In the Laboratory
Bioanalytical Experiments for the Undergraduate Laboratory: Monitoring Glucose in Sports Drinks
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J. Justin Gooding,* Wenrong Yang, and Manihar Situmorang School of Chemistry, The University of New South Wales, Sydney, NSW 2052, Australia; *
[email protected] At the University of New South Wales, students in the final year are offered an optional subject called Chemistry in Biological Systems. This course is designed to introduce our students to the biological emphasis in modern chemistry, whether that be using biological molecules for synthesis or analysis, designing molecules that mimic the function of biological molecules, or synthesizing biologically active molecules. Part of the course involves the application of biological molecules in analytical chemistry, so-called bioanalytical chemistry. The students taking this course have already completed their final-year analytical chemistry course and are familiar with many traditional methods of conducting quantitative analysis. The goal of bioanalytical chemistry is to exploit the extraordinary specificity of some biological molecules for the analysis of target molecules by linking the biorecognition event with some method of signal transduction. This is what is achieved with bioassays and biosensors. The great advantage of bioanalytical chemistry is that, in some cases, the specificity of the biorecognition molecule allows the analysis of complex samples, such as body fluids and food, with little sample preparation. The teaching of bioanalytical chemistry requires appropriate laboratory experiments that illustrate the ease of conducting analyses in complex samples as well as some of the drawbacks of such techniques. However, there is currently a scarcity of literature on bioanalytical experiments for the undergraduate laboratory class. Most of those described either have been immunoassay experiments (1, 2) or have outlined the use of enzyme electrodes (3–8). Surprisingly, despite some soluble enzyme assay experiments with a biochemical focus (see Pope et al. [9] and references therein), we are unaware of any undergraduate experiments employing solution enzyme assays for analytical purposes. This paper presents two complementary bioanalytical experiments that we have developed for our final-year undergraduate students. Each experiment can be completed in a three-hour laboratory class. The first involves the use of a solution-based enzyme assay for the analysis of glucose concentrations in sports drinks. The second analyses the same solutions using an enzyme electrode. Sports drinks were chosen as the unknown because the labelling indicated the presence of more than one sugar (usually sucrose and glucose) and because their high media profile stimulated the students’ interest in the analyses to be performed. Both experiments rely on the ability of glucose oxidase (GOx) to react with β-D-glucose with a high degree of specificity. We emphasize to the students that GOx will react almost exclusively with β-D-glucose even in the presence of the many other compounds in the drink.
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Experiment 1. Solution Enzyme Assay for Glucose The enzyme assay used in this experiment is a variant of the standard assay for the determination of glucose oxidase activity (10). The assay involves coupling the oxidation of β-D-glucose by GOx with a second enzyme reaction involving horseradish peroxidase (HRP) as outlined below: GOx
β-D-glucose + O2 → β-D-gluconolactone + H2O2 (1) HRP
H2O2 + ferrocyanide → H2O + ferricyanide
(2)
The extent of reaction 1, and hence the concentration of glucose, is determined by spectroscopically monitoring the production of ferricyanide in the second enzyme reaction. The use of ferrocyanide as the chromogenic species is an important variation from the usual glucose assay in which the highly carcinogenic o-dianisidine is used. Ferrocyanide is safer, cheaper, and more readily available. The only equipment required for the experiment is a UV–vis spectrophotometer and cuvettes, micropipets, and volumetric flasks. The most expensive item in the whole experiment is the enzymes. In the experiment students establish a calibration curve between 0 and 0.6 mM glucose. Each group of students then conducts triplicate measurements on two unknowns, one a sports drink and the other an ordinary soft drink. In analyzing the unknown sports drinks and soft drinks, students are expected to find the appropriate dilution values. The fact that the glucose levels in high-energy sports drinks are not necessarily higher than those in ordinary soft drinks certainly stimulates the students’ interest. The students investigate the influence of possible interferences by comparing the results obtained from the calibration curve with results from an analysis conducted using the standard addition method. This shows that there are no significant interferences for the drinks used. The calibration curves from all the groups are combined to give the students a measure of the reproducibility. We have found the between-group variability in calibration curves to be less than 10%. This good between-group agreement highlights the robustness of the assay for teaching purposes. When the same students repeat the analyses, the within-group relative standard deviation is about 3%. Before conducting these experiments, the students are made aware through lectures of the potential advantages of bioanalytical methods for reducing the complexity of preparation prior to analysis. The solution assay is an excellent experiment for highlighting this advantage, as it is easy to perform and highly reliable. From experimental work in previous courses the students are familiar with more complex
Journal of Chemical Education • Vol. 78 No. 6 June 2001 • JChemEd.chem.wisc.edu
In the Laboratory
Experiment 2. Enzyme Biosensor The solution assay experiment is followed in the next week by the fabrication of an enzyme electrode. The enzyme electrode uses only a single enzyme, glucose oxidase. The concentration of glucose in the sample is determined electrochemically via the oxidation of H2O2 produced in reaction 1 above. To do this, the GOx is simply immobilized onto a platinum electrode by entrapment behind a dialysis membrane. The specialized equipment required for this experiment is a potentiostat with a platinum working electrode and the usual reference and auxiliary electrodes. The time for this experiment is not as generous as that for the solution assay, but the experiment can still be easily performed within three hours provided the enzyme electrodes are prepared as soon as the students start the class. After the enzyme electrode has been prepared, it is preconditioned in a buffer solution. The students then record a calibration curve by adding aliquots from a glucose stock solution until the onset of saturation. A representative calibration curve showing the saturation of the enzyme electrode is shown in Figure 1. Before the unknowns are analyzed, the enzyme electrode must be reconditioned to remove any glucose remaining within the enzyme layer. The sports and soft drinks are then analyzed in triplicate and the glucose concentration is determined using the calibration curve. Analysis of the sports drinks using the calibrated enzyme electrodes usually gives a slightly higher glucose concentration than the solution assay. The higher glucose reading is due to interference from other species in the sports drinks that can be oxidized directly at the electrode. By also performing the analysis using the standard addition method students observed the extent of these matrix effects. The standard addition method gives the same results as the solution assay. The triplicate analysis of the unknown gives the students an indication of the repeatability of the analysis for a calibrated enzyme electrode, which is typically less than 5% RSD (see Fig. 1). The simple but somewhat crude way of preparing the enzyme electrodes was originally chosen for its ease and speed. However, combining the calibration curves recorded by different groups gave the students a good indication of the between-group reproducibility of this method of fabricating enzyme electrodes (represented in Fig. 1 by the upper and lower lines). The large variability in the enzyme electrodes made by different groups serves a powerful educational purpose, emphasizing to the students the difficulty in manufacturing many devices without the need to individually calibrate each one. The large variability in response obtained with this method of fabricating enzyme electrodes does raise the question of whether a more reproducible method would serve the experiment better. Data on the reproducibility of enzyme electrodes fabricated with different methods of enzyme immobilization is scarce. However, a review by Hall et al. (11) indicates that very few methods will give lower than 10% variability between biosensors. Even commercial home glucose
meters do not achieve the 15% accuracy that the American Diabetes Association would like (12). Therefore, while more reproducible methods of fabricating the enzyme electrode exist, we feel that the advantages of the dialysis membrane approach in speed, simplicity, and reliability outweigh any small gains in reproducibility gains. We do, however, believe that it is imperative that the laboratory be supplemented by lectures that teach students about the effect of fabrication parameters on enzyme electrode performance and thus the importance of being able to control the enzyme immobilization (13, 14). Hazards This experiment contains no significant hazards. Discussion The advantage of doing both experiments is that it allows students to compare two bioanalytical techniques and to consider the advantages and disadvantages of each. The students are asked to compare the analytical methods with regard to ease of use, sensitivity, measurable concentration range, influence of the co-substrate, and possible errors and interferences. For the solution assay, the measurable concentration range is determined by a Lineweaver–Burk plot to determine the Michaelis constant, Km. The apparent Km for the enzyme electrode is also estimated from the calibration curve. The difference in the Km values emphasizes the partitioning effect of the dialysis membrane in the enzyme electrode. The student response to both experiments has been very positive. The enzyme electrode experiment gets a particularly favorable response because students feel they are conducting modern progressive chemistry. The experiments make clear some of the issues in bioanalytical chemistry, as was reflected in the students’ performance on examinations and assignments. We have found the experiments illustrate not only the potential advantages of bioanalytical chemistry for analyzing complex samples, but also important issues such as the sensitivity of
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sample preparation steps required for analysis of other foodstuffs, such as the analysis of calcium in milk, and therefore express surprise at how easy these enzyme assays are to perform.
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[Glucose] / (mmol/L) Figure 1. A typical calibration curve obtained with the enzyme electrode. The background solution was 0.05 M KCl and 0.05 M phosphate buffer at pH 7.0. The error bars give the 95% confidence limits for the repeatability of a single enzyme electrode. The upper and lower lines reflect the range of responses from enzyme electrodes fabricated by 10 different groups of students.
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the biological molecules, the role of mass transport in dictating the response, and the difficulties in taking an analytical technique from experienced operators in a laboratory to the lay person working in the field. WSupplemental
Material
Notes for the instructor and detailed background information and instructions for students are available in this issue of JCE Online. Literature Cited 1. Inda, L. A.; Razquin, P.; Pampreave, F.; Alava, M. A.; Calvo, M. J. Chem. Educ. 1998, 75, 1618. 2. Anderson, G. L.; McNellis, L. A. J. Chem. Educ. 1998, 75, 1275. 3. Martinez-Fabregas, E.; Alegret, S. J. Chem. Educ. 1994, 71, A67.
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4. Riechel, T. L. J. Chem. Educ. 1984, 61, 640. 5. Sadik, O. A.; Brenda, S.; Joasil, P.; Lord, J. J. Chem. Educ. 1999, 76, 967. 6. Sittampalam, G.; Wilson, G. S. J. Chem. Educ. 1982, 59, 70. 7. Wang, J.; Macca, C. J. Chem. Educ. 1996, 73, 797. 8. Eggins, B. R.; McAteer, G. Educ. Chem. 1997, 20. 9. Pope, S. R.; Tolleson, T. D.; Williams, R. J.; Underhill, R. D.; Deal, S. T. J. Chem. Educ. 1998, 75, 761. 10. Methods in Enzymatic Assays; Bergmeyer, H. U., Ed.; Verlag Chemie: Weinheim, 1974; Vol. 3. 11. Hall, E. A. H.; Gooding, J. J.; Hall, C. E. Mikrochim. Acta 1995, 121, 119. 12. Henning, T. P.; Cunningham, D. D. In Commercial Biosensors; Ramsey, G., Ed.; Wiley: New York, 1998; Vol. 148, p 304. 13. Gooding, J. J.; Hall, E. A. H. Electroanalysis 1996, 8, 407. 14. Gooding, J. J.; Hall, E. A. H.; Hibbert, D. B. Electroanalysis 1998, 10, 1130.
Journal of Chemical Education • Vol. 78 No. 6 June 2001 • JChemEd.chem.wisc.edu
