting a fresh enzyme to the bottom flow chamber. The oxidation reaction of glucose and the decomposition of hydrogen peroxide are catalyzed by aqueous GOD and catalase compound solution. Coenzymes can more effectively catalyze the electrochemistry reaction, which has been testified in our early work [6]. (3) Interval microdialysis perfusion and control system, the pump perfuses at the rate of 6 l/min for about 15 min. The pump stops perfusing for 15 min after the fresh glucose dialysate has replaced the waste solution in the upper flow chamber. In the period of stopping pumping, the equilibrium of the glucose molecule between the s.c. tissue and the dialysate can be completely established, so that the concentration of the
M. Pan et al. / Sensors and Actuators A 108 (2003) 258–262
259
trochemical character, such as promise of further enhancements of signal/noise ration and selectivity. Fig. 1 shows the schematic diagram of detection system. The perfusion fluid is pumped from container through a microdialysis probe, which is drenched in a analyte solution and the glucose dialysate is imported to the “sandwich” sensor chip, the amperometric signal is measured by potentiostat. Here using this glucose sensor system, continuous glucose concentration can be measured in real time.
3. Materials and methods 3.1. Materials
Fig. 1. The schematic diagram of glucose sensor system.
dialysate has a conform correlation to the real concentration of the s.c. tissue. Meanwhile, the reaction in the bottom flow chamber can finish completely and the sensor can be recovered well. Moreover, the pump is controlled by computer, through which this system can adjust the pumping speed and the interval time easily. (4) The electrochemistry analysis system is developed in our work. It includes high precision potentiostat with low noise, control unit for pump and ca/dc, dc/ac convertors controlled by micro-computing unit (MCU). Via RS232, PC is connected to MCU for continuous glucose monitoring and further processing (see Fig. 1).
2. Detection principle and system Glucose measurements based on hydrogen peroxide electrodes have been widely used [7,8]. First, the hydrogen peroxide is derived from Eq. (1) when the reaction between glucose and oxygen is catalyzed by enzyme GOD. GOD
glucose + O2 −→ gluconic acid + H2 O2
(1)
Second, when the polarizing voltage between the electrodes is set to about 600 mV, the following reaction occurs at the anode. catalase
H2 O2 −−−→ 2H+ + O2 + 2e−
(2)
The amperometric signal at Au IDA microelectrodes is a result of the electrochemical oxidation of hydrogen peroxide, the concentration of which is proportional to the glucose concentration according to (1) and (2). This work presents a novel glucose sensor system to examine the amperometric value for H2 O2 detection based on Au IDA microelectrodes. The microelectrode can be defined as the electrodes with dimensions of few micrometers. Due to the advantages of the microelectrode, such as high current densities and high diffusion rates, the importance of the use of microelectrodes has been widely accepted in the field of electroanalytcal chemistry. In recent years the IDA was widely used due to its convenient fabrication and good elec-
The following chemicals were used: Glucose oxidase (GOx , EC 1.1.3.4, from Aspergillus niger, 250 units/mg) and catalase (EC 1.11.1.6, from bovine liver, 26,000 units/ml) were purchased from Roche. -d-Glucose was purchased from Sigma. NaH2 PO4 ·2H2 O, Na2 HPO4 ·12H2 O, and NaCl (Pudong Gaonan reagent factory, Shanghai, China) were used to prepare phosphate buffered saline (PBS). All other chemicals were of analytical grade, and aqueous solutions were prepared in doubly distilled water. 3.2. Microdialysis probe A commercially available microdialysis probe was used with a molecule cut-off 20,000 Da, so the biologic molecule in analyte solution such as protein cannot penetrate the membrane whereas the glucose molecule can traverse it and reach to the perfusion fluid. The principle of microdialysis procedure and the structure of the microdialysis probe are shown in Fig. 2. 3.3. Sensor structure and microelectrode The outlet of the dialysis probe was connected to the inlet of the flow chamber (Fig. 3b and c). Biocompatible microdialysis membrane, between upper and bottom silicon chip (Fig. 3a), can prevent the GOD and catalase enzyme from diffusing into the glucose dialysate. The upper silicon chip and the bottom silicon chip are cross-bracing placed to avoid middle microdialysis membrane being crushed. Materials after reaction are disposed through the outlet of the upper chamber (Fig. 3b). Au IDA electrodes and microgrooves are fabricated in the silicon substrate (Fig. 3c). Au IDA microelectrodes buildup is made up of 20 pairs
Fig. 2. The structure of microdialysis probe.
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M. Pan et al. / Sensors and Actuators A 108 (2003) 258–262
Fig. 4. Continuous current–time response of the glucose sensor at different glucose concentrations. Numbers on each current plateau indicate the glucose concentration (mM), perfusion rate = 6 l/min.
Fig. 3. (a) “Sandwich” structure of sensor system; (b) 3D view of upper silicon chip; (c) 3D view of bottom silicon chip.
of interdigital electrodes, and each interdigital electrode length is 12 mm.
Continuous current–time response of the glucose sensor at different standard glucose concentrations is shown in Fig. 4. This sensor has short response time (reach steady-state less than 8–10 s during the different concentration glucose determinations), and there is little fluctuating of current values for inhomogeneous of diffusion. The relationship between the concentration and the current is shown in Fig. 5, in which every current corresponding a certain concentration is the maximum of the reaction. According to Fig. 5 (䊏), this sensor has an approximately 3.4 nA/mM sensitivity, the curve also have a good linearity when detecting range is from 0.8 to 30 mM. For in invo determinations using enzyme sensor, regular calibration of the sensor is crucial. On the principle of interval perfusion design of the sensor, the reaction can finish completely and the sensor can be recovered well in the period of stopping pumping. Moreover, before refreshing enzyme solution each time, the flow channels and microgrooves were rinsed using PBS. The bias current was recorded and eliminated by the hardware of the device to calibrate to zero each time before refreshing enzyme solution. Though aqueous enzymes are easy to be replaced and keep up activity for long time, have short response time, which suits for microdialysis technique, the stability of aqueous enzymes is still one problem compared with immobilized enzymes.
4. Experiments and results We prepared 5 mg/ml GOD and a proper amount of catalase in PBS (pH = 6.8), and variant glucose solution with concentration range from 0.1 to 50 mM. PBS was used as perfusion fluid. Before the experiments, the flow chambers were rinsed using PBS and glucose enzyme solution was refreshed. A stepping-motor-controlled syringe pump, which could be controlled by computer to fix the perfusion rate for permeating the chambers, was made for the micro dialysis probe. Then the amperometric detection of glucose was performed by applying a potential of 500 mV. The device measured the current at different concentration ranged from 0.5 to 50 mM.
Fig. 5. The relationship between the glucose concentration and the measured current.
M. Pan et al. / Sensors and Actuators A 108 (2003) 258–262
Fig. 6. (a) Single GOD + glucose or catalase + H2 O2 ; (b) GOD and catalase mixed with cAu.
Colloidal metal nanoparticles are of great interest due to their special properties, which can be utilized in many applications, such as catalysis [9,10]. Moreover, duo to gold nanoparticles has biocompatible property, and can agglomerate and adsorb biologic molecule, gold nanoparticles can stabilize and “immobilize” the enzymes, which could improve the stability of aqueous enzymes. In a typical experiment, colloidal gold nanoparticles was synthesized by borohydride reduction of aqueous HAuCl4 solution (1.5 mg of HAuCl4 in 100 ml of water) as described in detail elsewhere [11,12]. Then after different experimental try, a proper amount of aqueous colloidal gold nanoparticles (about 30 nm diameter) was mixed with aqueous enzymes solution in PBS and was shaked homogeneously for about 2–3 min. After being placed in room temperature for about 10–15 min, the mixture was transferred into the bottom silicon chip for experiments. The result of experiments is shown in Fig. 5 (䊉) and the sensitivity of sensor had been enhanced. The sensor has higher sensitivity (approximately 9.8 nA/mM), which is nearly three (≈2.8) times as the sensitivity of sensor when only coenzymes catalyzing the reaction. The curve also have a good linearity when glucose concentration range is from 0.5 to 38 mM. Fig. 6 shows the difference of the single GOD with glucose or catalase with H2 O2 and the effect of gold nanoparticles mixed to GOD and catalase with glucose or H2 O2 .
5. Discussion and conclusion This novel sensor has large contact surface area between the microdialysis membrane and the Au IDA in the solution of the flow chambers, which increases the number of reacting molecula and decreases the molecular diffusing time. In our glucose sensor system, two microdialysis membranes are applied to microdialysis probe and “sandwich” structure sensor. This can prevent electrodes from being insulated and clogged, and it also provides enough time for glucose molecule traversing the microdialysis membrane to establish a dynamic balance. It is easy to replace the inactive enzyme solution and the interval pump scheme can well deal with the problem of glucose depletion region around the measur-
261
ing site. From the point of view of reusability, this kind of sensor has a long lifespan with continuous supplementary enzyme solution. In addition, this microdialysis-based glucose sensor is highly biocompatibility. In order to solve the problem of the stability and activity of aqueous enzymes, aqueous colloidal gold nanoparticles were mixed with aqueous coenzymes solution. To the best of our knowledge, this is the first demonstration that aqueous colloidal gold nanoparticles enhance the activity of aqueous glucose oxidase enzyme and catalase enzyme. Because gold nanoparticles also can adsorb and agglomerate enzyme molecules, it can stabilize and “immobilize” the enzymes, which improve the stability of aqueous enzymes. During the procedure of our 2-month continuous determinations, the results of experiments have confirmed it. It is worth mentioning that the sensitivity of sensor would not be improved if colloidal gold solution were perfused after coenzymes solution was perfused. Diameter and quantity of colloidal gold, pH value, time for mixing colloidal gold with enzymes and for placing mixture, etc. are the factors, which decide the result of experiments. In the further work and research, the mechanism of gold nanoparticles enhancing the sensitivity of biosensor will be studied through realizing the direct electrons transfer between the enzyme activity center and microelectrode. And more experiments will be done to test the mechanism hypothesis. This system is being developed in the laboratory and the present experiments is in vitro, the preliminary research work demonstrates that it is useful for monitoring diabetes in vivo and it has a good prospect when miniaturized into portable device.
Acknowledgements This project is financially supported by National Natural Science Foundation and Zhejiang Province Natural Science Foundation.
References [1] W. Kerner, M. Kiwit, B. Linke, et al., The function of a H2 O2 detecting electroenzymatic glucose electrode is markedly impaired in human sub-cutaneous tissue and plasma, Biosens. Bioelectron. (1993) 473–482. [2] Y. Hashiguchi, M. Sakakida, K. Nishida, et al., Development of a miniaturized glucose monitoring system by combining a needle-type glucose sensor with microdialysis sampling method: long-term subcutaneous tissue glucose monitoring in ambulatory diabetic patients, Diabetes Care (1994) 387–396. [3] H. Hinkers, C. Dumschat, R. Sundermeier, et al., Microdialysis system for continuous glucose monitoring, in: Proceedings of the 8th International Conference on Solid-State Sensors and Actuators, Stockholm, Sweden, 1995, pp. 470–473. [4] E.F. Pfeiffer, C. Meyerhoff, F. Bischof, et al., On line continuous monitoring of subcutaneous tissue glucose is feasible by combining
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[7]
[8]
[9] [10] [11]
[12]
M. Pan et al. / Sensors and Actuators A 108 (2003) 258–262 portable glucose sensor with microdialysis, Horm. Metab. Res. 25 (1993) 121–124. E. Csöregi, T. Laurell, L. Gorton, On-line glucose monitoring by using microdialysis sampling and amperometric detection based on ‘wired’ glucose oxidase in carbon paste, Mikrochim. Acta 121 (1995) 31–40. P. Yu, W.T. Liu, L. Guang, Y. Chen, et al., A novel microdialysis glucose sensor system based on co-immobilizing on Au microelectrode by sol–gel technique, IEEE-EMBS 2001,Turkey, 2001. L. Gorton, J. Gunilla, Amperometric bio-sensors based on an apparent direct electron transfer between electrodes and immobilized peroxidases plenary lecture, Analyst 117 (1992) 1235–1241. P. Atanasov, S. Gamburzev, A.L. Ghindilis, et al., Bifunctional hydrogen peroxide electrode as an amperometric transducer for biosensors, Sens. Actuators B 43 (1997) 70–77. A. Fulishima, K. Honda, Electrochemical photolysis of water at a semiconductor electrode, Nature 37 (1972) 238. A.P. Alivisatos, Semiconductor clusters, nanocrystals and quantum dots, Science 271 (1996) 933–937. V. Patil, R.B. Malvankar, M. Sastry, Role of particle size in individual and competitive diffusion of carboxylic acid derivatized colloidal gold particles in thermally evaporated fatty amine films, Langmuir 15 (1999) 8197. S.L. Pan, M. Chen, H.L. Li, Aqueous gold sols of rod-shaped particles prepared by the template method, Colloids Surf. A: Physicochem. Eng. Aspects 180 (2001) 55–62.
Biographies Min Pan: lecture of BME, Zhejiang University. Graduated from Zhejiang University, PR China, and obtained the BS and master’s degree of BME in 1992 and 2000, respectively. As visiting researcher, he researched in
USA at 1995, and in Pisa University from 1999 to 2000. Now he is teaching and researching in the Biosensor National Special Lab, Zhejiang University. His interest fields include: biosensor and chemical sensor, transducer and system integration. Xishan Guo: PhD candidate, senior research assistant of Biosensor National Special Lab, PR China, he received the bachelor degree and master degree in biomedical engineering, respectively, in 1999 and in 2002 from the department of biomedical engineering, Zhejiang University, PR China. He researched at “E. Piaggio” of Pisa University from October 2002 to February 2003. His research direction and interest mainly include biosensor, electronic nose and electronic tongue, protein biochip, MEMS, etc. Yuquan Chen: professor of BME, director of Biosensor National Special Lab, Zhejiang University. He obtained the master degree in Zhejiang University, 1982. From 1984 to 1987, he researched in the Case Western Reserve University, USA. From 1999 to 2000, he researched at Department of Chemistry, Harvard University and his research field includes biosensor, chemical sensor, signal measurement and analysis, micro structure, etc. Cai Qiang: PhD student of BME, Zhejiang University. He was born in 1974, and get B.S. and M.S. in Zhejiang University at 1996, 1999 respectively. His interested field includes: biosensor, chemical sensor and intelligent system. Li Guang: received his Bachelor and Master Degrees of BME at Zhejiang University, China in 1987 and 1991, respectively. He got his PhD degree of BME at Imperial College of Science, Technology and Medicine, London, UK in 1998. After his PhD study, he used to work at University of Glasgow and Moor Instruments Ltd, UK for three years. He is current a professor of Biomedical Engineering at Zhejiang University. His research interests include biosensors, biomedical instruments and neuroiformatics.
A novel glucose sensor system with Au nanoparticles based on microdialysis and coenzymes for continuous glucose monitoring Min Pan∗ , Xishan Guo, Qiang Cai, Guang Li, Yuquan Chen Biosensor National Special Laboratory, Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, PR China Received 12 September 2002; received in revised form 12 August 2003; accepted 25 August 2003
Abstract A novel glucose sensor based on microdialysis technique has been developed for continuous glucose monitoring. It includes: (1) The microdialysis sampling and interval perfusion system, it can prevent electrode from being fouled and it also provides enough time for glucose molecule traversing the microdialysis membrane to establish a dynamic balance, and the sensor can be recovered well, too. (2) A “sandwich” structure glucose sensor based on an aqueous glucose oxidase (GOD) and catalase solution, it is easy to replace the inactive enzyme solution. Au interdigital array (IDA) microelectrodes and microgrooves were fabricated on the silicon wafer for electrochemistry determinations. (3) Continuous glucose monitoring system. The results of in vitro experiments show this glucose sensor has short response time, high sensitivity and good linearity. To improve the stability of liquid enzyme, aqueous colloidal gold nanoparticles was mixed with coenzymes solution. The result of experiments shows the repeatability of sensor was improved, and the sensitivity of sensor was enhanced. To the best of our knowledge, this is the first demonstration that aqueous colloidal gold nanoparticles enhance the activity of aqueous enzymes. © 2003 Elsevier B.V. All rights reserved. Keywords: Microdialysis; Microelectrode; Glucose sensor; Electrochemistry; Enzyme; Gold nanoparticles
1. Introduction Recently many studies have been done for in vivo continuous glucose monitoring system. A feasible method to monitor the blood glucose concentration is the implantation of a biosensor in the subcutaneous (s.c.) tissue. However, the function of glucose electrode such as a hydrogen peroxide detecting electroenzymatic electrode is markedly impaired in human s.c. tissue and plasma. Even if the surface of the electrode were coated with biocompatible membrane, the insulating protein shell surrounding the sensor would also affect sensor sensitivity soon after it was implanted in s.c. tissue, and the glucose sensor would be clogged [1]. Moreover, the implanted glucose sensor is usually fabricated by immobilized glucose oxidase (GOD) membrane technique, and this kind of sensor leads to decreasing enzyme’s activity and sensitivity. It also takes a long response time for this kind of sensor to reach steady-state and the enzyme is not easy to replace when losing activity. To cope with these problems, a new glucose sensor system developed here includes: (1) The microdialysis sampling ∗ Corresponding author. Fax: +86-571-87951676. E-mail address: [email protected] (M. Pan).
0924-4247/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.sna.2003.08.009
system, it can prevent electrode from being insulated by protein in the s.c. tissue for longtime monitoring, this technique was reported somewhere [2–5]. (2) The “sandwich” structure microfabricated sensor system (Fig. 3a), it integrates three layers: upper silicon chip, biocompatible microdialysis membrane and bottom silicon chip with Au interdigital array (IDA) electrodes. In order to avoid the middle dialysis membrane is crushed, the upper silicon chip and the bottom silicon chip are cross-bracing placed together. Two flow chambers with microgrooves are separately fabricated on the upper and bottom silicon wafers. It is easy to change the inactive enzyme solution just by injecting a fresh enzyme to the bottom flow chamber. The oxidation reaction of glucose and the decomposition of hydrogen peroxide are catalyzed by aqueous GOD and catalase compound solution. Coenzymes can more effectively catalyze the electrochemistry reaction, which has been testified in our early work [6]. (3) Interval microdialysis perfusion and control system, the pump perfuses at the rate of 6 l/min for about 15 min. The pump stops perfusing for 15 min after the fresh glucose dialysate has replaced the waste solution in the upper flow chamber. In the period of stopping pumping, the equilibrium of the glucose molecule between the s.c. tissue and the dialysate can be completely established, so that the concentration of the
M. Pan et al. / Sensors and Actuators A 108 (2003) 258–262
259
trochemical character, such as promise of further enhancements of signal/noise ration and selectivity. Fig. 1 shows the schematic diagram of detection system. The perfusion fluid is pumped from container through a microdialysis probe, which is drenched in a analyte solution and the glucose dialysate is imported to the “sandwich” sensor chip, the amperometric signal is measured by potentiostat. Here using this glucose sensor system, continuous glucose concentration can be measured in real time.
3. Materials and methods 3.1. Materials
Fig. 1. The schematic diagram of glucose sensor system.
dialysate has a conform correlation to the real concentration of the s.c. tissue. Meanwhile, the reaction in the bottom flow chamber can finish completely and the sensor can be recovered well. Moreover, the pump is controlled by computer, through which this system can adjust the pumping speed and the interval time easily. (4) The electrochemistry analysis system is developed in our work. It includes high precision potentiostat with low noise, control unit for pump and ca/dc, dc/ac convertors controlled by micro-computing unit (MCU). Via RS232, PC is connected to MCU for continuous glucose monitoring and further processing (see Fig. 1).
2. Detection principle and system Glucose measurements based on hydrogen peroxide electrodes have been widely used [7,8]. First, the hydrogen peroxide is derived from Eq. (1) when the reaction between glucose and oxygen is catalyzed by enzyme GOD. GOD
glucose + O2 −→ gluconic acid + H2 O2
(1)
Second, when the polarizing voltage between the electrodes is set to about 600 mV, the following reaction occurs at the anode. catalase
H2 O2 −−−→ 2H+ + O2 + 2e−
(2)
The amperometric signal at Au IDA microelectrodes is a result of the electrochemical oxidation of hydrogen peroxide, the concentration of which is proportional to the glucose concentration according to (1) and (2). This work presents a novel glucose sensor system to examine the amperometric value for H2 O2 detection based on Au IDA microelectrodes. The microelectrode can be defined as the electrodes with dimensions of few micrometers. Due to the advantages of the microelectrode, such as high current densities and high diffusion rates, the importance of the use of microelectrodes has been widely accepted in the field of electroanalytcal chemistry. In recent years the IDA was widely used due to its convenient fabrication and good elec-
The following chemicals were used: Glucose oxidase (GOx , EC 1.1.3.4, from Aspergillus niger, 250 units/mg) and catalase (EC 1.11.1.6, from bovine liver, 26,000 units/ml) were purchased from Roche. -d-Glucose was purchased from Sigma. NaH2 PO4 ·2H2 O, Na2 HPO4 ·12H2 O, and NaCl (Pudong Gaonan reagent factory, Shanghai, China) were used to prepare phosphate buffered saline (PBS). All other chemicals were of analytical grade, and aqueous solutions were prepared in doubly distilled water. 3.2. Microdialysis probe A commercially available microdialysis probe was used with a molecule cut-off 20,000 Da, so the biologic molecule in analyte solution such as protein cannot penetrate the membrane whereas the glucose molecule can traverse it and reach to the perfusion fluid. The principle of microdialysis procedure and the structure of the microdialysis probe are shown in Fig. 2. 3.3. Sensor structure and microelectrode The outlet of the dialysis probe was connected to the inlet of the flow chamber (Fig. 3b and c). Biocompatible microdialysis membrane, between upper and bottom silicon chip (Fig. 3a), can prevent the GOD and catalase enzyme from diffusing into the glucose dialysate. The upper silicon chip and the bottom silicon chip are cross-bracing placed to avoid middle microdialysis membrane being crushed. Materials after reaction are disposed through the outlet of the upper chamber (Fig. 3b). Au IDA electrodes and microgrooves are fabricated in the silicon substrate (Fig. 3c). Au IDA microelectrodes buildup is made up of 20 pairs
Fig. 2. The structure of microdialysis probe.
260
M. Pan et al. / Sensors and Actuators A 108 (2003) 258–262
Fig. 4. Continuous current–time response of the glucose sensor at different glucose concentrations. Numbers on each current plateau indicate the glucose concentration (mM), perfusion rate = 6 l/min.
Fig. 3. (a) “Sandwich” structure of sensor system; (b) 3D view of upper silicon chip; (c) 3D view of bottom silicon chip.
of interdigital electrodes, and each interdigital electrode length is 12 mm.
Continuous current–time response of the glucose sensor at different standard glucose concentrations is shown in Fig. 4. This sensor has short response time (reach steady-state less than 8–10 s during the different concentration glucose determinations), and there is little fluctuating of current values for inhomogeneous of diffusion. The relationship between the concentration and the current is shown in Fig. 5, in which every current corresponding a certain concentration is the maximum of the reaction. According to Fig. 5 (䊏), this sensor has an approximately 3.4 nA/mM sensitivity, the curve also have a good linearity when detecting range is from 0.8 to 30 mM. For in invo determinations using enzyme sensor, regular calibration of the sensor is crucial. On the principle of interval perfusion design of the sensor, the reaction can finish completely and the sensor can be recovered well in the period of stopping pumping. Moreover, before refreshing enzyme solution each time, the flow channels and microgrooves were rinsed using PBS. The bias current was recorded and eliminated by the hardware of the device to calibrate to zero each time before refreshing enzyme solution. Though aqueous enzymes are easy to be replaced and keep up activity for long time, have short response time, which suits for microdialysis technique, the stability of aqueous enzymes is still one problem compared with immobilized enzymes.
4. Experiments and results We prepared 5 mg/ml GOD and a proper amount of catalase in PBS (pH = 6.8), and variant glucose solution with concentration range from 0.1 to 50 mM. PBS was used as perfusion fluid. Before the experiments, the flow chambers were rinsed using PBS and glucose enzyme solution was refreshed. A stepping-motor-controlled syringe pump, which could be controlled by computer to fix the perfusion rate for permeating the chambers, was made for the micro dialysis probe. Then the amperometric detection of glucose was performed by applying a potential of 500 mV. The device measured the current at different concentration ranged from 0.5 to 50 mM.
Fig. 5. The relationship between the glucose concentration and the measured current.
M. Pan et al. / Sensors and Actuators A 108 (2003) 258–262
Fig. 6. (a) Single GOD + glucose or catalase + H2 O2 ; (b) GOD and catalase mixed with cAu.
Colloidal metal nanoparticles are of great interest due to their special properties, which can be utilized in many applications, such as catalysis [9,10]. Moreover, duo to gold nanoparticles has biocompatible property, and can agglomerate and adsorb biologic molecule, gold nanoparticles can stabilize and “immobilize” the enzymes, which could improve the stability of aqueous enzymes. In a typical experiment, colloidal gold nanoparticles was synthesized by borohydride reduction of aqueous HAuCl4 solution (1.5 mg of HAuCl4 in 100 ml of water) as described in detail elsewhere [11,12]. Then after different experimental try, a proper amount of aqueous colloidal gold nanoparticles (about 30 nm diameter) was mixed with aqueous enzymes solution in PBS and was shaked homogeneously for about 2–3 min. After being placed in room temperature for about 10–15 min, the mixture was transferred into the bottom silicon chip for experiments. The result of experiments is shown in Fig. 5 (䊉) and the sensitivity of sensor had been enhanced. The sensor has higher sensitivity (approximately 9.8 nA/mM), which is nearly three (≈2.8) times as the sensitivity of sensor when only coenzymes catalyzing the reaction. The curve also have a good linearity when glucose concentration range is from 0.5 to 38 mM. Fig. 6 shows the difference of the single GOD with glucose or catalase with H2 O2 and the effect of gold nanoparticles mixed to GOD and catalase with glucose or H2 O2 .
5. Discussion and conclusion This novel sensor has large contact surface area between the microdialysis membrane and the Au IDA in the solution of the flow chambers, which increases the number of reacting molecula and decreases the molecular diffusing time. In our glucose sensor system, two microdialysis membranes are applied to microdialysis probe and “sandwich” structure sensor. This can prevent electrodes from being insulated and clogged, and it also provides enough time for glucose molecule traversing the microdialysis membrane to establish a dynamic balance. It is easy to replace the inactive enzyme solution and the interval pump scheme can well deal with the problem of glucose depletion region around the measur-
261
ing site. From the point of view of reusability, this kind of sensor has a long lifespan with continuous supplementary enzyme solution. In addition, this microdialysis-based glucose sensor is highly biocompatibility. In order to solve the problem of the stability and activity of aqueous enzymes, aqueous colloidal gold nanoparticles were mixed with aqueous coenzymes solution. To the best of our knowledge, this is the first demonstration that aqueous colloidal gold nanoparticles enhance the activity of aqueous glucose oxidase enzyme and catalase enzyme. Because gold nanoparticles also can adsorb and agglomerate enzyme molecules, it can stabilize and “immobilize” the enzymes, which improve the stability of aqueous enzymes. During the procedure of our 2-month continuous determinations, the results of experiments have confirmed it. It is worth mentioning that the sensitivity of sensor would not be improved if colloidal gold solution were perfused after coenzymes solution was perfused. Diameter and quantity of colloidal gold, pH value, time for mixing colloidal gold with enzymes and for placing mixture, etc. are the factors, which decide the result of experiments. In the further work and research, the mechanism of gold nanoparticles enhancing the sensitivity of biosensor will be studied through realizing the direct electrons transfer between the enzyme activity center and microelectrode. And more experiments will be done to test the mechanism hypothesis. This system is being developed in the laboratory and the present experiments is in vitro, the preliminary research work demonstrates that it is useful for monitoring diabetes in vivo and it has a good prospect when miniaturized into portable device.
Acknowledgements This project is financially supported by National Natural Science Foundation and Zhejiang Province Natural Science Foundation.
References [1] W. Kerner, M. Kiwit, B. Linke, et al., The function of a H2 O2 detecting electroenzymatic glucose electrode is markedly impaired in human sub-cutaneous tissue and plasma, Biosens. Bioelectron. (1993) 473–482. [2] Y. Hashiguchi, M. Sakakida, K. Nishida, et al., Development of a miniaturized glucose monitoring system by combining a needle-type glucose sensor with microdialysis sampling method: long-term subcutaneous tissue glucose monitoring in ambulatory diabetic patients, Diabetes Care (1994) 387–396. [3] H. Hinkers, C. Dumschat, R. Sundermeier, et al., Microdialysis system for continuous glucose monitoring, in: Proceedings of the 8th International Conference on Solid-State Sensors and Actuators, Stockholm, Sweden, 1995, pp. 470–473. [4] E.F. Pfeiffer, C. Meyerhoff, F. Bischof, et al., On line continuous monitoring of subcutaneous tissue glucose is feasible by combining
262
[5]
[6]
[7]
[8]
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Biographies Min Pan: lecture of BME, Zhejiang University. Graduated from Zhejiang University, PR China, and obtained the BS and master’s degree of BME in 1992 and 2000, respectively. As visiting researcher, he researched in
USA at 1995, and in Pisa University from 1999 to 2000. Now he is teaching and researching in the Biosensor National Special Lab, Zhejiang University. His interest fields include: biosensor and chemical sensor, transducer and system integration. Xishan Guo: PhD candidate, senior research assistant of Biosensor National Special Lab, PR China, he received the bachelor degree and master degree in biomedical engineering, respectively, in 1999 and in 2002 from the department of biomedical engineering, Zhejiang University, PR China. He researched at “E. Piaggio” of Pisa University from October 2002 to February 2003. His research direction and interest mainly include biosensor, electronic nose and electronic tongue, protein biochip, MEMS, etc. Yuquan Chen: professor of BME, director of Biosensor National Special Lab, Zhejiang University. He obtained the master degree in Zhejiang University, 1982. From 1984 to 1987, he researched in the Case Western Reserve University, USA. From 1999 to 2000, he researched at Department of Chemistry, Harvard University and his research field includes biosensor, chemical sensor, signal measurement and analysis, micro structure, etc. Cai Qiang: PhD student of BME, Zhejiang University. He was born in 1974, and get B.S. and M.S. in Zhejiang University at 1996, 1999 respectively. His interested field includes: biosensor, chemical sensor and intelligent system. Li Guang: received his Bachelor and Master Degrees of BME at Zhejiang University, China in 1987 and 1991, respectively. He got his PhD degree of BME at Imperial College of Science, Technology and Medicine, London, UK in 1998. After his PhD study, he used to work at University of Glasgow and Moor Instruments Ltd, UK for three years. He is current a professor of Biomedical Engineering at Zhejiang University. His research interests include biosensors, biomedical instruments and neuroiformatics.