In the Laboratory
Application of a Datalogger in Biosensing: A Glucose Biosensor Martin M. F. Choi* and Pui Shan Wong Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, PRC; *
[email protected] A biosensor is normally defined as a device incorporating a biological molecular recognition component connected to a transducer that can output an electronic signal proportional to the concentration of the analyte being sensed (1). The high selectivity in biosensors provided by the biological recognition systems including antibodies, enzymes, nucleic acids, receptors, and cells has been used to detect important biological molecules such as antigens, nucleic acids, amino acids, creatinine, urea, and glucose. Enzymes are biological recognition molecules commonly employed in research and development because most chemical reactions in living systems are catalyzed by enzymes. Enzymes are often immobilized on solid substrates so that they can be reused. Methods for enzyme immobilization include physisorption, covalent attachment, and entrapment. This article describes how an eggshell membrane can be employed as a platform for the immobilization of glucose oxidase. The shelf-life of the immobilized enzyme is long and the eggshell membrane has excellent permeability to gases and water. This makes it an ideal bioplatform for enzyme immobilization. Glucose is a major component of animal and plant carbohydrates. Quantitative determination of glucose is of paramount importance in biochemistry, clinical chemistry, and food analysis. Techniques such as spectrophotometry (2– 7 ) and amperometry (8–10) for glucose determination have been studied. Most often glucose oxidase is used to catalyze the oxidation of glucose by oxygen to produce gluconic acid and hydrogen peroxide (8). D-glucose
glucose oxidase
+ O2 + H2O → D-gluconic acid + H2O2
The application of a datalogger (a computer interfaced to one or more sensors) in biology, chemistry, and physics laboratories in Hong Kong schools is becoming popular. The major advantage of a datalogger is that students can simultaneously monitor, in real time, system parameters such as temperature, electric current, electric potential, pressure, light intensity, pH, and oxygen concentration. We have reported the successful application of an oxygen sensor in conjunction with a datalogger in monitoring the liberation of dissolved oxygen in the photosynthesis of seaweed (11). The concentration of glucose in a sample can be indirectly determined by monitoring either the depletion of oxygen or the increase in hydrogen peroxide concentration. In this work we combine our novel immobilization technique with an oxygen sensor and a datalogger to demonstrate the fabrication of a glucose biosensor for the analysis of glucose in some beverage samples. Glucose oxidase was immobilized on the surface of a fresh eggshell membrane and the glucose oxidase– immobilized eggshell membrane was mounted onto an oxygen electrode. The oxygen electrode was then placed in contact with a sample solution for measurement of glucose concentration. The detection of glucose is based on the depletion of the dissolved oxygen in the sample solution. When glucose is present 982
Figure 1. Top: A glucose oxidase–immobilized eggshell membrane covering an oxygen electrode. Bottom: Experimental setup for the glucose biosensor. 1, glucose oxidase–immobilized eggshell membrane; 2, oxygen electrode; 3, sample in a 50-mL conical flask; 4, ScienceWorkshop 500 interface; 5, notebook PC.
in the sample solution, it will be enzymatically oxidized by the dissolved oxygen with a concomitant decrease in the dissolved oxygen concentration in the sample solution (see the previous chemical equation). The proposed method is simple, safe, and convenient to use. Experimental Procedure The experimental setup is illustrated in Figure 1. A fresh egg was purchased from a local market. The membrane was carefully peeled from the broken eggshell after the albumen and yolk had been removed. It was cleaned with a copious volume of deionized water. The membrane was placed in a clean watch glass and cut into a circle ca. 2 cm in diameter. Two hundred microliters of a solution of glucose oxidase1 (0.8% w/v) in phosphate2 buffer (25 mM) at pH 7.0 was added. After 20 min, 10 µL of 25% (w/w) glutaraldehyde solution3 as a cross-linking agent was dropped onto the surface of the membrane and left to stand for 5 min. A glass rod was gently used to spread the glutaraldehyde solution thoroughly on the membrane surface. The membrane was then immersed
Journal of Chemical Education • Vol. 79 No. 8 August 2002 • JChemEd.chem.wisc.edu
In the Laboratory
Decrease in Oxygen Level (ppm)
Dissolved Oxygen (ppm)
1.4 8
4 5 6
6
7 8
4
0
1
2 3
2
0 0
200
400
600
800
1.2
y = 1.1989x + 0.0072 1.0 0.8 0.6 0.4 0.2 0.0
1000
0
Time / s Figure 2. The dissolved oxygen content of a phosphate buffer (pH 7.0) after each succesive addition of various volumes of glucose standard (0.5 M) and samples. (0) No standard or glucose solution was added; (1) 10 µL; (2) 20 µL; (3) 50 µL; (4) 80 µL; (5) 100 µL of glucose standard were added; 50 µL of (6) sample 1; (7) sample 2; and (8) sample 3 were added.
R 2 = .9991
0.2
0.4
0.6
0.8
1.0
Concentration of glucose / (mmol/L) Figure 3. Calibration plot of the decrease in the oxygen level in the solution against concentration of glucose.
Table 1. Concentration of Glucose in Some Commercial Beverages
in and washed with a pH 7.0 phosphate buffer for 5 min. After washing, the glucose oxidase–immobilized eggshell membrane was stored in a pH 7.0 phosphate buffer at 4 °C until further use. The glucose oxidase–immobilized eggshell membrane was positioned on the surface of a Pasco CI-6542 oxygen sensor4 and kept in a steady position by an O-ring (Figure 1, upper panel). The electrode was immersed into a stirred 50-mL phosphate buffer solution (pH 7.0). Various volumes (10– 100 µL) of standard (0.5 M) or sample glucose solution5 were injected into the phosphate buffer with the use of a 0.2-mL syringe. The dissolved oxygen signal was captured at a sampling rate of 10 per second and processed by a datalogger system consisting of a ScienceWorkshop 500 interface, serial cables, a power supply, and control software.4 The data were logged in a notebook PC for real-time display and processing as shown in the lower panel of Figure 1. Hazards As glutaraldehyde is irritating and corrosive, students must wear splash goggles and gloves. Results and Discussion The oxygen electrode acting as an oxygen transducer was employed to measure the rate of oxygen consumption in the enzymatic oxidation of glucose. The analytical signal of the glucose biosensor is the decrease in the dissolved oxygen content upon exposure to glucose solution. A typical response curve of the glucose biosensor is shown in Figure 2. The decrease in the oxygen level was found to be proportional to the glucose concentration. A linear calibration curve plotting the decrease in the oxygen level against the concentration of glucose is shown in Figure 3. The glucose concentration of some commercial beverage samples was also determined by our glucose biosensor. The results are displayed in Table 1. All the samples except Diet Sprite contained glucose, in various concentrations. The use of a glucose oxidase immobilized–eggshell membrane, an oxygen electrode, and a datalogger provides a convenient and simple method for glucose determination. In the
Beverage
Concentration of Glucose/M
Vita lemon tea
0.305
Vita guava juice
0.0930
Sprite
0.0700
Lucozade
0.246
Diet Sprite
not detected
experiment described here we can introduce students to the concept of biosensor technology. The glucose biosensor is easy to fabricate and can be used repeatedly for at least several months. Furthermore, this experiment can readily be modified to design other enzyme-based biosensors associated with enzymatic reactions involving oxygen as a reactant or product. For instance, catalase can be immobilized on an eggshell membrane for the determination of hydrogen peroxide. catalase
2H2O2 → O2 + 2H2O The increase in the oxygen level can be simply monitored by an oxygen electrode and a datalogger. Acknowledgments We would like to express our sincere thanks to Raymond W.-H. Fong of the Curriculum Development Institute of the Education Department, Hong Kong SAR, for the loan of the ScienceWorkshop system and a notebook PC. We also express our gratitude to the editor and the reviewers for valuable suggestions on the manuscript. Notes 1. Glucose oxidase (EC 1.1.3.4 from Aspergillus niger) with a specific activity of 25,000 units per gram of solid was obtained from Sigma (St. Louis, MO). 2. Monosodium dihydrogen phosphate and disodium hydrogen phosphate were from Farco Chemical Supplies (Beijing). 3. Glutaraldehyde solution, 50 wt % in water, was purchased from Aldrich (Milwaukee, WI).
JChemEd.chem.wisc.edu • Vol. 79 No. 8 August 2002 • Journal of Chemical Education
983
In the Laboratory 4. Pasco CI-6542 oxygen sensor and ScienceWorkshop 500 interface were purchased from Pasco Scientific, Roseville, CA (http:// www.pasco.com). The total cost is approximately $720 U.S. Alternative sources for oxygen probes and their accessories are Vernier Software & Technology, Beaverton, OR (http://www.vernier. com) and PP Systems, Haverhill, MA(http://www.ppsystems.com). 5. β-D-Glucose was from Acros Organics (Geel, Belgium).
Literature Cited 1. Leech, D. Chem. Soc. Rev. 1994, 23, 205. 2. Toren, E. C. Jr. J. Chem. Educ. 1967, 44, 172. 3. Daines, T. L.; Morse, K. W. J. Chem. Educ. 1976, 53, 126.
984
4. Bateman, R. C. Jr.; Evans, J. A. J. Chem. Educ. 1995, 72, A240. 5. Mullis, T. C.; Winge, J. T.; Deal, S. T. J. Chem. Educ. 1999, 76, 1711. 6. Edmiston, P. L.; Williams, T. R. J. Chem. Educ. 2000, 77, 377. 7. Vasilarou, A.-M. G.; Georgiou, C. A. J. Chem. Educ. 2000, 77, 1327. 8. Sittampalam, G.; Wilson, G. S. J. Chem. Educ. 1982, 59, 70. 9. Wang, J.; Maccà, C. J. Chem. Educ. 1996, 73, 797. 10. Sadik, O. A.; Brenda, S.; Joasil, P.; Lord, J. J. Chem. Educ. 1999, 76, 967. 11. Choi, M. M. F.; Wong, P. S.; Yiu, T. P. J. Chem. Educ. 2002, 79, 980.
Journal of Chemical Education • Vol. 79 No. 8 August 2002 • JChemEd.chem.wisc.edu
